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The molecular chaperone calnexin facilitates folding and assembly of class I histocompatibility molecules.

The molecular chaperone calnexin facilitates folding and assembly of class I histocompatibility... The EMBO Journal vol.15 no.7 pp.1495-1506, 1996 chaperone calnexin facilitates folding The molecular and assembly of class histocompatibility molecules that calnexin that are unable to assemble, suggesting may Aikaterini Vassilakos, Myrna F.Cohen-Doyle, ER control machinery that be a component of the quality Per A.Peterson", Michael R.Jackson1 and export of incompletely folded or prevents premature David B.Williams2 unassembled proteins to the Golgi apparatus (Helenius, of of Toronto, Toronto, Department Biochemistry, University in 1995). Indeed, recent studies 1994; reviewed Williams, M5S Canada and 1R.W.Johnson Pharmaceutical Research Ontario 1A8, the export of have demonstrated that calnexin impedes Institute. La CA 92121. USA Jolla. of the class I histo- incompletely assembled subunits 2Corresponding author and the T cell from the compatibility molecule receptor and ER (Jackson et al., 1994; Rajagopalan Brenner, 1994; a membrane of the Calnexin, protein endoplasmic Rajagopalan et al., 1994). reticulum, is generally thought to function as a molecu- Unlike other molecular chaperones, calnexin displays a lar chaperone, based on indirect or correlative marked preference for Asn-linked glycoproteins. Treat- evidence. To examine calnexin's functions more ment of cultured cells with tunicamycin to block glycosyla- directly, we reconstituted the assembly of class I histo- tion (Ou et al., 1993) or with the oligosaccharide compatibility molecules in the absence or presence of processing inhibitors castanospermine and 1-deoxynojiri- calnexin in Drosophila melanogaster cells. Calnexin mycin, prevents calnexin binding to most newly synthe- enhanced the assembly of class I heavy chains with sized (Hammond and Helenius, 1994; Hammond proteins as much as 5-fold. The f2-microglobulin improved et al., 1994). The latter compounds inhibit glucosidases I assembly appeared largely due to more efficient folding remove glucose residues from the and II, enzymes that of heavy chains, as evidenced by increased reactivity that is attached to oligosaccharide Glc3Man9GlcNAc2 with a conformation-sensitive monoclonal antibody and chains as enter the ER. These nascent polypeptide they by a reduction in the level of aggregates. Similar that calnexin and other observations led to the suggestion findings were obtained in mouse or human cells when on specifically recognizes the Glc structure IMan9GlcNAc2 the interaction of calnexin with class I heavy chains glycoproteins (Hammond et al., 1994). Proof that calnexin by treatment with the oligosaccharide was prevented functions as a lectin was subsequently provided by demon- processing inhibitor castanospermine. The ability of ER strating direct binding of the luminal domain of folding and to prevent calnexin to facilitate heavy chain calnexin to the Glc oligosaccharide (Ware MangGlcNAc2 the formation of aggregates provides compelling evid- et al., 1995). However, there are many examples of ence that calnexin functions as a bona fide molecular calnexin binding to proteins that lack Asn-linked oligo- chaperone. saccharides, suggesting that calnexin also recognizes the Keywords: calnexin/class I histocompatibility molecules/ polypeptide portion of incompletely folded proteins endoplasmic reticulumlmolecular chaperones/protein (Kearse et al., 1994; Loo and Clarke, 1994; Rajagopalan folding et al., 1994; Arunachalam and Cresswell, 1995; Carreno et al., 1995). Further evidence supporting polypeptide interactions was obtained by digesting complexes of calnexin and with endo- newly synthesized glycoproteins Introduction glycosidase H. All oligosaccharides could be removed by the without dissociation of the enzyme deglycosylated Proteins that are translocated into the endoplasmic reticu- from calnexin and Cresswell, 1995; proteins (Arunachalam lum (ER) fold and undergo subunit assembly with the Ware et al., 1995; Zhang et al., 1995). of a diverse of soluble assistance group folding factors, There is intense interest in establishing whether calnexin disulfide iso- including protein isomerase, peptidylprolyl indeed functions as a molecular chaperone that facilitates merase and molecular of the hsp7O (BiP) chaperones folding and, if so, how it utilizes its apparent dual and families in and protein hsp9O (GRP94) (reviewed Gething Hammond mode of binding to effect its function. Recently, Helenius et In the Sambrook, 1992; al., 1992). addition, to and Helenius castanospermine prevent ER contains a putative molecular chaperone known as (1994) employed stomatitis virus G a I membrane the of calnexin to vesicular calnexin. Calnexin is non-glycosylated, Type binding maturation of G homo- glycoprotein and found that glycoprotein protein that contains a 461 residue luminal domain form was to calreticulin et to a fully disulfide-bonded substantially logous (Wada al., 1991). result is of a binds to a diverse of both impaired. Although this suggestive chaperone Calnexin transiently array function for caution should be exercised in and soluble their calnexin, membrane proteins, shortly following the effects of this inhibitor since it have translocation into the and its dissociation can interpreting may ER, usually effects on other ER and be correlated with some in pleiotropic chaperones folding stage polypeptide folding interacts in For which shares extensive or subunit calnexin factors. example, calreticulin, assembly. Furthermore, or with subunits with the luminal domain of fashion with misfolded sequence identity calnexin, prolonged proteins Press C Oxford University A.Vassilakos et aL binds transiently to immature forms of myeloperoxidase Kb Kb + 32m 32m (Nauseef et al., 1995) as well as other glycoproteins calnexin (Peterson et al., 1995). This chaperone-like binding is KO r Endo H also inhibited by castanospermine. Tb4w4mAD,-q ol0 ____\__ Kb Endo H We have been studying calnexin functions during the 3rnm associated 4, biogenesis of mouse class I histocompatibility molecules 4w am (Degen and Williams, 1991; Degen et al., 1992; Margolese et al., 1993). - Class I molecules are cell surface glyco- m __ga urlclssembfie-~a P 20 40 80 P 20 40 80 chase proteins that are responsible for presenting cytosolically min.) generated peptides to B cytotoxic T lymphocytes (Germain and Margulies, 1993). They consist of a highly poly- Kb 32m + Kb t2m morphic calnexin transmembrane heavy chain that possesses 1-3 120 l Asn-linked .._, oligosaccharides, a soluble, non-glycosylated '001 subunit termed and a peptide 02-microglobulin (02m), ligand that is 80 8-10 residues in length. Immediately follow- 0FU T ing synthesis, I class heavy chains bind quantitatively to calnexin and assembly with occurs shortly thereafter P2m (Degen and Williams, 1991). The heavy hetero- chain-P2m dimers then associate with the TAP peptide transporter -i that transports cytosolic peptides into the ER lumen .-. et 20 (Ortmann al., 1994; Suh et 40 60 80 al., 1994). Subsequently, 20 40 60 80 peptide binding, release from TAP and dissociation from chase min) chase (min) calnexin all appear to occur at about the same time (Degen u92m-assocated -A-- 32pm-associated et et al., 1992; Suh al., 1994). _--- unassemnbled unassembled We I previously expressed mouse class chains heavy and ,B2m in cells. Fig. 1. Effects of calnexin on Kb assembly with f32m in Drosophila melanogaster These cells Drosophila cells. (A) Drosophila cells expressing Kb and f2m in the absence (left are functionally calnexin deficient since no endogenous panels) or presence (right panels) of mammalian calnexin were calnexin could be in homolog detected association with radiolabeled with [35S]Met for 5 min and subsequently incubated in the mouse chains. heavy Upon co-expressing mammalian media containing excess unlabeled Met for the indicated times. Cells calnexin we demonstrated a role for calnexin in were the lysed in digitonin lysis buffer and Kb molecules were isolated with antibodies total recognizing (anti-8 antiserum), 12m-associated intracellular retention and stabilization of class I assembly (mAb unassembled 20-8-4S), or (anti-HC antiserum) heavy chains. intermediates et An obvious (Jackson al., 1994). advantage Following digestion with endoglycosidase H (endo H), proteins were of this system is that it does not on non- rely potentially SDS-PAGE. analyzed by reducing Only the heavy chain of regions the specific chemical to a calnexin-deficient agents produce gels are shown. The mobilities of endo H-sensitive (endo HS) and endo H-resistant (endo Kb environment. In this we examined the effects of Hr) heavy chains are indicated on the top panel report only. The letter P signifies pulse-labeled samples. (B) The results from calnexin I on class heavy chain and subunit folding five independent experiments were densitometric quantified by analysis assembly using the Drosophila expression Further- system. of weakly exposed and the amounts fluorographs of f2m-associated we these data more, compared with the effects observed and unassembled heavy chains Hs and endo Hr (endo combined) were following treatment of mouse and human cells with expressed as a percentage of the total chains in heavy present the pulse samples. Data points mean values and error The represent bars reflect castanospermine. results indicated that calnexin does the range of the replicate samples. The data from the 20 min time indeed function as a molecular chaperone and is an points were excluded from the because were analysis they obtained important in the and component folding assembly pathway from only a single experiment. of class I molecules. left The panels). small population of Kb molecules that Results assembled was gradually transported out of the ER as Calnexin facilitates of class I molecules assessed by the maturation of assembly Asn-linked oligosaccharides in expressed Drosophila cells to a form resistant to with digestion endoglycosidase H Drosophila cells expressing either Kb or Db chains 80 Unassembled Kb chains heavy min). remained heavy (t1/2 and J2m in the or absence of at a presence co-expressed high level throughout the chase and were exported calnexin were subjected to a pulse-chase analysis to very slowly from the ER. No further assembly with f2m determine the kinetics and of of these was observed efficiency assembly beyond 80 min of chase (data not shown). mouse class I heavy chains with 2m. The results obtained In marked of contrast, co-expression calnexin resulted in for Kb are in depicted Figure IA. of heterodimer quantitative of Kb Efficiency assembly with f32m (Figure 1, right formation was assessed by comparing heavy-chain reac- panels). As a consequence of increased assembly more tivity to three antibodies: a mAb, an heterodimers were transported out of the ER but, consistent 02m-dependent that antibody recognizes total Kb with the heavy chains, and an findings of an earlier study (Jackson et al., 1994), antibody that recognizes unassembled Kb the heterodimers that molecules. The had been associated with calnexin results were quantified by densitometry and both were transported more ,B2m- slowly than heterodimers assembled associated and unassembled heavy chains were expressed in the absence of calnexin endo H resistance (t112 as a of total chains in percentage heavy the pulse 120 min). sample (Figure iB). In the absence of co-expressed calnexin, only Db heavy chains share 81% sequence identity with Kb ~20% of Kb chains assembled with heavy f32m (Figure 1, heavy chains, yet an of Db analysis assembly with ,2m 1496 Molecular chaperone functions of calnexin A in a 5 min pulse were present as a series of degradation DO ft2m Db 32m t cainexin products of faster mobility than the full-length Db heavy chain (Figure 2A, left panel, total). At this time point totai .32m-associated total B2m-associated little assembly with had occurred (Figure 2A, left P2m first two lanes). Degradation continued panel, compare and, to a lesser extent, included cainex r during chase incubations Db-,32m heterodimers. Co-expression of calnexin dramatically inhibited degradation (Figure 2A, right - _ ___ -_ -,irav OM 0 panel). The observation that a substantial proportion of 4- aLwe DD heavy chains was degraded very early after synthesis, before assembly, raised the possibility that a decreased Dr efficiency of assembly in the absence of calnexin could OF be masked by the rapid loss of unassembled molecules. To minimize degradation, Drosophila cells were incuba- ted with the membrane-permeable protease inhibitor, P 30 chase 2C LLnL, which slowed, but did not block, the degradation D' - O 2C P ',' rn Db in of calnexin. these of expressed the absence Under of the total Db heavy chains conditions, only ~60-70% DO Db 32m -calnexir R2M present in the pulse sample assembled with in the P2m 32m assoc totai 32m-assoc. total absence of calnexin (Figure 2B, left panel and 2C). The value of 60-70% assembly is most likely an overestimate D C maLe given that degradation was not completely blocked by LLnL treatment. This residual degradation appeared restricted to heavy chains that did not assemble since, at n ^ 20 20 P 1 I 20 chase rnir P ; 0 20 P the 10 and 20 min time points, the remaining heavy chains were f2m-associated and relatively stable (Figure 2B, compare total and lanes). Inefficient IP2m-associated assembly was due not to degradation simply limiting in the availability of heavy chains but rather, as detailed of next section, to the inability of a population heavy - calnexin --* In chains to fold correctly (see below). cells expressing ----}--U A - calirexr efficient with all of the Db calnexin, assembly was more e-< / 2'_. heavy chains forming heterodimers (Figure 2B and C). for Kb 1), the effect of calnexin As was observed (Figure on Db was manifested rapidly; as early as the assembly .--_ 2r 5 min pulse sample calnexin enhanced assembly by ~3-fold. A difference between these two class I chase (min) major isotypes is the fate of unassembled heavy chains in the Fig. 2. Effects of calnexin on Db assembly with in Drosophila P2m absence of calnexin. Db chains that do not assemble heavy cells. (A) Drosophila cells expressing Db and in the absence or P2m are whereas unassembled Kb heavy rapidly degraded of were radiolabeled with for 5 min and presence calnexin [35S]Met chains are stable and are largely retained intracellularly incubated in media containing unlabeled Met for the subsequently indicated times. Cells were lysed in digitonin lysis buffer and Db in a conformation that is not competent for binding P2m. molecules were isolated with antibodies recognizing total (mAb 28-14- 8S and anti-HC combined) or fB2m-associated (mAbs B22-249.Rl and facilitates of Db chains Calnexin folding heavy 27-11-13S combined) molecules. Proteins were analyzed by reducing in cells expressed Drosophila and SDS-PAGE and the mobilities of co-isolated calnexin, immature for the more efficient of class Db chains are indicated. One explanation assembly mature Db heavy chains, and degraded heavy of H mature Db chains In the absence endo digestion, heavy I molecules in the of calnexin is that calnexin presence to the more than their transported Golgi apparatus migrate rapidly of chains to a conformation that promotes folding heavy immature due to the of to precursors processing oligosaccharides For Db is competent for interaction with j32m. molecules, forms in cells. The smaller, complex Drosophila (B) experiment a folded an mAb (28-14-8S) exists that recognizes epitope in was except that cells were pre-incubated for performed (A) repeated chains. 40 and all in the of the protease in the a3 domain of free or 2m-associated heavy min, radiolabeled, chased, presence LLnL (100 lM, Sigma). (C) The results in (B) were inhibitor, this was used as an indication of Acquisition of epitope quantified by densitometry and the amount of 12m-associated heavy Db heavy chain folding. of the total chains at each time point was expressed as a percentage cells Db chains in Drosophila expressing only heavy in heavy chains present the pulse sample. the absence or of calnexin were to presence subjected immunoisolation followed pulse-chase radiolabeling by chains 3A and cells somewhat different results of total or 28-14-8S reactive in Drosophila yielded heavy (Figure of total chains occurred In the absence or of calnexin there degradation heavy (Figure 2A). presence B). Significant Db in the absence of calnexin to be of during the chase period although appeared quantitative assembly with P2m chase the characteristic intermediates seen in the total versus 20 min lanes). degradation (Figure 2A, compare not detected in these cells of calnexin there was considerable were However, in the absence previous experiments left since Db chains even in the that lacked degradation of heavy apparent pulse I2m (Figure 3A, panel). However, was it was of total chains labeled of the 28-14-8S sample. Typically, 20-40% heavy acquisition epitope very rapid, 1497 A.Vassilakos et al. Db + cainexin Calnexin prevents aggregation of Kb heavy chains Db 28-1 4-8S total 28-14-8S total expressed in Drosophila cells We next examined whether the dramatic effect of calnexin _- i nfl.. c<, :e x.r- on assembly of Kb heavy chains with (Figure 1) P2m could also be attributed in part to differences in the folding state of Kb heavy chains. It was not possible to monitor acquisition of a conformational epitope as was done for Db, because no conformation-specific mAbs for un- 46, p assembled Kb molecules exist. Instead, we took advantage P l IC 2r) i; ) a.2C 212C D 0 20- chase mir of the fact that Kb heavy chains not competent for assembly with were relatively stable in the absence of co- P2m expressed calnexin (Figure lA, left panel). This permitted an assessment of the aggregation state of Kb. 1 . Drosophila cells expressing Kb and ,B2m with or without P-----....... -, fj - ci lex calnexin were pulse radiolabeled and chased for 80 min, at which time assembly was complete and any remaining free heavy chains were not competent for further assembly (Figure 1). To determine if unassembled heavy chains existed in an aggregated state, isolated heavy chains were - -.-- x c a: r' .* subjected to non-reducing SDS-PAGE. Using an antiserum that recognizes all folding states of Kb, a high molecular weight aggregate was detected that barely entered a 7.5% resolving gel (Figure 4A, total, -DTT). The aggregate 0 1 c, 20 30 was stable to heating in SDS at 55°C but could be partially chase (minli dissociated upon heating at 70°C. Complete dissociation was effected only by heating at 70°C under reducing conditions (Figure 4A, +DTT). Thus, the aggregate Db in Fig. 3. Calnexin enhances folding of heavy chains expressed to contain chains associated Db appeared heavy through Drosophila cells. (A) Drosophila cells expressing heavy chain ± calnexin were to as described subjected pulse-chase radiolabeling non-covalent interactions. The relative both and disulfide in the legend to Figure 2A. Digitonin lysates were incubated with amount of was determined as aggregate densitometrically antibodies total Db chains 28-14-8S and anti- recognizing heavy (mAb a percentage of the total heavy chain recovered in both HC combined) or heavy chains with a folded a3 domain (mAb 28-14- free and aggregated forms see % (Figure 4A, aggregates). 8S) and isolated proteins were analyzed by reducing SDS-PAGE. The mobilities of the Db heavy chain and co-isolated calnexin are Importantly, there was a 3-fold increase in the percentage indicated. (B) The results in (A) were quantified by densitometry and of aggregates in cells lacking calnexin, with as much as the amount of 28-14-8S reactive heavy chains at each time point was 43% of Kb chains recovered in the form of heavy aggre- expressed as a percentage of the total heavy chains present in the gates. That the aggregates contained heavy chains that pulse sample. were incompetent for assembly with ,32m was confirmed by comparing total chains with assembled heavy Kb_t2m possible to observe the effects of calnexin on this process heterodimers recovered selectively with mAb 20-8-4S. even in the presence of degradation. Whereas 40% of the Heterodimers recovered with this mAb were found exclu- total Db chains heavy present in the pulse sample folded sively in a non-aggregated state (Figure 4A, P2m- into an mAb-reactive conformation in the absence of associated). 90% of when To confirm calnexin, heavy chains acquired the epitope that calnexin influences the extent of co-expressed with calnexin calnexin of Kb cell (Figure 3B). Again, aggregation heavy chains, lysates (radiolabeled exerted its effects at an early stage, the as above) were fractionated on 10-40% enhancing glycerol gradients efficiency of heavy-chain folding from 24-65% during prior to immunoisolation of total Kb molecules and analysis the 5 min pulse radiolabeling. Heavy chains that did not by reducing SDS-PAGE (Figure 4B and C). In cells fold into an mAb-reactive conformation in the absence of expressing calnexin, 97% of the Kb molecules that could calnexin were selectively degraded and by 20 min of be recovered from the gradient were present in fractions chains Kb chase, only heavy possessing the 28-14-8S epitope 1-3. Only 3% of molecules were recovered in denser remained (Figure 3A, left panel, compare total and 28- fractions (fractions 4-16). This was the same distribution 14-8S lanes). Degradation did not limit the opportunity as that observed for assembled Kb-,B2m heterodimers when for heavy chains to fold since after 10 min in the absence mAb 20-8-4S was used for the isolation (data not shown). of calnexin, only 40% of heavy chains had acquired the In the absence of calnexin, 78% of heavy chains that even unfolded epitope though heavy chains were available could be recovered from the gradient were present in (Figure 3A, left panel, compare 28-14-8S and total lanes). fractions 1-3 and a substantial proportion (22%) was Clearly, most heavy chains synthesized in the absence of found in the denser (4-16) fractions (Figure 4B and C). calnexin were incapable of correct folding. To confirm the Overall, findings suggest that one function of this conclusion, cells lacking calnexin were treated with calnexin is to prevent inappropriate molecular interactions LLnL to retard degradation and no increase in heavy- that lead to aggregation. The extent of aggregation assessed chain was observed folding (data not shown). Thus, by the two techniques (22-43%) was somewhat less than interaction with calnexin increases the efficiency of heavy- expected, based on the low efficiency of subunit assembly chain in cells calnexin folding. lacking (Figure 1). It is possible that 1498 functions of calnexin Molecular chaperone during cell lysis and immune isolation some non-covalent of calnexin with class I heavy chains in mouse cells. were disrupted, leading to an underestimation Although the potential existed for castanospermine to interactions the amount of aggregates. Alternatively, in addition to affect processes other than those mediated by calnexin, of there may have been significant amounts of validity of data obtained could be assessed in the aggregates, the of the results obtained using the Drosophila misfolded heavy-chain monomers that were incompetent context To confirm that castanospermine prevents calnexin for assembly. system. mouse EL4 cells were radio- binding to heavy chains, calnexin interaction presence or absence of the inhibitor and Castanospermine prevents labeled in the class I molecules in mouse cells or class I heavy chains were immunoisolated with then calnexin the results obtained in the Drosophila (Figure 5A). In the absence of castano- In addition to from cell lysates we wished to determine whether cal- treatment, anti-calnexin antibodies recovered expression system spermine in a similar manner in mouse cells where calnexin and a series of associated proteins, two of which nexin functions of class I calnexin interactions were originally electrophoretic mobilities as Kb and Db the details had the same established (Degen and Williams, 1991; Degen et al., heavy chains (Figure 5A, left panel, compare lane 1 with We used castanospermine to prevent the interaction lanes 2-4). Disruption of anti-calnexin immune complexes 1992). with SDS and re-isolation using antibodies specific for A I chains showed that both Kb and Db molecules '< "2'- - alex class heavy were present in complexes with calnexin (Figure 5A, left panel, lane 6). Castanospermine treatment resulted in a dramatic reduction in the amount of proteins co-immuno- -,. -.rz -:1,:, precipitating with calnexin and, in particular, the two bands corresponding to class I heavy chains were absent (Figure 5A, right panel, lane 1). Disruption of anti-calnexin immune complexes and re-isolation with anti-heavy chain I complexes Ab confirmed the absence of calnexin-class of class I heavy (Figure 5A, right panel, lane 6). Synthesis cells as chains was normal in castanospermine-treated isolation with anti-class I Ab evidenced by direct immune lanes 2-4 in both panels). (Figure 5A, compare inhibits assembly of class I Castanospermine molecules ab * * treated with or without castanospermine EL4 cells were the of class I assembly was determined by and efficiency immunoisolation of both total and heavy 02m-associated Castanospermine treatment resulted chains (Figure 5B). in a decrease in the assembly of both Kb and Db dramatic W. .. _m chains with Densitometric analysis revealed heavy P2m. whereas both Kb and Db molecules assembled almost that for Kb and 80% for Db) during quantitatively (100% of Kb chains in 4. Calnexin inhibits aggregation heavy Drosophila Fig. Kb and + calnexin were cells. (A) Drosophila cells expressing f2m for 10 chased for radiolabeled with [35S]Met min, subsequently in RIPA buffer. RIPA buffer to be 80 min, and then lysed lysis proved chains than digitonin more effective in solubilizing aggregated heavy were isolated with antibodies recognizing lysis buffer. Kb molecules either total chains antiserum and 20-8-4S mAb heavy (anti-8 or heterodimers (mAb 20-8-4S). Immune combined) .. Kb_P2m were incubated in SDS-PAGE sample buffer ± DTT at complexes or for 15 min and were analyzed by non-reducing 55C 70°C proteins of the Kb chain and 4. SDS-PAGE The mobilities heavy (7.5% gel). The of total heavy heavy chain aggregates are indicated. percentage ~-4--A - and is chains as was determined densitometrically present aggregates IF lanes. cells were * a included at the bottom of the gel (B) Drosophila as in Cell were applied to the top radiolabeled and lysed (A). lysates and the gradients were centrifuged for of a 10-40% glycerol gradient in an SW-41 rotor. Gradients were fractionated, 15 h at 34 000 r.p.m. from each fraction total Kb chains were heavy immunoprecipitated were with anti-8 antiserum, and isolated proteins analyzed by reducing that to the bottom SDS-PAGE. P fractions represent proteins pelleted the total of Kb chains of the T samples represent pool heavy gradient. immune of an amount of to obtained by precipitation equivalent lysate the The data from were by that loaded on gradient. (C) (B) quantified chain recovered in each and the relative amount of densitometry heavy of the total chain was as heavy applied fraction expressed percentage f-actior' jmber to the gradient. 1499 A.Vassilakos et al. min of chase under normal conditions, there was a 4- to - stlrnospirmire +castanospermine 5-fold decrease in assembly (25% for Kb and 15% for Db) when calnexin interactions were inhibited with castano- cal'fX spermine (Figure SC). Similar experiments were C. .:,: performed _. using murine MDAY-D2 cells that express class I mole- cules of d-haplotype. Castanospermine treatment resulted f.-'i in a 2-3-fold decrease in the assembly of Ld and Dd 'a_1 ) molecules as well (data not shown). As shown in it was Figure SB, apparent that the small of Kb and Db population heavy chains that assembled in B castanospermine-treated cells was transported more rapidly -,,::astano r-1 +castarnospernine soerm to the Golgi apparatus relative to assembled molecules in total associated 32r0.[at untreated cells endo H resistance = 20 min for Kb (t1/2 and 30 min for in Db treated cells compared with 30 min for Kb and >>30 min for Db in control cells). The formation of endo H-resistant molecules in castano- spermine-treated cells requires the action of a Golgi endo-mannosidase to convert glucosylated precursor oligo- saccharides to a truncated form that can be acted on by Golgi glycosyltransferases (Moore and Spiro, 1990). Since __b the endo-mannosidase may be inefficient (Moore and Spiro, 1992), ER to Golgi transport rates may be even more ; -1 :: l, F' r1 1 ; C 2W' chase8 ,:! c h ( rapid in the drug-treated cells. The enhanced ;s transport observed in the presence of castanospermine was con- sistent with our earlier findings in cells Drosophila where, in the absence of calnexin, heterodimers were transported more rapidly out of the ER than when calnexin was co- expressed (Jackson et al., 1994). /3'!IIZ/-------. C2b EL4 -castanospermYlinel Castanospermine impairs folding of class I heavy q ,.' ,/ '~~~~~K' chains In Drosophila cells lacking calnexin, the inefficient assem- | -castanosperrnine bly of class I molecules could be attributed largely to EL4 inefficient 20 folding of the heavy chain as evidenced by a reduction in a conformational epitope defined by mAb 0 20 30 28-14-8S Db) and increased (for by aggregation of heavy chase (min) chains To determine (for Kb). if a similar situation existed in mouse cells, 28-14-8S was in reactivity examined EL4 cells incubated in the absence or of presence castano- spermine. As shown in 6A and Figure B, the 28-14-8S Fig. 5. Effect of castanospermine on heavy in assembly epitope was acquired rapidly in untreated cells, being chain-P2m mouse cells. (A) Murine EL4 cells that express Kb, Db and f2m were present in 80% of total chains heavy following the 5 min 1 h in ± 1 pre-incubated for Met-free medium mM castanospermine. pulse and 100% between radiolabeling reaching 10 and Cells were subsequently radiolabeled in the presence of min 20 of chase. By contrast, only about 45% of total castanospermine with [35S]Met for 20 min and then were lysed in digitonin buffer. Calnexin-associated molecules were isolated lysis heavy chains acquired the epitope in castanospermine- with anti-calnexin antiserum Class I molecules were isolated (lane 1). treated cells. This result was remarkably similar to the with antibodies recognizing either Kb alone lane (anti-8 antiserum, 2), -2-fold reduction in the formation of the 28-14-8S epitope Db alone (mAb lane or both Kb and Db 28-14-8S, 3) (anti-HC observed in cells Drosophila lacking calnexin, further In antiserum, lane 4). lane 6, calnexin immunoprecipitates were to in supporting a role for calnexin in disrupted by heating 90°C 0.2% SDS/PBS and, facilitating heavy-chain following addition of a 10-fold excess of NP-40, reprecipitated with a folding. combination of anti-HC and anti-8, 28-14-8S antibodies (lane 6). Lane The reduction in heavy-chain folding as defined by the 5 is blank. The mobilities of Kb calnexin, and Db molecules are 28-14-8S epitope was considerably less than the 5-fold indicated. (B) EL4 cells were pre-incubated for I radiolabeled h, with reduction in Db assembly with f32m observed following [35S]Met for 5 min, and then chased for the indicated times + castanospermine. Cells were lysed in digitonin lysis buffer and Kb treatment castanospermine (Figure SC). If the reduced molecules were isolated with antibodies recognizing either total assembly was due primarily to the inability of heavy (anti-8) or r2m-associated (mAbs Y3 and B8-24-3 combined) heavy chains to acquire a conformation competent for association chains (top panel). Db molecules were isolated from replicate samples with f2m, then the 28-14-8S epitope was not an accurate using antibodies recognizing either total (mAb 28-14-8S and anti-HC combined) or f2m-associated (mAb B22-249-RI) heavy chains reflection of an assembly-competent conformation. To (bottom panel). Proteins were digested with endo H and analyzed by determine whether additional indications could be obtained reducing SDS-PAGE. The mobilities of the endo H-sensitive and for improper folding of Db heavy chains following castano- -resistant chains are heavy indicated. (C) The results in (B) were Db spermine treatment, molecules were isolated with mAb quantified and by densitometry the amount of heavy 2r2m-associated 28-14-8S from control chains at each time and drug-treated EL4 cell lysates point was expressed as a percentage of the total chains in heavy present the pulse sample. and were analyzed by non-reducing SDS-PAGE. Interest- 1500 Molecular chaperone functions of calnexin - castanospermine castanospermine -cas!anospermi7,e ,castanospermine non- non- 28-14-BS 28-14-8S ota total reducing reducing reducing reducing 46 Db Enao i-r 97 - hc dimers mm. - - DO Erac Hs -- .- -. 69 - P 2C g 'C 'C 20 2C chase 20 'C imimn 46-1 J hc monomers P 10 P 10 P P 10 10 chase min A-A----------- cs- Fig. 7. Effect of castanospermine treatment on the state aggregation of Db molecules. Mouse EL4 cells were radiolabeled + castanospermine as described for 6. At the Figure indicated chase times, aliquots of cells were lysed in buffer and Db molecules digitonin lysis were isolated with mAb 28-14-8S. Proteins were resolved by reducing and non-reducing SDS-PAGE and the mobilities of dimeric and monomeric Db heavy chains are indicated. chase -i - Furthermore, the differential of Db versus stability Kb Fig. Castanospermine treatment impairs folding of Db heavy chains heavy chains was apparent in castanospermine-treated in mouse cells. (A) Mouse EL4 cells were pre-incubated for I h, radiolabeled for min 5 with [35S]Met, and chased for the indicated EL4 cells. Whereas both heavy chains were degraded times, all + castanospermine. Digitonin lysates of cells were incubated very slowly under control conditions, their half-times of with antibodies recognizing either total Db heavy chains (mAb 28-14- turnover were 65 min and 100 for Db and min Kb 8S and anti-HC or Db combined) heavy chains with a folded a3 respectively, following incubation with castanospermine domain (mAb 28-14-8S) and isolated proteins were digested with endo H and analyzed by reducing SDS-PAGE. Note that (data not shown). since the anti-HC antiserum does not discriminate between Db and Kb, both heavy Collectively, the functions of calnexin that were estab- chains appear in the total heavy chain pool but can be discriminated lished in Drosophila cells (preventing degradation, following endo H digestion. Only the mobilities of the endo facilitating heavy chain folding and assembly with H-sensitive and -resistant Db heavy chains are indicated. (B) The P2m, and retaining assembly intermediates) are reproducible in results in (A) were quantified by densitometry and the amount of 28-14-8S reactive heavy chains at each time point was expressed as a mouse cells treated with castanospermine. This congruency percentage of the total heavy chains present in the pulse sample. that the suggests effects of castanospermine are due primarily to inhibition of calnexin interactions. Further ingly, heavy chain dimers formed rapidly in castano- for this view comes support from our finding that calreticu- spermine-treated cells (Figure 7, compare lin, the only other non-reducing chaperone known to be affected by panels -/+ castanospermine). These dimers were sensitive does not bind to castanospermine, mouse class I molecules to reduction, indicating that they were covalently linked at in their early stages biogenesis (A.Vassilakos and via inter-chain disulfide bonds (Figure 7). Similar disulfide- D.B.Williams, unpublished observations). linked homodimers have been described previously for non j32m-associated class I heavy chains (Capps et al., Castanospermine reduces cell-surface expression 1993). Therefore, at least some of the Db heavy chains of class I molecules that a acquired 28-14-8S reactive conformation exhibited Given that calnexin's influence is detectable at multiple characteristics of improperly folded molecules. No higher levels, it was of interest to determine how these various order oligomers or were aggregates observed in this effects are ultimately manifested in the of class expression analysis. I molecules at the cell surface. EL4 cells were treated in Velocity density gradient centrifugation was used to the absence or presence of castanospermine for 24 h and assess whether of Kb the of aggregates heavy chains, analogous expression molecules at the cell 02m-associated to those observed in Drosophila cells calnexin surface was lacking determined by flow cytometry (Figure The 8). (Figure 4B), formed in extended castanospermine-treated EL4 cells. treatment with castanospermine was required to Approximately 15% of Kb chains were allow for turnover of class I molecules at the heavy recovered present cell as aggregates in fractions 4-16 with in surface to treatment. There was no reduction in compared none prior untreated cells (data not shown). chain or this time not heavy 12m synthesis during (data In Drosophila cells lacking calnexin, the degradation shown). Figure 8 depicts the fluorescence data obtained of Db heavy chains was markedly accelerated relative to incubation of cells with mAbs following 32m-dependent cells calnexin 2 expressing (Figures and 3). In contrast, followed by A FITC-conjugated secondary antibody. Kb molecules were in was observed relatively stable in the absence of reduction fluorescence intensity following calnexin A similar was observed and surface (Figure 1). phenomenon castanospermine treatment. For both Kb Db, in mouse cells. Free Db chains in RIE- decreased to 30% of control values. The heavy expressed expression Db cells were castano- reduced surface was not due to some castano- degraded very rapidly following expression spermine treatment = 20 with no of the (t112 min) compared spermine-induced impairment secretory pathway detectable over 20 min in untreated cells. since treatment increased intracellular trans- degradation drug actually 1501 A.Vassilakos et al. 20 min not A (data shown). less dramatic but reproducible antibody culture condition difference was also observed in reactivity to mAb 5117. About 45% of chains heavy from drug-treated cells B22-249.R1 (Db) acquired this epitope in contrast to 70% of heavy chains castanospermine from control cells. These results suggest that calnexin is Y3 (Kb) required for efficient heavy chain folding and assem- P2m a) bly of both human and murine class I .0 molecules. Whether B22-249.R1 (Db) there are species differences in how calnexin functions in no additions cJ downstream events, such as in peptide loading or inter- Y3 (Kb) a) action with the TAP peptide transporter, remains to be no Ab established. controls A recent study provided evidence that castanospermine second Ab only treatment of CIR cells HLA-B*0702 expressing impaired '.. heavy chain folding but did not decrease assembly 1. I 1 1 with -. II. . . .1I I le le0 loe0 ... and even in the Salter, However, (Tector 1995). 12m fluorescence (FITC) absence of castanospermine, assembly of B*0702 heavy chains with was only 20% efficient. It is possible P2m Fig. 8. Effect of castanospermine on cell surface expression of class I that the B*0702 heavy chain was to over-expressed the molecules. EL4 Mouse cells were cultured under normal conditions extent that any effects of calnexin on assembly were either with no additions or with two additions of castanospermine masked by limiting availability of (50 at 24 h and 16 h to flow tg/ml) prior cytometric analysis. P2m. 02m- associated Kb and Db molecules at the cell surface were detected by incubation, first with mAb Y3 (for Kb) or with mAb B22-249.Rl (for Discussion Db) followed by FITC-conjugated goat anti-mouse IgG. Control samples omitting either the first or both antibodies in were included In this study two independent approaches were used to the analysis. assess the functions of calnexin in the biogenesis of class histocompatibility molecules. The results obtained by rates of assembled class I molecules I port (Figure 5). heterologous expression of class molecules in Drosophila decreased was most cells or Rather, expression likely due to by treating mouse or human cells with castano- impaired class I and These results were similar. In the folding assembly. spermine remarkably absence of that calnexin interactions in the ER translate into calnexin suggest interactions, assembly of both mouse and human more efficient cell surface of class I molecules. heavy chains with was expression ,32m substantially impaired. Since surface expression is a for Reduced assembly was largely due to of the prerequisite presentation misfolding of to T calnexin is heavy chains as evidenced by a reduction in mAb-defined peptide ligands cytotoxic cells, likely to contribute to the with which a conformational or the formation of significantly efficiency epitopes by oligomers cell can present endogenous or These the antigens. aggregates. findings provide most direct evidence to date that calnexin functions as a bona fide Castanospermine inhibits and of molecular chaperone. folding assembly the human HLA-B27 molecule How calnexin promotes protein or is folding assembly There is some controversy as to whether calnexin inter- the subject of considerable debate. Helenius and co- actions are as important in the biogenesis of human class workers have proposed that the most important component I molecules as are for mouse class I This of calnexin's they molecules. interactions is its binding to Glc,Mang- is the highlighted by inability to demonstrate an interaction GlcNAc2 oligosaccharides on nascent glycoproteins. This between calnexin and heterodimers in heavy oligosaccharide structure is maintained by a cycle of chain-N2m human cells such an interaction is detected glucose removal and which is although easily reglucosylation thought to in mouse cells (Sugita and and continue as as the Brenner, 1994; Nossner long glycoprotein remains incompletely Parham, 1995). Calnexin association with unassembled folded. When is the folding complete glycoprotein is no human heavy chains has, however, been clearly demon- longer a substrate for the glucosyltransferase that catalyzes strated (Degen et al., 1992; and reglucosylation and the cycle ends Rajagopalan Brenner, (Hammond and 1994). Human CIR cells, transfected with the class I Helenius, 1994). In this view the main function of calnexin HLA-B27 gene, were used to determine whether the is simply to retain incompletely folded or unassembled functions of calnexin established for murine class I mole- glycoproteins within the ER where folding enzymes and cules can be extended to the human molecular system. The folding chaperones such as BiP and GRP94 can assist of B27 heavy chains and assembly with 32m were assessed the folding process (Helenius, 1994; Hebert et al., 1995). by comparing reactivity with antibodies that recognize it has been shown Although that calnexin retains non- unassembled chains heavy (mAb HC-10), heavy chains native proteins in the ER (Jackson et al., 1994; Rajagopalan with folded a3-domains regardless of association with et al., 1994), its ability to facilitate class I heavy-chain and chains (mAb folding and assembly with ,2m as demonstrated in the 5H7), heavy (mAb P2m 02m-associated W6/32). As in and depicted Figure 9A B, castanospermine present study cannot be attributed only to retention. For treatment resulted in a 3.5-fold decrease in assembly of example, the enhanced folding of Db heavy chains in the the B27 heavy chain with Whereas 70% of presence of heavy calnexin is evident by the end of a 5 min N2m. chains assembled over a 20 min period in control cells, in both pulse radiolabeling Drosophila cells and in castano- 20% assembled in only the presence of castanospermine. spermine-treated mouse cells (Figures 3 and 6). The effect No further was in assembly observed either case beyond of calnexin on increasing the assembly of heavy chains 1502 Molecular chaperone functions of calnexin -castanospermine unassem. 5H7 total 2n2m-assoc. 46 - aMW -0 am am am am0 qwam castanospermi ne 46 - a Om 2C am P aPm am 2 '0- 20 10 20 P 10 20 P 10 20 chase 1 (min) jnassemtlied folded u3 domain - 5H7 132m-associated 8"- 6c. --- :~~~~~ 2, r 2C chase imin! ----U-- - castarospe,m:ne castarospeorlnle Fig. 9. Effect of castanospermine on the folding and assembly of the human HLA-B27 molecule. (A) Human CIR cells expressing HLA-B27 were for 1 h, radiolabeled for 5 min with [35S]Met and then chased for the indicated times, all castanospermine. B27 molecules were pre-incubated isolated from digitonin lysates using antibodies recognizing either unfolded (mAb HC-1O), a3 domain-folded (mAb 5H7), (mAb W6/ 02m-associated 32) or total (all mAbs combined) heavy chains. Isolated proteins were analyzed by reducing SDS-PAGE and the mobility of the B27 heavy chain is of the B27 chain indicated. The data from the experiment in (A) were quantified by densitometry and the various conformational forms heavy (B) were expressed as percentages of the total heavy-chain signal present at each chase time. or stabilize the with is also obvious immediately following the pulse may prevent the formation of aggregates P2m also in Drosophila cells (Figures 1 and 2) and during the first polypeptide chain against proteolytic attack. Calnexin a mechanism other 10 min of chase in mouse or human cells (Figures 5 and enhanced Db heavy-chain folding by The 2- to 3-fold enhancement 9). Within these short time periods there are negligible than preventing degradation. 5 occurred differences in export of class I molecules from the ER in of folding observed during a min radiolabeling to differential in the absence or presence of calnexin. too rapidly to be attributed degradation functions? There is or of calnexin 3 and How then does calnexin effect its the absence presence (Figures 6). associations due calnexin now considerable evidence that polypeptide This effect may be to stabilizing partially a of the overall inter- folded conformations that are otherwise labile in the constitute substantial component action between calnexin and glycoproteins. Indeed, sites absence of calnexin interactions or in the continuing of interaction between calnexin and class I heavy chains absence of binding to P2m. have been mapped in proximity to the transmembrane Our model of how calnexin functions involves a working of chain where no N-linked glycans are mechanism that incorporates both oligosaccharide region the heavy two-step located et 1993; Carreno et al., 1995). and components in calnexin interactions (Ware (Margolese al., polypeptide it is that calnexin possesses a polypeptide et 1995). Initially, calnexin utilizes its lectin site to Therefore, likely al., site in addition to its site for binding bind oligosaccharides thereby bringing binding Glc,Man9- GlcIMangGlcNAc2 with the membrane- Calnexin function to other nascent glycoproteins into proximity GlcNAc2. may analogous calnexin associates molecular chaperones, facilitating protein folding by bind- constrained chaperone. Subsequently, and at this ing to structural elements exposed in unfolded proteins with the unfolded polypeptide stage directly interaction becomes less as preventing in- the lectin-oligosaccharide impor- (such hydrophobic surfaces) thereby the We that calnexin intermolecular associations that lead to tant for maintaining complex. propose appropriate as a molecular aggregation. functions in this second step typical for this view. In the or Our current results provide support chaperone, preventing aggregation rapid degradation of Kb or intermediates. It is not absence of calnexin, extensive aggregation heavy and stabilizing folding assembly in in cells to a lesser whether calnexin functions like other chains was observed Drosophila and, clear chaperones if what mouse cells. Formation of and release extent, in castanospermine-treated cycles binding and, so, regulates that the ER luminal domain of disulfide-linked Db chain dimers was such The recent aberrant heavy cycles. finding ATP raises the that in mouse cells treated with the of calnexin binds observed castanospermine; possibility binding be nucleotide or of may have been precluded could regulated by binding production larger aggregates cycles Db chains. et Such a the of unassembled exchange (Ou al., 1995). hypothetical cycle by rapid degradation heavy dissociation of the of a involve nucleotide-induced on the intrinsic sensitivity might Therefore, depending interaction with calnexin from calnexin followed to glycoprotein by two-step rebinding glycoprotein degradation, 1503 A.Vassilakos et al. in the domain of free or Db and and would continue until all polypeptide sites for o3 heavy chains, mAb calnexin P2m-associated 5H7 (Smith et al., 1994) which recognizes a folded determinant in the binding are buried in the folded molecule. In this scenario, a3 domain of free or B or HLA-A, C heavy chains (a 2r2m-associated the deglucosylation-reglucosylation cycle maintains the from Dr A rabbit antiserum gift J.Bluestone). (anti-8) directed against structure necessary for the first step in Glc1MangGlcNAc2 the C-terminus of Kb was provided by Dr B.Barber and was used to calnexin binding (or rebinding) and also facilitates all final isolate conformational states of Kb (Smith et al., 1986). Unassembled mouse class heavy chains were isolated with a rabbit antiserum dissociation when the glucosyltransferase fails to regluco- (anti- Dr and R.Machold et HC) provided by H.Ploegh (Machold al., 1995). sylate the native glycoprotein. mAb HC- 10, obtained from Dr H.Ploegh, was used to isolate unassembled It is apparent that calnexin's interactions are not abso- HLA-B27 chains et For the heavy (Stam al., 1986). isolation of calnexin lutely essential for class I heavy-chain folding and assem- and associated a rabbit antiserum proteins, raised against the C-terminus bly with In the absence of calnexin, ~40% of canine calnexin was used of mouse et (Jackson al., 1994). P2m. or human heavy chains acquire a correctly folded epitope in their domains and about 20-30% of heavy chains Pulse-chase and immune isolation OC3 radiolabeling Transfected cells were to assemble with These findings suggest Drosophila subjected pulse-chase radiolabeling that the diver- P2m. as described essentially previously (Jackson et al., 1994). Cells were sity of folding enzymes and molecular chaperones within in ml of buffer lysed 1 lysis containing either 1% digitonin (Sigma) or the ER may provide a degree of redundancy in assisting 1% Nonidet P40 (BDH) in PBS, pH 7.4, supplemented with 10 mM protein folding. The recent demonstration that class I iodoacetamide, 1% aprotinin, 0.25 mM 4-(2-aminoethyl)benzene-sul- biogenesis appears normal in a calnexin-deficient mutant and 10 each of fonyl fluoride, chymostatin, leupeptin, antipain gg/ml cell line also and incubations and suggests that, in the chronic pepstatin. Antibody collection of immune absence of complexes with isolation were protein A-agarose performed as described calnexin, alternative previously chaperone pathways can compensate et (Jackson Bead-bound were washed three times al., 1994). complexes for the loss of calnexin function (Sadasivan et al., 1995; with either 0.2% digitonin/PBS, pH 7.4 or NTSE (0.5% NP-40, 10 mM Scott and Dawson, 1995). One candidate for a compensat- Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA and 0.02% sodium azide) ory chaperone is BiP, which associates with several depending on the buffer used for lysis. Most immune isolations recovered unglycosylated in excess of 90% of the respective class I (and presumably not calnexin-associated) species in a single clearance. When mAb B22-249.R I was used, a second clearance was incorporated mouse class I molecules, but not their glycosylated to isolate all of the Db heavy chains. Digestion with counterparts (Fraser et P2m-associated al., 1987; Degen, 1995). Further- H was endoglycosidase as described previously (Jackson et al., 1994). more, a recent report has demonstrated that prolonged Immune complexes were analyzed by SDS-PAGE using 10% gels treatment of cells with castanospermine results in increased (Laemmli, 1970) and radioactive were proteins visualized by fluoro- expression of BiP mRNA (Pahl graphy. and Baeuerle, 1995). Murine EL4 and RIE-Db cells and human I C R cells were radiolabeled, An examination of calnexin's role in class I biogenesis and to lysed subjected immune isolation as described for Drosophila should ultimately address what the implications are for cells with the following exceptions. Pre-incubation was conducted for class I-restricted antigen presentation. For the 20-30% of min in 60 Met-free RPMI 1640 supplemented with 9 mM HEPES, 2 mM mouse class I molecules that are able to assemble in the + glutamine, 1 mM castanospermine at 37°C. Cells were radiolabeled in absence of calnexin, we have the same medium at 37°C + castanospermine after which the preliminary data indicating cells were incubated for various chase times in that they medium supplemented with 1 mM associate with the TAP peptide transporter and unlabeled Met ± castanospermine. Detergent lysates of human Cl R apparently acquire peptide ligands based on their stability cells were 10% incubated with formalin-treated Staphylococcus aureus in detergent lysates (W.-K.Suh, A.Vassilakos and D.B. cell suspension (Sigma) prior to immune isolation to remove endo- Williams, unpublished data). Whether or not calnexin genously expressed immunoglobulin. influences the spectrum of peptides bound is currently under investigation. Collectively, the results demonstrate Velocity density gradient centrifugation that calnexin, while not absolutely necessary for class I Drosophila cells expressing H-2 ± Kb and ,B2m calnexin were radio- labeled with biogenesis, plays an [35S]Met for 5 min and then chased for 80 min. important role in the efficient folding, Cells (IX 107) were lysed in 1 ml RIPA buffer (25 mM HEPES, pH 7.4, assembly and surface expression of murine and human 150 mM NaCl, 1% NP-40, 1% deoxycholate, 0.1% SDS, 20 mM N- class I molecules and hence, in a cell's ability to present ethylmaleimide and protease inhibitors), centrifuged to remove insoluble endogenous antigens. material, and 0.25 ml was loaded onto a 10-40% (w/v) linear glycerol gradient. Gradients were centrifuged at 4°C for 15 h at 35 000 r.p.m. using a Beckman SW41 rotor and 0.75 ml fractions were collected. Kb Materials and methods heavy chains were immunoisolated from each fraction with anti-8 serum and analyzed by SDS-PAGE. As a measure of total Kb molecules applied Cells and antibodies to the gradient, an additional 0.25 ml sample of lysate was treated Drosophila melanogaster Schneider cells transfected with cDNAs encod- directly with anti-8 and analyzed along with the gradient fractions. ing murine H-2 Kb or Db class I heavy chains and fB2m (either alone or Gradients were calibrated with Bio-Rad molecular mass standards with canine calnexin) have been described previously (Jackson et al., consisting of thyroglobulin (670 kDa), IgG (158 kDa), ovalbumin 1994). Murine EL4 cells (H-2b lymphoma; Gorer, 1950) and RIE-Db (44 kDa) and myoglobin (17 kDa). cells thymoma transfected with the Db heavy chain gene; (P2rm-deficient Allen et 1986) were al., grown in RPMI 1640 medium supplemented Flow cytometric with 10% fetal bovine serum and analysis antibiotics. Human C R cells transfected EL4 cells were with the HLA-B27 gene were obtained cultured for 24 h either alone or with the addition from Dr P.Cresswell and main- of castanospermine tained as described for murine cells (0.27 mM) at 24 h and at 16 h prior to with the addition of 0.5 processing for mg/ml G418 (Alexander et al., 1989). flow cytometry. Cells (I 106) were washed with PBS-BSA (PBS The following containing conformation-dependent monoclonal 0.1I% BSA and 0.1I% NaN3), resuspended in 0.1 antibodies (mAbs) ml PBS- were used: mAbs BSA, and 20-8-4S (Ozato and Sachs, 1981), Y3 (Jones incubated for I h with 1-2 ,ug of either mAb and B22-249.RI or Janeway, 1981) and B8-24-3 (Kohler et al., 1981) for mouse Y3 (all at 4°C). Cells were washed in PBS-BSA H-2 Kb and incubated for 45 heavy chains associated with mAbs B22-249.R1 (Lemke et min in 1/50 dilution of FITC-conjugated al., goat anti-mouse IgG (Sigma). N32m, 1979) and 27-11-13S (Ozato and Sachs, 1981) for mouse H-2 Db heavy The cells were washed in PBS and resuspended at 2-5x 106 in cells/ml chains associated with R2m, mAb W6/32 for human HLA-A, B or PBS containing 0.1 NaN3. C Incubations omitting either the first antibody chains heavy associated with et or both antibodies were j32m (Barnstable al., 1978), mAb 28- included as controls. Fluorescence data were 14-8S et (Ozato which a conformational collected and on al., 1980) recognizes a Coulter Elite epitope analyzed Epics flow cytometer. 1504 functions of calnexin Molecular chaperone TCR3 proteins with the molecular chaperone calnexin and specifically Acknowledgements results in accelerated degradation of nascent TCRtx proteins within We thank Peter Cresswell. Brian Barber, Jeff Bluestone, Hidde Ploegh the endoplasmic reticulum. EMBO J., 13, 3678-3686. and Robert Machold for their generous gifts of antibodies and cell lines. Kohler,G., Fischer-Lindahl,K. and Heusser,C. 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Quant. in a calnexin negative of MHC class I transport by the molecular (1994) Regulation Biol.. in press. calnexin IP90). Science. 263, 384-387. chaperone. (p88. MHC class I expression and transport Scott,J.E. and Dawson.J.R. (1995) and Janeway.C.A. (1981) Cooperative interaction of B Jones.B. cell line. J. lInmulinol.. 155, 143-148. T is MHC in a calnexin deficient with antigen-specific helper lymphocytes lymphocytes Smith,D.M.. BluestoneJA.. JeyarajahD.R. NewbergM.H. , restricted. 292. 547-549. Natuxre. Persistence of glucose Thistlethwaite.J.R..Jr and Woodle,S.E. (1994) and (1994) Engelhard,V.H.. Kearse.K.P.. Williams,D.B. Singer,A. association of TCRax and Inhibition of T cell activation by a monoclonal antibody against the residues on core oligosaccharides prevents 1505 A.Vassilakos et al. a3 domain of human I MHC class molecules. J. Immunol., 153, 1054-1067. Smith,M.H., Parker,J.R.M., Hodges,R.S. and Barber,B.H. (1986) The preparation and characterization of anti-peptide heteroantisera recognizing subregions of the intracytoplasmic domain of class I H-2 antigens. Mol. Immunol., 23, 1077-1092. Stam,N.J., Spits,H. and Ploegh,H.L. (1986) Monoclonal antibodies raised against denatured HLA-B locus heavy chains permit biochemical characterization of certain HLA-C locus products. J. Immunol., 137, 2299-2306. Sugita,M. and Brenner,M.B. (1994) An unstable Major P2-microglobulin: histocompatibility complex class I heavy chain intermediate dissociates from calnexin and then is stabilized by binding peptide. J. Exp. Med., 180, 2163-2171. Suh,W.K., Cohen-Doyle,M.F., and Fruh,K., Wang,K., Peterson,P.A. Williams,D.B. (1994) Interaction of MHC class I molecules with the transporter associated with antigen processing. Science, 264, 1322-1326. Tector,M. and Salter,R.D. (1995) Calnexin influences folding of human class I histocompatibility proteins but not their assembly with P2- microglobulin. J. Biol. Chem., 270, 19638-19642. Wada,I. et al. (1991) SSRa and associated calnexin are major calcium binding proteins of the endoplasmic reticulum membrane. J. Biol. Chem., 266, 19599-19610. Ware,F, Vassilakos,A., Peterson,P.A., Jackson,M.R., Lehrman,M.A. and Williams,D.B. (1995) The molecular chaperone calnexin binds oligosaccharide as an initial step in recognizing Glc1Man9GlcNAc2 unfolded glycoproteins. J. Biol. Chem., 270, 4697-4704. Williams,D.B. (1995) Calnexin: a molecular chaperone with a taste for carbohydrate. Biochem. Cell Biol., 73, 123-132. Zang,Q., Tector,M. and Salter,R.D. (1995) Calnexin recognizes of I carbohydrate and protein determinants class major histocompatibility complex molecules. J. Biol. Chem., 270,3944-3948. on 1995 Received on September 4, 1995; revised November 20, http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The EMBO Journal Springer Journals

The molecular chaperone calnexin facilitates folding and assembly of class I histocompatibility molecules.

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Springer Journals
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Copyright © European Molecular Biology Organization 1996
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0261-4189
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1460-2075
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10.1002/j.1460-2075.1996.tb00493.x
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Abstract

The EMBO Journal vol.15 no.7 pp.1495-1506, 1996 chaperone calnexin facilitates folding The molecular and assembly of class histocompatibility molecules that calnexin that are unable to assemble, suggesting may Aikaterini Vassilakos, Myrna F.Cohen-Doyle, ER control machinery that be a component of the quality Per A.Peterson", Michael R.Jackson1 and export of incompletely folded or prevents premature David B.Williams2 unassembled proteins to the Golgi apparatus (Helenius, of of Toronto, Toronto, Department Biochemistry, University in 1995). Indeed, recent studies 1994; reviewed Williams, M5S Canada and 1R.W.Johnson Pharmaceutical Research Ontario 1A8, the export of have demonstrated that calnexin impedes Institute. La CA 92121. USA Jolla. of the class I histo- incompletely assembled subunits 2Corresponding author and the T cell from the compatibility molecule receptor and ER (Jackson et al., 1994; Rajagopalan Brenner, 1994; a membrane of the Calnexin, protein endoplasmic Rajagopalan et al., 1994). reticulum, is generally thought to function as a molecu- Unlike other molecular chaperones, calnexin displays a lar chaperone, based on indirect or correlative marked preference for Asn-linked glycoproteins. Treat- evidence. To examine calnexin's functions more ment of cultured cells with tunicamycin to block glycosyla- directly, we reconstituted the assembly of class I histo- tion (Ou et al., 1993) or with the oligosaccharide compatibility molecules in the absence or presence of processing inhibitors castanospermine and 1-deoxynojiri- calnexin in Drosophila melanogaster cells. Calnexin mycin, prevents calnexin binding to most newly synthe- enhanced the assembly of class I heavy chains with sized (Hammond and Helenius, 1994; Hammond proteins as much as 5-fold. The f2-microglobulin improved et al., 1994). The latter compounds inhibit glucosidases I assembly appeared largely due to more efficient folding remove glucose residues from the and II, enzymes that of heavy chains, as evidenced by increased reactivity that is attached to oligosaccharide Glc3Man9GlcNAc2 with a conformation-sensitive monoclonal antibody and chains as enter the ER. These nascent polypeptide they by a reduction in the level of aggregates. Similar that calnexin and other observations led to the suggestion findings were obtained in mouse or human cells when on specifically recognizes the Glc structure IMan9GlcNAc2 the interaction of calnexin with class I heavy chains glycoproteins (Hammond et al., 1994). Proof that calnexin by treatment with the oligosaccharide was prevented functions as a lectin was subsequently provided by demon- processing inhibitor castanospermine. The ability of ER strating direct binding of the luminal domain of folding and to prevent calnexin to facilitate heavy chain calnexin to the Glc oligosaccharide (Ware MangGlcNAc2 the formation of aggregates provides compelling evid- et al., 1995). However, there are many examples of ence that calnexin functions as a bona fide molecular calnexin binding to proteins that lack Asn-linked oligo- chaperone. saccharides, suggesting that calnexin also recognizes the Keywords: calnexin/class I histocompatibility molecules/ polypeptide portion of incompletely folded proteins endoplasmic reticulumlmolecular chaperones/protein (Kearse et al., 1994; Loo and Clarke, 1994; Rajagopalan folding et al., 1994; Arunachalam and Cresswell, 1995; Carreno et al., 1995). Further evidence supporting polypeptide interactions was obtained by digesting complexes of calnexin and with endo- newly synthesized glycoproteins Introduction glycosidase H. All oligosaccharides could be removed by the without dissociation of the enzyme deglycosylated Proteins that are translocated into the endoplasmic reticu- from calnexin and Cresswell, 1995; proteins (Arunachalam lum (ER) fold and undergo subunit assembly with the Ware et al., 1995; Zhang et al., 1995). of a diverse of soluble assistance group folding factors, There is intense interest in establishing whether calnexin disulfide iso- including protein isomerase, peptidylprolyl indeed functions as a molecular chaperone that facilitates merase and molecular of the hsp7O (BiP) chaperones folding and, if so, how it utilizes its apparent dual and families in and protein hsp9O (GRP94) (reviewed Gething Hammond mode of binding to effect its function. Recently, Helenius et In the Sambrook, 1992; al., 1992). addition, to and Helenius castanospermine prevent ER contains a putative molecular chaperone known as (1994) employed stomatitis virus G a I membrane the of calnexin to vesicular calnexin. Calnexin is non-glycosylated, Type binding maturation of G homo- glycoprotein and found that glycoprotein protein that contains a 461 residue luminal domain form was to calreticulin et to a fully disulfide-bonded substantially logous (Wada al., 1991). result is of a binds to a diverse of both impaired. Although this suggestive chaperone Calnexin transiently array function for caution should be exercised in and soluble their calnexin, membrane proteins, shortly following the effects of this inhibitor since it have translocation into the and its dissociation can interpreting may ER, usually effects on other ER and be correlated with some in pleiotropic chaperones folding stage polypeptide folding interacts in For which shares extensive or subunit calnexin factors. example, calreticulin, assembly. Furthermore, or with subunits with the luminal domain of fashion with misfolded sequence identity calnexin, prolonged proteins Press C Oxford University A.Vassilakos et aL binds transiently to immature forms of myeloperoxidase Kb Kb + 32m 32m (Nauseef et al., 1995) as well as other glycoproteins calnexin (Peterson et al., 1995). This chaperone-like binding is KO r Endo H also inhibited by castanospermine. Tb4w4mAD,-q ol0 ____\__ Kb Endo H We have been studying calnexin functions during the 3rnm associated 4, biogenesis of mouse class I histocompatibility molecules 4w am (Degen and Williams, 1991; Degen et al., 1992; Margolese et al., 1993). - Class I molecules are cell surface glyco- m __ga urlclssembfie-~a P 20 40 80 P 20 40 80 chase proteins that are responsible for presenting cytosolically min.) generated peptides to B cytotoxic T lymphocytes (Germain and Margulies, 1993). They consist of a highly poly- Kb 32m + Kb t2m morphic calnexin transmembrane heavy chain that possesses 1-3 120 l Asn-linked .._, oligosaccharides, a soluble, non-glycosylated '001 subunit termed and a peptide 02-microglobulin (02m), ligand that is 80 8-10 residues in length. Immediately follow- 0FU T ing synthesis, I class heavy chains bind quantitatively to calnexin and assembly with occurs shortly thereafter P2m (Degen and Williams, 1991). The heavy hetero- chain-P2m dimers then associate with the TAP peptide transporter -i that transports cytosolic peptides into the ER lumen .-. et 20 (Ortmann al., 1994; Suh et 40 60 80 al., 1994). Subsequently, 20 40 60 80 peptide binding, release from TAP and dissociation from chase min) chase (min) calnexin all appear to occur at about the same time (Degen u92m-assocated -A-- 32pm-associated et et al., 1992; Suh al., 1994). _--- unassemnbled unassembled We I previously expressed mouse class chains heavy and ,B2m in cells. Fig. 1. Effects of calnexin on Kb assembly with f32m in Drosophila melanogaster These cells Drosophila cells. (A) Drosophila cells expressing Kb and f2m in the absence (left are functionally calnexin deficient since no endogenous panels) or presence (right panels) of mammalian calnexin were calnexin could be in homolog detected association with radiolabeled with [35S]Met for 5 min and subsequently incubated in the mouse chains. heavy Upon co-expressing mammalian media containing excess unlabeled Met for the indicated times. Cells calnexin we demonstrated a role for calnexin in were the lysed in digitonin lysis buffer and Kb molecules were isolated with antibodies total recognizing (anti-8 antiserum), 12m-associated intracellular retention and stabilization of class I assembly (mAb unassembled 20-8-4S), or (anti-HC antiserum) heavy chains. intermediates et An obvious (Jackson al., 1994). advantage Following digestion with endoglycosidase H (endo H), proteins were of this system is that it does not on non- rely potentially SDS-PAGE. analyzed by reducing Only the heavy chain of regions the specific chemical to a calnexin-deficient agents produce gels are shown. The mobilities of endo H-sensitive (endo HS) and endo H-resistant (endo Kb environment. In this we examined the effects of Hr) heavy chains are indicated on the top panel report only. The letter P signifies pulse-labeled samples. (B) The results from calnexin I on class heavy chain and subunit folding five independent experiments were densitometric quantified by analysis assembly using the Drosophila expression Further- system. of weakly exposed and the amounts fluorographs of f2m-associated we these data more, compared with the effects observed and unassembled heavy chains Hs and endo Hr (endo combined) were following treatment of mouse and human cells with expressed as a percentage of the total chains in heavy present the pulse samples. Data points mean values and error The represent bars reflect castanospermine. results indicated that calnexin does the range of the replicate samples. The data from the 20 min time indeed function as a molecular chaperone and is an points were excluded from the because were analysis they obtained important in the and component folding assembly pathway from only a single experiment. of class I molecules. left The panels). small population of Kb molecules that Results assembled was gradually transported out of the ER as Calnexin facilitates of class I molecules assessed by the maturation of assembly Asn-linked oligosaccharides in expressed Drosophila cells to a form resistant to with digestion endoglycosidase H Drosophila cells expressing either Kb or Db chains 80 Unassembled Kb chains heavy min). remained heavy (t1/2 and J2m in the or absence of at a presence co-expressed high level throughout the chase and were exported calnexin were subjected to a pulse-chase analysis to very slowly from the ER. No further assembly with f2m determine the kinetics and of of these was observed efficiency assembly beyond 80 min of chase (data not shown). mouse class I heavy chains with 2m. The results obtained In marked of contrast, co-expression calnexin resulted in for Kb are in depicted Figure IA. of heterodimer quantitative of Kb Efficiency assembly with f32m (Figure 1, right formation was assessed by comparing heavy-chain reac- panels). As a consequence of increased assembly more tivity to three antibodies: a mAb, an heterodimers were transported out of the ER but, consistent 02m-dependent that antibody recognizes total Kb with the heavy chains, and an findings of an earlier study (Jackson et al., 1994), antibody that recognizes unassembled Kb the heterodimers that molecules. The had been associated with calnexin results were quantified by densitometry and both were transported more ,B2m- slowly than heterodimers assembled associated and unassembled heavy chains were expressed in the absence of calnexin endo H resistance (t112 as a of total chains in percentage heavy the pulse 120 min). sample (Figure iB). In the absence of co-expressed calnexin, only Db heavy chains share 81% sequence identity with Kb ~20% of Kb chains assembled with heavy f32m (Figure 1, heavy chains, yet an of Db analysis assembly with ,2m 1496 Molecular chaperone functions of calnexin A in a 5 min pulse were present as a series of degradation DO ft2m Db 32m t cainexin products of faster mobility than the full-length Db heavy chain (Figure 2A, left panel, total). At this time point totai .32m-associated total B2m-associated little assembly with had occurred (Figure 2A, left P2m first two lanes). Degradation continued panel, compare and, to a lesser extent, included cainex r during chase incubations Db-,32m heterodimers. Co-expression of calnexin dramatically inhibited degradation (Figure 2A, right - _ ___ -_ -,irav OM 0 panel). The observation that a substantial proportion of 4- aLwe DD heavy chains was degraded very early after synthesis, before assembly, raised the possibility that a decreased Dr efficiency of assembly in the absence of calnexin could OF be masked by the rapid loss of unassembled molecules. To minimize degradation, Drosophila cells were incuba- ted with the membrane-permeable protease inhibitor, P 30 chase 2C LLnL, which slowed, but did not block, the degradation D' - O 2C P ',' rn Db in of calnexin. these of expressed the absence Under of the total Db heavy chains conditions, only ~60-70% DO Db 32m -calnexir R2M present in the pulse sample assembled with in the P2m 32m assoc totai 32m-assoc. total absence of calnexin (Figure 2B, left panel and 2C). The value of 60-70% assembly is most likely an overestimate D C maLe given that degradation was not completely blocked by LLnL treatment. This residual degradation appeared restricted to heavy chains that did not assemble since, at n ^ 20 20 P 1 I 20 chase rnir P ; 0 20 P the 10 and 20 min time points, the remaining heavy chains were f2m-associated and relatively stable (Figure 2B, compare total and lanes). Inefficient IP2m-associated assembly was due not to degradation simply limiting in the availability of heavy chains but rather, as detailed of next section, to the inability of a population heavy - calnexin --* In chains to fold correctly (see below). cells expressing ----}--U A - calirexr efficient with all of the Db calnexin, assembly was more e-< / 2'_. heavy chains forming heterodimers (Figure 2B and C). for Kb 1), the effect of calnexin As was observed (Figure on Db was manifested rapidly; as early as the assembly .--_ 2r 5 min pulse sample calnexin enhanced assembly by ~3-fold. A difference between these two class I chase (min) major isotypes is the fate of unassembled heavy chains in the Fig. 2. Effects of calnexin on Db assembly with in Drosophila P2m absence of calnexin. Db chains that do not assemble heavy cells. (A) Drosophila cells expressing Db and in the absence or P2m are whereas unassembled Kb heavy rapidly degraded of were radiolabeled with for 5 min and presence calnexin [35S]Met chains are stable and are largely retained intracellularly incubated in media containing unlabeled Met for the subsequently indicated times. Cells were lysed in digitonin lysis buffer and Db in a conformation that is not competent for binding P2m. molecules were isolated with antibodies recognizing total (mAb 28-14- 8S and anti-HC combined) or fB2m-associated (mAbs B22-249.Rl and facilitates of Db chains Calnexin folding heavy 27-11-13S combined) molecules. Proteins were analyzed by reducing in cells expressed Drosophila and SDS-PAGE and the mobilities of co-isolated calnexin, immature for the more efficient of class Db chains are indicated. One explanation assembly mature Db heavy chains, and degraded heavy of H mature Db chains In the absence endo digestion, heavy I molecules in the of calnexin is that calnexin presence to the more than their transported Golgi apparatus migrate rapidly of chains to a conformation that promotes folding heavy immature due to the of to precursors processing oligosaccharides For Db is competent for interaction with j32m. molecules, forms in cells. The smaller, complex Drosophila (B) experiment a folded an mAb (28-14-8S) exists that recognizes epitope in was except that cells were pre-incubated for performed (A) repeated chains. 40 and all in the of the protease in the a3 domain of free or 2m-associated heavy min, radiolabeled, chased, presence LLnL (100 lM, Sigma). (C) The results in (B) were inhibitor, this was used as an indication of Acquisition of epitope quantified by densitometry and the amount of 12m-associated heavy Db heavy chain folding. of the total chains at each time point was expressed as a percentage cells Db chains in Drosophila expressing only heavy in heavy chains present the pulse sample. the absence or of calnexin were to presence subjected immunoisolation followed pulse-chase radiolabeling by chains 3A and cells somewhat different results of total or 28-14-8S reactive in Drosophila yielded heavy (Figure of total chains occurred In the absence or of calnexin there degradation heavy (Figure 2A). presence B). Significant Db in the absence of calnexin to be of during the chase period although appeared quantitative assembly with P2m chase the characteristic intermediates seen in the total versus 20 min lanes). degradation (Figure 2A, compare not detected in these cells of calnexin there was considerable were However, in the absence previous experiments left since Db chains even in the that lacked degradation of heavy apparent pulse I2m (Figure 3A, panel). However, was it was of total chains labeled of the 28-14-8S sample. Typically, 20-40% heavy acquisition epitope very rapid, 1497 A.Vassilakos et al. Db + cainexin Calnexin prevents aggregation of Kb heavy chains Db 28-1 4-8S total 28-14-8S total expressed in Drosophila cells We next examined whether the dramatic effect of calnexin _- i nfl.. c<, :e x.r- on assembly of Kb heavy chains with (Figure 1) P2m could also be attributed in part to differences in the folding state of Kb heavy chains. It was not possible to monitor acquisition of a conformational epitope as was done for Db, because no conformation-specific mAbs for un- 46, p assembled Kb molecules exist. Instead, we took advantage P l IC 2r) i; ) a.2C 212C D 0 20- chase mir of the fact that Kb heavy chains not competent for assembly with were relatively stable in the absence of co- P2m expressed calnexin (Figure lA, left panel). This permitted an assessment of the aggregation state of Kb. 1 . Drosophila cells expressing Kb and ,B2m with or without P-----....... -, fj - ci lex calnexin were pulse radiolabeled and chased for 80 min, at which time assembly was complete and any remaining free heavy chains were not competent for further assembly (Figure 1). To determine if unassembled heavy chains existed in an aggregated state, isolated heavy chains were - -.-- x c a: r' .* subjected to non-reducing SDS-PAGE. Using an antiserum that recognizes all folding states of Kb, a high molecular weight aggregate was detected that barely entered a 7.5% resolving gel (Figure 4A, total, -DTT). The aggregate 0 1 c, 20 30 was stable to heating in SDS at 55°C but could be partially chase (minli dissociated upon heating at 70°C. Complete dissociation was effected only by heating at 70°C under reducing conditions (Figure 4A, +DTT). Thus, the aggregate Db in Fig. 3. Calnexin enhances folding of heavy chains expressed to contain chains associated Db appeared heavy through Drosophila cells. (A) Drosophila cells expressing heavy chain ± calnexin were to as described subjected pulse-chase radiolabeling non-covalent interactions. The relative both and disulfide in the legend to Figure 2A. Digitonin lysates were incubated with amount of was determined as aggregate densitometrically antibodies total Db chains 28-14-8S and anti- recognizing heavy (mAb a percentage of the total heavy chain recovered in both HC combined) or heavy chains with a folded a3 domain (mAb 28-14- free and aggregated forms see % (Figure 4A, aggregates). 8S) and isolated proteins were analyzed by reducing SDS-PAGE. The mobilities of the Db heavy chain and co-isolated calnexin are Importantly, there was a 3-fold increase in the percentage indicated. (B) The results in (A) were quantified by densitometry and of aggregates in cells lacking calnexin, with as much as the amount of 28-14-8S reactive heavy chains at each time point was 43% of Kb chains recovered in the form of heavy aggre- expressed as a percentage of the total heavy chains present in the gates. That the aggregates contained heavy chains that pulse sample. were incompetent for assembly with ,32m was confirmed by comparing total chains with assembled heavy Kb_t2m possible to observe the effects of calnexin on this process heterodimers recovered selectively with mAb 20-8-4S. even in the presence of degradation. Whereas 40% of the Heterodimers recovered with this mAb were found exclu- total Db chains heavy present in the pulse sample folded sively in a non-aggregated state (Figure 4A, P2m- into an mAb-reactive conformation in the absence of associated). 90% of when To confirm calnexin, heavy chains acquired the epitope that calnexin influences the extent of co-expressed with calnexin calnexin of Kb cell (Figure 3B). Again, aggregation heavy chains, lysates (radiolabeled exerted its effects at an early stage, the as above) were fractionated on 10-40% enhancing glycerol gradients efficiency of heavy-chain folding from 24-65% during prior to immunoisolation of total Kb molecules and analysis the 5 min pulse radiolabeling. Heavy chains that did not by reducing SDS-PAGE (Figure 4B and C). In cells fold into an mAb-reactive conformation in the absence of expressing calnexin, 97% of the Kb molecules that could calnexin were selectively degraded and by 20 min of be recovered from the gradient were present in fractions chains Kb chase, only heavy possessing the 28-14-8S epitope 1-3. Only 3% of molecules were recovered in denser remained (Figure 3A, left panel, compare total and 28- fractions (fractions 4-16). This was the same distribution 14-8S lanes). Degradation did not limit the opportunity as that observed for assembled Kb-,B2m heterodimers when for heavy chains to fold since after 10 min in the absence mAb 20-8-4S was used for the isolation (data not shown). of calnexin, only 40% of heavy chains had acquired the In the absence of calnexin, 78% of heavy chains that even unfolded epitope though heavy chains were available could be recovered from the gradient were present in (Figure 3A, left panel, compare 28-14-8S and total lanes). fractions 1-3 and a substantial proportion (22%) was Clearly, most heavy chains synthesized in the absence of found in the denser (4-16) fractions (Figure 4B and C). calnexin were incapable of correct folding. To confirm the Overall, findings suggest that one function of this conclusion, cells lacking calnexin were treated with calnexin is to prevent inappropriate molecular interactions LLnL to retard degradation and no increase in heavy- that lead to aggregation. The extent of aggregation assessed chain was observed folding (data not shown). Thus, by the two techniques (22-43%) was somewhat less than interaction with calnexin increases the efficiency of heavy- expected, based on the low efficiency of subunit assembly chain in cells calnexin folding. lacking (Figure 1). It is possible that 1498 functions of calnexin Molecular chaperone during cell lysis and immune isolation some non-covalent of calnexin with class I heavy chains in mouse cells. were disrupted, leading to an underestimation Although the potential existed for castanospermine to interactions the amount of aggregates. Alternatively, in addition to affect processes other than those mediated by calnexin, of there may have been significant amounts of validity of data obtained could be assessed in the aggregates, the of the results obtained using the Drosophila misfolded heavy-chain monomers that were incompetent context To confirm that castanospermine prevents calnexin for assembly. system. mouse EL4 cells were radio- binding to heavy chains, calnexin interaction presence or absence of the inhibitor and Castanospermine prevents labeled in the class I molecules in mouse cells or class I heavy chains were immunoisolated with then calnexin the results obtained in the Drosophila (Figure 5A). In the absence of castano- In addition to from cell lysates we wished to determine whether cal- treatment, anti-calnexin antibodies recovered expression system spermine in a similar manner in mouse cells where calnexin and a series of associated proteins, two of which nexin functions of class I calnexin interactions were originally electrophoretic mobilities as Kb and Db the details had the same established (Degen and Williams, 1991; Degen et al., heavy chains (Figure 5A, left panel, compare lane 1 with We used castanospermine to prevent the interaction lanes 2-4). Disruption of anti-calnexin immune complexes 1992). with SDS and re-isolation using antibodies specific for A I chains showed that both Kb and Db molecules '< "2'- - alex class heavy were present in complexes with calnexin (Figure 5A, left panel, lane 6). Castanospermine treatment resulted in a dramatic reduction in the amount of proteins co-immuno- -,. -.rz -:1,:, precipitating with calnexin and, in particular, the two bands corresponding to class I heavy chains were absent (Figure 5A, right panel, lane 1). Disruption of anti-calnexin immune complexes and re-isolation with anti-heavy chain I complexes Ab confirmed the absence of calnexin-class of class I heavy (Figure 5A, right panel, lane 6). Synthesis cells as chains was normal in castanospermine-treated isolation with anti-class I Ab evidenced by direct immune lanes 2-4 in both panels). (Figure 5A, compare inhibits assembly of class I Castanospermine molecules ab * * treated with or without castanospermine EL4 cells were the of class I assembly was determined by and efficiency immunoisolation of both total and heavy 02m-associated Castanospermine treatment resulted chains (Figure 5B). in a decrease in the assembly of both Kb and Db dramatic W. .. _m chains with Densitometric analysis revealed heavy P2m. whereas both Kb and Db molecules assembled almost that for Kb and 80% for Db) during quantitatively (100% of Kb chains in 4. Calnexin inhibits aggregation heavy Drosophila Fig. Kb and + calnexin were cells. (A) Drosophila cells expressing f2m for 10 chased for radiolabeled with [35S]Met min, subsequently in RIPA buffer. RIPA buffer to be 80 min, and then lysed lysis proved chains than digitonin more effective in solubilizing aggregated heavy were isolated with antibodies recognizing lysis buffer. Kb molecules either total chains antiserum and 20-8-4S mAb heavy (anti-8 or heterodimers (mAb 20-8-4S). Immune combined) .. Kb_P2m were incubated in SDS-PAGE sample buffer ± DTT at complexes or for 15 min and were analyzed by non-reducing 55C 70°C proteins of the Kb chain and 4. SDS-PAGE The mobilities heavy (7.5% gel). The of total heavy heavy chain aggregates are indicated. percentage ~-4--A - and is chains as was determined densitometrically present aggregates IF lanes. cells were * a included at the bottom of the gel (B) Drosophila as in Cell were applied to the top radiolabeled and lysed (A). lysates and the gradients were centrifuged for of a 10-40% glycerol gradient in an SW-41 rotor. Gradients were fractionated, 15 h at 34 000 r.p.m. from each fraction total Kb chains were heavy immunoprecipitated were with anti-8 antiserum, and isolated proteins analyzed by reducing that to the bottom SDS-PAGE. P fractions represent proteins pelleted the total of Kb chains of the T samples represent pool heavy gradient. immune of an amount of to obtained by precipitation equivalent lysate the The data from were by that loaded on gradient. (C) (B) quantified chain recovered in each and the relative amount of densitometry heavy of the total chain was as heavy applied fraction expressed percentage f-actior' jmber to the gradient. 1499 A.Vassilakos et al. min of chase under normal conditions, there was a 4- to - stlrnospirmire +castanospermine 5-fold decrease in assembly (25% for Kb and 15% for Db) when calnexin interactions were inhibited with castano- cal'fX spermine (Figure SC). Similar experiments were C. .:,: performed _. using murine MDAY-D2 cells that express class I mole- cules of d-haplotype. Castanospermine treatment resulted f.-'i in a 2-3-fold decrease in the assembly of Ld and Dd 'a_1 ) molecules as well (data not shown). As shown in it was Figure SB, apparent that the small of Kb and Db population heavy chains that assembled in B castanospermine-treated cells was transported more rapidly -,,::astano r-1 +castarnospernine soerm to the Golgi apparatus relative to assembled molecules in total associated 32r0.[at untreated cells endo H resistance = 20 min for Kb (t1/2 and 30 min for in Db treated cells compared with 30 min for Kb and >>30 min for Db in control cells). The formation of endo H-resistant molecules in castano- spermine-treated cells requires the action of a Golgi endo-mannosidase to convert glucosylated precursor oligo- saccharides to a truncated form that can be acted on by Golgi glycosyltransferases (Moore and Spiro, 1990). Since __b the endo-mannosidase may be inefficient (Moore and Spiro, 1992), ER to Golgi transport rates may be even more ; -1 :: l, F' r1 1 ; C 2W' chase8 ,:! c h ( rapid in the drug-treated cells. The enhanced ;s transport observed in the presence of castanospermine was con- sistent with our earlier findings in cells Drosophila where, in the absence of calnexin, heterodimers were transported more rapidly out of the ER than when calnexin was co- expressed (Jackson et al., 1994). /3'!IIZ/-------. C2b EL4 -castanospermYlinel Castanospermine impairs folding of class I heavy q ,.' ,/ '~~~~~K' chains In Drosophila cells lacking calnexin, the inefficient assem- | -castanosperrnine bly of class I molecules could be attributed largely to EL4 inefficient 20 folding of the heavy chain as evidenced by a reduction in a conformational epitope defined by mAb 0 20 30 28-14-8S Db) and increased (for by aggregation of heavy chase (min) chains To determine (for Kb). if a similar situation existed in mouse cells, 28-14-8S was in reactivity examined EL4 cells incubated in the absence or of presence castano- spermine. As shown in 6A and Figure B, the 28-14-8S Fig. 5. Effect of castanospermine on heavy in assembly epitope was acquired rapidly in untreated cells, being chain-P2m mouse cells. (A) Murine EL4 cells that express Kb, Db and f2m were present in 80% of total chains heavy following the 5 min 1 h in ± 1 pre-incubated for Met-free medium mM castanospermine. pulse and 100% between radiolabeling reaching 10 and Cells were subsequently radiolabeled in the presence of min 20 of chase. By contrast, only about 45% of total castanospermine with [35S]Met for 20 min and then were lysed in digitonin buffer. Calnexin-associated molecules were isolated lysis heavy chains acquired the epitope in castanospermine- with anti-calnexin antiserum Class I molecules were isolated (lane 1). treated cells. This result was remarkably similar to the with antibodies recognizing either Kb alone lane (anti-8 antiserum, 2), -2-fold reduction in the formation of the 28-14-8S epitope Db alone (mAb lane or both Kb and Db 28-14-8S, 3) (anti-HC observed in cells Drosophila lacking calnexin, further In antiserum, lane 4). lane 6, calnexin immunoprecipitates were to in supporting a role for calnexin in disrupted by heating 90°C 0.2% SDS/PBS and, facilitating heavy-chain following addition of a 10-fold excess of NP-40, reprecipitated with a folding. combination of anti-HC and anti-8, 28-14-8S antibodies (lane 6). Lane The reduction in heavy-chain folding as defined by the 5 is blank. The mobilities of Kb calnexin, and Db molecules are 28-14-8S epitope was considerably less than the 5-fold indicated. (B) EL4 cells were pre-incubated for I radiolabeled h, with reduction in Db assembly with f32m observed following [35S]Met for 5 min, and then chased for the indicated times + castanospermine. Cells were lysed in digitonin lysis buffer and Kb treatment castanospermine (Figure SC). If the reduced molecules were isolated with antibodies recognizing either total assembly was due primarily to the inability of heavy (anti-8) or r2m-associated (mAbs Y3 and B8-24-3 combined) heavy chains to acquire a conformation competent for association chains (top panel). Db molecules were isolated from replicate samples with f2m, then the 28-14-8S epitope was not an accurate using antibodies recognizing either total (mAb 28-14-8S and anti-HC combined) or f2m-associated (mAb B22-249-RI) heavy chains reflection of an assembly-competent conformation. To (bottom panel). Proteins were digested with endo H and analyzed by determine whether additional indications could be obtained reducing SDS-PAGE. The mobilities of the endo H-sensitive and for improper folding of Db heavy chains following castano- -resistant chains are heavy indicated. (C) The results in (B) were Db spermine treatment, molecules were isolated with mAb quantified and by densitometry the amount of heavy 2r2m-associated 28-14-8S from control chains at each time and drug-treated EL4 cell lysates point was expressed as a percentage of the total chains in heavy present the pulse sample. and were analyzed by non-reducing SDS-PAGE. Interest- 1500 Molecular chaperone functions of calnexin - castanospermine castanospermine -cas!anospermi7,e ,castanospermine non- non- 28-14-BS 28-14-8S ota total reducing reducing reducing reducing 46 Db Enao i-r 97 - hc dimers mm. - - DO Erac Hs -- .- -. 69 - P 2C g 'C 'C 20 2C chase 20 'C imimn 46-1 J hc monomers P 10 P 10 P P 10 10 chase min A-A----------- cs- Fig. 7. Effect of castanospermine treatment on the state aggregation of Db molecules. Mouse EL4 cells were radiolabeled + castanospermine as described for 6. At the Figure indicated chase times, aliquots of cells were lysed in buffer and Db molecules digitonin lysis were isolated with mAb 28-14-8S. Proteins were resolved by reducing and non-reducing SDS-PAGE and the mobilities of dimeric and monomeric Db heavy chains are indicated. chase -i - Furthermore, the differential of Db versus stability Kb Fig. Castanospermine treatment impairs folding of Db heavy chains heavy chains was apparent in castanospermine-treated in mouse cells. (A) Mouse EL4 cells were pre-incubated for I h, radiolabeled for min 5 with [35S]Met, and chased for the indicated EL4 cells. Whereas both heavy chains were degraded times, all + castanospermine. Digitonin lysates of cells were incubated very slowly under control conditions, their half-times of with antibodies recognizing either total Db heavy chains (mAb 28-14- turnover were 65 min and 100 for Db and min Kb 8S and anti-HC or Db combined) heavy chains with a folded a3 respectively, following incubation with castanospermine domain (mAb 28-14-8S) and isolated proteins were digested with endo H and analyzed by reducing SDS-PAGE. Note that (data not shown). since the anti-HC antiserum does not discriminate between Db and Kb, both heavy Collectively, the functions of calnexin that were estab- chains appear in the total heavy chain pool but can be discriminated lished in Drosophila cells (preventing degradation, following endo H digestion. Only the mobilities of the endo facilitating heavy chain folding and assembly with H-sensitive and -resistant Db heavy chains are indicated. (B) The P2m, and retaining assembly intermediates) are reproducible in results in (A) were quantified by densitometry and the amount of 28-14-8S reactive heavy chains at each time point was expressed as a mouse cells treated with castanospermine. This congruency percentage of the total heavy chains present in the pulse sample. that the suggests effects of castanospermine are due primarily to inhibition of calnexin interactions. Further ingly, heavy chain dimers formed rapidly in castano- for this view comes support from our finding that calreticu- spermine-treated cells (Figure 7, compare lin, the only other non-reducing chaperone known to be affected by panels -/+ castanospermine). These dimers were sensitive does not bind to castanospermine, mouse class I molecules to reduction, indicating that they were covalently linked at in their early stages biogenesis (A.Vassilakos and via inter-chain disulfide bonds (Figure 7). Similar disulfide- D.B.Williams, unpublished observations). linked homodimers have been described previously for non j32m-associated class I heavy chains (Capps et al., Castanospermine reduces cell-surface expression 1993). Therefore, at least some of the Db heavy chains of class I molecules that a acquired 28-14-8S reactive conformation exhibited Given that calnexin's influence is detectable at multiple characteristics of improperly folded molecules. No higher levels, it was of interest to determine how these various order oligomers or were aggregates observed in this effects are ultimately manifested in the of class expression analysis. I molecules at the cell surface. EL4 cells were treated in Velocity density gradient centrifugation was used to the absence or presence of castanospermine for 24 h and assess whether of Kb the of aggregates heavy chains, analogous expression molecules at the cell 02m-associated to those observed in Drosophila cells calnexin surface was lacking determined by flow cytometry (Figure The 8). (Figure 4B), formed in extended castanospermine-treated EL4 cells. treatment with castanospermine was required to Approximately 15% of Kb chains were allow for turnover of class I molecules at the heavy recovered present cell as aggregates in fractions 4-16 with in surface to treatment. There was no reduction in compared none prior untreated cells (data not shown). chain or this time not heavy 12m synthesis during (data In Drosophila cells lacking calnexin, the degradation shown). Figure 8 depicts the fluorescence data obtained of Db heavy chains was markedly accelerated relative to incubation of cells with mAbs following 32m-dependent cells calnexin 2 expressing (Figures and 3). In contrast, followed by A FITC-conjugated secondary antibody. Kb molecules were in was observed relatively stable in the absence of reduction fluorescence intensity following calnexin A similar was observed and surface (Figure 1). phenomenon castanospermine treatment. For both Kb Db, in mouse cells. Free Db chains in RIE- decreased to 30% of control values. The heavy expressed expression Db cells were castano- reduced surface was not due to some castano- degraded very rapidly following expression spermine treatment = 20 with no of the (t112 min) compared spermine-induced impairment secretory pathway detectable over 20 min in untreated cells. since treatment increased intracellular trans- degradation drug actually 1501 A.Vassilakos et al. 20 min not A (data shown). less dramatic but reproducible antibody culture condition difference was also observed in reactivity to mAb 5117. About 45% of chains heavy from drug-treated cells B22-249.R1 (Db) acquired this epitope in contrast to 70% of heavy chains castanospermine from control cells. These results suggest that calnexin is Y3 (Kb) required for efficient heavy chain folding and assem- P2m a) bly of both human and murine class I .0 molecules. Whether B22-249.R1 (Db) there are species differences in how calnexin functions in no additions cJ downstream events, such as in peptide loading or inter- Y3 (Kb) a) action with the TAP peptide transporter, remains to be no Ab established. controls A recent study provided evidence that castanospermine second Ab only treatment of CIR cells HLA-B*0702 expressing impaired '.. heavy chain folding but did not decrease assembly 1. I 1 1 with -. II. . . .1I I le le0 loe0 ... and even in the Salter, However, (Tector 1995). 12m fluorescence (FITC) absence of castanospermine, assembly of B*0702 heavy chains with was only 20% efficient. It is possible P2m Fig. 8. Effect of castanospermine on cell surface expression of class I that the B*0702 heavy chain was to over-expressed the molecules. EL4 Mouse cells were cultured under normal conditions extent that any effects of calnexin on assembly were either with no additions or with two additions of castanospermine masked by limiting availability of (50 at 24 h and 16 h to flow tg/ml) prior cytometric analysis. P2m. 02m- associated Kb and Db molecules at the cell surface were detected by incubation, first with mAb Y3 (for Kb) or with mAb B22-249.Rl (for Discussion Db) followed by FITC-conjugated goat anti-mouse IgG. Control samples omitting either the first or both antibodies in were included In this study two independent approaches were used to the analysis. assess the functions of calnexin in the biogenesis of class histocompatibility molecules. The results obtained by rates of assembled class I molecules I port (Figure 5). heterologous expression of class molecules in Drosophila decreased was most cells or Rather, expression likely due to by treating mouse or human cells with castano- impaired class I and These results were similar. In the folding assembly. spermine remarkably absence of that calnexin interactions in the ER translate into calnexin suggest interactions, assembly of both mouse and human more efficient cell surface of class I molecules. heavy chains with was expression ,32m substantially impaired. Since surface expression is a for Reduced assembly was largely due to of the prerequisite presentation misfolding of to T calnexin is heavy chains as evidenced by a reduction in mAb-defined peptide ligands cytotoxic cells, likely to contribute to the with which a conformational or the formation of significantly efficiency epitopes by oligomers cell can present endogenous or These the antigens. aggregates. findings provide most direct evidence to date that calnexin functions as a bona fide Castanospermine inhibits and of molecular chaperone. folding assembly the human HLA-B27 molecule How calnexin promotes protein or is folding assembly There is some controversy as to whether calnexin inter- the subject of considerable debate. Helenius and co- actions are as important in the biogenesis of human class workers have proposed that the most important component I molecules as are for mouse class I This of calnexin's they molecules. interactions is its binding to Glc,Mang- is the highlighted by inability to demonstrate an interaction GlcNAc2 oligosaccharides on nascent glycoproteins. This between calnexin and heterodimers in heavy oligosaccharide structure is maintained by a cycle of chain-N2m human cells such an interaction is detected glucose removal and which is although easily reglucosylation thought to in mouse cells (Sugita and and continue as as the Brenner, 1994; Nossner long glycoprotein remains incompletely Parham, 1995). Calnexin association with unassembled folded. When is the folding complete glycoprotein is no human heavy chains has, however, been clearly demon- longer a substrate for the glucosyltransferase that catalyzes strated (Degen et al., 1992; and reglucosylation and the cycle ends Rajagopalan Brenner, (Hammond and 1994). Human CIR cells, transfected with the class I Helenius, 1994). In this view the main function of calnexin HLA-B27 gene, were used to determine whether the is simply to retain incompletely folded or unassembled functions of calnexin established for murine class I mole- glycoproteins within the ER where folding enzymes and cules can be extended to the human molecular system. The folding chaperones such as BiP and GRP94 can assist of B27 heavy chains and assembly with 32m were assessed the folding process (Helenius, 1994; Hebert et al., 1995). by comparing reactivity with antibodies that recognize it has been shown Although that calnexin retains non- unassembled chains heavy (mAb HC-10), heavy chains native proteins in the ER (Jackson et al., 1994; Rajagopalan with folded a3-domains regardless of association with et al., 1994), its ability to facilitate class I heavy-chain and chains (mAb folding and assembly with ,2m as demonstrated in the 5H7), heavy (mAb P2m 02m-associated W6/32). As in and depicted Figure 9A B, castanospermine present study cannot be attributed only to retention. For treatment resulted in a 3.5-fold decrease in assembly of example, the enhanced folding of Db heavy chains in the the B27 heavy chain with Whereas 70% of presence of heavy calnexin is evident by the end of a 5 min N2m. chains assembled over a 20 min period in control cells, in both pulse radiolabeling Drosophila cells and in castano- 20% assembled in only the presence of castanospermine. spermine-treated mouse cells (Figures 3 and 6). The effect No further was in assembly observed either case beyond of calnexin on increasing the assembly of heavy chains 1502 Molecular chaperone functions of calnexin -castanospermine unassem. 5H7 total 2n2m-assoc. 46 - aMW -0 am am am am0 qwam castanospermi ne 46 - a Om 2C am P aPm am 2 '0- 20 10 20 P 10 20 P 10 20 chase 1 (min) jnassemtlied folded u3 domain - 5H7 132m-associated 8"- 6c. --- :~~~~~ 2, r 2C chase imin! ----U-- - castarospe,m:ne castarospeorlnle Fig. 9. Effect of castanospermine on the folding and assembly of the human HLA-B27 molecule. (A) Human CIR cells expressing HLA-B27 were for 1 h, radiolabeled for 5 min with [35S]Met and then chased for the indicated times, all castanospermine. B27 molecules were pre-incubated isolated from digitonin lysates using antibodies recognizing either unfolded (mAb HC-1O), a3 domain-folded (mAb 5H7), (mAb W6/ 02m-associated 32) or total (all mAbs combined) heavy chains. Isolated proteins were analyzed by reducing SDS-PAGE and the mobility of the B27 heavy chain is of the B27 chain indicated. The data from the experiment in (A) were quantified by densitometry and the various conformational forms heavy (B) were expressed as percentages of the total heavy-chain signal present at each chase time. or stabilize the with is also obvious immediately following the pulse may prevent the formation of aggregates P2m also in Drosophila cells (Figures 1 and 2) and during the first polypeptide chain against proteolytic attack. Calnexin a mechanism other 10 min of chase in mouse or human cells (Figures 5 and enhanced Db heavy-chain folding by The 2- to 3-fold enhancement 9). Within these short time periods there are negligible than preventing degradation. 5 occurred differences in export of class I molecules from the ER in of folding observed during a min radiolabeling to differential in the absence or presence of calnexin. too rapidly to be attributed degradation functions? There is or of calnexin 3 and How then does calnexin effect its the absence presence (Figures 6). associations due calnexin now considerable evidence that polypeptide This effect may be to stabilizing partially a of the overall inter- folded conformations that are otherwise labile in the constitute substantial component action between calnexin and glycoproteins. Indeed, sites absence of calnexin interactions or in the continuing of interaction between calnexin and class I heavy chains absence of binding to P2m. have been mapped in proximity to the transmembrane Our model of how calnexin functions involves a working of chain where no N-linked glycans are mechanism that incorporates both oligosaccharide region the heavy two-step located et 1993; Carreno et al., 1995). and components in calnexin interactions (Ware (Margolese al., polypeptide it is that calnexin possesses a polypeptide et 1995). Initially, calnexin utilizes its lectin site to Therefore, likely al., site in addition to its site for binding bind oligosaccharides thereby bringing binding Glc,Man9- GlcIMangGlcNAc2 with the membrane- Calnexin function to other nascent glycoproteins into proximity GlcNAc2. may analogous calnexin associates molecular chaperones, facilitating protein folding by bind- constrained chaperone. Subsequently, and at this ing to structural elements exposed in unfolded proteins with the unfolded polypeptide stage directly interaction becomes less as preventing in- the lectin-oligosaccharide impor- (such hydrophobic surfaces) thereby the We that calnexin intermolecular associations that lead to tant for maintaining complex. propose appropriate as a molecular aggregation. functions in this second step typical for this view. In the or Our current results provide support chaperone, preventing aggregation rapid degradation of Kb or intermediates. It is not absence of calnexin, extensive aggregation heavy and stabilizing folding assembly in in cells to a lesser whether calnexin functions like other chains was observed Drosophila and, clear chaperones if what mouse cells. Formation of and release extent, in castanospermine-treated cycles binding and, so, regulates that the ER luminal domain of disulfide-linked Db chain dimers was such The recent aberrant heavy cycles. finding ATP raises the that in mouse cells treated with the of calnexin binds observed castanospermine; possibility binding be nucleotide or of may have been precluded could regulated by binding production larger aggregates cycles Db chains. et Such a the of unassembled exchange (Ou al., 1995). hypothetical cycle by rapid degradation heavy dissociation of the of a involve nucleotide-induced on the intrinsic sensitivity might Therefore, depending interaction with calnexin from calnexin followed to glycoprotein by two-step rebinding glycoprotein degradation, 1503 A.Vassilakos et al. in the domain of free or Db and and would continue until all polypeptide sites for o3 heavy chains, mAb calnexin P2m-associated 5H7 (Smith et al., 1994) which recognizes a folded determinant in the binding are buried in the folded molecule. In this scenario, a3 domain of free or B or HLA-A, C heavy chains (a 2r2m-associated the deglucosylation-reglucosylation cycle maintains the from Dr A rabbit antiserum gift J.Bluestone). (anti-8) directed against structure necessary for the first step in Glc1MangGlcNAc2 the C-terminus of Kb was provided by Dr B.Barber and was used to calnexin binding (or rebinding) and also facilitates all final isolate conformational states of Kb (Smith et al., 1986). Unassembled mouse class heavy chains were isolated with a rabbit antiserum dissociation when the glucosyltransferase fails to regluco- (anti- Dr and R.Machold et HC) provided by H.Ploegh (Machold al., 1995). sylate the native glycoprotein. mAb HC- 10, obtained from Dr H.Ploegh, was used to isolate unassembled It is apparent that calnexin's interactions are not abso- HLA-B27 chains et For the heavy (Stam al., 1986). isolation of calnexin lutely essential for class I heavy-chain folding and assem- and associated a rabbit antiserum proteins, raised against the C-terminus bly with In the absence of calnexin, ~40% of canine calnexin was used of mouse et (Jackson al., 1994). P2m. or human heavy chains acquire a correctly folded epitope in their domains and about 20-30% of heavy chains Pulse-chase and immune isolation OC3 radiolabeling Transfected cells were to assemble with These findings suggest Drosophila subjected pulse-chase radiolabeling that the diver- P2m. as described essentially previously (Jackson et al., 1994). Cells were sity of folding enzymes and molecular chaperones within in ml of buffer lysed 1 lysis containing either 1% digitonin (Sigma) or the ER may provide a degree of redundancy in assisting 1% Nonidet P40 (BDH) in PBS, pH 7.4, supplemented with 10 mM protein folding. The recent demonstration that class I iodoacetamide, 1% aprotinin, 0.25 mM 4-(2-aminoethyl)benzene-sul- biogenesis appears normal in a calnexin-deficient mutant and 10 each of fonyl fluoride, chymostatin, leupeptin, antipain gg/ml cell line also and incubations and suggests that, in the chronic pepstatin. Antibody collection of immune absence of complexes with isolation were protein A-agarose performed as described calnexin, alternative previously chaperone pathways can compensate et (Jackson Bead-bound were washed three times al., 1994). complexes for the loss of calnexin function (Sadasivan et al., 1995; with either 0.2% digitonin/PBS, pH 7.4 or NTSE (0.5% NP-40, 10 mM Scott and Dawson, 1995). One candidate for a compensat- Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA and 0.02% sodium azide) ory chaperone is BiP, which associates with several depending on the buffer used for lysis. Most immune isolations recovered unglycosylated in excess of 90% of the respective class I (and presumably not calnexin-associated) species in a single clearance. When mAb B22-249.R I was used, a second clearance was incorporated mouse class I molecules, but not their glycosylated to isolate all of the Db heavy chains. Digestion with counterparts (Fraser et P2m-associated al., 1987; Degen, 1995). Further- H was endoglycosidase as described previously (Jackson et al., 1994). more, a recent report has demonstrated that prolonged Immune complexes were analyzed by SDS-PAGE using 10% gels treatment of cells with castanospermine results in increased (Laemmli, 1970) and radioactive were proteins visualized by fluoro- expression of BiP mRNA (Pahl graphy. and Baeuerle, 1995). Murine EL4 and RIE-Db cells and human I C R cells were radiolabeled, An examination of calnexin's role in class I biogenesis and to lysed subjected immune isolation as described for Drosophila should ultimately address what the implications are for cells with the following exceptions. Pre-incubation was conducted for class I-restricted antigen presentation. For the 20-30% of min in 60 Met-free RPMI 1640 supplemented with 9 mM HEPES, 2 mM mouse class I molecules that are able to assemble in the + glutamine, 1 mM castanospermine at 37°C. Cells were radiolabeled in absence of calnexin, we have the same medium at 37°C + castanospermine after which the preliminary data indicating cells were incubated for various chase times in that they medium supplemented with 1 mM associate with the TAP peptide transporter and unlabeled Met ± castanospermine. Detergent lysates of human Cl R apparently acquire peptide ligands based on their stability cells were 10% incubated with formalin-treated Staphylococcus aureus in detergent lysates (W.-K.Suh, A.Vassilakos and D.B. cell suspension (Sigma) prior to immune isolation to remove endo- Williams, unpublished data). Whether or not calnexin genously expressed immunoglobulin. influences the spectrum of peptides bound is currently under investigation. Collectively, the results demonstrate Velocity density gradient centrifugation that calnexin, while not absolutely necessary for class I Drosophila cells expressing H-2 ± Kb and ,B2m calnexin were radio- labeled with biogenesis, plays an [35S]Met for 5 min and then chased for 80 min. important role in the efficient folding, Cells (IX 107) were lysed in 1 ml RIPA buffer (25 mM HEPES, pH 7.4, assembly and surface expression of murine and human 150 mM NaCl, 1% NP-40, 1% deoxycholate, 0.1% SDS, 20 mM N- class I molecules and hence, in a cell's ability to present ethylmaleimide and protease inhibitors), centrifuged to remove insoluble endogenous antigens. material, and 0.25 ml was loaded onto a 10-40% (w/v) linear glycerol gradient. Gradients were centrifuged at 4°C for 15 h at 35 000 r.p.m. using a Beckman SW41 rotor and 0.75 ml fractions were collected. Kb Materials and methods heavy chains were immunoisolated from each fraction with anti-8 serum and analyzed by SDS-PAGE. As a measure of total Kb molecules applied Cells and antibodies to the gradient, an additional 0.25 ml sample of lysate was treated Drosophila melanogaster Schneider cells transfected with cDNAs encod- directly with anti-8 and analyzed along with the gradient fractions. ing murine H-2 Kb or Db class I heavy chains and fB2m (either alone or Gradients were calibrated with Bio-Rad molecular mass standards with canine calnexin) have been described previously (Jackson et al., consisting of thyroglobulin (670 kDa), IgG (158 kDa), ovalbumin 1994). Murine EL4 cells (H-2b lymphoma; Gorer, 1950) and RIE-Db (44 kDa) and myoglobin (17 kDa). cells thymoma transfected with the Db heavy chain gene; (P2rm-deficient Allen et 1986) were al., grown in RPMI 1640 medium supplemented Flow cytometric with 10% fetal bovine serum and analysis antibiotics. Human C R cells transfected EL4 cells were with the HLA-B27 gene were obtained cultured for 24 h either alone or with the addition from Dr P.Cresswell and main- of castanospermine tained as described for murine cells (0.27 mM) at 24 h and at 16 h prior to with the addition of 0.5 processing for mg/ml G418 (Alexander et al., 1989). flow cytometry. Cells (I 106) were washed with PBS-BSA (PBS The following containing conformation-dependent monoclonal 0.1I% BSA and 0.1I% NaN3), resuspended in 0.1 antibodies (mAbs) ml PBS- were used: mAbs BSA, and 20-8-4S (Ozato and Sachs, 1981), Y3 (Jones incubated for I h with 1-2 ,ug of either mAb and B22-249.RI or Janeway, 1981) and B8-24-3 (Kohler et al., 1981) for mouse Y3 (all at 4°C). Cells were washed in PBS-BSA H-2 Kb and incubated for 45 heavy chains associated with mAbs B22-249.R1 (Lemke et min in 1/50 dilution of FITC-conjugated al., goat anti-mouse IgG (Sigma). N32m, 1979) and 27-11-13S (Ozato and Sachs, 1981) for mouse H-2 Db heavy The cells were washed in PBS and resuspended at 2-5x 106 in cells/ml chains associated with R2m, mAb W6/32 for human HLA-A, B or PBS containing 0.1 NaN3. C Incubations omitting either the first antibody chains heavy associated with et or both antibodies were j32m (Barnstable al., 1978), mAb 28- included as controls. Fluorescence data were 14-8S et (Ozato which a conformational collected and on al., 1980) recognizes a Coulter Elite epitope analyzed Epics flow cytometer. 1504 functions of calnexin Molecular chaperone TCR3 proteins with the molecular chaperone calnexin and specifically Acknowledgements results in accelerated degradation of nascent TCRtx proteins within We thank Peter Cresswell. Brian Barber, Jeff Bluestone, Hidde Ploegh the endoplasmic reticulum. EMBO J., 13, 3678-3686. and Robert Machold for their generous gifts of antibodies and cell lines. Kohler,G., Fischer-Lindahl,K. and Heusser,C. 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Published: Apr 1, 1996

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