Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

O-Glycosylated MUC2 Monomer and Dimer from LS 174T Cells Are Water-soluble, whereas Larger MUC2 Species Formed Early during Biosynthesis Are Insoluble and Contain Nonreducible Intermolecular Bonds

O-Glycosylated MUC2 Monomer and Dimer from LS 174T Cells Are Water-soluble, whereas Larger MUC2... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 30, Issue of July 24, pp. 18864 –18870, 1998 © 1998 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. O-Glycosylated MUC2 Monomer and Dimer from LS 174T Cells Are Water-soluble, whereas Larger MUC2 Species Formed Early during Biosynthesis Are Insoluble and Contain Nonreducible Intermolecular Bonds* (Received for publication, April 13, 1998, and in revised form, May 7, 1998) Magnus A. B. Axelsson, Noomi Asker, and Gunnar C. Hansson‡ Department of Medical Biochemistry, University of Go ¨ teborg, Medicinaregatan 9, 413 90 Gothenburg, Sweden The MUC2 mucin is the major gel-forming mucin in the mucosal surfaces is the mucus layer, the gel properties of the small and large intestine. Due to its sequence simi- which are due to macromolecules called mucins. A majority of larities with the von Willebrand factor, it has been sug- the mucins known today belong to this classical gel-forming gested to dimerize in the endoplasmic reticulum and type, although a few glycoproteins defined as mucins are mem- polymerize in the trans-Golgi network. Using an O-gly- brane-bound with yet unknown physiological functions (1). The cosylation-sensitive MUC2 antiserum, a dimerization present definition of mucin includes all glycoproteins that con- has been shown to occur in the endoplasmic reticulum sist of more than 50% O-linked oligosaccharides and that have of LS 174T cells (Asker, N., Axelsson, M. A. B., Olofsson, a majority of these oligosaccharides localized to mucin do- S.-O., and Hansson, G. C. (1998) J. Biol. Chem. 273, 18857– mains. These domains have a high number of O-glycosylated 18863). Using an antiserum immunoprecipitating O-gly- Ser and Thr amino acids, often appearing in tandem repeat cosylated MUC2 mucin, monomers and dimers were sequences. Gel-forming mucins are probably altered in several shown to occur in soluble form in the lysate of LS 174T diseases. Thus, alterations in the mucus barrier are probably cells. The amount of O-glycosylated dimer was small, essential in the pathogenesis of, for example, infections, peptic and no larger species were found even after long chase ulcers, and inflammatory bowel disease. Diseases such as cys- periods. However, most of the labeled MUC2 mucin was tic fibrosis and chronic bronchitis, and also trivial infections, found in pelleted debris of the cell lysate. This insoluble are characterized by increased mucus viscosity. Despite its MUC2 mucin was recovered by immunoprecipitation af- medical interest, the biochemical nature of these altered mucin ter reduction of disulfide bonds. Analysis by agarose gel electrophoresis revealed two bands, of which the properties is still poorly understood, largely due to the difficul- smaller migrated as the O-glycosylated monomer and ties associated with the large size of these molecules. the larger migrated as the O-glycosylated dimer of the The gel-forming mucins are proposed to be disulfide bond- M guani- cell lysis supernatant. Mucins insoluble in 6 stabilized linear polymers of highly glycosylated proteins (2), dinium chloride could also be obtained from LS 174T although other models are discussed (3). A typical example of cells. Such mucins have earlier been found in the small such a mucin is encoded by the MUC2 gene, one of the few intestine (Carlstedt, I., Herrmann, A., Karlsson, H., Shee- mucin genes fully sequenced (4). The MUC2 mucin occurs in han, J., Fransson, L.-Å., and Hansson, G. C. (1993) J. Biol. small and large intestine (5, 6) and probably also in the airways Chem. 268, 18771–18781). Reduction of the mucins fol- upon epithelial stress, such as infection (7). The primary trans- lowed by purification by isopycnic density gradient ul- lation product, the mucin apoprotein, has a size of about 600 tracentrifugation and analysis by agarose gel electro- kDa, including N-glycans. It is composed of five major regions; phoresis revealed two bands reacting with an anti- three of these, one N-terminal, one C-terminal, and one central, MUC2 tandem repeat antibody after deglycosylation. are rich in Cys, whereas the two others are rich in Thr, Ser, and These bands migrated identically to the bands shown by Pro. The two latter regions are the mucin domains, also called metabolic labeling, and they could also be separated by tandem repeat domains, and they become heavily O-glycosy- rate zonal ultracentrifugation. These results suggest that the MUC2 mucin is forming nonreducible intermo- lated in the Golgi apparatus, thus obtaining extreme proteo- lecular bonds early in biosynthesis, but after initial lytic resistance. The Cys rich N- and C-terminal regions show O-glycosylation. large similarities in the Cys positions to the corresponding parts of the von Willebrand factor (4, 8). This protein is known to form dimers stabilized by C-terminal disulfide bonds (9). The The mucosal surfaces comprise a 1000-fold larger interface dimerization of the von Willebrand factor takes place in the between the external and internal milieu than the skin. At the endoplasmic reticulum, whereas a further N-terminal oli- same time, the chemical and microbiological challenges are gomerization occurs in the late Golgi compartments because it more demanding. The most important protective component of requires low pH (9). Due to the sequence similarities, a similar assembly procedure has been proposed for the MUC2 mucin. Using an antiserum (a-MUC2TR) directed against protein * This work was supported by Swedish Medical Research Council epitopes in the MUC2 tandem repeat, and thus only immuno- Grants 7461 and 10443, the IngaBritt and Arne Lundbergs Stiftelse, precipitating non-O-glycosylated MUC2 mucin species, we the Go ¨ teborg Medical Society, and the Swedish Cystic Fibrosis Foun- dation. The costs of publication of this article were defrayed in part by have previously shown that non-O-glycosylated MUC2 dimers the payment of page charges. This article must therefore be hereby are formed in LS 174T cells (10). Because O-glycosylation marked “advertisement” in accordance with 18 U.S.C. Section 1734 starts in the cis-Golgi or earlier, this indicates an endoplasmic solely to indicate this fact. reticulum-based dimerization in analogy with the von Will- ‡ To whom correspondence should be addressed. Tel.: 46-31-7733488; Fax: 46-31-416108; E-mail: [email protected]. ebrand factor. This assumption has now been verified by sub- 18864 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Nonreducible Bonds of Insoluble MUC2 Mucin 18865 a-MUC2C2, or 25 mlof a-MUC2TR. 300 ml of immunoprecipitin was cellular fractionation (11). By the help of an antiserum directed then added before further incubation for 30 min. The immunoprecipi- against protein epitopes unaffected by O-glycosylation, the tates were pelleted by centrifugation and washed four times in washing monomer and dimer were identified in O-glycosylated form. solution (10 mM Tris-HCl, pH 7.4, 2 mM EDTA, 0.1% Triton X-100, 0.1% However, the amount of especially O-glycosylated dimers was SDS). The samples were released from the immunoprecipitin for 5 min small, suggesting that these disappeared somewhere. The at 95 °C in 50 mM Tris-HCl, pH 6.8, 20% glycerol, 5% SDS and, if dimers were found to form insoluble MUC2 mucin occurring in reducing conditions denoted, also 5% b-mercaptoethanol and 10 mM dithiothreitol. The immunoprecipitin was pelleted, and bromphenol cell debris pelleted from cell lysate. The oligomeric stage of this blue (0.015%) was added to the supernatant prior to electrophoresis. insoluble MUC2 mucin could not be analyzed, as reduction was Agarose gel electrophoresis (1.5 mm thick) was run on a vertical gel necessary for solubilization. When analyzing the reduced insol- apparatus (140 3 160-mm gels, Hoefer) casted on agarose gel support uble MUC2 mucin, not only the monomeric forms expected medium (Gel Bond Film, FMC). The separation gel was 1% Ultrapure from the von Willebrand factor analogy were found. Up to half (Life Technologies, Inc.) and 1% Sea Plaque low gelling temperature the amount of monomers were linked together with nonreduc- (FMC) or 2% Ultrapure (Life Technologies, Inc.). The buffer contained 0.378 M Tris-HCl, pH 8.8, and 0.1% SDS. The stacking gel was made of ible bonds and migrated as O-glycosylated dimers on agarose 0.8% agarose (SeaKem Gold, FMC) in 0.126 M Tris-HCl, pH 6.8, and gels. 0.1% SDS. The electrode buffer at the cathode was 0.05 M Tris, 0.384 M Mucins insoluble in chaotropic solutions, such as 6 M guani- glycine, and 0.1% SDS, and at the anode 0.025 M Tris, 0.192 M glycine, dinium chloride, have earlier been reported in the small intes- and 0.05% SDS. The current for electrophoresis was about 10 mA. Gels tine (12, 13), and in the gall bladder bile of some patients (14). were fixed for3hin30% ethanol and 10% acetic acid, soaked in Amplify Here we report that the cell line LS 174T can also form mucins fluorographic reagent (Amersham Pharmacia Biotech) with 5% glycerol for 30 min, dried on slab gel dryer for2hat50 °C,and exposed at insoluble in 6 M guanidinium chloride. These were found to 280 °C to a Biomax MS film (Kodak). contain the MUC2 mucin with nonreducible bonds, migrating Purification of Insoluble Mucins Followed by Western Blot—LS 174T similarly to the metabolically labeled insoluble MUC2 mucin cells were cultured in roller bottles for 10 days with daily medium from cell debris pellets. changes and washed twice in cold PBS. The purification was performed as described before (13). In short, the attached cells were extracted with MATERIALS AND METHODS guanidinium chloride (6 M guanidinium chloride, 5 mM EDTA, 10 mM Antibodies and Cell Lines—The polyclonal sera a-MUC2TR and NaH PO ,pH6.5,5mM N-ethylmaleimide, and 0.5 mM phenylmethyl- 2 4 a-MUC2N3 have been described (10, 11). An antiserum called sulfonyl fluoride), homogenized in a Dounce homogenizer (loose pestle), a-MUC2C2 was raised in a rabbit against a synthetic peptide, CIIKRP- and incubated for1hat room temperature under agitation. Insoluble DNQHVILKPGDFK, based on amino acids 4995–5013 on the C termi- material was pelleted by centrifugation for 25 min at 40,000 3 g, and nus of the human MUC2 apoprotein (4). A New Zealand White rabbit the pellet was washed and centrifuged six times in similar guanidinium was immunized once with 500 mg of peptide conjugated via Cys to 400 chloride buffer. The pellets were brought into solution by reduction of mg of keyhole limpet hemocyanin in Freund’s complete adjuvant, and disulfide bonds in reduction guanidinium chloride (6 M guanidinium twice with 250 mg of peptide conjugated to 200 mg of keyhole limpet chloride, 100 mM Tris, 5 mM EDTA, 10 mM freshly added dithiothreitol, hemocyanin in Freund’s incomplete adjuvant. The interval between the pH 8.0) for 5 h at 37 °C under gentle agitation. Cysteine groups were immunizations was 2 weeks. The specificity of the a-MUC2C2 anti- alkylated by the addition of 150 mM iodoacetamide (powder) followed by serum was tested as described for the a-MUC2N3 antiserum (11) using incubation overnight at room temperature under agitation in the dark. 1 mg of immunizing peptide/25 ml of antiserum for inhibiting immuno- The mucins were then purified by three rounds of isopycnic density precipitation. The colon adenocarcinoma cell line LS 174T (ATCC CL gradient ultracentrifugation (15), two with 4 M guanidinium chloride 188), producing MUC2 mucin, was cultivated as described before (10). and one with 0.2 M guanidinium chloride. After unloading the gradient Pulse-Chase, Immunoprecipitation, and Electrophoresis Proce- into fractions, the mucin peaks were identified by periodic acid-Schiff dures—Newly confluent LS 174T cells were starved for1hin2mlof slot blot, dialyzed against water, lyophilized, redissolved, and boiled for methionine-free minimum essential medium (Life Technologies, Inc.) 5 min at 95 °C in 50 mM Tris-HCl, pH 6.8, 20% glycerol, and 5% SDS. with 10% fetal bovine serum and 2 mM glutamine per 27-cm Petri dish After electrophoresis performed as above (without Gel Bond Film and and metabolically labeled using 150 mCi of [ S]methionine (Redivue without stacking gel), the proteins were electrophoretically transferred Promix [ S] Cell Labeling Mix, Amersham Pharmacia Biotech) per (1 mA/cm for3hat 14 °C, Sartoblot II-S) to a nylon blotting membrane Petri dish. Brefeldin A (Epicentre Technologies), 10 mg (solubilized in 5 (Immobilon-P, Millipore) using 48 mM Tris, 39 mM glycine, 0.0375% ml of ethanol) per ml of medium, was added 15 min before metabolic SDS, and 10% methanol (pH 8.3) as transfer buffer. The membrane was labeling. Ammonium chloride (25 mM) was added at the start of star- washed several times in water after blotting and dried before deglyco- vation. In pulse-chase experiments, the cells were chased with culture sylation with gaseous hydrogen fluoride at room temperature overnight medium, and when chase time exceeded 1 day, the medium was re- (16). The membrane was then blocked in saturated casein (Sigma) in placed daily. The cells were washed twice in cold PBS (137 mM NaCl, 150 mM NaCl, 20 mM Tris-HCl, pH 7.5, 0.1% Tween 20 for1hat room 2.7 mM KCl, 1.8 mM KH PO , 10.1 mM Na HPO ) before lysis on ice by 2 4 2 4 temperature and stained with 1% a-MUC2TR antiserum for1hat room the help of a cell scraper in 1 ml (per Petri dish) of PBS with 50 mM temperature, followed by incubation for1hat room temperature with Tris-HCl, pH 7.4, 5 mM EDTA, 1% Triton X-100, 5 mM N-ethylmaleim- peroxidase-conjugated anti-rabbit antibody (DAKO) diluted 1:1000. ide, 0.5 mM phenylmethylsulfonyl fluoride (Sigma), 20 mg/ml aprotinin Both antibody incubations were in 10% of saturated casein in 150 mM (Trasylol, Bayer), 60 mg/ml leupeptin (Sigma), 3.8 mg/ml calpain inhib- NaCl, 20 mM Tris-HCl, pH 7.5, 0.01% Tween 20. The assay was devel- itor I (Boehringer Mannheim), 0.7 mg/ml pepstatin (Boehringer Mann- oped by the ECL reagent (Amersham Pharmacia Biotech), according to heim), 0.02% sodium azide. The lysate was sonicated three times for 2 s the manufacturers recommendations and using Biomax MS film each (intensity 15, MSE Soniprep 100) and clarified by centrifugation. (Kodak). The cell debris pellet thus obtained was washed several times in PBS, Rate Zonal Ultracentrifugation—MUC2 mucin insoluble in 6 M gua- and its mucin content was solubilized by reduction in 200 ml of PBS, 100 nidinium chloride was purified as described above, lyophilized, and mM Tris-HCl, pH 8, 50 mM dithiothreitol (Merck) for1hat37 °C under redissolved in water (2 mg/ml). 20 ml of this solution was layered on top agitation. Material not solubilized by the reduction was pelleted by of a linear 22– 43% (w/w) sucrose gradient in 150 mM NaCl, 20 mM centrifugation and discarded, and the supernatant was incubated with Tris-HCl, pH 7.5, 0.02% NaN , with a volume of 5 ml in a 13 3 51-mm 150 mM iodoacetamide (Sigma) for 30 min at room temperature under ultracentrifuge tube (Beckman). The ultracentrifugation was per- agitation in the dark, and then diluted with PBS to 1 ml. This solution formed in a Beckman swinging bucket rotor (SW55Ti) for 15 h at 40,000 and the clarified cell lysate were then handled in similar ways. They rpm at 15 °C. Fractions (200 ml) were recovered from the top, and the were incubated with 25 ml of preimmune rabbit serum for 30 min water was evaporated in a vacuum centrifuge (Heto, Allerød, Den- followed by 30 min with 300 ml of 10% (v/v) immunoprecipitin (Life mark). The material was dissolved and boiled in 50 mM Tris-HCl, pH Technologies, Inc.). After pelleting the immunoprecipitin by centrifuga- 6.8, 20% glycerol, 5% SDS and subjected to SDS-agarose gel electro- tion, the supernatant was incubated under agitation for2hat room phoresis, Western blotting, hydrogen fluoride treatment, and assay as temperature or overnight at 4 °C with 50 mlof a-MUC2N3, 50 mlof described above. The intensity of the bands obtained on the film was measured by video densitometry using a video camera from KAPPA Messtechnik (Gleichen, Germany) and software from Bildanalys (Stock- The abbreviation used is: PBS, phosphate-buffered saline. holm, Sweden). 18866 Nonreducible Bonds of Insoluble MUC2 Mucin had been assembled. When the cells were cultured for 3 h after radioactive labeling (Fig. 1C), O-glycosylated monomers (O-M) and dimers (O-D) were found by both the a-MUC2N3 and the a-MUC2C2 antisera. The O-glycosylation-sensitive a- MUC2TR antiserum, however, did not precipitate these spe- cies, but only the remaining non-O-glycosylated monomer and dimer. The O-glycosylated monomer migrated to a position slightly above the non-O-glycosylated dimer on the gel, whereas the O-glycosylated dimer migrated only a short dis- tance from the stacking gel-separation gel interface. No larger bands specifically reacting with the a-MUC2N3 and a-MUC2C2 antisera could be found in the separation or stack- ing gels. Cells were also labeled for 30 min in the presence of Brefeldin A (Fig. 1B). This drug blocks the traffic of vesicles from the endoplasmic reticulum to the Golgi apparatus but not the traffic in the opposite direction, resulting in a transport of Golgi stack enzymes into the endoplasmic reticulum (17). As a result of this, glycosyltransferases from the proximal parts of FIG.1. Comparison of the mobility of non-O-glycosylated, par- tially O-glycosylated, and native O-glycosylated MUC2 species the Golgi occur in the endoplasmic reticulum O-glycosylating on SDS-agarose gel electrophoresis. Fluororadiography of SDS- proteins prematurely. Such O-glycosylation, however, is incom- agarose gel electrophoresis on immunoprecipitations from LS 174T cells plete, giving smaller sized mucins than the native O-glycosy- labeled for 20 min (panel A), Brefeldin A treated and labeled for 30 min lated molecule. Monomers (Fig. 1, o-M) and dimers (o-D) gly- (panel B), and not treated, labeled for 2 h, and chased for1h(panel C). After cell lysis, cell debris discarding, and precipitation with preim- cosylated in this way were precipitated by all of the antisera. mune serum, immunoprecipitations were performed with a-MUC2N3, This included the a-MUC2TR antiserum, reacting with a se- a-MUC2C2, or a-MUC2TR. The separation gel was 1% Ultrapure (Life quence repeated about 100 times in MUC2, whereas the Brefel- Technologies, Inc.) and 1% Sea Plaque low gelling temperature (FMC); din A treatment probably gave a glycosylation of only some of the gel was analyzed under nonreducing conditions. The designation M refers to non-O-glycosylated monomer, D to non-O-glycosylated dimer, the tandem repeats, allowing the antiserum to react with re- o-M to partially O-glycosylated monomer obtained by Brefeldin A treat- maining non-O-glycosylated repeats. The partially O-glycosy- ment, o-D to partially O-glycosylated dimer obtained by Brefeldin A lated bands (Fig. 1, o-M and o-D), but not the non-O-glycosy- treatment, O-M to O-glycosylated monomer, and O-D to O-glycosylated lated mono- and dimer bands (M and D), reacted with the Helix dimer. pomatia lectin (not shown). The monomers and dimers ob- served after Brefeldin A treatment migrated to a position be- Subcellular Fractionation—Subcellular fractionation was performed tween the non-O-glycosylated and the native O-glycosylated as described elsewhere (11). An alternative homogenization technique was also used. Cells were resuspended in ice-cold 130 mM KCl, 25 mM species. This shows that O-glycans decrease the electrophoretic Tris-HCl, pH 7.5, and protease inhibitors (11) and homogenized by mobility of the MUC2 mucin on SDS-agarose electrophoresis passing through syringe needles (5 3 22 G/0.7 mm, 5 3 25 G/0.5 mm, dependent on the amount of glycosylation and that migration 3 3 27 G/0.4 mm). on agarose gels could be used for interpreting the glycosylation status of mucins. RESULTS Larger MUC2 Species than O-Glycosylated Dimer Are Insol- Migration of Non-O-glycosylated and O-Glycosylated MUC2 uble and Contain Nonreducible Intermolecular Bonds—No Species on SDS-Agarose Gel Electrophoresis—We have previ- MUC2 species larger than O-glycosylated dimer were observed ously shown that the a-MUC2TR antiserum precipitates only on the agarose gels irrespective of the labeling and chase times non-O-glycosylated MUC2 species (10), whereas the used. To find out where the predicted larger forms of MUC2 a-MUC2N3 antiserum also precipitates species migrating were lost, the cell debris pelleted from the metabolically labeled slower on SDS-agarose gels. These were regarded as O-glyco- cells, lysed with detergent and ultrasonicated, was reduced by sylated also because they occurred in the Golgi apparatus but dithiothreitol. The obtained solubilized material was immuno- not in the endoplasmic reticulum, of LS 174T cells (11). In order precipitated with the a-MUC2N3 antiserum, followed by aga- to further relate these different bands on SDS-agarose gel rose gel electrophoresis (Fig. 2C). Two major MUC2 bands were electrophoresis to each other, partially O-glycosylated MUC2 found, of which the larger showed a partial separation into two. molecules, with intermediate sizes between non-O-glycosylated As a comparison, nonreduced MUC2 immunoprecipitated in and O-glycosylated species, were obtained by Brefeldin A treat- the normal way from the clarified cell lysate is shown (Fig. 2B). ment of the LS174T cells and analyzed by SDS-agarose gel This contains O-glycosylated monomer (O-M) and O-glycosy- electrophoresis. An additional antiserum, supposed to immu- lated dimer (O-D). A corresponding reduced lane was also in- noprecipitate both non-O-glycosylated and O-glycosylated cluded to demonstrate that the dimer (O-D) is reducible disap- MUC2, called a-MUC2C2, was also used to further verify that pearing upon thiol reduction (Fig. 2A). The monomer (O-M) and the different bands were indeed the MUC2 mucin. dimer (O-D) correspond in size to the two main bands of the Lysates from metabolically labeled LS174T cells were sub- reduced insoluble portion. It seems therefore reasonable to jected to immunoprecipitation with the three different antisera believe that the upper band from the insoluble portion (Fig. 2C, mentioned, and the precipitates were analyzed by SDS-agarose gel electrophoresis and autoradiography (Fig. 1). Radioactive O-X) consists of two monomers held together with bonds that are nonreducible by mercaptoethanol and dithiothreitol. This labeling for 20 min (Fig. 1A) revealed no O-glycosylated spe- cies, but the non-O-glycosylated monomer (M) was found by the nonreducible material (O-X) is different from the in vivo-occur- ring reducible dimer (O-D) and will therefore be referred to as O-glycosylation-sensitive antiserum directed against a MUC2 tandem repeat peptide (a-MUC2TR) (10) and by the X-dimer in this paper. Bands migrating to the same positions a-MUC2N3 and the a-MUC2C2 antisera. The a-MUC2TR an- as monomer and X-dimer could also be precipitated from the tiserum, reacting strongly compared with the two other sera, debris pellets by the a-MUC2C2 antiserum or by the H. poma- revealed that some non-O-glycosylated dimers (Fig. 1, D), also, tia lectin (not shown). No material at all in the reduced debris Nonreducible Bonds of Insoluble MUC2 Mucin 18867 from small intestine (12, 13), where the major part in the rat has been shown to be due to Muc2 (6, 8). Cultivated LS 174T cells were extracted with 6 M guanidinium chloride, and the insoluble mucin complexes were pelleted by centrifugation and washed six times. The material was solubilized by dithiothre- itol reduction and purified by three rounds of isopycnic density gradient ultracentrifugation in guanidinium chloride (15). The periodic acid-Schiff slot blot intensity of the fractions from the third preparative ultracentrifugation step, in 0.5 M guani- dinium chloride, is shown in Fig. 2E, having a peak within the density interval between 1.53 and 1.49 g/ml, as expected for mucins (15). The material from these fractions was pooled and analyzed by SDS-agarose gel electrophoresis to compare the electrophoresis pattern to that obtained by immunoprecipita- tion. The mucins were Western blotted onto nylon membranes and deglycosylated by gaseous hydrogen fluoride before stain- ing with the tandem repeat antiserum a-MUC2TR. Two bands were found, migrating to the same positions on the gel as monomer and X-dimer, respectively (Fig. 2D). No staining at all was found with a-MUC2TR on a non-deglycosylated control membrane (not shown). Because this antiserum does not bind O-glycosylated MUC2, this finding is further evidence that the species were O-glycosylated. This procedure also confirms that the bands at monomer and X-dimer position on the gels are indeed the MUC2 mucin, because the deglycosylation exposed the epitopes detected by the a-MUC2TR antiserum. Separation of Monomer and X-dimer by Rate Zonal Ultra- centrifugation—As an alternative molecular size separation method to SDS-agarose gel electrophoresis, not dependent on electrical charge, the monomer and X-dimer were also sub- jected to rate zonal ultracentrifugation. MUC2 mucin insoluble in 6 M guanidinium chloride solubilized by reduction was lay- ered on top of a sucrose gradient, which, after the centrifuga- FIG.2. Comparison of nonreduced and reduced soluble and tion, was recovered as 25 fractions run on agarose gel and reduced insoluble MUC2 mucin on SDS-agarose gel electro- phoresis. Fluororadiography of SDS-agarose gel electrophoresis on assayed by Western blot (Fig. 3A). The intensity of the obtained immunoprecipitations with a-MUC2N3 of LS 174T cell lysate (panel A, monomer and X-dimer bands in every fraction was measured reduced; panel B, nonreduced) and of the reduced corresponding cell by video densitometry (Fig. 3B). As expected for heteroge- debris pellet (panel C). Cells were labeled for 2 h, lysed, and ultrasoni- neously glycosylated molecules, both monomer and X-dimer cated. Insoluble material was pelleted by centrifugation. The pellet obtained was washed in PBS, and the mucin content was solubilized by showed a broad distribution over the gradient. Some of the reduction in 50 m M dithiothreitol and alkylated in 150 mM iodoacet- material migrated to the bottom of the ultracentrifuge tube, amide. Both the cell lysate and the reduced pellet were then subjected probably due to aggregation, but not by covalent linkages be- to preimmune immunoprecipitation followed by specific immunopre- cause it could be dissolved by boiling in SDS prior to electro- cipitation with a-MUC2N3. Shown is also a a-MUC2TR-stained West- ern blot of SDS-agarose gel electrophoresis on mucins insoluble in 6 M phoresis. Such aggregation might also have contributed to the guanidinium chloride obtained from LS 174T cells (panel D). These broad distribution over the gradient. Both species, however, mucins were solubilized by reduction in 10 mM dithiothreitol and alky- showed fairly distinct intensity maxima, probably representing lated in 30 mM iodoacetamide, prior to purification by three rounds of similar glycosylation stages of nonaggregated molecules. These isopycnic CsCl density gradient ultracentrifugation, in which the frac- tions were assayed by periodic acid-Schiff slot blot. The result from the maxima were localized at 29.25% sucrose for the monomer and third centrifugation in 0.5 M guanidinium chloride is shown in panel E. 31.5% sucrose for the X-dimer, giving sedimentation values of Marked are the fractions pooled and subjected to dialysis, lyophiliza- 14 S and 19 S, respectively, as calculated by the method of tion, and SDS-agarose gel electrophoresis followed by Western blot. McEwen (18). As shown in Fig. 2E, both species had the same After deglycosylation, the Western blot was assayed using a-MUC2TR, peroxidase-conjugated secondary antibody, and the ECL reagent (panel density, about 1.51 g/ml, as expected for O-glycosylated mucins D). For D, the same sample as in C was included, and it migrated (15). If the diffusion constant of the O-glycosylated MUC2 identically on the gel (not shown). All gels used were 2% Ultrapure (Life monomer is similar to that of monomer units from human Technologies, Inc.). The designation O-M refers to O-glycosylated mon- 28 2 cervical mucins in water (19), 4.7 3 10 cm /s, our results omer, O-D to O-glycosylated dimer (in panels A and B), and O-X to pairs of O-glycosylated monomers linked by nonreducible bonds, i.e. X-dimers would give a mass of the MUC2 monomer of about 2.7 3 10 . (in panels C and D). The diffusion constant of the X-dimer is not known, making a calculation of its absolute mass impossible. However, together with the SDS-agarose gel electrophoresis results, the rate zonal pellets could be precipitated using the a-MUC2TR antiserum (not shown). This shows that non-O-glycosylated MUC2 species ultracentrifugation makes it likely that the X-dimer indeed is a dimer. It also shows that the separation observed on the aga- are soluble and not incorporated into insoluble material. It should also be noted that no signs of nonreducible bonds have rose gels is due to size differences and not, for example, charge. Pulse-Chase Analysis of the Appearance of Soluble and In- ever been observed in non-O-glycosylated MUC2 species (10, 11). soluble MUC2 Mucin—In order to study the appearance of MUC2 Mucin, Which Was Insoluble in 6 M Guanidinium non-O-glycosylated and O-glycosylated soluble MUC2 species, Chloride, Also Contained Nonreducible Bonds—Mucins insol- immunoprecipitation with the a-MUC2N3 antiserum (Fig. 4A) uble in 6 M guanidinium chloride have earlier been reported serially followed by immunoprecipitation with the a-MUC2TR 18868 Nonreducible Bonds of Insoluble MUC2 Mucin FIG.4. Pulse-chase analysis of the appearance of soluble MUC2 mucin. Immunoprecipitations with the a-MUC2N3 antiserum (panel A), serially followed by immunoprecipitations with the a-MUC2TR (panel B), both on nonreduced SDS-agarose gel electro- phoresis. LS147T cells were labeled with [ S]methionine for the time denoted under the lanes, or, when this exceeded 2 h, labeled for2hand chased with new medium for the rest of the time. The cells were then lysed, insoluble cell debris were pelleted, and precipitation with preim- mune serum was performed on the supernatants, followed by specific precipitation. The separation gel used in panel A was 2% Ultrapure (Life Technologies, Inc.) and in panel B was 1% Ultrapure (Life Tech- nologies, Inc.) and 1% Sea Plaque low gelling temperature (FMC). The gels were assayed by fluororadiography. O-D, O-M, D, and M are as in Fig. 1. FIG.3. Separation of monomer and X-dimer by rate zonal ul- tracentrifugation. Western blot, performed as in Fig. 2D, showing every second fraction from a sucrose density gradient ultracentrifuga- tion of the reduced and alkylated 6 M guanidinium chloride insoluble mucins from LS 174T cells (panel A). Also shown (panel B) are video densitometry measurements of monomer (l ) and X-dimer (E)ofall fractions from the ultracentrifugation, which were recovered from the top. Sucrose concentrations (f) according to refractive index measure- ments are shown. O-X and O-M are as in Fig. 2. antiserum (Fig. 4B) was performed on the soluble parts of the lysates from LS 174T cells that had been pulse-chased for up to 3 days. The O-glycosylated molecules appeared after about 1 h and reached maximum radiolabeling after about 2 h (Fig. 4A). The amounts of both species then quickly decreased to levels FIG.5. Pulse-chase analysis of the appearance of insoluble that still remained constant in the cells after three days. As MUC2 mucin. Fluororadiography of SDS-agarose gel electrophoresis shown previously (10, 11), the non-O-glycosylated precursors on immunoprecipitations with a-MUC2N3 on the reduced cell debris pellets from the experiment in Fig. 4 (panel A) and from LS 174T cells appeared within 0.5 h and showed maximum intensity after metabolically labeled for 2 h and chased for 24 h in the presence of 25 1 h, as is also shown in Fig. 4B. The non-O-glycosylated mon- mM ammonium chloride (panel B). The pellets were washed in PBS, the omer decreased to low levels after only a few hours, whereas mucin content solubilized by reduction in 50 mM dithiothreitol and the dimer, formed from the monomer, was still present in alkylated in 150 mM iodoacetamide prior to preimmune immunoprecipi- reasonable amounts after 24 h. tation followed by specific immunoprecipitation with a-MUC2N3. The gel was 2% Ultrapure (Life Technologies, Inc.). O-X and O-M are as in The appearance of the insoluble MUC2 mucin was studied by Fig. 2. immunoprecipitation with the a-MUC2N3 antiserum on re- duced debris pellets from the immunoprecipitations in Fig. 4. Both the monomers and X-dimers turned up after 1 h (Fig. 5), 8 h is, to our experience, not real but rather a randomly occur- i.e. at the same time as the O-glycosylated bands in solution ring artifact, probably caused by incomplete reduction (incom- (Fig. 4A). The appearance of these insoluble forms of MUC2 plete solubilization) or destruction of the antibodies by the this early suggests a fast synthesis of the nonreducible bonds reducing or alkylating agents. and a cellular location relatively early in the biosynthesis path- Subcellular Fractionation—In an attempt to localize the cel- way. The bands remained in the cells in the same intensity lular location for the formation of the insoluble MUC2 mole- range after 3 days. In other experiments, high activity was still cules, subcellular fractionation was performed, using two dif- observed after 5 days, and weaker activity was observed after 7 ferent types of homogenization. However, all of the insoluble days (not shown). The lowered radiolabeling intensity at 3 and material after homogenization was recovered in the pellet ob- Nonreducible Bonds of Insoluble MUC2 Mucin 18869 tained by centrifugation at 1,400 3 g prior to subcellular frac- whereas the epitopes of a-MUC2TR are tandemly repeated tionation (not shown). No nonreducible X-dimers were found in about 100-fold in every molecule. This might also contribute to the subcellular fractions. This means that the appearance of the differences wherein the MUC2TR antiserum should be less insoluble MUC2 could not be studied by subcellular sensitive for the peptide conformation as caused by, for exam- fractionation. ple, the high number of probable Cys-linkages in the nontan- Nonreducible Bond Formation Does Not Require Low pH— dem repeat regions. Because the MUC2 apomucin is very large, The N terminus of the MUC2 mucin shows similarities in the repeated binding sites might also minimize mechanical disso- localizations of the Cys residues with the von Willebrand fac- ciation from the immunoprecipitin during the washing steps. tor. Because the von Willebrand factor dimers are known to The two bands observed on the agarose gels of reduced in- form polymers stabilized by N-terminal disulfide bonds by a soluble MUC2 mucin were regarded as a monomer and a dimer process that requires an acidic pH (9), a similar mechanism has (called X-dimer), because they migrated similarly on the gel to been proposed for the MUC2 mucin. No disulfide-stabilized the monomer and reducible dimer of the soluble MUC2 mucin. MUC2 species larger than dimers were detected in this study. Because mobility comparisons are uncertain when dealing with To study whether a lowered pH could influence the formation of broad bands caused by heterogeneous O-glycosylation, and be- the nonreducible bonds in MUC2, LS 174T cells were cultured cause separation could be due also to charge, rate zonal ultra- in the presence of ammonium chloride. This neutralizes the centrifugation was performed to verify that the monomer and acidic pH in the distal Golgi apparatus and secretory vesicles X-dimer were indeed of different size. This analysis suggested (20). The ratio between monomer and X-dimer was constant a mass of 2.7 MDa for the monomer using a previously pub- after ammonia treatment (Fig. 5B), although the amount of lished diffusion coefficient for human cervical mucins (19). both species was decreased, which we have noticed before. With a predicted size for the non-O-glycosylated MUC2 mono- This indicates that the formation of the nonreducible bonds in mer of 600 kDa, this suggests that the mass is made up of about MUC2 is not pH-sensitive. 80% O-glycans, which is within the usual range of mucin gly- cosylation (13, 21). The lack of information on the diffusion DISCUSSION constant for the X-dimer makes it impossible to calculate its The striking similarities in the localizations of the Cys resi- mass and state that it is indeed a dimer. For human cervical dues between the N and C termini of MUC2 and the von mucins, which have MUC5AC, MUC5B, MUC2, and MUC6 as Willebrand factor made it likely that the assembly followed known gel-forming components (22, 23), it has been shown that similar pathways. The present results show that this is not the oligomers, with the average size of four monomer units, have a case, and instead an insoluble form of MUC2 is formed early sedimentation coefficient about twice that of the monomers via intermolecular bonding that cannot be reduced. The pres- (40.4 for oligomers and 19.2 S for monomers in water) (19, 21). ent results are dependent on the specificity of the anti-MUC2 The sedimentation coefficient of the X-dimer was only 1.3–1.4 antisera available. The antisera a-MUC2N3 and a-MUC2C2 times larger than that of monomer, and if oligomerization does were able to precipitate glycosylated MUC2 monomers and not affect the diffusion properties of MUC2 and human cervical reducible dimers from the soluble cell lysate portion and mono- mucins in very different ways, these figures may suggest that mers and nonreducible X-dimers from reduced insoluble cell the X-dimer consists of two or, less likely, three monomer units. debris pellet. The conclusion that the immunoprecipitated spe- The ratio between sedimentation coefficients for non-O-glyco- cies were O-glycosylated was based on the observations that sylated, SDS-treated MUC2 dimer and monomer was just they (i) were larger than the non-O-glycosylated species and above 1.2, as was the ratio between apoB100 and apoB53/48 the incompletely glycosylated species produced upon Brefeldin (11). Dimerization of O-glycosylated and non-O-glycosylated A treatment (Fig. 1), (ii) appeared later in time during pulse- MUC2 apomucins may affect the diffusion constants in propor- chase than the non-O-glycosylated species (Fig. 4A), (iii) tional ways, if O-glycans and bound SDS, respectively, cause showed nonreducible size polydispersity on the gels, most prob- similar stretching of the tandem repeat regions. If this is the ably due to heterogeneous glycosylation, (iv) were localized to case, one should thus expect identical ratios. The small differ- the Golgi apparatus according to subcellular fractionation (11), ences found for the ratios, together with the results from the (v) reacted with the H. pomatia lectin (11), and (vi) were not immunoprecipitated by a-MUC2TR (Fig. 1). In addition, mono- SDS agarose gel electrophoresis support the interpretation that the X-dimer is indeed a dimer. mers and X-dimers from the guanidinium chloride insoluble preparation were regarded as O-glycosylated because they (vii) The finding of nonreducible bonds associated with MUC2 reacted with the a-MUC2TR antiserum on Western blot only mucin insolubility was an unexpected phenomenon. Because after deglycosylation (Fig. 2D), (viii) had a density of 1,51 g/ml the insoluble MUC2 portion had to be solubilized by reduction (Fig. 2E), as expected for O-glycosylated mucins (15), and (ix) prior to analysis, the rate of disulfide bond-stabilized oligomer- had higher sedimentation coefficients on ultracentrifugation ization could not be investigated. It is therefore impossible to than (SDS-treated) non-O-glycosylated species, as for mono- say whether nonreducible links between disulfide bond-stabi- mers 14 S (Fig. 3B) and 8 S (11), respectively. It should be noted lized dimers are enough to give the insolubility or whether the that the a-MUC2TR antiserum is much more efficient in im- mucins are further oligomerized by disulfide bonds before the munoprecipitating MUC2 than the O-glycosylation-insensitive nonreducible links are assembled. If the von Willebrand factor antisera, as shown in Fig. 1. This makes immediate kinetic analogy is correct, no further oligomerization than dimeriza- comparisons between the amounts of O-glycosylated and non- tion should occur until the trans-Golgi network, because such O-glycosylated MUC2 mucin, immunoprecipitated with the dif- oligomerization requires low pH. It seems possible that the ferent types of antisera, impossible. The reason for the differ- nonreducible bonds are assembled earlier because their forma- ence in effectiveness is not totally clear, but it might include tion does not require low pH, as shown by ammonium chloride different antibody titers and a large consumption of O-glycosy- treatment (Fig. 5B). As already mentioned, no nonreducible lation-insensitive antibodies by epitopes on nonlabeled MUC2 a-MUC2TR either in supernatant bonds could be seen using mucin stored in the cells. The O-glycosylation-insensitive anti- (10, 11) or pellet (not shown), showing that no non-O-glycosy- sera are directed against nonrepeated protein epitopes, lated species were linked with such bonds. This indicates a localization of the nonreducible bond formation in the Golgi M. A. B. Axelsson, N. Asker, and G. C. Hansson, unpublished data. rather than in the endoplasmic reticulum. Attempts to verify 18870 Nonreducible Bonds of Insoluble MUC2 Mucin this theory by two different subcellular fractionation protocols electrophoresis, suggesting that more than two nonreducible failed, as the insoluble MUC2 mucin was found already in the bonds can be formed in vivo. cell debris routinely pelleted after homogenization, and thus Mucins that are insoluble in 6 M guanidinium chloride are was excluded from the ultracentrifugation step. It is not clear consistently found in the small intestine (12, 13) and, to a more why the insoluble mucins were pelleted in these experiments variable extent, in for example the gall bladder (14). The phys- when they should have been encapsulated into membrane ves- iological function of mucin insolubility could be to improve the icles. One possibility for this could be that they gave the whole mucus barrier in water-filled lumen organs. The finding that vesicle a density high enough for pelleting; another possibility both guanidinium chloride insoluble MUC2 mucins and the is that they in some way caused the vesicles to rupture. How- metabolically labeled water-insoluble MUC2 mucins contain ever, vesicles containing Golgi markers were obtained at sub- the unexpected X-dimers suggests that water and guanidinium cellular fractionation and contained soluble O-glycosylated chloride insolubilities are reflecting an identical phenomenon. mono- and dimers (11). This might suggest that there could This could propose that one of the key events in the formation exist different vesicles containing soluble and insoluble MUC2 of the very resistant and insoluble MUC2 is the formation of mucins. nonreducible bonds. How the bonds give rise to insolubility is The nature of the nonreducible bonds is presently unknown. not clear. It could also be the case, of course, that they are not The fact that the linkages are nonreducible and survive 6 M vital for insolubility but are only synchronized with disulfide guanidinium chloride or boiling in 5% SDS suggests an uncom- bond-stabilized polymerization and/or oligosaccharide elonga- plicated covalent nature. This linkage could be between amino tion, yielding the insolubility. An attractive hypothesis, how- acids within the primary MUC2 sequence. Another possibility ever, could be that the nonreducible bonds serve as cross-links is that the bonding is via smaller linking proteins or peptides. between linear MUC2 oligomers. Mucins have previously been proposed to be polymerized via a linking peptide, but an isolated linking peptide has been shown REFERENCES to be encoded by the C terminus of Muc2 (24, 25). 1. Gendler, S. J., and Spicer, A. P. (1995) Annu. Rev. Physiol. 57, 604 – 634 2. Carlstedt, I., Sheehan, J. K., Corfield, A. P., and Gallagher, J. T. (1985) The X-dimers, obtained by reduction, are obviously soluble Biochemistry 20, 40 –76 because they can be immunoprecipitated. Our results contra- 3. Forstner, J. F., and Forstner, G. G. (1994) in Physiology of the Gastrointestinal dict that such soluble particles exist in vivo in substantial Tract (Johnson, L. R., ed) 3rd Ed., pp. 1255–1283, Raven Press, New York 4. Gum, J. R., Jr., Hicks, J. W., Toribara, N. W., Siddiki, B., and Kim, Y. S. (1994) amounts (Fig. 2A). Thus, nonreducible bonds between mono- J. Biol. Chem. 269, 2440 –2446 mers seem to be absent or rare in vivo. If monomers are not 5. Chang, S. K., Dohrman, AF, Basbaum, C. B., Ho, S. B., Tsuda, T., Toribara, N. W., Gum, J. R., and Kim, Y. S. (1994) Gastroenterology 107, 28 –36 forming nonreducible links, this suggests a conformational 6. Hansson, G. C., Baeckstro ¨ m, D., Carlstedt, I., and Klinga-Levan, K. (1994) change of the MUC2 molecule upon disulfide-stabilized dimer Biochem. Biophys. Res. Commun. 198, 181–190 formation, allowing the formation of the nonreducible bond. 7. Li, J.-D., Dohrman, A. F., Gallup, M., Miyata, S., Gum, J. R., Kim, Y. S., Nadel, J. A., Prince, A., and Basbaum, C. B. (1997) Proc. Natl. Acad. Sci. U. S. A. This would imply that the sites for the two different kinds of 94, 967–972 bonds are localized near each other on the molecule, in the C 8. Gum, J. R., Jr., Hicks, J. W., Toribara, N. W., Rothe, E. M., Lagace, R. E., and terminus if the von Willebrand factor analogy is correct. If the Kim, Y. S. (1992) J. Biol. Chem. 267, 21375–21383 9. Wagner, D. D. (1990) Annu. Rev. Cell Biol. 6, 217–246 nonreducible bonds are C-terminal and if they are formed in 10. Asker, N., Baeckstro ¨ m, D., Axelsson, M. A. B., Carlstedt, I., and Hansson, G. C. the Golgi, one may speculate on the presence of a specific (1995) Biochem. J. 308, 873– 880 11. Asker, N., Axelsson, M. A. B., Olofsson, S.-O., and Hansson, G. C. (1998) enzyme system catalyzing their formation. Attempts were J. Biol. Chem. 273, 18857–18863 made to move an eventual bond formation catalyzing enzyme 12. Karlsson, H., Sundler, F., and Fransson, L.-Å. (1982) Adv. Exp. Med. Biol. 144, system from the Golgi to the endoplasmic reticulum by the help 155–157 13. Carlstedt, I., Herrmann, A., Karlsson, H., Sheehan, J., Fransson, L.-Å., and of Brefeldin A. This did not, however, give rise to any nonre- Hansson, G. C. (1993) J. Biol. Chem. 268, 18771–18781 ducible bonds after 30 min either in the soluble or insoluble 14. Baeckstro ¨ m, D., Karlsson, N., and Hansson, G. C. (1994) J. Biol. Chem. 269, portion of the cell lysate (not shown). If the formation of the 14430 –14437 15. Carlstedt, I., Lindgren, H., Sheehan, J. K., Ulmsten, U., and Wingerup, L. nonreducible bonds takes place in the early Golgi, the C termi- (1983) Biochem. J. 211, 13–22 nus heparin binding domain on the MUC2 mucin could partic- 16. Axelsson, M. A. B., Hansson, E. M., Sikut, R., and Hansson, G. C. (1998) Glycoconj. J., in press ipate in some way, because this domain is cleaved off already in 17. Pelham, H. R. B. (1991) Cell 67, 449 – 451 the cis-Golgi apparatus (26). Such a function of this domain 18. McEwen, C. R. (1967) Anal. Biochem. 20, 114 –149 might be to bind heparan sulfate and thus orient the MUC2 19. Sheehan, J. K., and Carlstedt, I. (1984) Biochem. J. 217, 93–101 20. Wagner, D. D., Mayadas, T., and Marder, V. J. (1986) J. Cell Biol. 102, dimers in a way facilitating nonreducible bond formation. 1320 –1324 In Fig. 2B, the X-dimer band consists of two equally strong 21. Carlstedt, I., Lindgren, H., and Sheehan, J. K. (1983) Biochem. J. 213, 427– 435 adjacent bands. The presence and intensity of the upper of 22. Audie, J. P., Janin, A., Porchet, N., Copin, M. C., Gosselin, B., and Aubert, J. P. these is, in our experience, highly variable, and often only one (1993) J. Histochem. Cytochem. 10, 1479 –1485 X-dimer band can be found. However, the intensity of this 23. Gipson, I. K., Ho, S. B., Spurr-Michaud, S. J., Tisdale, A. S., Zhan, Q., Torlakovic, E., Pudney, J., Anderson, D. J., Toribara, N. W., and Hill, J. A. additional band had a tendency to increase upon prolonged (1997) Biol. Reprod. 56, 999 –1011 pulse-chase times (Fig. 5A). The variable additional bands in- 24. Roberton, A. M., Mantle, M., Fahim, R. E. F., Specian, R. D., Bennick, A., Kawagashi, S., Sherman, P., and Forstner, J. F. (1989) Biochem. J. 261, dicate that some MUC2 monomer residues might be involved in 637– 647 more than one nonreducible bond and that the formation of the 25. Xu, G., Huan, L.-J., Khatri, I. A., Wang, D., Bennick, A., Fahim, R. E. F., second bond could be delayed. The presence of nonreducible Forstner, G. G., and Forstner, J. F. (1992) J. Biol. Chem. 267, 5401–5407 26. Xu, G., Forstner, G. G., and Forstner, J. F. (1996) Glycoconj. J. 13, 81– 89 bonds within MUC2 from the intestine has been proposed by 27. Carlstedt, I., Davies, J., Herrmann, A., Lindell, G., Mårtensson, S., and Carlstedt et al. (27). They have shown that reduced MUC2 from Nordman, H. (1996) 4th International Workshop on Carcinoma-Associated intestinal tissue can form a ladder of bands upon agarose gel Mucins, July 27–31, Cambridge, p. 17, ICRF, London http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

O-Glycosylated MUC2 Monomer and Dimer from LS 174T Cells Are Water-soluble, whereas Larger MUC2 Species Formed Early during Biosynthesis Are Insoluble and Contain Nonreducible Intermolecular Bonds

Axelsson, Magnus A.B.; Asker, Noomi; Hansson, Gunnar C.
Journal of Biological Chemistry – Jul 1, 1998
Download PDF
7 pages

Loading next page...
 
/lp/unpaywall/o-glycosylated-muc2-monomer-and-dimer-from-ls-174t-cells-are-water-xc00F9FclS

References

References for this paper are not available at this time. We will be adding them shortly, thank you for your patience.

Publisher
Unpaywall
ISSN
0021-9258
DOI
10.1074/jbc.273.30.18864
Publisher site
See Article on Publisher Site

Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 30, Issue of July 24, pp. 18864 –18870, 1998 © 1998 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. O-Glycosylated MUC2 Monomer and Dimer from LS 174T Cells Are Water-soluble, whereas Larger MUC2 Species Formed Early during Biosynthesis Are Insoluble and Contain Nonreducible Intermolecular Bonds* (Received for publication, April 13, 1998, and in revised form, May 7, 1998) Magnus A. B. Axelsson, Noomi Asker, and Gunnar C. Hansson‡ Department of Medical Biochemistry, University of Go ¨ teborg, Medicinaregatan 9, 413 90 Gothenburg, Sweden The MUC2 mucin is the major gel-forming mucin in the mucosal surfaces is the mucus layer, the gel properties of the small and large intestine. Due to its sequence simi- which are due to macromolecules called mucins. A majority of larities with the von Willebrand factor, it has been sug- the mucins known today belong to this classical gel-forming gested to dimerize in the endoplasmic reticulum and type, although a few glycoproteins defined as mucins are mem- polymerize in the trans-Golgi network. Using an O-gly- brane-bound with yet unknown physiological functions (1). The cosylation-sensitive MUC2 antiserum, a dimerization present definition of mucin includes all glycoproteins that con- has been shown to occur in the endoplasmic reticulum sist of more than 50% O-linked oligosaccharides and that have of LS 174T cells (Asker, N., Axelsson, M. A. B., Olofsson, a majority of these oligosaccharides localized to mucin do- S.-O., and Hansson, G. C. (1998) J. Biol. Chem. 273, 18857– mains. These domains have a high number of O-glycosylated 18863). Using an antiserum immunoprecipitating O-gly- Ser and Thr amino acids, often appearing in tandem repeat cosylated MUC2 mucin, monomers and dimers were sequences. Gel-forming mucins are probably altered in several shown to occur in soluble form in the lysate of LS 174T diseases. Thus, alterations in the mucus barrier are probably cells. The amount of O-glycosylated dimer was small, essential in the pathogenesis of, for example, infections, peptic and no larger species were found even after long chase ulcers, and inflammatory bowel disease. Diseases such as cys- periods. However, most of the labeled MUC2 mucin was tic fibrosis and chronic bronchitis, and also trivial infections, found in pelleted debris of the cell lysate. This insoluble are characterized by increased mucus viscosity. Despite its MUC2 mucin was recovered by immunoprecipitation af- medical interest, the biochemical nature of these altered mucin ter reduction of disulfide bonds. Analysis by agarose gel electrophoresis revealed two bands, of which the properties is still poorly understood, largely due to the difficul- smaller migrated as the O-glycosylated monomer and ties associated with the large size of these molecules. the larger migrated as the O-glycosylated dimer of the The gel-forming mucins are proposed to be disulfide bond- M guani- cell lysis supernatant. Mucins insoluble in 6 stabilized linear polymers of highly glycosylated proteins (2), dinium chloride could also be obtained from LS 174T although other models are discussed (3). A typical example of cells. Such mucins have earlier been found in the small such a mucin is encoded by the MUC2 gene, one of the few intestine (Carlstedt, I., Herrmann, A., Karlsson, H., Shee- mucin genes fully sequenced (4). The MUC2 mucin occurs in han, J., Fransson, L.-Å., and Hansson, G. C. (1993) J. Biol. small and large intestine (5, 6) and probably also in the airways Chem. 268, 18771–18781). Reduction of the mucins fol- upon epithelial stress, such as infection (7). The primary trans- lowed by purification by isopycnic density gradient ul- lation product, the mucin apoprotein, has a size of about 600 tracentrifugation and analysis by agarose gel electro- kDa, including N-glycans. It is composed of five major regions; phoresis revealed two bands reacting with an anti- three of these, one N-terminal, one C-terminal, and one central, MUC2 tandem repeat antibody after deglycosylation. are rich in Cys, whereas the two others are rich in Thr, Ser, and These bands migrated identically to the bands shown by Pro. The two latter regions are the mucin domains, also called metabolic labeling, and they could also be separated by tandem repeat domains, and they become heavily O-glycosy- rate zonal ultracentrifugation. These results suggest that the MUC2 mucin is forming nonreducible intermo- lated in the Golgi apparatus, thus obtaining extreme proteo- lecular bonds early in biosynthesis, but after initial lytic resistance. The Cys rich N- and C-terminal regions show O-glycosylation. large similarities in the Cys positions to the corresponding parts of the von Willebrand factor (4, 8). This protein is known to form dimers stabilized by C-terminal disulfide bonds (9). The The mucosal surfaces comprise a 1000-fold larger interface dimerization of the von Willebrand factor takes place in the between the external and internal milieu than the skin. At the endoplasmic reticulum, whereas a further N-terminal oli- same time, the chemical and microbiological challenges are gomerization occurs in the late Golgi compartments because it more demanding. The most important protective component of requires low pH (9). Due to the sequence similarities, a similar assembly procedure has been proposed for the MUC2 mucin. Using an antiserum (a-MUC2TR) directed against protein * This work was supported by Swedish Medical Research Council epitopes in the MUC2 tandem repeat, and thus only immuno- Grants 7461 and 10443, the IngaBritt and Arne Lundbergs Stiftelse, precipitating non-O-glycosylated MUC2 mucin species, we the Go ¨ teborg Medical Society, and the Swedish Cystic Fibrosis Foun- dation. The costs of publication of this article were defrayed in part by have previously shown that non-O-glycosylated MUC2 dimers the payment of page charges. This article must therefore be hereby are formed in LS 174T cells (10). Because O-glycosylation marked “advertisement” in accordance with 18 U.S.C. Section 1734 starts in the cis-Golgi or earlier, this indicates an endoplasmic solely to indicate this fact. reticulum-based dimerization in analogy with the von Will- ‡ To whom correspondence should be addressed. Tel.: 46-31-7733488; Fax: 46-31-416108; E-mail: [email protected]. ebrand factor. This assumption has now been verified by sub- 18864 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Nonreducible Bonds of Insoluble MUC2 Mucin 18865 a-MUC2C2, or 25 mlof a-MUC2TR. 300 ml of immunoprecipitin was cellular fractionation (11). By the help of an antiserum directed then added before further incubation for 30 min. The immunoprecipi- against protein epitopes unaffected by O-glycosylation, the tates were pelleted by centrifugation and washed four times in washing monomer and dimer were identified in O-glycosylated form. solution (10 mM Tris-HCl, pH 7.4, 2 mM EDTA, 0.1% Triton X-100, 0.1% However, the amount of especially O-glycosylated dimers was SDS). The samples were released from the immunoprecipitin for 5 min small, suggesting that these disappeared somewhere. The at 95 °C in 50 mM Tris-HCl, pH 6.8, 20% glycerol, 5% SDS and, if dimers were found to form insoluble MUC2 mucin occurring in reducing conditions denoted, also 5% b-mercaptoethanol and 10 mM dithiothreitol. The immunoprecipitin was pelleted, and bromphenol cell debris pelleted from cell lysate. The oligomeric stage of this blue (0.015%) was added to the supernatant prior to electrophoresis. insoluble MUC2 mucin could not be analyzed, as reduction was Agarose gel electrophoresis (1.5 mm thick) was run on a vertical gel necessary for solubilization. When analyzing the reduced insol- apparatus (140 3 160-mm gels, Hoefer) casted on agarose gel support uble MUC2 mucin, not only the monomeric forms expected medium (Gel Bond Film, FMC). The separation gel was 1% Ultrapure from the von Willebrand factor analogy were found. Up to half (Life Technologies, Inc.) and 1% Sea Plaque low gelling temperature the amount of monomers were linked together with nonreduc- (FMC) or 2% Ultrapure (Life Technologies, Inc.). The buffer contained 0.378 M Tris-HCl, pH 8.8, and 0.1% SDS. The stacking gel was made of ible bonds and migrated as O-glycosylated dimers on agarose 0.8% agarose (SeaKem Gold, FMC) in 0.126 M Tris-HCl, pH 6.8, and gels. 0.1% SDS. The electrode buffer at the cathode was 0.05 M Tris, 0.384 M Mucins insoluble in chaotropic solutions, such as 6 M guani- glycine, and 0.1% SDS, and at the anode 0.025 M Tris, 0.192 M glycine, dinium chloride, have earlier been reported in the small intes- and 0.05% SDS. The current for electrophoresis was about 10 mA. Gels tine (12, 13), and in the gall bladder bile of some patients (14). were fixed for3hin30% ethanol and 10% acetic acid, soaked in Amplify Here we report that the cell line LS 174T can also form mucins fluorographic reagent (Amersham Pharmacia Biotech) with 5% glycerol for 30 min, dried on slab gel dryer for2hat50 °C,and exposed at insoluble in 6 M guanidinium chloride. These were found to 280 °C to a Biomax MS film (Kodak). contain the MUC2 mucin with nonreducible bonds, migrating Purification of Insoluble Mucins Followed by Western Blot—LS 174T similarly to the metabolically labeled insoluble MUC2 mucin cells were cultured in roller bottles for 10 days with daily medium from cell debris pellets. changes and washed twice in cold PBS. The purification was performed as described before (13). In short, the attached cells were extracted with MATERIALS AND METHODS guanidinium chloride (6 M guanidinium chloride, 5 mM EDTA, 10 mM Antibodies and Cell Lines—The polyclonal sera a-MUC2TR and NaH PO ,pH6.5,5mM N-ethylmaleimide, and 0.5 mM phenylmethyl- 2 4 a-MUC2N3 have been described (10, 11). An antiserum called sulfonyl fluoride), homogenized in a Dounce homogenizer (loose pestle), a-MUC2C2 was raised in a rabbit against a synthetic peptide, CIIKRP- and incubated for1hat room temperature under agitation. Insoluble DNQHVILKPGDFK, based on amino acids 4995–5013 on the C termi- material was pelleted by centrifugation for 25 min at 40,000 3 g, and nus of the human MUC2 apoprotein (4). A New Zealand White rabbit the pellet was washed and centrifuged six times in similar guanidinium was immunized once with 500 mg of peptide conjugated via Cys to 400 chloride buffer. The pellets were brought into solution by reduction of mg of keyhole limpet hemocyanin in Freund’s complete adjuvant, and disulfide bonds in reduction guanidinium chloride (6 M guanidinium twice with 250 mg of peptide conjugated to 200 mg of keyhole limpet chloride, 100 mM Tris, 5 mM EDTA, 10 mM freshly added dithiothreitol, hemocyanin in Freund’s incomplete adjuvant. The interval between the pH 8.0) for 5 h at 37 °C under gentle agitation. Cysteine groups were immunizations was 2 weeks. The specificity of the a-MUC2C2 anti- alkylated by the addition of 150 mM iodoacetamide (powder) followed by serum was tested as described for the a-MUC2N3 antiserum (11) using incubation overnight at room temperature under agitation in the dark. 1 mg of immunizing peptide/25 ml of antiserum for inhibiting immuno- The mucins were then purified by three rounds of isopycnic density precipitation. The colon adenocarcinoma cell line LS 174T (ATCC CL gradient ultracentrifugation (15), two with 4 M guanidinium chloride 188), producing MUC2 mucin, was cultivated as described before (10). and one with 0.2 M guanidinium chloride. After unloading the gradient Pulse-Chase, Immunoprecipitation, and Electrophoresis Proce- into fractions, the mucin peaks were identified by periodic acid-Schiff dures—Newly confluent LS 174T cells were starved for1hin2mlof slot blot, dialyzed against water, lyophilized, redissolved, and boiled for methionine-free minimum essential medium (Life Technologies, Inc.) 5 min at 95 °C in 50 mM Tris-HCl, pH 6.8, 20% glycerol, and 5% SDS. with 10% fetal bovine serum and 2 mM glutamine per 27-cm Petri dish After electrophoresis performed as above (without Gel Bond Film and and metabolically labeled using 150 mCi of [ S]methionine (Redivue without stacking gel), the proteins were electrophoretically transferred Promix [ S] Cell Labeling Mix, Amersham Pharmacia Biotech) per (1 mA/cm for3hat 14 °C, Sartoblot II-S) to a nylon blotting membrane Petri dish. Brefeldin A (Epicentre Technologies), 10 mg (solubilized in 5 (Immobilon-P, Millipore) using 48 mM Tris, 39 mM glycine, 0.0375% ml of ethanol) per ml of medium, was added 15 min before metabolic SDS, and 10% methanol (pH 8.3) as transfer buffer. The membrane was labeling. Ammonium chloride (25 mM) was added at the start of star- washed several times in water after blotting and dried before deglyco- vation. In pulse-chase experiments, the cells were chased with culture sylation with gaseous hydrogen fluoride at room temperature overnight medium, and when chase time exceeded 1 day, the medium was re- (16). The membrane was then blocked in saturated casein (Sigma) in placed daily. The cells were washed twice in cold PBS (137 mM NaCl, 150 mM NaCl, 20 mM Tris-HCl, pH 7.5, 0.1% Tween 20 for1hat room 2.7 mM KCl, 1.8 mM KH PO , 10.1 mM Na HPO ) before lysis on ice by 2 4 2 4 temperature and stained with 1% a-MUC2TR antiserum for1hat room the help of a cell scraper in 1 ml (per Petri dish) of PBS with 50 mM temperature, followed by incubation for1hat room temperature with Tris-HCl, pH 7.4, 5 mM EDTA, 1% Triton X-100, 5 mM N-ethylmaleim- peroxidase-conjugated anti-rabbit antibody (DAKO) diluted 1:1000. ide, 0.5 mM phenylmethylsulfonyl fluoride (Sigma), 20 mg/ml aprotinin Both antibody incubations were in 10% of saturated casein in 150 mM (Trasylol, Bayer), 60 mg/ml leupeptin (Sigma), 3.8 mg/ml calpain inhib- NaCl, 20 mM Tris-HCl, pH 7.5, 0.01% Tween 20. The assay was devel- itor I (Boehringer Mannheim), 0.7 mg/ml pepstatin (Boehringer Mann- oped by the ECL reagent (Amersham Pharmacia Biotech), according to heim), 0.02% sodium azide. The lysate was sonicated three times for 2 s the manufacturers recommendations and using Biomax MS film each (intensity 15, MSE Soniprep 100) and clarified by centrifugation. (Kodak). The cell debris pellet thus obtained was washed several times in PBS, Rate Zonal Ultracentrifugation—MUC2 mucin insoluble in 6 M gua- and its mucin content was solubilized by reduction in 200 ml of PBS, 100 nidinium chloride was purified as described above, lyophilized, and mM Tris-HCl, pH 8, 50 mM dithiothreitol (Merck) for1hat37 °C under redissolved in water (2 mg/ml). 20 ml of this solution was layered on top agitation. Material not solubilized by the reduction was pelleted by of a linear 22– 43% (w/w) sucrose gradient in 150 mM NaCl, 20 mM centrifugation and discarded, and the supernatant was incubated with Tris-HCl, pH 7.5, 0.02% NaN , with a volume of 5 ml in a 13 3 51-mm 150 mM iodoacetamide (Sigma) for 30 min at room temperature under ultracentrifuge tube (Beckman). The ultracentrifugation was per- agitation in the dark, and then diluted with PBS to 1 ml. This solution formed in a Beckman swinging bucket rotor (SW55Ti) for 15 h at 40,000 and the clarified cell lysate were then handled in similar ways. They rpm at 15 °C. Fractions (200 ml) were recovered from the top, and the were incubated with 25 ml of preimmune rabbit serum for 30 min water was evaporated in a vacuum centrifuge (Heto, Allerød, Den- followed by 30 min with 300 ml of 10% (v/v) immunoprecipitin (Life mark). The material was dissolved and boiled in 50 mM Tris-HCl, pH Technologies, Inc.). After pelleting the immunoprecipitin by centrifuga- 6.8, 20% glycerol, 5% SDS and subjected to SDS-agarose gel electro- tion, the supernatant was incubated under agitation for2hat room phoresis, Western blotting, hydrogen fluoride treatment, and assay as temperature or overnight at 4 °C with 50 mlof a-MUC2N3, 50 mlof described above. The intensity of the bands obtained on the film was measured by video densitometry using a video camera from KAPPA Messtechnik (Gleichen, Germany) and software from Bildanalys (Stock- The abbreviation used is: PBS, phosphate-buffered saline. holm, Sweden). 18866 Nonreducible Bonds of Insoluble MUC2 Mucin had been assembled. When the cells were cultured for 3 h after radioactive labeling (Fig. 1C), O-glycosylated monomers (O-M) and dimers (O-D) were found by both the a-MUC2N3 and the a-MUC2C2 antisera. The O-glycosylation-sensitive a- MUC2TR antiserum, however, did not precipitate these spe- cies, but only the remaining non-O-glycosylated monomer and dimer. The O-glycosylated monomer migrated to a position slightly above the non-O-glycosylated dimer on the gel, whereas the O-glycosylated dimer migrated only a short dis- tance from the stacking gel-separation gel interface. No larger bands specifically reacting with the a-MUC2N3 and a-MUC2C2 antisera could be found in the separation or stack- ing gels. Cells were also labeled for 30 min in the presence of Brefeldin A (Fig. 1B). This drug blocks the traffic of vesicles from the endoplasmic reticulum to the Golgi apparatus but not the traffic in the opposite direction, resulting in a transport of Golgi stack enzymes into the endoplasmic reticulum (17). As a result of this, glycosyltransferases from the proximal parts of FIG.1. Comparison of the mobility of non-O-glycosylated, par- tially O-glycosylated, and native O-glycosylated MUC2 species the Golgi occur in the endoplasmic reticulum O-glycosylating on SDS-agarose gel electrophoresis. Fluororadiography of SDS- proteins prematurely. Such O-glycosylation, however, is incom- agarose gel electrophoresis on immunoprecipitations from LS 174T cells plete, giving smaller sized mucins than the native O-glycosy- labeled for 20 min (panel A), Brefeldin A treated and labeled for 30 min lated molecule. Monomers (Fig. 1, o-M) and dimers (o-D) gly- (panel B), and not treated, labeled for 2 h, and chased for1h(panel C). After cell lysis, cell debris discarding, and precipitation with preim- cosylated in this way were precipitated by all of the antisera. mune serum, immunoprecipitations were performed with a-MUC2N3, This included the a-MUC2TR antiserum, reacting with a se- a-MUC2C2, or a-MUC2TR. The separation gel was 1% Ultrapure (Life quence repeated about 100 times in MUC2, whereas the Brefel- Technologies, Inc.) and 1% Sea Plaque low gelling temperature (FMC); din A treatment probably gave a glycosylation of only some of the gel was analyzed under nonreducing conditions. The designation M refers to non-O-glycosylated monomer, D to non-O-glycosylated dimer, the tandem repeats, allowing the antiserum to react with re- o-M to partially O-glycosylated monomer obtained by Brefeldin A treat- maining non-O-glycosylated repeats. The partially O-glycosy- ment, o-D to partially O-glycosylated dimer obtained by Brefeldin A lated bands (Fig. 1, o-M and o-D), but not the non-O-glycosy- treatment, O-M to O-glycosylated monomer, and O-D to O-glycosylated lated mono- and dimer bands (M and D), reacted with the Helix dimer. pomatia lectin (not shown). The monomers and dimers ob- served after Brefeldin A treatment migrated to a position be- Subcellular Fractionation—Subcellular fractionation was performed tween the non-O-glycosylated and the native O-glycosylated as described elsewhere (11). An alternative homogenization technique was also used. Cells were resuspended in ice-cold 130 mM KCl, 25 mM species. This shows that O-glycans decrease the electrophoretic Tris-HCl, pH 7.5, and protease inhibitors (11) and homogenized by mobility of the MUC2 mucin on SDS-agarose electrophoresis passing through syringe needles (5 3 22 G/0.7 mm, 5 3 25 G/0.5 mm, dependent on the amount of glycosylation and that migration 3 3 27 G/0.4 mm). on agarose gels could be used for interpreting the glycosylation status of mucins. RESULTS Larger MUC2 Species than O-Glycosylated Dimer Are Insol- Migration of Non-O-glycosylated and O-Glycosylated MUC2 uble and Contain Nonreducible Intermolecular Bonds—No Species on SDS-Agarose Gel Electrophoresis—We have previ- MUC2 species larger than O-glycosylated dimer were observed ously shown that the a-MUC2TR antiserum precipitates only on the agarose gels irrespective of the labeling and chase times non-O-glycosylated MUC2 species (10), whereas the used. To find out where the predicted larger forms of MUC2 a-MUC2N3 antiserum also precipitates species migrating were lost, the cell debris pelleted from the metabolically labeled slower on SDS-agarose gels. These were regarded as O-glyco- cells, lysed with detergent and ultrasonicated, was reduced by sylated also because they occurred in the Golgi apparatus but dithiothreitol. The obtained solubilized material was immuno- not in the endoplasmic reticulum, of LS 174T cells (11). In order precipitated with the a-MUC2N3 antiserum, followed by aga- to further relate these different bands on SDS-agarose gel rose gel electrophoresis (Fig. 2C). Two major MUC2 bands were electrophoresis to each other, partially O-glycosylated MUC2 found, of which the larger showed a partial separation into two. molecules, with intermediate sizes between non-O-glycosylated As a comparison, nonreduced MUC2 immunoprecipitated in and O-glycosylated species, were obtained by Brefeldin A treat- the normal way from the clarified cell lysate is shown (Fig. 2B). ment of the LS174T cells and analyzed by SDS-agarose gel This contains O-glycosylated monomer (O-M) and O-glycosy- electrophoresis. An additional antiserum, supposed to immu- lated dimer (O-D). A corresponding reduced lane was also in- noprecipitate both non-O-glycosylated and O-glycosylated cluded to demonstrate that the dimer (O-D) is reducible disap- MUC2, called a-MUC2C2, was also used to further verify that pearing upon thiol reduction (Fig. 2A). The monomer (O-M) and the different bands were indeed the MUC2 mucin. dimer (O-D) correspond in size to the two main bands of the Lysates from metabolically labeled LS174T cells were sub- reduced insoluble portion. It seems therefore reasonable to jected to immunoprecipitation with the three different antisera believe that the upper band from the insoluble portion (Fig. 2C, mentioned, and the precipitates were analyzed by SDS-agarose gel electrophoresis and autoradiography (Fig. 1). Radioactive O-X) consists of two monomers held together with bonds that are nonreducible by mercaptoethanol and dithiothreitol. This labeling for 20 min (Fig. 1A) revealed no O-glycosylated spe- cies, but the non-O-glycosylated monomer (M) was found by the nonreducible material (O-X) is different from the in vivo-occur- ring reducible dimer (O-D) and will therefore be referred to as O-glycosylation-sensitive antiserum directed against a MUC2 tandem repeat peptide (a-MUC2TR) (10) and by the X-dimer in this paper. Bands migrating to the same positions a-MUC2N3 and the a-MUC2C2 antisera. The a-MUC2TR an- as monomer and X-dimer could also be precipitated from the tiserum, reacting strongly compared with the two other sera, debris pellets by the a-MUC2C2 antiserum or by the H. poma- revealed that some non-O-glycosylated dimers (Fig. 1, D), also, tia lectin (not shown). No material at all in the reduced debris Nonreducible Bonds of Insoluble MUC2 Mucin 18867 from small intestine (12, 13), where the major part in the rat has been shown to be due to Muc2 (6, 8). Cultivated LS 174T cells were extracted with 6 M guanidinium chloride, and the insoluble mucin complexes were pelleted by centrifugation and washed six times. The material was solubilized by dithiothre- itol reduction and purified by three rounds of isopycnic density gradient ultracentrifugation in guanidinium chloride (15). The periodic acid-Schiff slot blot intensity of the fractions from the third preparative ultracentrifugation step, in 0.5 M guani- dinium chloride, is shown in Fig. 2E, having a peak within the density interval between 1.53 and 1.49 g/ml, as expected for mucins (15). The material from these fractions was pooled and analyzed by SDS-agarose gel electrophoresis to compare the electrophoresis pattern to that obtained by immunoprecipita- tion. The mucins were Western blotted onto nylon membranes and deglycosylated by gaseous hydrogen fluoride before stain- ing with the tandem repeat antiserum a-MUC2TR. Two bands were found, migrating to the same positions on the gel as monomer and X-dimer, respectively (Fig. 2D). No staining at all was found with a-MUC2TR on a non-deglycosylated control membrane (not shown). Because this antiserum does not bind O-glycosylated MUC2, this finding is further evidence that the species were O-glycosylated. This procedure also confirms that the bands at monomer and X-dimer position on the gels are indeed the MUC2 mucin, because the deglycosylation exposed the epitopes detected by the a-MUC2TR antiserum. Separation of Monomer and X-dimer by Rate Zonal Ultra- centrifugation—As an alternative molecular size separation method to SDS-agarose gel electrophoresis, not dependent on electrical charge, the monomer and X-dimer were also sub- jected to rate zonal ultracentrifugation. MUC2 mucin insoluble in 6 M guanidinium chloride solubilized by reduction was lay- ered on top of a sucrose gradient, which, after the centrifuga- FIG.2. Comparison of nonreduced and reduced soluble and tion, was recovered as 25 fractions run on agarose gel and reduced insoluble MUC2 mucin on SDS-agarose gel electro- phoresis. Fluororadiography of SDS-agarose gel electrophoresis on assayed by Western blot (Fig. 3A). The intensity of the obtained immunoprecipitations with a-MUC2N3 of LS 174T cell lysate (panel A, monomer and X-dimer bands in every fraction was measured reduced; panel B, nonreduced) and of the reduced corresponding cell by video densitometry (Fig. 3B). As expected for heteroge- debris pellet (panel C). Cells were labeled for 2 h, lysed, and ultrasoni- neously glycosylated molecules, both monomer and X-dimer cated. Insoluble material was pelleted by centrifugation. The pellet obtained was washed in PBS, and the mucin content was solubilized by showed a broad distribution over the gradient. Some of the reduction in 50 m M dithiothreitol and alkylated in 150 mM iodoacet- material migrated to the bottom of the ultracentrifuge tube, amide. Both the cell lysate and the reduced pellet were then subjected probably due to aggregation, but not by covalent linkages be- to preimmune immunoprecipitation followed by specific immunopre- cause it could be dissolved by boiling in SDS prior to electro- cipitation with a-MUC2N3. Shown is also a a-MUC2TR-stained West- ern blot of SDS-agarose gel electrophoresis on mucins insoluble in 6 M phoresis. Such aggregation might also have contributed to the guanidinium chloride obtained from LS 174T cells (panel D). These broad distribution over the gradient. Both species, however, mucins were solubilized by reduction in 10 mM dithiothreitol and alky- showed fairly distinct intensity maxima, probably representing lated in 30 mM iodoacetamide, prior to purification by three rounds of similar glycosylation stages of nonaggregated molecules. These isopycnic CsCl density gradient ultracentrifugation, in which the frac- tions were assayed by periodic acid-Schiff slot blot. The result from the maxima were localized at 29.25% sucrose for the monomer and third centrifugation in 0.5 M guanidinium chloride is shown in panel E. 31.5% sucrose for the X-dimer, giving sedimentation values of Marked are the fractions pooled and subjected to dialysis, lyophiliza- 14 S and 19 S, respectively, as calculated by the method of tion, and SDS-agarose gel electrophoresis followed by Western blot. McEwen (18). As shown in Fig. 2E, both species had the same After deglycosylation, the Western blot was assayed using a-MUC2TR, peroxidase-conjugated secondary antibody, and the ECL reagent (panel density, about 1.51 g/ml, as expected for O-glycosylated mucins D). For D, the same sample as in C was included, and it migrated (15). If the diffusion constant of the O-glycosylated MUC2 identically on the gel (not shown). All gels used were 2% Ultrapure (Life monomer is similar to that of monomer units from human Technologies, Inc.). The designation O-M refers to O-glycosylated mon- 28 2 cervical mucins in water (19), 4.7 3 10 cm /s, our results omer, O-D to O-glycosylated dimer (in panels A and B), and O-X to pairs of O-glycosylated monomers linked by nonreducible bonds, i.e. X-dimers would give a mass of the MUC2 monomer of about 2.7 3 10 . (in panels C and D). The diffusion constant of the X-dimer is not known, making a calculation of its absolute mass impossible. However, together with the SDS-agarose gel electrophoresis results, the rate zonal pellets could be precipitated using the a-MUC2TR antiserum (not shown). This shows that non-O-glycosylated MUC2 species ultracentrifugation makes it likely that the X-dimer indeed is a dimer. It also shows that the separation observed on the aga- are soluble and not incorporated into insoluble material. It should also be noted that no signs of nonreducible bonds have rose gels is due to size differences and not, for example, charge. Pulse-Chase Analysis of the Appearance of Soluble and In- ever been observed in non-O-glycosylated MUC2 species (10, 11). soluble MUC2 Mucin—In order to study the appearance of MUC2 Mucin, Which Was Insoluble in 6 M Guanidinium non-O-glycosylated and O-glycosylated soluble MUC2 species, Chloride, Also Contained Nonreducible Bonds—Mucins insol- immunoprecipitation with the a-MUC2N3 antiserum (Fig. 4A) uble in 6 M guanidinium chloride have earlier been reported serially followed by immunoprecipitation with the a-MUC2TR 18868 Nonreducible Bonds of Insoluble MUC2 Mucin FIG.4. Pulse-chase analysis of the appearance of soluble MUC2 mucin. Immunoprecipitations with the a-MUC2N3 antiserum (panel A), serially followed by immunoprecipitations with the a-MUC2TR (panel B), both on nonreduced SDS-agarose gel electro- phoresis. LS147T cells were labeled with [ S]methionine for the time denoted under the lanes, or, when this exceeded 2 h, labeled for2hand chased with new medium for the rest of the time. The cells were then lysed, insoluble cell debris were pelleted, and precipitation with preim- mune serum was performed on the supernatants, followed by specific precipitation. The separation gel used in panel A was 2% Ultrapure (Life Technologies, Inc.) and in panel B was 1% Ultrapure (Life Tech- nologies, Inc.) and 1% Sea Plaque low gelling temperature (FMC). The gels were assayed by fluororadiography. O-D, O-M, D, and M are as in Fig. 1. FIG.3. Separation of monomer and X-dimer by rate zonal ul- tracentrifugation. Western blot, performed as in Fig. 2D, showing every second fraction from a sucrose density gradient ultracentrifuga- tion of the reduced and alkylated 6 M guanidinium chloride insoluble mucins from LS 174T cells (panel A). Also shown (panel B) are video densitometry measurements of monomer (l ) and X-dimer (E)ofall fractions from the ultracentrifugation, which were recovered from the top. Sucrose concentrations (f) according to refractive index measure- ments are shown. O-X and O-M are as in Fig. 2. antiserum (Fig. 4B) was performed on the soluble parts of the lysates from LS 174T cells that had been pulse-chased for up to 3 days. The O-glycosylated molecules appeared after about 1 h and reached maximum radiolabeling after about 2 h (Fig. 4A). The amounts of both species then quickly decreased to levels FIG.5. Pulse-chase analysis of the appearance of insoluble that still remained constant in the cells after three days. As MUC2 mucin. Fluororadiography of SDS-agarose gel electrophoresis shown previously (10, 11), the non-O-glycosylated precursors on immunoprecipitations with a-MUC2N3 on the reduced cell debris pellets from the experiment in Fig. 4 (panel A) and from LS 174T cells appeared within 0.5 h and showed maximum intensity after metabolically labeled for 2 h and chased for 24 h in the presence of 25 1 h, as is also shown in Fig. 4B. The non-O-glycosylated mon- mM ammonium chloride (panel B). The pellets were washed in PBS, the omer decreased to low levels after only a few hours, whereas mucin content solubilized by reduction in 50 mM dithiothreitol and the dimer, formed from the monomer, was still present in alkylated in 150 mM iodoacetamide prior to preimmune immunoprecipi- reasonable amounts after 24 h. tation followed by specific immunoprecipitation with a-MUC2N3. The gel was 2% Ultrapure (Life Technologies, Inc.). O-X and O-M are as in The appearance of the insoluble MUC2 mucin was studied by Fig. 2. immunoprecipitation with the a-MUC2N3 antiserum on re- duced debris pellets from the immunoprecipitations in Fig. 4. Both the monomers and X-dimers turned up after 1 h (Fig. 5), 8 h is, to our experience, not real but rather a randomly occur- i.e. at the same time as the O-glycosylated bands in solution ring artifact, probably caused by incomplete reduction (incom- (Fig. 4A). The appearance of these insoluble forms of MUC2 plete solubilization) or destruction of the antibodies by the this early suggests a fast synthesis of the nonreducible bonds reducing or alkylating agents. and a cellular location relatively early in the biosynthesis path- Subcellular Fractionation—In an attempt to localize the cel- way. The bands remained in the cells in the same intensity lular location for the formation of the insoluble MUC2 mole- range after 3 days. In other experiments, high activity was still cules, subcellular fractionation was performed, using two dif- observed after 5 days, and weaker activity was observed after 7 ferent types of homogenization. However, all of the insoluble days (not shown). The lowered radiolabeling intensity at 3 and material after homogenization was recovered in the pellet ob- Nonreducible Bonds of Insoluble MUC2 Mucin 18869 tained by centrifugation at 1,400 3 g prior to subcellular frac- whereas the epitopes of a-MUC2TR are tandemly repeated tionation (not shown). No nonreducible X-dimers were found in about 100-fold in every molecule. This might also contribute to the subcellular fractions. This means that the appearance of the differences wherein the MUC2TR antiserum should be less insoluble MUC2 could not be studied by subcellular sensitive for the peptide conformation as caused by, for exam- fractionation. ple, the high number of probable Cys-linkages in the nontan- Nonreducible Bond Formation Does Not Require Low pH— dem repeat regions. Because the MUC2 apomucin is very large, The N terminus of the MUC2 mucin shows similarities in the repeated binding sites might also minimize mechanical disso- localizations of the Cys residues with the von Willebrand fac- ciation from the immunoprecipitin during the washing steps. tor. Because the von Willebrand factor dimers are known to The two bands observed on the agarose gels of reduced in- form polymers stabilized by N-terminal disulfide bonds by a soluble MUC2 mucin were regarded as a monomer and a dimer process that requires an acidic pH (9), a similar mechanism has (called X-dimer), because they migrated similarly on the gel to been proposed for the MUC2 mucin. No disulfide-stabilized the monomer and reducible dimer of the soluble MUC2 mucin. MUC2 species larger than dimers were detected in this study. Because mobility comparisons are uncertain when dealing with To study whether a lowered pH could influence the formation of broad bands caused by heterogeneous O-glycosylation, and be- the nonreducible bonds in MUC2, LS 174T cells were cultured cause separation could be due also to charge, rate zonal ultra- in the presence of ammonium chloride. This neutralizes the centrifugation was performed to verify that the monomer and acidic pH in the distal Golgi apparatus and secretory vesicles X-dimer were indeed of different size. This analysis suggested (20). The ratio between monomer and X-dimer was constant a mass of 2.7 MDa for the monomer using a previously pub- after ammonia treatment (Fig. 5B), although the amount of lished diffusion coefficient for human cervical mucins (19). both species was decreased, which we have noticed before. With a predicted size for the non-O-glycosylated MUC2 mono- This indicates that the formation of the nonreducible bonds in mer of 600 kDa, this suggests that the mass is made up of about MUC2 is not pH-sensitive. 80% O-glycans, which is within the usual range of mucin gly- cosylation (13, 21). The lack of information on the diffusion DISCUSSION constant for the X-dimer makes it impossible to calculate its The striking similarities in the localizations of the Cys resi- mass and state that it is indeed a dimer. For human cervical dues between the N and C termini of MUC2 and the von mucins, which have MUC5AC, MUC5B, MUC2, and MUC6 as Willebrand factor made it likely that the assembly followed known gel-forming components (22, 23), it has been shown that similar pathways. The present results show that this is not the oligomers, with the average size of four monomer units, have a case, and instead an insoluble form of MUC2 is formed early sedimentation coefficient about twice that of the monomers via intermolecular bonding that cannot be reduced. The pres- (40.4 for oligomers and 19.2 S for monomers in water) (19, 21). ent results are dependent on the specificity of the anti-MUC2 The sedimentation coefficient of the X-dimer was only 1.3–1.4 antisera available. The antisera a-MUC2N3 and a-MUC2C2 times larger than that of monomer, and if oligomerization does were able to precipitate glycosylated MUC2 monomers and not affect the diffusion properties of MUC2 and human cervical reducible dimers from the soluble cell lysate portion and mono- mucins in very different ways, these figures may suggest that mers and nonreducible X-dimers from reduced insoluble cell the X-dimer consists of two or, less likely, three monomer units. debris pellet. The conclusion that the immunoprecipitated spe- The ratio between sedimentation coefficients for non-O-glyco- cies were O-glycosylated was based on the observations that sylated, SDS-treated MUC2 dimer and monomer was just they (i) were larger than the non-O-glycosylated species and above 1.2, as was the ratio between apoB100 and apoB53/48 the incompletely glycosylated species produced upon Brefeldin (11). Dimerization of O-glycosylated and non-O-glycosylated A treatment (Fig. 1), (ii) appeared later in time during pulse- MUC2 apomucins may affect the diffusion constants in propor- chase than the non-O-glycosylated species (Fig. 4A), (iii) tional ways, if O-glycans and bound SDS, respectively, cause showed nonreducible size polydispersity on the gels, most prob- similar stretching of the tandem repeat regions. If this is the ably due to heterogeneous glycosylation, (iv) were localized to case, one should thus expect identical ratios. The small differ- the Golgi apparatus according to subcellular fractionation (11), ences found for the ratios, together with the results from the (v) reacted with the H. pomatia lectin (11), and (vi) were not immunoprecipitated by a-MUC2TR (Fig. 1). In addition, mono- SDS agarose gel electrophoresis support the interpretation that the X-dimer is indeed a dimer. mers and X-dimers from the guanidinium chloride insoluble preparation were regarded as O-glycosylated because they (vii) The finding of nonreducible bonds associated with MUC2 reacted with the a-MUC2TR antiserum on Western blot only mucin insolubility was an unexpected phenomenon. Because after deglycosylation (Fig. 2D), (viii) had a density of 1,51 g/ml the insoluble MUC2 portion had to be solubilized by reduction (Fig. 2E), as expected for O-glycosylated mucins (15), and (ix) prior to analysis, the rate of disulfide bond-stabilized oligomer- had higher sedimentation coefficients on ultracentrifugation ization could not be investigated. It is therefore impossible to than (SDS-treated) non-O-glycosylated species, as for mono- say whether nonreducible links between disulfide bond-stabi- mers 14 S (Fig. 3B) and 8 S (11), respectively. It should be noted lized dimers are enough to give the insolubility or whether the that the a-MUC2TR antiserum is much more efficient in im- mucins are further oligomerized by disulfide bonds before the munoprecipitating MUC2 than the O-glycosylation-insensitive nonreducible links are assembled. If the von Willebrand factor antisera, as shown in Fig. 1. This makes immediate kinetic analogy is correct, no further oligomerization than dimeriza- comparisons between the amounts of O-glycosylated and non- tion should occur until the trans-Golgi network, because such O-glycosylated MUC2 mucin, immunoprecipitated with the dif- oligomerization requires low pH. It seems possible that the ferent types of antisera, impossible. The reason for the differ- nonreducible bonds are assembled earlier because their forma- ence in effectiveness is not totally clear, but it might include tion does not require low pH, as shown by ammonium chloride different antibody titers and a large consumption of O-glycosy- treatment (Fig. 5B). As already mentioned, no nonreducible lation-insensitive antibodies by epitopes on nonlabeled MUC2 a-MUC2TR either in supernatant bonds could be seen using mucin stored in the cells. The O-glycosylation-insensitive anti- (10, 11) or pellet (not shown), showing that no non-O-glycosy- sera are directed against nonrepeated protein epitopes, lated species were linked with such bonds. This indicates a localization of the nonreducible bond formation in the Golgi M. A. B. Axelsson, N. Asker, and G. C. Hansson, unpublished data. rather than in the endoplasmic reticulum. Attempts to verify 18870 Nonreducible Bonds of Insoluble MUC2 Mucin this theory by two different subcellular fractionation protocols electrophoresis, suggesting that more than two nonreducible failed, as the insoluble MUC2 mucin was found already in the bonds can be formed in vivo. cell debris routinely pelleted after homogenization, and thus Mucins that are insoluble in 6 M guanidinium chloride are was excluded from the ultracentrifugation step. It is not clear consistently found in the small intestine (12, 13) and, to a more why the insoluble mucins were pelleted in these experiments variable extent, in for example the gall bladder (14). The phys- when they should have been encapsulated into membrane ves- iological function of mucin insolubility could be to improve the icles. One possibility for this could be that they gave the whole mucus barrier in water-filled lumen organs. The finding that vesicle a density high enough for pelleting; another possibility both guanidinium chloride insoluble MUC2 mucins and the is that they in some way caused the vesicles to rupture. How- metabolically labeled water-insoluble MUC2 mucins contain ever, vesicles containing Golgi markers were obtained at sub- the unexpected X-dimers suggests that water and guanidinium cellular fractionation and contained soluble O-glycosylated chloride insolubilities are reflecting an identical phenomenon. mono- and dimers (11). This might suggest that there could This could propose that one of the key events in the formation exist different vesicles containing soluble and insoluble MUC2 of the very resistant and insoluble MUC2 is the formation of mucins. nonreducible bonds. How the bonds give rise to insolubility is The nature of the nonreducible bonds is presently unknown. not clear. It could also be the case, of course, that they are not The fact that the linkages are nonreducible and survive 6 M vital for insolubility but are only synchronized with disulfide guanidinium chloride or boiling in 5% SDS suggests an uncom- bond-stabilized polymerization and/or oligosaccharide elonga- plicated covalent nature. This linkage could be between amino tion, yielding the insolubility. An attractive hypothesis, how- acids within the primary MUC2 sequence. Another possibility ever, could be that the nonreducible bonds serve as cross-links is that the bonding is via smaller linking proteins or peptides. between linear MUC2 oligomers. Mucins have previously been proposed to be polymerized via a linking peptide, but an isolated linking peptide has been shown REFERENCES to be encoded by the C terminus of Muc2 (24, 25). 1. Gendler, S. J., and Spicer, A. P. (1995) Annu. Rev. Physiol. 57, 604 – 634 2. Carlstedt, I., Sheehan, J. K., Corfield, A. P., and Gallagher, J. T. (1985) The X-dimers, obtained by reduction, are obviously soluble Biochemistry 20, 40 –76 because they can be immunoprecipitated. Our results contra- 3. Forstner, J. F., and Forstner, G. G. (1994) in Physiology of the Gastrointestinal dict that such soluble particles exist in vivo in substantial Tract (Johnson, L. R., ed) 3rd Ed., pp. 1255–1283, Raven Press, New York 4. Gum, J. R., Jr., Hicks, J. W., Toribara, N. W., Siddiki, B., and Kim, Y. S. (1994) amounts (Fig. 2A). Thus, nonreducible bonds between mono- J. Biol. Chem. 269, 2440 –2446 mers seem to be absent or rare in vivo. If monomers are not 5. Chang, S. K., Dohrman, AF, Basbaum, C. B., Ho, S. B., Tsuda, T., Toribara, N. W., Gum, J. R., and Kim, Y. S. (1994) Gastroenterology 107, 28 –36 forming nonreducible links, this suggests a conformational 6. Hansson, G. C., Baeckstro ¨ m, D., Carlstedt, I., and Klinga-Levan, K. (1994) change of the MUC2 molecule upon disulfide-stabilized dimer Biochem. Biophys. Res. Commun. 198, 181–190 formation, allowing the formation of the nonreducible bond. 7. Li, J.-D., Dohrman, A. F., Gallup, M., Miyata, S., Gum, J. R., Kim, Y. S., Nadel, J. A., Prince, A., and Basbaum, C. B. (1997) Proc. Natl. Acad. Sci. U. S. A. This would imply that the sites for the two different kinds of 94, 967–972 bonds are localized near each other on the molecule, in the C 8. Gum, J. R., Jr., Hicks, J. W., Toribara, N. W., Rothe, E. M., Lagace, R. E., and terminus if the von Willebrand factor analogy is correct. If the Kim, Y. S. (1992) J. Biol. Chem. 267, 21375–21383 9. Wagner, D. D. (1990) Annu. Rev. Cell Biol. 6, 217–246 nonreducible bonds are C-terminal and if they are formed in 10. Asker, N., Baeckstro ¨ m, D., Axelsson, M. A. B., Carlstedt, I., and Hansson, G. C. the Golgi, one may speculate on the presence of a specific (1995) Biochem. J. 308, 873– 880 11. Asker, N., Axelsson, M. A. B., Olofsson, S.-O., and Hansson, G. C. (1998) enzyme system catalyzing their formation. Attempts were J. Biol. Chem. 273, 18857–18863 made to move an eventual bond formation catalyzing enzyme 12. Karlsson, H., Sundler, F., and Fransson, L.-Å. (1982) Adv. Exp. Med. Biol. 144, system from the Golgi to the endoplasmic reticulum by the help 155–157 13. Carlstedt, I., Herrmann, A., Karlsson, H., Sheehan, J., Fransson, L.-Å., and of Brefeldin A. This did not, however, give rise to any nonre- Hansson, G. C. (1993) J. Biol. Chem. 268, 18771–18781 ducible bonds after 30 min either in the soluble or insoluble 14. Baeckstro ¨ m, D., Karlsson, N., and Hansson, G. C. (1994) J. Biol. Chem. 269, portion of the cell lysate (not shown). If the formation of the 14430 –14437 15. Carlstedt, I., Lindgren, H., Sheehan, J. K., Ulmsten, U., and Wingerup, L. nonreducible bonds takes place in the early Golgi, the C termi- (1983) Biochem. J. 211, 13–22 nus heparin binding domain on the MUC2 mucin could partic- 16. Axelsson, M. A. B., Hansson, E. M., Sikut, R., and Hansson, G. C. (1998) Glycoconj. J., in press ipate in some way, because this domain is cleaved off already in 17. Pelham, H. R. B. (1991) Cell 67, 449 – 451 the cis-Golgi apparatus (26). Such a function of this domain 18. McEwen, C. R. (1967) Anal. Biochem. 20, 114 –149 might be to bind heparan sulfate and thus orient the MUC2 19. Sheehan, J. K., and Carlstedt, I. (1984) Biochem. J. 217, 93–101 20. Wagner, D. D., Mayadas, T., and Marder, V. J. (1986) J. Cell Biol. 102, dimers in a way facilitating nonreducible bond formation. 1320 –1324 In Fig. 2B, the X-dimer band consists of two equally strong 21. Carlstedt, I., Lindgren, H., and Sheehan, J. K. (1983) Biochem. J. 213, 427– 435 adjacent bands. The presence and intensity of the upper of 22. Audie, J. P., Janin, A., Porchet, N., Copin, M. C., Gosselin, B., and Aubert, J. P. these is, in our experience, highly variable, and often only one (1993) J. Histochem. Cytochem. 10, 1479 –1485 X-dimer band can be found. However, the intensity of this 23. Gipson, I. K., Ho, S. B., Spurr-Michaud, S. J., Tisdale, A. S., Zhan, Q., Torlakovic, E., Pudney, J., Anderson, D. J., Toribara, N. W., and Hill, J. A. additional band had a tendency to increase upon prolonged (1997) Biol. Reprod. 56, 999 –1011 pulse-chase times (Fig. 5A). The variable additional bands in- 24. Roberton, A. M., Mantle, M., Fahim, R. E. F., Specian, R. D., Bennick, A., Kawagashi, S., Sherman, P., and Forstner, J. F. (1989) Biochem. J. 261, dicate that some MUC2 monomer residues might be involved in 637– 647 more than one nonreducible bond and that the formation of the 25. Xu, G., Huan, L.-J., Khatri, I. A., Wang, D., Bennick, A., Fahim, R. E. F., second bond could be delayed. The presence of nonreducible Forstner, G. G., and Forstner, J. F. (1992) J. Biol. Chem. 267, 5401–5407 26. Xu, G., Forstner, G. G., and Forstner, J. F. (1996) Glycoconj. J. 13, 81– 89 bonds within MUC2 from the intestine has been proposed by 27. Carlstedt, I., Davies, J., Herrmann, A., Lindell, G., Mårtensson, S., and Carlstedt et al. (27). They have shown that reduced MUC2 from Nordman, H. (1996) 4th International Workshop on Carcinoma-Associated intestinal tissue can form a ladder of bands upon agarose gel Mucins, July 27–31, Cambridge, p. 17, ICRF, London

Journal

Journal of Biological Chemistry – Unpaywall

Published: Jul 1, 1998

There are no references for this article.