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The primary structure of the beta‐subunit of the cell surface adhesion glycoproteins LFA‐1, CR3 and p150,95 and its relationship to the fibronectin receptor.

The primary structure of the beta‐subunit of the cell surface adhesion glycoproteins LFA‐1, CR3... The EMBO Journal vol.6 no.4 pp.915-919, 1987 The primary structure of the f-subunit of the cell surface adhesion glycoproteins LFA-1, CR3 and p150,95 and its relationship to the fibronectin receptor S.K.A.Lawl, J.Gagnon2, J.E.K.Hildreth3, C.E.Wells, Arnaout et al., 1985; Springer et al., 1985a). Invariably, they A.C.Willis and A.J.Wong4 are deficient in all three complexes suggesting that the deficien- cy is in the common fl-subunit. Furthermore, it is apparent that 'The MRC Immunochemistry Unit, Department of Biochemistry, University the absence of the f-subunit interferes with the maturation and of Oxford, South Parks Road, Oxford OXI 3QU, UK, and 3The Department of Pharmacology and Experimental Theurapeutics, The expression of the a-subunits Johns (Springer et al., 1985a). Hopkins University School of Medicine, Baltimore, MD 21205, USA Recently, it has been speculated that this group of adhesion proteins may belong to a more extended family of 2Present address: National Research Council Canada, Biotechnology Research cell-surface Institute, 6100 Avenue Royalmount, Montreal, Quebec, Canada H4P 2R2 proteins including the fibronectin receptor (Tamkun et al., 1986), the vitronectin receptor (Suzuki et al., 1986), and the gpIIb/IIIa 4Present address: Paediatric Research Unit, UMDS, Guy's Tower, London Bridge, SE1 9RT, UK glycoprotein of platelets (Cosgrove et al., 1986; Pytela et al., 1986; Pierce et al., 1987), all of which have specific affinity Communicated by A.F.Williams for peptides containing the amino acid sequence Arg Gly Asp The lymphocyte-function-associated antigen-i (LFA-1), the as found in (RGD) the binding region of the fibronectin molecule complement receptor type 3 (CR3) and the antigen p150,95 (Pytela et al., 1985; Pierschbacher and Ruoslahti, 1984; Wright are cell-surface glycoproteins. They are heterodimeric com- and Meyer, 1985). Such speculation is reinforced by the obser- plexes, each containing a unique a-subunit noncovalently vation that a synthetic peptide from C3, which contains the se- associated with a common f-subunit. We have purified the quence RGD, can bind to CR3 (Wright et al., 1987). f-subunit from human spleen and obtained limited peptide We focused our study on the f-subunit of this set of adhesion sequences. What appears to be the complete primary struc- glycoproteins with the aim of determining the primary structure ture for the fully processed :-subunit was obtained by cDNA by a combination of protein and cDNA sequencing. The fl-subunit sequencing of clones from a phorbol ester (PMA) stimulated protein sequence was found to be very similar to chicken integrin, U937 cDNA library. There are five possible glycosylation sites a subunit of a fibronectin binding molecule on fibroblasts and a transmembrane segment. The sequence contains a high (Tamkun et al., 1986). level of cysteine (7.6%), with 24 of the 57 cysteine residues being found in three repeating units each with eight residues. Results The entire primary structure has 47% identity to a subunit The purification of the fl-subunit LFA-J of of a fibronectin binding protein from chicken fibroblasts. It The f-subunit of the cell-surface seems that adhesion glycoproteins was LFA-1, CR3 and p150,95 antigens to may belong from human an extended family of cell surface purified spleen by affinity chromatography using molecules including the the H-52 monoclonal fibronectin binding protein. antibody (Hildreth and The August, 1985). methodology employed was basically that of Williams and Key words: re- f-subunit/CR3, LFA-1, plSO,95/fibronectin Barclay (1986). The detergent [Nonidet P40 (NP40)] extract of ceptors spleen membranes was passed the through H-52 column twice. The bound materials eluted at pH 11.5 in the first passage con- tained dissociated a- and fl-subunits since the Introduction interaction between the subunits is broken in the irreversibly alkaline conditions The lymphocyte-function-associated antigen-l (LFA-1), the com- et second (Sanchez-Madrid al., 1983). Upon passage only the plement 3 and the are cell- receptor type (CR3) antigen p50,95 fl-subunit was bound to the H-52 column and it was eluted at surface a- glycoprotein complexes each a containing unique pH 2.2. About 50% of the of the extract antigenic activity NP40 subunit and presumably an identical f-subunit (Sanchez-Madrid was recovered at this point. The eluted material was succinylated et The mol. wts of the as al., 1983). apparent a-subunits, deter- and before further reduced/alkylated purification by gel-filtration mined are 175 165 000 and 150 000 by SDS-PAGE, 000, a S-400 column in the of SDS. through Sephacryl presence 0.2% of daltons respectively, and that the f-subunit is 95 000 daltons. About 0.5 mg pf as determined the method of protein, by Lowry Each of the three in form of complexes participates some cell- et al. was obtained from 70 of human (1951), g spleen. The LFA-1 mediates interactions between adhesion activities; T-cells material eluted from the column had an mol. affinity apparent and their et Hildreth et targets (Sanchez-Madrid al., 1982; al., wt of 95 000 daltons on SDS -PAGE and this increased to CR3 has for a natural 1983); affinity iC3b, degradation product 120 000 after and 135 000 daltons succinylation mol. apparent of C3 factors I and H in the et by complement system (Ross al., wt after not succinylation/reduction/alkylation (data shown). and the interaction 1983) may promote phagocytic activity Tryptic peptides and has a similar bin- (Wright Silverstein, 1982, 1983); p150,95 to CR3 but the resultant manifestation of this The fl-subunit was with ding specificity succinylated/reduced/alkylated digested interaction is not known and Malhotra et and the were (Micklem Sim, trypsin peptides separated filtration follow- 1985; by gel ed reverse Twelve of the al., 1986). by phase h.p.l.c. peptides yielded uni- of suffer from Patients deficient in this Two T3-9 and T7-14 group glycoproteins NH2-terminal sequences. peptides, que infections et of and recurrent bacterial and contain sequences CNVCEC Two fungal (Anderson al., 1985; CDGVQI respectively. Press IRL Oxford, Limited, England S.K.A.Law et al. I I I I II I I I AVAI 1 BAfHI BAN1 ECORI ~~~~I- 1 1 7 HINFI I I I I I I PVUI I I I I RSA 1 Il II I I STYI CLONES J-9 I - X- 1 J-19 Fig. The restriction maps of clones X-1 and J-19. The restriction sites for used to for are J-9, enzyme enzymes generate shown. fragments sequencing The map was from obtained clone J-9 except for the 5'-extension on X-1 and where the 5' of the site was J-19, characterised. at the fragment StyI (----) 3'-ends of represent segments the clones that have not been characterised or sequenced. anti-sense oligonucleotide 17 bases in and CTCGCCCTGGTGGGGCTGCTCTCCCTCGGGTGCGTCCTCTCTCAGGAGTGCACGAAGTTC 60 mixtures, length, com- AAGGTCAGCAGCTGCCGGGAATGCATCGAGTCGGGGCCCGGCTGCACCTGGTGCCAGAAG plexity of 64 and 128 respectively, were synthesised according 180 CTGAACTTCACAGGGCCGGGGGATCCTGACTCCATTCGCTGCGACACCCGGCCACAGCTG CTCATGAGGGGCTGTGCGGCTGACGACATCATGGACCCCACAAGCCTCGCTGAAACCCAG to the sequences. GAAGACCACAATGGGGGCCAGAAGCAGCTGTCCCCACAAAAAGTGACGCTTTACCTGCGA 300 CCAGGCCAGGCAGCAGCGTTCAACGTGACCTTCCGGCGGGCCAAGGGCTACCCCATCGAC 360 cDNA cloning CTGTACTATCTGATGGACCTCTCCTACTCCATGCTTGATGACCTCAGGAATGTCAAGAAG CTAGGTGGCGACCTGCTCCGGGCCCTCAACGAGATCACCGAGTCCGGCCGCATTGGCTTC The two oligonucleotide mixtures were used to screen a cDNA 540 GGGTCCTTCGTGGACAAGACCGTGCTGCCGTTCGTGAACACGCACCCTGATAAGCTGCGA AACCCATGCCCCAACAAGGAGAAAGAGTGCCAGCCCCCGTTTGCCTTCAGGCACGTGCTG library from phorbol ester stimulated (PMA) U-937 which cells, AAGCTGACCAACAACTCCAACCAGTTTCAGACCGAGGTCGGGAAGCAGCTGATTTCCGGA 660 AACCTGGATGCACCCGAGGGTGGGCTGGACGCCATGATGCAGGTCGCCGCCTGCCCGGAG 720 are known to express all three cell surface adhesion glycopro- GAAATCGGCTGGCGCAACGTCACGCGGCTGCTGGTGTTTGCCACTGATGACGGCTTCCAT 780 TTCGCGGGCGACGGAAAGCTGGGCGCCATCCTGACCCCCAACGACGGCCGCTGTCACCTG 840 teins (Sanchez-Madrid et Six al., 1983; Malhotra, 1986). positive GAGGACAACTTGTACAAGAGGACCAACGAATTCGACTACCCATCGGTCGGCCAGCTGGCG 900 CACAAGCTGGCTGAAAACAACATCCACCCCATCTTCGCGGTGACCAGTAGGATGGTGAAG 960 clones which to both were hybridised oligonucleotides obtained ACCTACGAGAAACTCACCGAGATCATCCCCAAGTCAGCCGTGGGGGAGCTGTCTGAGGAC 1020 from - 35 000 colonies in the first Four of the clones 1080 screening. TCCAGCAATGTGGTCCATCTCATTAAGAATGCTTACAATAAACTCTCCTCCAGGGTCTTC CTGGATCACAACGCCCTCCCCGACACCCTGAAAGTCACCTACGACTCCTTCTGCAGCAAT 1140 - - had inserts of 2.7 kb; the other two had inserts of 1.8 kb. GGAGTGACGCACAGGAACCAGCCCAGAGGTGACTGTGATGGCGTGCAGATCAATGTCCCG 1200 ATCACCTTCCAGGTGAAGGTCACGGCCACAGAGTGCATCCAGGAGCAGTCGTTTGTCATC The six clones were not of each other. duplicates 1 3 2 0 CGGGCGCTGGGCTTCACGGACATAGTGACCGTGCAGGTCCTTCCCCAGTGTGAGTGCCGG TGCCGGGACCAGACCAGAGACCGCAGCCTCTGCCATGGCAAGGGCTTCTTGGAGTGCGGC One of the was studied It clones, J-9, contains extensively. ATCTGCAGGTGTGACACTGGCTACATTGGGAAAAACTGTGAGTGCCAGACACAGGGCCGG 1440 a poly(A) tail but does not to be full since a Nor- AGCAGCCAGGAGCTGGAAGGAAGCTGCCGGAAGGACAACAACTCCATCATCTGCTCAGGG 1 500 appear length CTGGGGGACTGTGTCTGCGGGCAGTGCCTGTGCCACACCAGCGACGTCCCCGGCAAGCTG 1 560 thern blot of the RNA with J-9 showed a original probed 1620 single ATATACGGGCAGTACTGCGAGTGTGACACCATCAACTGTGAGCGCTACAACGGCCAGGTC 16 80 TGCGGCGGCCCGGGGACGGCGCTCTGCTTCTGCGGGAAGTGCCCGCTGCCACCCGGGCTTT band between 3 and 3.5 kb. The most 5'-end Styl fragment (see 1 740 GAGGGCTCAGCGTGCCAGTGCGAGAGGACCACTGAGGGCTGCCTGAACCCGCGGCGTGTT 1 800 GAGTGTAGTGGTCCTGGCCGGTGCCGCTGCAACGTATGCCGAGTGCCATTCAGGCTACCAG restriction maps on was used to rescreen the cDNA Figure 1) CTGCCTCTGTGCCAGGAGTGCCCCGGCTGCCCCTCACCCTGTGGCAAGTACATCTCCTGC 1860 GCCGAGTGCCTGAAGTTCGAAAAGGGCCCCTTTGGGAAGAACTGCAGCGCGGCGTGTCCG 1920 library. Two X-1 and included extra on clones, J-19, sequence GGCCTGCAGCTGTCGAACAACCCCGTGAAGGGCAGGACCTGCAAGGAGAGGGACTCAGAG 1980 the 5'-side of J-9. Their restriction are also included in 2040 maps GGCTGCTGGGTGGCCTACACGCTGGAGCAGCAGGACGGGATGGACCGCTACCTCATCTAT GTGGATGAGAGCCGAGAGTGTGTGGCAGGCCCCAACATCGCCGCCATCGTCGGGGGCACC Figure GTGGCAGGCATCGTGCTCATCGGCATTCTCCTGCTGGTCATCTGGAAGGCTCTGATCCAC CTGAGCGACCTCCGGGAGTACAGGCGCTTTGAGAAGGAGAAGCTCAAGTCCCAGTGGAAC 2220 7he primary structure of the ,B-subunit AATGATAATCCCCTTTTCAAGAGCGCCACCACGACGGTCATGAACCCCAAGTTTGCTGAG 2280 AGTTAGGAGCA The primary structure of the f-subunit was determined a com- by bination of protein and cDNA Most of the cDNA sequencing. Fig. 2. The nucleotide obtained from sequence clones J-9, X-1 and J-19 was obtained from sequence clone J-9 for the 5' end which except covering an open reading frame. The first StyI site, starting at position 341, from X-1 where were clones and J-19. The was to fragments 5' to it were obtained and strategy employed sequenced for clones X-1 and J-19 is marked. Also marked is the termination codon with TAG at position cleave restriction clone into and enzymes, M13 sequence clones at random In picked the later (Figure 1). stages, specific fragments were to obtain purified sequences between The gaps. chicken fibroblasts (Tamkun et Extensive 5 al., 1986). identity is '-end of clones X- 1 sequences and J- 19 were obtained from the observed between the $-chain of the LFA-1 of family glycopro- fragment 5' to the first restriction site StyI with and without the teins and integrin. BamHI cut in the middle. A tail poly(A) was found at the 3'-end but the clones do not include that could code the sequence leader Discussion sequence since no codon for methionine is present. However, an open frame is evident and the A combination of and cDNA reading nucleotide peptide sequencing has what sequence yielded this covering open frame is shown in 2. appears to be the reading Figure complete primary structure for the Except fully pro- for a region from residue numbers cessed 2086-2 140 and the 23 bases f-subunit of the cell-surface adhesion glycoproteins at the most 3'-end, sequence data from both directions were ob- LFA-1, CR3 and p150,95. tained. In those two regions, the The protein was sequence was confirmed from purified from human spleen by virtue of its at least three different gel readings. On the average, each posi- affinity to the monoclonal antibody H-52. This antibody, though tion was covered 3.47 times. to initially reported have specificity for the fl-subunit of the three The translated protein sequence is shown in cell-surface Figure 3 together glycoprotein complexes (Hildreth and August, 1985), with the positions of the 12 tryptic peptides, which are turns out to be more in dispers- complicated its interaction with the an- ed the throughout sequence. tigens. It binds to the dissociated f-subunits from all three com- Also shown in 3 is Figure the sequence of the plexes, but only to the LFA-l integrin antigen, and not CR3 and p 150,95, molecule, a subunit of the fibronectin binding in their intact (x-f forms protein from (Micklem et al., 1986). Hence, the 916 The ,3-subunit of the LFA-1, CR3 and p150,95 cell surface antigens 1 36 -----LALVGLLSLGCVL---------SQECTKFKVSSCRECIESGPGCT be the assertion that the on could explained by simple epitope 1 MAETNLTLLTWAGILCCLIWSGSAQQGGSDCIKANAKSCGECIQAGPNCG is masked the a-subunits of the CR3 and the fl-subunit by p150,95 complexes. 37 RCDTRPQLLMRGCAADD 87 WCQKLNFTGPGDPDS MDPTSLAETQEDHNGG The cDNA clones X-1 and J-19 have insert sizes between 100 J-9, 51 WCKKTDFLQEGEPTSARCDDLAALKSKGCPEQDIENPRGSKRVLEDREVT 2.7 and 2.9 kb and are close to the size of the mRNA from the PMA-stimulated U-937 cells An frame 88 OK-------------- 122 (3-3.5 kb). open reading QLSPQKVTLYLRPGQAAAFNVTFRRAKGYPIDLY 101 150 NRKIGAAEKLKPEAITQIQPQKLVLQLRVGEPQTFSLKFKRAEDYPIDLY was found in the consensus from the 5' end sequence starting 4 to nucleotides number 2284-2286 where a termination codon +-T11-14--' FT12-12 -b GFGSFVDKTVLPFV 172 123 YLMDLSYSMLDDLRNVKKLGCDLLRALNE TESGR TAG is found. The contains five N-linked car- sequence possible 151 YLMDLSYSMKDDLENVKSLGTALMREMEKITSDFRIGFGSFVEKTVMPYI sites at 246 and in bohydrate positions 42, 108, 204, 493, agree- ment with the of Dahms and Hart who findings (1985) +-aT9-1 173 222 from the NTHPDKLRNPCPNKEKECQPPFAFRHVLKLTNNSNOFQTEVGKOLISGNL demonstrated the of five presence glycosylated peptides 201 STTPAKLRNPCTG-DQNCTSPFSYKNVLSLTSEGNKFNELVGKQHISGNL The also contains a $-subunit. sequence hydrophobic segment that the N-terminal from residues 693 -715. It is therefore likely 223 DAPEGGLDAMMQVAACPEEIGWRNVTRLLVFATDDGFHFAGDGKLGAILT 272 of the is the extracellular domain and the C- portion protein 250 DSPEGGFDAIMOVAVCGDQIGWRNVTRLLVFSTDAGFHFAGDGKLGGIVL 299 in bet- terminal with one transmembrane cytoplasmic segment 4-T 11 -1 6+ ween. The clones do not include the ATG for the initia- 322 signal 273 PNDGRCHLEDNLYKRSNEFDYPSVGQLAHKLAENNIQPIFAVTSRMVKTY 300 PNDGKCHLENNMYTMSHYYDYPSIAHLVQKLSENNIQTIFAVTEEFQAVY tion methionine residue. it is that a of However, likely portion the leader is included in clone J-19. Since the N-terminal sequence 323 EKLTEI IPKSAVGELSEDSSNVVHLIKNAYNKLSSRVFLDHNALPDTLKV 372 of the fl-subunit is blocked et it is not (Springer al., 1985b), possi- 350 KELKNLIPKSAVGTLSSNSSNVIOLI IDAYNSLSSEVILENSKLPKEVTI ble to the exact N-terminal of the mature but pinpoint protein *-T7-14 it is to that it be the residue tempting speculate might glutamine 373 ---QEQSFV TYDSFCSNGVTHRNQPRGDCDCVQINVPITFQVKVTATECI 400 449 after the leader If that SYKSYCKNGVNDTQEDGRKCSNISIGDEVRFEINVTANECPKKGQNETIK (position 15) putative sequence fragment. is the the f-subunit would contain 747 residues and would case, -T9-19 +-Tl0 420 IRALGFTDIVTVQVLPQCECRCRDQS-RDRSLCH-GKGFLECGICRCDTG have a mol. wt of 82 542 daltons. with the Together carbohydrate 450 IKPLGFTEEVEIHLQFICDCLCQSEGEPNSPACHDGNGTFECGACRCNEG at the N-linked estimated to have a mol. wt glycosylation sites, -2 4<T3-7 of 3000 daltons the mol. wt of the fl-subunit is estimated per site, 468 YIGKNCECQTQGRSSQELEGSCRKDNNSI ICSGLGDCVCGOCLCHTSDVP to be 97 542 daltons which is in reasonable with the 549 agreement S00 RIGRLCECSTDEVNSEDMDAYCRRENSTEICSNNGECICGQCVCKKRENT mol. wt SDS -PAGE. Our most N-terminal apparent by pep- 1 -3 +-T1 Q Ti marked the first definitive at residue 567 tide, 1-14, sequence 518 GKLIYGOYCECDTINCERYNGQVCGGPGRGLCFCGKCRCHPGFEGSACQC 550 NEVYSGKYCECDNFNCDRSNGLICGG--NGICKCRVCECFPNFTGSACDC number 137 (Figure 3). A most unusual feature of the structure is its primary high cys- -T3-99 teine content. A total of 57 residues were identified with cysteine 568 ERTTEGCLNPRRVECSGRGRCRCNVCEC-HSGYQLPLCOECPGCPSPCGK 647 42 found within a stretch of 256 residues 437 598 SLDTTPCMAGNGOICNGRGTCECGTCNCTDPKFQGPTCEMCQTCLGVCAE (residues -692) N-terminal to the transmembrane Within this sequence. cysteine- 655 rich there to be three units 617 YISCAECLKFEKGPFGKNCSAACPGLQLSNNPVKGR-------T----TCK region, appear repeating arbitarily 648 HKDCVOCRAFEKGEKKETCSQECMHFNMTRVESRGKLPQPVHPDPLSHCK defined from residues 473 and 565-603 to con- -525, 526-564, tain residues in a of C X C eight cysteine arranged pattern ( ) +T10-9 T13-17 C( ) CXCXX CX C( ) where each X C( ) 656 ERDSEGCWVAYTLEQQDGMDRYLIYVDESRECVAGPNIAAIVGGTVAGIV 705 stands for an amino acid and the stretches of 698 EKDVGDCWFYFTYSVNSNGEAS-VHVVETPECPSGPDI IPIVAGVVAGIV 746 ( ) represents amino acids with from 4 to residues. length ranging 706 LICILLLVIWKALIHLSDLREYRRFEKEKLKSQWNN-DNPLFKSATTTVM 754 The three were to statistical repeating segments subjected 747 LIGLALLLIWKLLMIIHDRREFAKFEKEKMNAKWDTCENPIYKSAVTTVV 796 the ALIGN et analysis by program (Dayhoff al., 1983; Staden, The scores of 1986). pairwise comparison yielded alignment 3.59, NPKFAES 761 and 4.29 SD that the of a chance 5.45 units; indicating probability 797 NPKYEGK 803 2 x 10-4 et is less than relationship (Dayhoff al., 1983). is The of residues in the pattern cysteine cysteine-rich Fig. 3. The primary structure of the 13-subunit of the cell surface adhesion region is shown. The of of a fibronectin glycoproteins. The derived amino acid sequence position similar to that first observed in a subunit very the 12 tryptic peptides (< >), the five potential N-linked glycosylation from fibroblasts et chicken binding protein (integrin) (Tamkun sites (A), and the putative transmembrane segment ( ) are marked. show a level of similari- In the two al., 1986). fact, proteins high The primary structure of integrin (Tamkun et al., 1986) is shown as the matched with a 47% overall The two are ty. sequences easily lower sequence. The alignment of the two sequences was made by the of amino acid residues from In a stretch 278 ALIGN program (Dayhoff et al., 1983). Identical residues are indicated by identity. positions below the are found at the level of 63%. (-) sequences. 90 to identities 367, However, site at a residue in proposed possible phosphorylation tyrosine here must be is not found in the fl-subunit. This fl-subunit purified and reported formally regarded integrin (position 788) aligned the of LFA-1 must be that consistent with the failure to as that the antigen. It stressed, however, observation is phosphorylate ,B- under various con- are to be of cell-surface adhesion the f-subunits of the three antigen complexes likely subunit glycoproteins on: the extensive of ditions identical, based (i) sharing antigenic epitopes, (Malhotra, 1986). and in their of monoclonal antibodies et The exact roles of the LFA-1 e.g. that IB4 (Wright al., 1983), p150,95 respective and cells are not clear. et and TSl/18 et between effector MHM23 (Hildreth al., 1983) (Sanchez-Madrid interaction target to in invariable that defective However, CR3 has been postulated participate phagocytic al., 1983); and (ii) the finding patients in all three on their cell and Silvers- in the f-subunit are deficient events mediated activated antigens (Wright by macrophages it serves as a communicative bet- not bind to CR3 and surfaces. The fact that H-52 does plS50,95 bridge tein, 1982). Possibly, 917 S.K.A.Law et al. ween iC3b on on the outside opsonised particles and the actin Acknowledgements network on the the of inside, perturbation which by drugs such We wish to the late Professor under whose director- acknowledge R.R.Porter, as B was known to inhibit cytochalasin the phagocytic capacity ship of the MRC Immunochemistry Unit this project was initiated. We would of the and cells (Axline Reaven, 1974). Thus, CR3 may be com- like to thank S.J.Dodsworth for technical assistance and Miss C.Brooks for prepara- with the fibronectin which tion of the Part of this was Lister the ex- manuscript. project supported by a Institute pared receptor, bridges of Preventive Medicine Research Fellowship to S.K.A.L. and by an MRC Pro- tracellular fibronection and the intracellular actin systems Grant to S.K.A.L. and ject A.J.W. The between the two (Hynes, 1981). analogy receptors may ex- tend to the between the specific recognition receptors and their extracellular It has been ligands. established that the type III do- References main of the fibronection a receptor recognises sequence ofRGD Anderson,D.C., Schmalstieg,F.C., Shearer,W., Becker-Freeman,K., Kohl,S., on the fibronectin molecule (Perschbacher and Ruoslahti, 1984). Smith,C.W., Tosi,M.F. and Springer,T. (1985) Fed. Proc., 44, 2671-2677. It is therefore to note that the Arnaout,M.A., Dana,N., Pitt,J. and Todd,R.F., III (1985) Fed. Proc., intriguing tripeptide RGD is also 44, 2664-2670. found in the structure of C3 primary (de Bruijn and Fey, 1985). and Cell. Axline,S.G. Reaven,E.P. (1974) J. Biol., 62, 647-659. a of C3 the RGD Furthermore, synthetic peptide covering region Biggin,M.D., Gibson,T.J. and Hong,G.F. (1983) Proc. Natl. Acad. Sci. USA, was shown to have for CR3 et affinity (Wright al., 1987). 80, 3963-3965. The structural between the in- high homology f-subunit and and Biochem Christie,D.L. Gagnon,J. (1982) J., 201, 555-567. Cosgrove,L.J., Sandrin,M.S., Rajasekariah,P. and Mckenzie,I.F.C. (1986) Proc. the that these are tegrin clearly strengthens conjecture proteins Natl. Acad. Sci. USA, 83, 752-756. related and to an extended of cell-surface belong family proteins and Dahms,N.M. Hart,G.W. (1985) J. Immunol., 134. 3978-3986. involved in various forms of adhesion reactions. and Methods Dayhoff,M.O., Barker,W.C. Hunt,L.T. (1983) Enzymol., 91, 524-545. de and Proc. Natl. Acad. Sci. Bruijn,M.H.L. Fey,G.H. (1985) USA, 82, Materials and methods 708-712. Gait,M.J., Singh,M., Sheppard,R.C., Edge,M.D., Greene,A.R., cell adhesion the (3-subunit the Purification of of surface glycoproteins and Heathcliffe,G.R., Atkinson,T.C., Newton,C.R. Markham,A.F. (1980) and The H-52 The methods were those of Williams essentially Barclay (1986). Nucleic Acids Res., 8, 1081-1096. was to monoclonal and 4B antibody (Hildreth August, 1985) coupled Sepharose and J. Immunol., Hildreth,J.E.K. August,J.T. (1985) 134, 3272-3280. extract of 70 of human CL at 8 ml of beads. The NP40 mg IgG per g spleen and Hildreth,J.E.K., Gotch,F.M., Hildreth,P.D.K. McMichael,A.J. (1983) Eur. eluted at 11.5 was a 35 ml H-52 column. The material was passed through pH J. 202-208. Immunol., 13, volume. The a second H-52 column of 8 ml was passed through packed antigen In and Hynes,R.O. (1981) Poste,G. Nicholson,G.L. (eds), Cytoskeletal Elements 2.2. All buffers contain eluted at used mM 0.2 mM 1 iodoacetamide, pH and Plasma Cell Membrane Organisation, Surface Reviews, Vol. 7. Elsevier 1 1 phenylmethanesulphonyl fluoride, 1,10-phenathroline, leupeptin, Biomedical New York. tsM /4M Press, A mM and 3 sodium azide. activities the pepstatin Antigenic throughout /M 680-685. Laemmli,U.K. (1970) Nature, 227, of an were determined inhibition indirect radioactive purification procedure by and Lowry,O.H., Rosebrough,H.J., Farr,A.L. Randall,R.J. (1951) J. Biol. was and The of the assessed binding assay (Williams Barclay, 1986). purity antigen 265-275. Chem., 193, SDS-PAGE by (Laemmli, 1970). D. Phil Malhotra,V. (1986) Thesis, University of Oxford, UK. Protein and Malhotra,V., Hogg,N. Sim,R.B. (1986) Eur. J. Immunol., 16, 1117 -1123. chemistry and In Molecular A The (3-subunit obtained from was reduced Maniatis,T., Fritsch,E.F. Sambrook,J. (1982) Cloning: affinity chromatography succinylated, Manual. Cold Harbor Cold Har- Laboratory Spring Laboratory Press, Spring and with acid and alkylated iodo[2-3H]acetic (Christie Gagnon, 1982). Tryptic New York. were obtained and fractionated followed bor, filtration peptides by gel (Sephadex G-75) and Biochem. 233-236. reverse and Williams and Micklem,K.J. Sim,R.B. (1985) J., 231, by phase h.p.l.c. (Christie Gagnon, 1982; Gagnon, and In the 7hird N-terminal was carried out in an Micklem,K.J., Law,S.K.A. Mason,D.Y. (1986) Proceedings of 1982). peptide sequencing Applied Biosystems International and on Human Gas Phase with PTH amino acid detected Workshop Conference Leucocyte Differentiation Sequencer by h.p.l.c. in Antigens, press. cDNA library Pierce,M.W., Remold-O'Donnel,E., Todd,R.F., III and Arnaout,M.A. (1987) RNA was to sucrose and the 28S 18S+ subjected gradient ultracentrifugation +, Biochim. in Biophys. Acta, press. to standard et The (Malhotra, 1986) according procedures (Maniatis al., 1982). and Pierschbacher,M.D. Ruoslahti,E. (1984) Nature, 309, 30-33. RNA was to sucrose and the + 18S subjected gradient ultracentrifugation 20S+, and Pytela,R., Pierschbacher,M.D. Ruoslahti,E. (1985) Proc. Natl. Acad. Sci. and fractions were and selected an 5S- pooled poly(A)+ by passing through 5766-5770. USA, 82, cellulose column. cDNA was made from of the and oligo(dT) 5 ,4g 28S+, 18S+ Pytela,R., Pierschbacher,M.D., Ginsberg,M.H., Plow,E.F., Rouslahti,E. (1986) 5S- RNA a cDNA kit International poly(A)+ using synthesis (Amersham UK) 1559-1562. Science, 231, to the manufacturers The cDNA was blunt-end to according procedures. ligated and Ross,G.D., Newman,S.L., Lambris,J.D., Devery-Pocius,J.E., Cain,J.A. the vector PAT-X and transfected to Escherichia coli MC-1061. The plasmid J. Lachmann,P.J. (1983) Exp. Med., 158, 334-352. was 50-fold before at The of the library amplified storage -70°C. complexity Sanchez-Madrid,F., Krensky,A.M., Ware,C.F., Robbins,E., Strominger,J.L., is - 300 000. About 62.5% of the colonies contain sizeable inserts which library and Proc. Burakoff,S.J. Springer,T.A. (1982) Natl. Acad. Sci. USA, 79, have an of 1.5 1.0 kb. average length + 7489-7493. cDNA and Sanchez-Madrid,F., Nagy,J.A., Robbins,E., Simon,P. and Springer,T.A. (1983) cloning sequencing J. 1785-1803. Exp. Med., 158, were the method et Oligonucleotides synthesised by phosphodiester (Gait al., 1980). and Proc. Natl. Initial Sanger,F., Nicklen,S. Coulson,A.R. (1977) Acad. Sci. USA, of the cDNA was with the screening library 32P-labelled oligonucleotide 5463-5466. 74, of the for further clones was with probes. Subsequent re-screening library cDNA and inserts obtained from Springer,T.A., Sastre,L. Anderson,D.C. (1985a) Biochem. Soc. Trans., 13, the initial clones et positive (Maniatis al., 1982). Sequenc- 3-6. of cDNA was the M13 ing by method (Sanger et al., 1977; Biggin et al., 1983). and Springer,T.A., Teplow,D.B. Dreyer,W.J. (1985b) Nature, 314, 540-542. Data and handling analysis Nucleic Acids Staden,R. (1986) Res., 14, 217-231. data were handled the Sequence by computer program of Staden (1986). Assess- Suzuki,S., Argraves,W.S., Pytela,R., Arai,H., Krusius,T., Pierschbacher,M.D. ment of similarities was made sequence using the ALIGN program (Dayhoff et and Proc. Acad. Ruoslahti,E. (1986) Natl. Sci. USA, 83, 8614-8618. scores were calculated al., 1983). Alignment using the Mutation Data Matrix Tamkun,J.W., DeSimone,D.W., Fonda,D., Patel,R.S., Buck,C., Horwitz,A.F. with a bias of +6 and of (250 PAMS) gap penalty +6. 100 random runs were and Hynes,R.O. (1986) Cell, 46, 271-282. to establish the mean random scores for performed the sequences. and Williams,A.F. Gagnon,J. (1982) Science, 216, 696-703. 918 of the LFA-1, CR3 and p150,95 cell surface antigens The ,B-subunit Williams,A.F. and Barclay,A.N. (1986) In Weir,D.M. (ed.), Handbook of Ex- perimental Immunology, Vol. 1 Immunochemistry. Blackwell Scientific Publica- tions, Oxford, UK, pp. 22.1-22.24. Wright,S.D. and Meyer,B.C. (1985) J. Exp. Med., 162, 762-767. Wright,S.D. and Silverstein,S.C. (1982) J. Exp. Med., 156, 1149-1164. Wright,S.D. and Silverstein,S.C. (1983) J. Exp. Med., 158, 2016-2023. Wright,S.D., Rao,P.E., Van Voorhis,W.C., Craigmyle,L.S., Iida,K., Talle, M.A., Westburg,E.F., Goldstein,G., Silverstein,S.C. (1983) Proc. Natl. Acad. USA, 80, 5699-5703. Sci. Wright,S.D., Reddy,P.A., Jong,M.T.C. and Erickson,B.W. (1987) Proc. Natl. Acad. Sci. USA, (in press). Received on 28, 1987 January in proof Note added These sequence data have been submitted to the EMBL/GenBank Data Libraries under the accession number Y00057. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The EMBO Journal Springer Journals

The primary structure of the beta‐subunit of the cell surface adhesion glycoproteins LFA‐1, CR3 and p150,95 and its relationship to the fibronectin receptor.

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Springer Journals
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Copyright © European Molecular Biology Organization 1987
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0261-4189
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10.1002/j.1460-2075.1987.tb04838.x
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Abstract

The EMBO Journal vol.6 no.4 pp.915-919, 1987 The primary structure of the f-subunit of the cell surface adhesion glycoproteins LFA-1, CR3 and p150,95 and its relationship to the fibronectin receptor S.K.A.Lawl, J.Gagnon2, J.E.K.Hildreth3, C.E.Wells, Arnaout et al., 1985; Springer et al., 1985a). Invariably, they A.C.Willis and A.J.Wong4 are deficient in all three complexes suggesting that the deficien- cy is in the common fl-subunit. Furthermore, it is apparent that 'The MRC Immunochemistry Unit, Department of Biochemistry, University the absence of the f-subunit interferes with the maturation and of Oxford, South Parks Road, Oxford OXI 3QU, UK, and 3The Department of Pharmacology and Experimental Theurapeutics, The expression of the a-subunits Johns (Springer et al., 1985a). Hopkins University School of Medicine, Baltimore, MD 21205, USA Recently, it has been speculated that this group of adhesion proteins may belong to a more extended family of 2Present address: National Research Council Canada, Biotechnology Research cell-surface Institute, 6100 Avenue Royalmount, Montreal, Quebec, Canada H4P 2R2 proteins including the fibronectin receptor (Tamkun et al., 1986), the vitronectin receptor (Suzuki et al., 1986), and the gpIIb/IIIa 4Present address: Paediatric Research Unit, UMDS, Guy's Tower, London Bridge, SE1 9RT, UK glycoprotein of platelets (Cosgrove et al., 1986; Pytela et al., 1986; Pierce et al., 1987), all of which have specific affinity Communicated by A.F.Williams for peptides containing the amino acid sequence Arg Gly Asp The lymphocyte-function-associated antigen-i (LFA-1), the as found in (RGD) the binding region of the fibronectin molecule complement receptor type 3 (CR3) and the antigen p150,95 (Pytela et al., 1985; Pierschbacher and Ruoslahti, 1984; Wright are cell-surface glycoproteins. They are heterodimeric com- and Meyer, 1985). Such speculation is reinforced by the obser- plexes, each containing a unique a-subunit noncovalently vation that a synthetic peptide from C3, which contains the se- associated with a common f-subunit. We have purified the quence RGD, can bind to CR3 (Wright et al., 1987). f-subunit from human spleen and obtained limited peptide We focused our study on the f-subunit of this set of adhesion sequences. What appears to be the complete primary struc- glycoproteins with the aim of determining the primary structure ture for the fully processed :-subunit was obtained by cDNA by a combination of protein and cDNA sequencing. The fl-subunit sequencing of clones from a phorbol ester (PMA) stimulated protein sequence was found to be very similar to chicken integrin, U937 cDNA library. There are five possible glycosylation sites a subunit of a fibronectin binding molecule on fibroblasts and a transmembrane segment. The sequence contains a high (Tamkun et al., 1986). level of cysteine (7.6%), with 24 of the 57 cysteine residues being found in three repeating units each with eight residues. Results The entire primary structure has 47% identity to a subunit The purification of the fl-subunit LFA-J of of a fibronectin binding protein from chicken fibroblasts. It The f-subunit of the cell-surface seems that adhesion glycoproteins was LFA-1, CR3 and p150,95 antigens to may belong from human an extended family of cell surface purified spleen by affinity chromatography using molecules including the the H-52 monoclonal fibronectin binding protein. antibody (Hildreth and The August, 1985). methodology employed was basically that of Williams and Key words: re- f-subunit/CR3, LFA-1, plSO,95/fibronectin Barclay (1986). The detergent [Nonidet P40 (NP40)] extract of ceptors spleen membranes was passed the through H-52 column twice. The bound materials eluted at pH 11.5 in the first passage con- tained dissociated a- and fl-subunits since the Introduction interaction between the subunits is broken in the irreversibly alkaline conditions The lymphocyte-function-associated antigen-l (LFA-1), the com- et second (Sanchez-Madrid al., 1983). Upon passage only the plement 3 and the are cell- receptor type (CR3) antigen p50,95 fl-subunit was bound to the H-52 column and it was eluted at surface a- glycoprotein complexes each a containing unique pH 2.2. About 50% of the of the extract antigenic activity NP40 subunit and presumably an identical f-subunit (Sanchez-Madrid was recovered at this point. The eluted material was succinylated et The mol. wts of the as al., 1983). apparent a-subunits, deter- and before further reduced/alkylated purification by gel-filtration mined are 175 165 000 and 150 000 by SDS-PAGE, 000, a S-400 column in the of SDS. through Sephacryl presence 0.2% of daltons respectively, and that the f-subunit is 95 000 daltons. About 0.5 mg pf as determined the method of protein, by Lowry Each of the three in form of complexes participates some cell- et al. was obtained from 70 of human (1951), g spleen. The LFA-1 mediates interactions between adhesion activities; T-cells material eluted from the column had an mol. affinity apparent and their et Hildreth et targets (Sanchez-Madrid al., 1982; al., wt of 95 000 daltons on SDS -PAGE and this increased to CR3 has for a natural 1983); affinity iC3b, degradation product 120 000 after and 135 000 daltons succinylation mol. apparent of C3 factors I and H in the et by complement system (Ross al., wt after not succinylation/reduction/alkylation (data shown). and the interaction 1983) may promote phagocytic activity Tryptic peptides and has a similar bin- (Wright Silverstein, 1982, 1983); p150,95 to CR3 but the resultant manifestation of this The fl-subunit was with ding specificity succinylated/reduced/alkylated digested interaction is not known and Malhotra et and the were (Micklem Sim, trypsin peptides separated filtration follow- 1985; by gel ed reverse Twelve of the al., 1986). by phase h.p.l.c. peptides yielded uni- of suffer from Patients deficient in this Two T3-9 and T7-14 group glycoproteins NH2-terminal sequences. peptides, que infections et of and recurrent bacterial and contain sequences CNVCEC Two fungal (Anderson al., 1985; CDGVQI respectively. Press IRL Oxford, Limited, England S.K.A.Law et al. I I I I II I I I AVAI 1 BAfHI BAN1 ECORI ~~~~I- 1 1 7 HINFI I I I I I I PVUI I I I I RSA 1 Il II I I STYI CLONES J-9 I - X- 1 J-19 Fig. The restriction maps of clones X-1 and J-19. The restriction sites for used to for are J-9, enzyme enzymes generate shown. fragments sequencing The map was from obtained clone J-9 except for the 5'-extension on X-1 and where the 5' of the site was J-19, characterised. at the fragment StyI (----) 3'-ends of represent segments the clones that have not been characterised or sequenced. anti-sense oligonucleotide 17 bases in and CTCGCCCTGGTGGGGCTGCTCTCCCTCGGGTGCGTCCTCTCTCAGGAGTGCACGAAGTTC 60 mixtures, length, com- AAGGTCAGCAGCTGCCGGGAATGCATCGAGTCGGGGCCCGGCTGCACCTGGTGCCAGAAG plexity of 64 and 128 respectively, were synthesised according 180 CTGAACTTCACAGGGCCGGGGGATCCTGACTCCATTCGCTGCGACACCCGGCCACAGCTG CTCATGAGGGGCTGTGCGGCTGACGACATCATGGACCCCACAAGCCTCGCTGAAACCCAG to the sequences. GAAGACCACAATGGGGGCCAGAAGCAGCTGTCCCCACAAAAAGTGACGCTTTACCTGCGA 300 CCAGGCCAGGCAGCAGCGTTCAACGTGACCTTCCGGCGGGCCAAGGGCTACCCCATCGAC 360 cDNA cloning CTGTACTATCTGATGGACCTCTCCTACTCCATGCTTGATGACCTCAGGAATGTCAAGAAG CTAGGTGGCGACCTGCTCCGGGCCCTCAACGAGATCACCGAGTCCGGCCGCATTGGCTTC The two oligonucleotide mixtures were used to screen a cDNA 540 GGGTCCTTCGTGGACAAGACCGTGCTGCCGTTCGTGAACACGCACCCTGATAAGCTGCGA AACCCATGCCCCAACAAGGAGAAAGAGTGCCAGCCCCCGTTTGCCTTCAGGCACGTGCTG library from phorbol ester stimulated (PMA) U-937 which cells, AAGCTGACCAACAACTCCAACCAGTTTCAGACCGAGGTCGGGAAGCAGCTGATTTCCGGA 660 AACCTGGATGCACCCGAGGGTGGGCTGGACGCCATGATGCAGGTCGCCGCCTGCCCGGAG 720 are known to express all three cell surface adhesion glycopro- GAAATCGGCTGGCGCAACGTCACGCGGCTGCTGGTGTTTGCCACTGATGACGGCTTCCAT 780 TTCGCGGGCGACGGAAAGCTGGGCGCCATCCTGACCCCCAACGACGGCCGCTGTCACCTG 840 teins (Sanchez-Madrid et Six al., 1983; Malhotra, 1986). positive GAGGACAACTTGTACAAGAGGACCAACGAATTCGACTACCCATCGGTCGGCCAGCTGGCG 900 CACAAGCTGGCTGAAAACAACATCCACCCCATCTTCGCGGTGACCAGTAGGATGGTGAAG 960 clones which to both were hybridised oligonucleotides obtained ACCTACGAGAAACTCACCGAGATCATCCCCAAGTCAGCCGTGGGGGAGCTGTCTGAGGAC 1020 from - 35 000 colonies in the first Four of the clones 1080 screening. TCCAGCAATGTGGTCCATCTCATTAAGAATGCTTACAATAAACTCTCCTCCAGGGTCTTC CTGGATCACAACGCCCTCCCCGACACCCTGAAAGTCACCTACGACTCCTTCTGCAGCAAT 1140 - - had inserts of 2.7 kb; the other two had inserts of 1.8 kb. GGAGTGACGCACAGGAACCAGCCCAGAGGTGACTGTGATGGCGTGCAGATCAATGTCCCG 1200 ATCACCTTCCAGGTGAAGGTCACGGCCACAGAGTGCATCCAGGAGCAGTCGTTTGTCATC The six clones were not of each other. duplicates 1 3 2 0 CGGGCGCTGGGCTTCACGGACATAGTGACCGTGCAGGTCCTTCCCCAGTGTGAGTGCCGG TGCCGGGACCAGACCAGAGACCGCAGCCTCTGCCATGGCAAGGGCTTCTTGGAGTGCGGC One of the was studied It clones, J-9, contains extensively. ATCTGCAGGTGTGACACTGGCTACATTGGGAAAAACTGTGAGTGCCAGACACAGGGCCGG 1440 a poly(A) tail but does not to be full since a Nor- AGCAGCCAGGAGCTGGAAGGAAGCTGCCGGAAGGACAACAACTCCATCATCTGCTCAGGG 1 500 appear length CTGGGGGACTGTGTCTGCGGGCAGTGCCTGTGCCACACCAGCGACGTCCCCGGCAAGCTG 1 560 thern blot of the RNA with J-9 showed a original probed 1620 single ATATACGGGCAGTACTGCGAGTGTGACACCATCAACTGTGAGCGCTACAACGGCCAGGTC 16 80 TGCGGCGGCCCGGGGACGGCGCTCTGCTTCTGCGGGAAGTGCCCGCTGCCACCCGGGCTTT band between 3 and 3.5 kb. The most 5'-end Styl fragment (see 1 740 GAGGGCTCAGCGTGCCAGTGCGAGAGGACCACTGAGGGCTGCCTGAACCCGCGGCGTGTT 1 800 GAGTGTAGTGGTCCTGGCCGGTGCCGCTGCAACGTATGCCGAGTGCCATTCAGGCTACCAG restriction maps on was used to rescreen the cDNA Figure 1) CTGCCTCTGTGCCAGGAGTGCCCCGGCTGCCCCTCACCCTGTGGCAAGTACATCTCCTGC 1860 GCCGAGTGCCTGAAGTTCGAAAAGGGCCCCTTTGGGAAGAACTGCAGCGCGGCGTGTCCG 1920 library. Two X-1 and included extra on clones, J-19, sequence GGCCTGCAGCTGTCGAACAACCCCGTGAAGGGCAGGACCTGCAAGGAGAGGGACTCAGAG 1980 the 5'-side of J-9. Their restriction are also included in 2040 maps GGCTGCTGGGTGGCCTACACGCTGGAGCAGCAGGACGGGATGGACCGCTACCTCATCTAT GTGGATGAGAGCCGAGAGTGTGTGGCAGGCCCCAACATCGCCGCCATCGTCGGGGGCACC Figure GTGGCAGGCATCGTGCTCATCGGCATTCTCCTGCTGGTCATCTGGAAGGCTCTGATCCAC CTGAGCGACCTCCGGGAGTACAGGCGCTTTGAGAAGGAGAAGCTCAAGTCCCAGTGGAAC 2220 7he primary structure of the ,B-subunit AATGATAATCCCCTTTTCAAGAGCGCCACCACGACGGTCATGAACCCCAAGTTTGCTGAG 2280 AGTTAGGAGCA The primary structure of the f-subunit was determined a com- by bination of protein and cDNA Most of the cDNA sequencing. Fig. 2. The nucleotide obtained from sequence clones J-9, X-1 and J-19 was obtained from sequence clone J-9 for the 5' end which except covering an open reading frame. The first StyI site, starting at position 341, from X-1 where were clones and J-19. The was to fragments 5' to it were obtained and strategy employed sequenced for clones X-1 and J-19 is marked. Also marked is the termination codon with TAG at position cleave restriction clone into and enzymes, M13 sequence clones at random In picked the later (Figure 1). stages, specific fragments were to obtain purified sequences between The gaps. chicken fibroblasts (Tamkun et Extensive 5 al., 1986). identity is '-end of clones X- 1 sequences and J- 19 were obtained from the observed between the $-chain of the LFA-1 of family glycopro- fragment 5' to the first restriction site StyI with and without the teins and integrin. BamHI cut in the middle. A tail poly(A) was found at the 3'-end but the clones do not include that could code the sequence leader Discussion sequence since no codon for methionine is present. However, an open frame is evident and the A combination of and cDNA reading nucleotide peptide sequencing has what sequence yielded this covering open frame is shown in 2. appears to be the reading Figure complete primary structure for the Except fully pro- for a region from residue numbers cessed 2086-2 140 and the 23 bases f-subunit of the cell-surface adhesion glycoproteins at the most 3'-end, sequence data from both directions were ob- LFA-1, CR3 and p150,95. tained. In those two regions, the The protein was sequence was confirmed from purified from human spleen by virtue of its at least three different gel readings. On the average, each posi- affinity to the monoclonal antibody H-52. This antibody, though tion was covered 3.47 times. to initially reported have specificity for the fl-subunit of the three The translated protein sequence is shown in cell-surface Figure 3 together glycoprotein complexes (Hildreth and August, 1985), with the positions of the 12 tryptic peptides, which are turns out to be more in dispers- complicated its interaction with the an- ed the throughout sequence. tigens. It binds to the dissociated f-subunits from all three com- Also shown in 3 is Figure the sequence of the plexes, but only to the LFA-l integrin antigen, and not CR3 and p 150,95, molecule, a subunit of the fibronectin binding in their intact (x-f forms protein from (Micklem et al., 1986). Hence, the 916 The ,3-subunit of the LFA-1, CR3 and p150,95 cell surface antigens 1 36 -----LALVGLLSLGCVL---------SQECTKFKVSSCRECIESGPGCT be the assertion that the on could explained by simple epitope 1 MAETNLTLLTWAGILCCLIWSGSAQQGGSDCIKANAKSCGECIQAGPNCG is masked the a-subunits of the CR3 and the fl-subunit by p150,95 complexes. 37 RCDTRPQLLMRGCAADD 87 WCQKLNFTGPGDPDS MDPTSLAETQEDHNGG The cDNA clones X-1 and J-19 have insert sizes between 100 J-9, 51 WCKKTDFLQEGEPTSARCDDLAALKSKGCPEQDIENPRGSKRVLEDREVT 2.7 and 2.9 kb and are close to the size of the mRNA from the PMA-stimulated U-937 cells An frame 88 OK-------------- 122 (3-3.5 kb). open reading QLSPQKVTLYLRPGQAAAFNVTFRRAKGYPIDLY 101 150 NRKIGAAEKLKPEAITQIQPQKLVLQLRVGEPQTFSLKFKRAEDYPIDLY was found in the consensus from the 5' end sequence starting 4 to nucleotides number 2284-2286 where a termination codon +-T11-14--' FT12-12 -b GFGSFVDKTVLPFV 172 123 YLMDLSYSMLDDLRNVKKLGCDLLRALNE TESGR TAG is found. The contains five N-linked car- sequence possible 151 YLMDLSYSMKDDLENVKSLGTALMREMEKITSDFRIGFGSFVEKTVMPYI sites at 246 and in bohydrate positions 42, 108, 204, 493, agree- ment with the of Dahms and Hart who findings (1985) +-aT9-1 173 222 from the NTHPDKLRNPCPNKEKECQPPFAFRHVLKLTNNSNOFQTEVGKOLISGNL demonstrated the of five presence glycosylated peptides 201 STTPAKLRNPCTG-DQNCTSPFSYKNVLSLTSEGNKFNELVGKQHISGNL The also contains a $-subunit. sequence hydrophobic segment that the N-terminal from residues 693 -715. It is therefore likely 223 DAPEGGLDAMMQVAACPEEIGWRNVTRLLVFATDDGFHFAGDGKLGAILT 272 of the is the extracellular domain and the C- portion protein 250 DSPEGGFDAIMOVAVCGDQIGWRNVTRLLVFSTDAGFHFAGDGKLGGIVL 299 in bet- terminal with one transmembrane cytoplasmic segment 4-T 11 -1 6+ ween. The clones do not include the ATG for the initia- 322 signal 273 PNDGRCHLEDNLYKRSNEFDYPSVGQLAHKLAENNIQPIFAVTSRMVKTY 300 PNDGKCHLENNMYTMSHYYDYPSIAHLVQKLSENNIQTIFAVTEEFQAVY tion methionine residue. it is that a of However, likely portion the leader is included in clone J-19. Since the N-terminal sequence 323 EKLTEI IPKSAVGELSEDSSNVVHLIKNAYNKLSSRVFLDHNALPDTLKV 372 of the fl-subunit is blocked et it is not (Springer al., 1985b), possi- 350 KELKNLIPKSAVGTLSSNSSNVIOLI IDAYNSLSSEVILENSKLPKEVTI ble to the exact N-terminal of the mature but pinpoint protein *-T7-14 it is to that it be the residue tempting speculate might glutamine 373 ---QEQSFV TYDSFCSNGVTHRNQPRGDCDCVQINVPITFQVKVTATECI 400 449 after the leader If that SYKSYCKNGVNDTQEDGRKCSNISIGDEVRFEINVTANECPKKGQNETIK (position 15) putative sequence fragment. is the the f-subunit would contain 747 residues and would case, -T9-19 +-Tl0 420 IRALGFTDIVTVQVLPQCECRCRDQS-RDRSLCH-GKGFLECGICRCDTG have a mol. wt of 82 542 daltons. with the Together carbohydrate 450 IKPLGFTEEVEIHLQFICDCLCQSEGEPNSPACHDGNGTFECGACRCNEG at the N-linked estimated to have a mol. wt glycosylation sites, -2 4<T3-7 of 3000 daltons the mol. wt of the fl-subunit is estimated per site, 468 YIGKNCECQTQGRSSQELEGSCRKDNNSI ICSGLGDCVCGOCLCHTSDVP to be 97 542 daltons which is in reasonable with the 549 agreement S00 RIGRLCECSTDEVNSEDMDAYCRRENSTEICSNNGECICGQCVCKKRENT mol. wt SDS -PAGE. Our most N-terminal apparent by pep- 1 -3 +-T1 Q Ti marked the first definitive at residue 567 tide, 1-14, sequence 518 GKLIYGOYCECDTINCERYNGQVCGGPGRGLCFCGKCRCHPGFEGSACQC 550 NEVYSGKYCECDNFNCDRSNGLICGG--NGICKCRVCECFPNFTGSACDC number 137 (Figure 3). A most unusual feature of the structure is its primary high cys- -T3-99 teine content. A total of 57 residues were identified with cysteine 568 ERTTEGCLNPRRVECSGRGRCRCNVCEC-HSGYQLPLCOECPGCPSPCGK 647 42 found within a stretch of 256 residues 437 598 SLDTTPCMAGNGOICNGRGTCECGTCNCTDPKFQGPTCEMCQTCLGVCAE (residues -692) N-terminal to the transmembrane Within this sequence. cysteine- 655 rich there to be three units 617 YISCAECLKFEKGPFGKNCSAACPGLQLSNNPVKGR-------T----TCK region, appear repeating arbitarily 648 HKDCVOCRAFEKGEKKETCSQECMHFNMTRVESRGKLPQPVHPDPLSHCK defined from residues 473 and 565-603 to con- -525, 526-564, tain residues in a of C X C eight cysteine arranged pattern ( ) +T10-9 T13-17 C( ) CXCXX CX C( ) where each X C( ) 656 ERDSEGCWVAYTLEQQDGMDRYLIYVDESRECVAGPNIAAIVGGTVAGIV 705 stands for an amino acid and the stretches of 698 EKDVGDCWFYFTYSVNSNGEAS-VHVVETPECPSGPDI IPIVAGVVAGIV 746 ( ) represents amino acids with from 4 to residues. length ranging 706 LICILLLVIWKALIHLSDLREYRRFEKEKLKSQWNN-DNPLFKSATTTVM 754 The three were to statistical repeating segments subjected 747 LIGLALLLIWKLLMIIHDRREFAKFEKEKMNAKWDTCENPIYKSAVTTVV 796 the ALIGN et analysis by program (Dayhoff al., 1983; Staden, The scores of 1986). pairwise comparison yielded alignment 3.59, NPKFAES 761 and 4.29 SD that the of a chance 5.45 units; indicating probability 797 NPKYEGK 803 2 x 10-4 et is less than relationship (Dayhoff al., 1983). is The of residues in the pattern cysteine cysteine-rich Fig. 3. The primary structure of the 13-subunit of the cell surface adhesion region is shown. The of of a fibronectin glycoproteins. The derived amino acid sequence position similar to that first observed in a subunit very the 12 tryptic peptides (< >), the five potential N-linked glycosylation from fibroblasts et chicken binding protein (integrin) (Tamkun sites (A), and the putative transmembrane segment ( ) are marked. show a level of similari- In the two al., 1986). fact, proteins high The primary structure of integrin (Tamkun et al., 1986) is shown as the matched with a 47% overall The two are ty. sequences easily lower sequence. The alignment of the two sequences was made by the of amino acid residues from In a stretch 278 ALIGN program (Dayhoff et al., 1983). Identical residues are indicated by identity. positions below the are found at the level of 63%. (-) sequences. 90 to identities 367, However, site at a residue in proposed possible phosphorylation tyrosine here must be is not found in the fl-subunit. This fl-subunit purified and reported formally regarded integrin (position 788) aligned the of LFA-1 must be that consistent with the failure to as that the antigen. It stressed, however, observation is phosphorylate ,B- under various con- are to be of cell-surface adhesion the f-subunits of the three antigen complexes likely subunit glycoproteins on: the extensive of ditions identical, based (i) sharing antigenic epitopes, (Malhotra, 1986). and in their of monoclonal antibodies et The exact roles of the LFA-1 e.g. that IB4 (Wright al., 1983), p150,95 respective and cells are not clear. et and TSl/18 et between effector MHM23 (Hildreth al., 1983) (Sanchez-Madrid interaction target to in invariable that defective However, CR3 has been postulated participate phagocytic al., 1983); and (ii) the finding patients in all three on their cell and Silvers- in the f-subunit are deficient events mediated activated antigens (Wright by macrophages it serves as a communicative bet- not bind to CR3 and surfaces. The fact that H-52 does plS50,95 bridge tein, 1982). Possibly, 917 S.K.A.Law et al. ween iC3b on on the outside opsonised particles and the actin Acknowledgements network on the the of inside, perturbation which by drugs such We wish to the late Professor under whose director- acknowledge R.R.Porter, as B was known to inhibit cytochalasin the phagocytic capacity ship of the MRC Immunochemistry Unit this project was initiated. We would of the and cells (Axline Reaven, 1974). Thus, CR3 may be com- like to thank S.J.Dodsworth for technical assistance and Miss C.Brooks for prepara- with the fibronectin which tion of the Part of this was Lister the ex- manuscript. project supported by a Institute pared receptor, bridges of Preventive Medicine Research Fellowship to S.K.A.L. and by an MRC Pro- tracellular fibronection and the intracellular actin systems Grant to S.K.A.L. and ject A.J.W. The between the two (Hynes, 1981). analogy receptors may ex- tend to the between the specific recognition receptors and their extracellular It has been ligands. established that the type III do- References main of the fibronection a receptor recognises sequence ofRGD Anderson,D.C., Schmalstieg,F.C., Shearer,W., Becker-Freeman,K., Kohl,S., on the fibronectin molecule (Perschbacher and Ruoslahti, 1984). Smith,C.W., Tosi,M.F. and Springer,T. (1985) Fed. Proc., 44, 2671-2677. It is therefore to note that the Arnaout,M.A., Dana,N., Pitt,J. and Todd,R.F., III (1985) Fed. Proc., intriguing tripeptide RGD is also 44, 2664-2670. found in the structure of C3 primary (de Bruijn and Fey, 1985). and Cell. Axline,S.G. Reaven,E.P. (1974) J. Biol., 62, 647-659. a of C3 the RGD Furthermore, synthetic peptide covering region Biggin,M.D., Gibson,T.J. and Hong,G.F. (1983) Proc. Natl. Acad. Sci. USA, was shown to have for CR3 et affinity (Wright al., 1987). 80, 3963-3965. The structural between the in- high homology f-subunit and and Biochem Christie,D.L. Gagnon,J. (1982) J., 201, 555-567. Cosgrove,L.J., Sandrin,M.S., Rajasekariah,P. and Mckenzie,I.F.C. (1986) Proc. the that these are tegrin clearly strengthens conjecture proteins Natl. Acad. Sci. USA, 83, 752-756. related and to an extended of cell-surface belong family proteins and Dahms,N.M. Hart,G.W. (1985) J. Immunol., 134. 3978-3986. involved in various forms of adhesion reactions. and Methods Dayhoff,M.O., Barker,W.C. Hunt,L.T. (1983) Enzymol., 91, 524-545. de and Proc. Natl. Acad. Sci. Bruijn,M.H.L. Fey,G.H. (1985) USA, 82, Materials and methods 708-712. Gait,M.J., Singh,M., Sheppard,R.C., Edge,M.D., Greene,A.R., cell adhesion the (3-subunit the Purification of of surface glycoproteins and Heathcliffe,G.R., Atkinson,T.C., Newton,C.R. Markham,A.F. (1980) and The H-52 The methods were those of Williams essentially Barclay (1986). Nucleic Acids Res., 8, 1081-1096. was to monoclonal and 4B antibody (Hildreth August, 1985) coupled Sepharose and J. Immunol., Hildreth,J.E.K. August,J.T. (1985) 134, 3272-3280. extract of 70 of human CL at 8 ml of beads. The NP40 mg IgG per g spleen and Hildreth,J.E.K., Gotch,F.M., Hildreth,P.D.K. McMichael,A.J. (1983) Eur. eluted at 11.5 was a 35 ml H-52 column. The material was passed through pH J. 202-208. Immunol., 13, volume. The a second H-52 column of 8 ml was passed through packed antigen In and Hynes,R.O. (1981) Poste,G. Nicholson,G.L. (eds), Cytoskeletal Elements 2.2. All buffers contain eluted at used mM 0.2 mM 1 iodoacetamide, pH and Plasma Cell Membrane Organisation, Surface Reviews, Vol. 7. Elsevier 1 1 phenylmethanesulphonyl fluoride, 1,10-phenathroline, leupeptin, Biomedical New York. tsM /4M Press, A mM and 3 sodium azide. activities the pepstatin Antigenic throughout /M 680-685. Laemmli,U.K. (1970) Nature, 227, of an were determined inhibition indirect radioactive purification procedure by and Lowry,O.H., Rosebrough,H.J., Farr,A.L. Randall,R.J. (1951) J. Biol. was and The of the assessed binding assay (Williams Barclay, 1986). purity antigen 265-275. Chem., 193, SDS-PAGE by (Laemmli, 1970). D. Phil Malhotra,V. (1986) Thesis, University of Oxford, UK. Protein and Malhotra,V., Hogg,N. Sim,R.B. (1986) Eur. J. Immunol., 16, 1117 -1123. chemistry and In Molecular A The (3-subunit obtained from was reduced Maniatis,T., Fritsch,E.F. Sambrook,J. (1982) Cloning: affinity chromatography succinylated, Manual. Cold Harbor Cold Har- Laboratory Spring Laboratory Press, Spring and with acid and alkylated iodo[2-3H]acetic (Christie Gagnon, 1982). Tryptic New York. were obtained and fractionated followed bor, filtration peptides by gel (Sephadex G-75) and Biochem. 233-236. reverse and Williams and Micklem,K.J. Sim,R.B. (1985) J., 231, by phase h.p.l.c. (Christie Gagnon, 1982; Gagnon, and In the 7hird N-terminal was carried out in an Micklem,K.J., Law,S.K.A. Mason,D.Y. (1986) Proceedings of 1982). peptide sequencing Applied Biosystems International and on Human Gas Phase with PTH amino acid detected Workshop Conference Leucocyte Differentiation Sequencer by h.p.l.c. in Antigens, press. cDNA library Pierce,M.W., Remold-O'Donnel,E., Todd,R.F., III and Arnaout,M.A. (1987) RNA was to sucrose and the 28S 18S+ subjected gradient ultracentrifugation +, Biochim. in Biophys. Acta, press. to standard et The (Malhotra, 1986) according procedures (Maniatis al., 1982). and Pierschbacher,M.D. Ruoslahti,E. (1984) Nature, 309, 30-33. RNA was to sucrose and the + 18S subjected gradient ultracentrifugation 20S+, and Pytela,R., Pierschbacher,M.D. Ruoslahti,E. (1985) Proc. Natl. Acad. Sci. and fractions were and selected an 5S- pooled poly(A)+ by passing through 5766-5770. USA, 82, cellulose column. cDNA was made from of the and oligo(dT) 5 ,4g 28S+, 18S+ Pytela,R., Pierschbacher,M.D., Ginsberg,M.H., Plow,E.F., Rouslahti,E. (1986) 5S- RNA a cDNA kit International poly(A)+ using synthesis (Amersham UK) 1559-1562. Science, 231, to the manufacturers The cDNA was blunt-end to according procedures. ligated and Ross,G.D., Newman,S.L., Lambris,J.D., Devery-Pocius,J.E., Cain,J.A. the vector PAT-X and transfected to Escherichia coli MC-1061. The plasmid J. Lachmann,P.J. (1983) Exp. Med., 158, 334-352. was 50-fold before at The of the library amplified storage -70°C. complexity Sanchez-Madrid,F., Krensky,A.M., Ware,C.F., Robbins,E., Strominger,J.L., is - 300 000. About 62.5% of the colonies contain sizeable inserts which library and Proc. Burakoff,S.J. Springer,T.A. (1982) Natl. Acad. Sci. USA, 79, have an of 1.5 1.0 kb. average length + 7489-7493. cDNA and Sanchez-Madrid,F., Nagy,J.A., Robbins,E., Simon,P. and Springer,T.A. (1983) cloning sequencing J. 1785-1803. Exp. Med., 158, were the method et Oligonucleotides synthesised by phosphodiester (Gait al., 1980). and Proc. Natl. Initial Sanger,F., Nicklen,S. Coulson,A.R. (1977) Acad. Sci. USA, of the cDNA was with the screening library 32P-labelled oligonucleotide 5463-5466. 74, of the for further clones was with probes. Subsequent re-screening library cDNA and inserts obtained from Springer,T.A., Sastre,L. Anderson,D.C. (1985a) Biochem. Soc. Trans., 13, the initial clones et positive (Maniatis al., 1982). Sequenc- 3-6. of cDNA was the M13 ing by method (Sanger et al., 1977; Biggin et al., 1983). and Springer,T.A., Teplow,D.B. Dreyer,W.J. (1985b) Nature, 314, 540-542. Data and handling analysis Nucleic Acids Staden,R. (1986) Res., 14, 217-231. data were handled the Sequence by computer program of Staden (1986). Assess- Suzuki,S., Argraves,W.S., Pytela,R., Arai,H., Krusius,T., Pierschbacher,M.D. ment of similarities was made sequence using the ALIGN program (Dayhoff et and Proc. Acad. Ruoslahti,E. (1986) Natl. Sci. USA, 83, 8614-8618. scores were calculated al., 1983). Alignment using the Mutation Data Matrix Tamkun,J.W., DeSimone,D.W., Fonda,D., Patel,R.S., Buck,C., Horwitz,A.F. with a bias of +6 and of (250 PAMS) gap penalty +6. 100 random runs were and Hynes,R.O. (1986) Cell, 46, 271-282. to establish the mean random scores for performed the sequences. and Williams,A.F. Gagnon,J. (1982) Science, 216, 696-703. 918 of the LFA-1, CR3 and p150,95 cell surface antigens The ,B-subunit Williams,A.F. and Barclay,A.N. (1986) In Weir,D.M. (ed.), Handbook of Ex- perimental Immunology, Vol. 1 Immunochemistry. Blackwell Scientific Publica- tions, Oxford, UK, pp. 22.1-22.24. Wright,S.D. and Meyer,B.C. (1985) J. Exp. Med., 162, 762-767. Wright,S.D. and Silverstein,S.C. (1982) J. Exp. Med., 156, 1149-1164. Wright,S.D. and Silverstein,S.C. (1983) J. Exp. Med., 158, 2016-2023. Wright,S.D., Rao,P.E., Van Voorhis,W.C., Craigmyle,L.S., Iida,K., Talle, M.A., Westburg,E.F., Goldstein,G., Silverstein,S.C. (1983) Proc. Natl. Acad. USA, 80, 5699-5703. Sci. Wright,S.D., Reddy,P.A., Jong,M.T.C. and Erickson,B.W. (1987) Proc. Natl. Acad. Sci. USA, (in press). Received on 28, 1987 January in proof Note added These sequence data have been submitted to the EMBL/GenBank Data Libraries under the accession number Y00057.

Journal

The EMBO Journal – Springer Journals

Published: Apr 1, 1987

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