The Structure of Anchorin CII, a Collagen Binding ... - Semantic Scholar

Vol. 263, No. 12, Issue of April 25, pp. 5921-5925, 1988

THEJOURNAL OF BIOLOGICAL CHEMISTRY

Printed in U.S.A.

The Structure of Anchorin CII, a Collagen BindingProtein Isolated from Chondrocyte Membrane* (Received for publication, December 3, 1987)

M. Pilar FernandezS, Ornella Selmins, George R. Martin, and Yoshihiko Yamadall From the Laboratory of Developmental Biology and Anomalies, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892

Michael Pfaffleq, Rainer Deutzmann, Jurgen Mollenhauer, and Klausvon der MarkT From the Departmentof Connective Tissue Research, Max-Planck-Institute furBiochemie, 0-8033 Martinsried, Federal Republic of Germany

cDNA clones for anchorin CII ( M . = 34,000), a collagen-binding protein, were isolated from a Xgt 11 cDNA library prepared from chick cartilage mRNA. Several overlapping clones were characterized which gave rise to an open reading frame coding for 329 residues and a 3”untranslated segment of 500 base pairs. The cloneswere identified as coding for anchorin by hybrid select translation analysis and by comparing the deduced amino acid sequence with the sequence of 10 tryptic peptides of the protein. A hydrophobic domain of 25 residues interrupted with 3 polar residues was identified with the carboxyl-terminal portion. There was no evidence for an aminoterminal signal peptide. Northern analysis revealed that the 5’ probe hybridizes to a single 1.7-kilobases (kb) mRNA species, whereas the 3’ probe hybridizes to two mRNA species of 1.7 kb and 5 kb, which are present in many cells including chondrocytes, crop cells, and fibroblasts. The level of anchorin mRNA in chick embryo fibroblasts was increased by infection with Rous sarcoma virus.

The interaction of cells with extracellular matrices influences their migration, adhesion, growth, and differentiation (for review, see Refs. 1-5). Such effects are probably specific and involve the binding of the cells to collagen, to proteoglycan,andtovarious glycoproteins suchasfibronectinand laminin. Specific receptors for laminin and fibronectin have been described (for review, see Refs. 6 and 7), and recent studies suggest that there are also distinct cell surface receptors for collagens (3, 8-14).’ One of these is anchorin CII, a collagen bindingproteinfirstisolated from chondrocyte * This work was supported in part by the Deutsche Forschungsgemeinschaft (Ma 534/6-7 and Ma 534/8-1). The costs of publication of this article were defrayed in partby the payment of page charges. This article must therefore be hereby marked“advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Present address: Departamento de Bioquimica, Facultad de Medicina, Universidad de Oviedo, 33006 Oviedo, Spain. 5 Present address: Dept. of Biology, Virginia Polytechnic Institute and State University, Blacksburg,VA 24061. ll Presentaddress:Klinische Arbeitsgruppen-Rheumatologie der MaxPlanck-Gesellschaft,Schwabachanlage 10, D-8520 Erlangen, FRG. 11 T o whom reprints requests shouldbe sent. The nucleotide sequence(s) reported in this paper has been submitted to theGenBankTM/EMBLDataBank with accession number(s) 503194. R. C. Ogle and C. D. Little, manuscript submittedfor publication.

plasma membranes and purified from detergent extracts by affinity chromatography oncollagen 11-Sepharose (15). It was also shown that chondrocytes utilize anchorin CII to bind to collagen substrates, and that antibody to anchorin CIIlocalizes the protein to the surface of these cells (16, 17). Subsequent studies suggested that fibroblasts may also contain an anchorin-like protein(18).In the present study, we report the isolation of cDNA clones for chick anchorin. We have characterized these clones by both DNAsequencing and Northern analyses. Tryptic peptides were isolated from anchorin CII and sequenced, and some 10 were placed in the amino acid sequence predicted from the nucleotide sequence data. MATERIALS AND METHODS

Isolation of Anchorin CII-Sterna from adult chickens were cut into small pieces and homogenized with an Ultraturrax homogenizer in 8.5% sucrose-TEA’ buffer (10 mM triethanolamine hydrochloride, pH 7.4, containing 0.1 mM EDTA, 0.1 mM phenylmethanesulfonyl fluoride, and 0.1 mM N-ethylmaleimide as protease inhibitors). The homogenate was centrifuged a t 8,000 X g for 1 h, and the resulting supernatant fluid was centrifuged at 53,900 X g for 3 h. The material isolated at this stepwas resuspended in 60 ml of 8.5% sucrose-TEA buffer and separatedby density gradient centrifugation a t 141,000 X g for 5 h in a Beckman SW 28 rotor according to Cates and Holland (19) and Schimmel et al. (20) using a stepwise gradient of 8.5, 17, and 40% sucrose. The plasma membranes of the chondrocyteswere present at the 17/40% interphase (15). The plasma membrane fraction was dialyzed against 50 mM Tris/HCl, pH 7.4, containing 75 mM NaCI, 2 M urea, 0.1% sodium deoxycholate (DEAE-column buffer), and homogenized with a Branson Sonifier. Some 250 ml of this membrane preparation was applied to a DEAE-cellulose column (2.5 X 18 cm) equilibrated in the same buffer. The column was washed with this buffer and then eluted stepwise with 0.5 M NaCl and then 1 M NaCl. The fractions were concentrated by ultrafiltration on an Amicon PM-10 membrane, dialyzed against 1 mM TEA/HCI, pH 7.4, containing 0.1% Nonidet P-40, and chromatographed oncollagen IISepharose as described (15). The material eluting between 50 and 100 mM NaCl was concentrated and separated by preparative SDSpolyacrylamide gel electrophoresis (15). In this procedure, approximately 100 pg of protein from the affinity column was dissolved in 1.5 ml of SDS-electrophoresis buffer and applied to a preparative 12% polyacrylamide-SDS slab gel (1.5 X 120 mm). After staining with Coomassie Blue, the portion of the gel containing anchorin CII was cut out, chopped into small pieces, washed in methanol, and dried in a Speed Vac concentrator. Tryptic Digestion and Peptide Purificationand Sequencing-Dried gel pieces were shakenin 0.5 ml of0.1 M (NH&COS, pH 8.5,

’The abbreviations used are: TEA, triethanolamine; Denhardt’s, 0.02% Ficoll, 0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone; SSC, 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0; SDS, sodium dodecyl sulfate; bp, base pairs; kb, kilobases; PIPES, 1,4-piperazinediethanesulfonicacid.

5921

5922

Anchorin CII cDNA

containing 0.1 mM CaCI2 and 30 pg of trypsin a t 37 "C for 14 h. The mRNA was achieved by boiling for 90 s in 1 mM EDTA, pH7.5. The reaction was stopped by adding 1.5 ml of 0.1% trifluoroacetic acid. supernatantfractioncontainingthe hybrid-selected mRNA was The tryptic peptides eluted from the gel slices were separated on a ethanol-precipitated using calf liver tRNA as carrier. The precipitate Gilson high performance liquid chromatographygradientsystem. was lyophilized and resuspended in 2.2 pl of sterile distilled water for They were first separated on a Vydac RP18 reverse-phase column translation in a rabbit reticulocyte lysate system in the presence of with a linear acetonitrile gradient (5-70%) in 0.1% trifluoroacetic ["'S]methionine. Translation was terminated by the addition of a acid. The resulting fractions were rechromatographed on a Merck solution of 125 mM Tris/HCl at pH 6.8, containing 5% SDS, 20% Hibar reverse-phasecolumn (LiChrospher CH18) with a more shallow glycerol, 0.1 mM p-mercaptoethanol, and 0.02% bromphenol blue; or acetonitrile gradientwhich was optimized for the purificationof each for immunoprecipitation in 0.1 M Tris-HC1, pH 7.2, containing 0.15 peptide. Peptidesisolated from the second reverse-phase column were M NaCI, 1% Triton X-100, 1%deoxycholate, 0.1% SDS, and 1% concentrated in a Speed Vac concentrator. Between100 and 1000 Trasylol. Polyclonal rabbit antiserum againstchick anchorin or norpmol of 13 trypticpeptides were sequencedusinga spinning-cup mal rabbit serumwas used along with proteinA coupled to Sepharose sequenator (Beckman) or a gas-phase sequenator (Applied Biosysheads to bind labeled proteins.These were subsequently released tems). from the beads by boiling in SDS-gel sample buffer and electrophoPreparation of RNA and cDNA Synthesis-Totalcellular RNA resed. was 'extracted from 10-day-old chick embryos and from 16-day-old chick sterna, crop, calvaria,andfibroblasts using guanidine HC1 RESULTS extraction (21). Poly(A) RNA wasisolatedby chromatography on oligo(dT)-cellulose (22). Poly(A) RNA from 16 day-old chick embryo Isolation of cDNA Clones for Anchorin CZZ-Initially,we sterna wasused as template for the synthesis of double-stranded screened a cDNA library prepared from chick chondrocyte cDNA. For this purpose, approximately 15 pg of poly(A) RNA was RNA using a polyclonal antibody against chick chondrocyte lyophilized and then incubated in the presence of 8 mM methylmercuric hydroxide and 10 units of RNasin for 10 min a t room temper- anchorin (16). Of approximately lo6 phages screened, 30 reacted positively withthe anti-anchorin antibody and clones 17 ature. After the addition of 90 mM 2-mercaptoethanol and 20 units more of RNase inhibitor, the mixture was further incubated for 15 remained stronglypositive after purification. Six of the clones min and then used as templatefor first strand synthesis. This reactionwere related,asdemdnstrated by Southernanalysis using was carried out with 15pg of oligo(dT) primer a t 37 "C for 3 h in the radiolabeled A1 clone as ahybridizationprobe (datanot presence of 30 units of reverse transcriptase in 0.1 M Tris-HC1, pH shown). A restriction map of these clones is shown in Fig. 1. 8.3, containing 0.14 M KC1, 0.01 M MgCl,, and 0.8 mM concentration To obtain larger cDNA clones, we screened the library by each of unlabeled deoxyribonucleotide triphosphates; 10 pCi each of hybridization with labeled DNA fragments from previously [ c Y - ~ ' P ] ~ G T P a n d [ C Y - ~were ~ P I ~used A T Pto quantitate cDNA synthesis. Second strand synthesis was performed as described by Oka- isolated clones and obtained several overlapping clones, two yama and Berg (23). When recombinant cDNA was packaged into of which (A4 and A6) are shown in Fig. 1. Both extend to the phage particles inuitro, approximately 25 X lo5plaques were obtained 3' untranslated regions. The 3'-end of the coding region was and about 95% of them contained cDNA inserts. confirmed both by nucleotide and by peptide sequencing. Screening of the Chick cDNA Librat-,-The chickchondrocyte Identification of Anchorin cDNA Clones by Hybrid Selected cDNA library was screened by antibody to anchorin as described Translation-Weutilizedhybridselection of chondrocyte elsewhere (24, 25). Rabbit anti-anchorin antibodies (16) were used at mRNA with the cloned DNA followed by translation as one a 1:300 dilution for the detection of fusion proteins induced with isopropyl-1-thio-p-o-galactopyranoside in Escherichia coli Y1090 in- method to establish the identity of anchorin. In these studies, fected with the recombinantphage. Subsequently, filterswere washed the insert from clone A1 was immobilized on activated diwith TBS and incubated with 1:lOOO horseradish peroxidase-conjuazobenzyloxymethyl paper and allowed to hybridize to chongated goat anti-rabbit antibody for 2 h and developed with 4-chloro1-naphthol(26).Positiveplaques were identified,rescreened, and drocyte poly(A) RNA. Bound mRNA was eluted and translated in a rabbit reticulocyte lysate in the presence of ["5S] purified as described (27). Characterization of Recombinant cDNA by Restriction Analysis and methionine. These studies showed that hybrid selection of Southern Blotting-Restriction enzymes were used under the condi- cartilage RNA by a 780-bp fragment from clone A1 allows the tions recommendedby the manufacturers. For Southern blotting, isolation of RNA which is translated predominantly to a single DNA was restricted, electrophoresed in agarose gels, transferred to protein ( M , = 32,000) (Fig. 2 A ) . Furthermore, this protein is nitrocellulose, and hybridized under standard conditions (28). Northern Analysis-Either poly(A) RNA (1 pg) or total RNA (5 immunoprecipitated by anti-anchorin antiserum as shown in Fig. 2B, lanes 3 and 4. The difference in size obtained from pg) was electrophoresed in1%agarose gels with formaldehyde, transferred to nitrocellulose (29), andhybridized with nick-translatedDNA the translated protein ( M , = 32,000), and authentic anchorin probes a t 37 "C in the presence of 5 X SSC, 50% formamide, 0.1% (Mr= 34,000) from chondrocytes on electrophoresis could be SDS, 250 pg/ml salmon sperm DNA,5 X Denhardt's solution, 50mM due todifferences in glycosylation or other post-translational Tris/HCl, pH 7.5, for 16-18 h. Filters were washed twice a t 40 "C modifications. In spite of the difference in size, however, the with 2 X SSC/O.2% SDS and twice with 2 X SSC. Labeled bands were detected by autoradiography. Taql EcoRl DNA Sequencing-Sequencing of selected clones was performed in Accl Sphl Pnl Taql M13mp18 or M13mp19 using thedideoxy method (30).The sequence I I I A4 P of both strands was determined, and eachnucleotide in sequence was A6 identified some five times. I I A15 Hybrid Select Translation and Immunoprecipitation-Small circles A14 (7 mm diameter) of ABM Trans-bind filter paper (Schleicher and Schuell) were activated by exposure to a freshly prepared solution of A22 NaN02 in 12 N HCl for 30 min a t 4 "C and washed three times in * A23 cold H,O and also in 0.2 M sodium acetate, pH4.0, at room temper! I A1 ature. The cDNA insert from clone A1 was denatured, spotted onto P A7 the activated paper, and allowed to dry overnight. The filters were then cut into 1-mm squares and washed sequentiallywithsterile I I I distilled water, 0.4 M NaOH, and then distilled water. Filters were 0 500 loo0 1500 bp prehybridized for 1 h a t 42 "C in a solution containing 60% formamFIG. 1. Restriction map and locations of cDNA clones for ide, 10 mM PIPES, pH 6.4, 0.4 M NaCI, and 20 mg/ml yeast tRNA. The hybridization mixture containing 25 pg of total RNA or poly(A) chick anchorin. A restriction map of the inserts of anchorin clones RNA, 60% formamide, 10 mM PIPES, pH 6.4, and 0.075 M NaCl was was constructed using several endonucleases asdescribed in the text. added and allowed to hybridize a t 42 "C overnight. The filters were The number below the bar indicates the distance in base pairs from extensively washed in 0.5 X SSC, 0.5% SDS a t 60 "C and once in 2 the 5'-end of the cDNA. The hatched bar from 987 bp to the end mM EDTA, pH 8.0, at room temperature. Elution of hybrid-selected represents the 3"untranslated region.

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Anchorin CII cDNA

A

5923

B 5'-probe

3"probe

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FIG.3. Northern analysis. The 5' probe, a 780-bp

EcoRI fragment of clone AI,and the 3' probe, a 600-bp EcoRI fragment of clone A4,were labeled by nick translation and hybridized toRNA which had been blotted a nitrocellulose onto membrane after electrophoresis -18 A, hybridization with 5' on a denaturing formaldehyde-agarose gel. probe. Twenty pg of total RNA,from different chick tissues, were '"".,b. blotted on each lane: sterna (laneI), fibroblasts (lane2), Rous 1 2 3 4 1 2 3 4 sarcoma virus-transformed fibroblast3),(lane and crop (lane 4 ) . B, hybridization with 3' probe: sterna (lane I ) , fibroblasts (lane 2), and FIG.2. Hybrid-selected translation. A 780-bp EcoRI fragment 3 ) . Positions in the gel 28 S and of 18 S ribosomal R N A s from cloneA1 was immobilized on diazobenzyloxymethylcellulose crop (lane paper and hybridized to total RNA or to poly(A+) RNA from chick are indicated. cartilage. Hybrid-selected RNA was eluted and translated in a rabbit reticulocyte lysate, and the productwas electrophoresed ina 10% SDS-polyacrylamidegel. A, translation products: in the absence of RNA (lane I ), from total cartilage RNA (lane2),from hybrid-selected C C C C C C C A A C C C C C G A A G A T G G C G A A G T A T A C A A G A G G C A C C G T G A C A G C A T T C T C T C C T P G E P G K M A K Y T R G T V T A F S P nitrocellulose paper alone (lane 3),or frommRNA hybrid-selected with cloneAI (lane 4 ) . B, immunoprecipitation with anti-anchorinTTTCATGCCAGAGCTGATGCAGAAGCCCTTCGCAAGGCCATGAAGGGAATGGGGACTGAT F D A R A D A E A L R K A M K G M G T D antiserum of the translation products: in RNA absence (lane I )of , CAAGAGACAATTCTGAACATCCTTACCAGCAGAAATAATGCTCAACGTCAAGAAATTGCA from total cartilage RNA (lane3),from mRNA hybrid selected with E E T I L K I L T S R N N A P R Q E I A TCTGCTTTTAAAACACTCTTTGGCAGGGATCTTGTGGATGACCTGAAATCAGAACTTACT clone A1 (lane4 ) . Lane 2 represents the control immunoprecipitation S A F K T L F G R D L V D D L K S E L T with nonimmune rabbit serum of the translation products fromC C C A total AGTTTGAAACACTGATGGTATCTTTGATGAGACCAGCACGTATTTTTGATGCTCAT G K F E T L M V S L M R P A R I F D A H cartilage RNA.Lower arrow points to migration of in uitro-translated GCACTCAACCATCCAATCAAGGGAGCAGGAACCAATGAGAAAGTGTTGACTGAAATTCTT anchorin. Upper arrow points the position at which purified A L K H anchorin A I K G A G T N E K V L T E I L GCCTCCACAACACTCCTGAAGT C A G A A T A T T A A A C A C G T T T A A T A T G C A A G A G T A T G A G migrated in same the gel. A S R T L L K 8 R I L N R F N M P E Y E I .

,

C A N L G R N K l T G R R H P A l F R D & TCCTGGTCCT CT C A G C C A A A T A C A G A T T C C T C A T G G C A G A G T T G A G G A G G C T C T T G T T W W S & & R Q I E I P D G R V E E A L V

GCCAACCTTGCAAGCAATAAGATCACAGGAAGAAGACATCAGGCCATTTTCAGAGACT

immunological cross-reactivity of the proteins indicates that they are both forms of anchorin and that cloneA1 codes for anchorin. Northern Analysis-Different portions of the cDNAclones for anchorin were used to analyze the mRNAspecies in chick cells. The 880-bp EcoRI fragment from the 5' portion of the cDNA hybridized to a single 1.7-kb mRNA species (Fig. 3A). This mRNA is found both in chondrocyte andin normal and Roussarcomavirus-transformed chick embryo fibroblasts. Interestingly, a 3-fold higher level of anchorin mRNA was found in the transformedchick cells. When the 3' probe was used forthe hybridization, an additional mRNA species about 5-kb in size was detected in chondrocytes, crop, and chick embryo fibroblasts (Fig. 3B). The relative amountsof the 5kb mRNA are somewhat lower than the 1.7-kb mRNA in these cells. Sequence of Anchorin-The nucleotide sequences of the cDNAcloneslisted in Fig. 1 were determined (Fig. 4). A continuous sequence with a single open reading frame of 987 nucleotides coding for a protein ( M , = 37,344) composed of 329 residues (Fig. 4) was determined. There is nopoly(A) tail in the first 250 nucleotides of the 3' untranslated segment. The deduced amino acid sequence does not contain Asn-XSer or Asn-Thr-Ser sequences, potential sites for N-linked

GAGAAGGATCCTCACGTCTTGTTTAGAGCTGGGGAGCTAAAATGGGGAACAGATGAAGAA

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TACATGACTATTTCTGGCTTTCAAATTGAAGAAACCATTGACCGTGAAACCTCTGGTGAT

Y M T l S G F Q l E E T l D R E T S C D TTGCAGAA TGCTTTTGGCAGTTCTGAAGT CATCCCAAGTGTGCCTGCTTATTTTGCT L E K L L L A V V K I R S V P A Y F A GAAACTTTGTATTATTCTAT A A C G G G C T G G C A C T G A T G A T G A T A C C C T G A T C A G A G T C E T L Y ( Y ) S M K C A G T D D , D T L I R V ATGGTTTCAAGAACTCAAATCGACCTGTTGGATATTACACATG'AATTCAGAAAGAATTTT

M

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CCCAAATCCTTGTATCAGATGATTCAGAAAGATACATCTGGGGACTACAGGAAGGCACTC

A K S L Y Q M I P K D T S G D Y R K A L CTCCTCCTCT T G G T G G A G A T G A T G A C T ~ A T G G T G G C A G C G A C G T G A A G G A T T T C T T G T A L L L 8 C G D D E ATCCAGCTTTGCACCCCTTCAGTTAGCATGCCTAGCTAAGATTTTGCATCTTAATGCTTT ATGGCTGTTCGAATTTATATTCATATCACACTTATTAAACACAAACATGTTACTACTAGC TGATAAACAGTCCCTCCTCCTCAGACGTCCTGACTCTGGGAATTTCAGTGCCTTCTGAGT GTATGCAAAGTCTCTCATGGAGTAGAGTAGTATCG

FIG.4. Nucleotide sequence of the cDNA coding for chick anchorin and the deduced amino acid sequence of the protein. Nucleotides arenumbered at the top right and amino acids at the bottom right.The translation intoamino acid sequenceis shown below aassingle open reading frame. Sequences underlined are to match peptide sequences obtained from anchorin tryptic 244-267 represents the hydrophobic reThe box enclosing residues gion. The tyrosine residue in parentheses a possible is phosphorylation site. Cysteines in the sequence are The circled. arrowhead at nucleotide 883 points at the EcoRI cleavage site in the anchorin

mRNA.

Anchorin CII cDNA

5924

oligosaccharides. However, there are several serine and thre- was subjected to preparative slab gel electrophoresis and a onine residues as possible sites for 0-linkedoligosaccharides. band ( M , = 34,000), identified as anchorin CII by immunoA tyrosine at residue 265 might be a potential phosphorylation blotting(16), was excised and digestedwith trypsin.The site since thesequence around thisresidue resembles a typical resulting peptides were eluted from the polyacrylamide gel and separatedby reverse-phase chromatography into 13 major tyrosinephosphorylationsignal(31).TheCOOH-terminal portion of the protein is rich in aspartic acid (9 out of 62 fractions (Fig. 5A). Each peak was rechromatographed by residues), whereas the NH,-terminal portion contains an un-reverse-phase chromatography using other conditions (Fig. 5B). Thirteen purified trypticpeptidesencompassing 113 usually high amount of glutamic acid (10 out of 68 residues). residues were sequenced (Table I). Isolation and Sequencing of Peptides from Anchorin CZZComparison of Peptide Sequences with the Sequence PreAnchorin CII preparedfrom chicken chondrocyte membranes dicted from the cDNA-The nucleotide sequence of clone A1 contains an open reading frame of 780 nucleotides which code A for 260 residues and include the sequences obtained for the 7 tryptic peptides underlined inFig. 4. This clone is located 100 bp away from an EcoRI cleavage site within the anchorin CII nucleotide sequence. Additional cDNA clones were obtained from the original Xgtll library by DNA hybridization with nick-translated 3'-end DNA fragments from clone A15 (see Fig. 1).Two of these clones, A4 and A6, were sequenced, and both were identified as anchorin CII, since they contained three other tryptic peptides(Fig. 4). The orientationof these clones as being COOH-terminal was established by the amino acid sequence of an overlapping tetrapeptide (HEFR) (Fig. 4) covering the internal EcoRI site. These clonesinclude the 00 IO 20 30 40 50 EFFLUENT VOL [ m l l continuation of an open reading frame of 207 base pairs coding for an additional 69 amino acids and a nontranslated region of 248 base pairs. The sequences of 10 out of the 13 peptides B are present in the deduced amino acid sequence (underlined I in Fig. 4). The sequence of three of the tryptic peptides on Table I is not contained in the sequence depicted in Fig. 4. They could arise from the 5'-endof the sequence or be derived from an alternativelyspliced exon which is not present in our cDNA clones. 0

DISCUSSION

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,

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I

12

20

FIG. 5. A , separation of tryptic peptides of anchorin CIIby reverse-phase chromatography on a Vydac RP18 column in 0.1%trifluoroacetic acid with a linear acetonitrile gradient. The numbers correspond to the sequenced peptides in Table I. B, rechromatography of peptide 11 by reverse-phase chromatographyon LiChrospher RP18 with a shallower acetonitrile gradient. All other fractions were rechromatographedusing the same procedure.

We have isolated several overlapping cDNA clones which span 1.5 kb encoding anchorin CII. The identification of the cloneswas established by hybridization toanchorinCII mRNA. Further, 10 peptides from anchorin havebeen sequenced and found to occur in the protein predicted by the sequence of our cDNA clones. Anchorin CII thus consistsof at least 329 residues (Mr= 37,344), some 10% larger than the size estimated by electrophoresis. Primer extension analysis (data not shown) revealed that the cDNA clones described here contain the 5'-end of anchorin mRNA, suggesting that the methionine at residue 7 could be the initiating residue. Additionally, thismethionine residue is implicated as the initiation site since sequences flanking the ATG codon con-

TABLE I A m i n o acid sequences derived from trypsin digestion of anchorin CII Position in Length sequence

Sequences

1. Gly-Thr-Val-Thr-Ala-Phe-Ser-Pro-Phe-Asp-Ala-Arg 13-24 2. Ile-Phe-Asp-Ala-His-Ala-Leu-Lys 3. Gln-Glu-Ile-Ala-Ser-Ala-Phe-Lys 4. Ser-Val-Ser-His-Leu-Arg 210-215 5. Ser-Leu-Tyr-Gln-Met-Ile-Gln-Lys 6. Val-Leu-Thr-Glu-Ile-Leu-Ala-Ser-Arg 7. His-Glu-Phe-Arg 8. Val-Asp-Glu-Ala-Leu-Val-Glu-Lys 9. Thr-Pro-Ala-Glu-Val-Gln-Asn-Ile-Lys 10. Gln-Val-Tyr-Met-Gln-Glu-Tyr-Glu-Ala-Asn-Leu-Glu-Asp-Lys 11. Ile-Thr-Gly-Glu-Thr-Ser-Gly-His-Phe-Gln-Arg 12. Gly-Ala-Gly-Thr-Asp-Asp-Asp-Thr-Leu-Ile-Arg 13. Thr-Leu-Phe-Gly-Arg Total

12 8 8

6 8 9 4

8 9 14 11 11 5 113

96-103 57-64 303-310 115-123 294-297 175-182 Not found Not found Not found 269-279 65-69

Anchorin CII cDNA tain the critical requirements for initiation of translation, an A in position -3 and a G in position +4 (32). If this is the case, there may not be an amino-terminal signal peptide in anchorin, although there areexamples of internal transmembrane segments being utilized as signal peptides (33, 35). The only hydrophobic sequence in anchorin (residues 244-267) contains 3charged residues, which is unusual for a transmembrane segment. There are several proteins, however, such as glycophorin A (36) and bacteriorhodopsin (37) whose transmembrane segments have charged amino acids. It is possible that anchorin CII may actually be a membrane-associated rather than a transmembrane protein. Inthe future, antibodies to synthetic peptides corresponding to NH2- and COOHterminal sequences could be generated and used to establish whether both endsof the protein are outside of the cell. Northern analysis revealed that the5’ cDNA probe hybridized to 1.7-kb mRNA species as expected for anchorin, whereas the 3’ cDNA probe hybridized to a 5-kb as well as the 1.7-kb mRNA species. These results suggest that the 1.7kb and 5-kb mRNAs are transcribed from different genes but contain homologous sequences or that thetwo transcripts are derived from the same gene via alternative splicing. Many genes have been reported to have undergone alternative splicing to generate a diversity of structures. It is also conceivable that anchorin belongs to a family of proteins and that the 5.5 mRNA species could code for a protein whose sequence is homologous to anchorin. Since some of the amino acid sequences from proteolytic fragments of anchorin are not present in the deduced sequence from cDNA clones, it is possible that they arise from another form of anchorin. Acknowledgments-We gratefully acknowledge the expert technical assistance by Marion van Menxel and SabineTolle. We also wish to thankDr. Klaus Kuhn for helpful advice during the course of this work and in the preparation of the manuscript and Dr. Marian Young for her advice on hybrid select translation. We thank Selma Jacobs and Lisa Wepasnick for secretarial help. REFERENCES 1. Kleinman, H. K., Klebe, R. J., and Martin, G. R. (1981) J . Cell Biol. 88,473-485 2. Yamada, K. M. (1983) Annu. Reu. Biochem. 52, 761-799 3. Montesano, K. M. (1986) Experentia (Basel) 42,977-985 4. Hay, E. D. (1981) in Cell Biology of Extracellulnr Matrix (Hay, E. D., ed) pp. 379-409, Plenum Publishing Corp., New York 5. Timpl, R., and Dziadek, M. (1986) Znt.Reu. Exp. Pathol. 2 9 , 1-112 6. Liotta, L. A., Wewer, U. M., Rao, C. N., and Bryant, G. (1985) in The Cell in Contuct (Edelman, G. M., and Thiery, J. P. eds)

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