The Human Type VI Collagen Gene - The Journal of Biological ...

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a3(VI) chain shows the presence of 1 additional cys- teine in this portion of ... contain a short triple helix flanked by amino- and carboxyl- terminal globular ... peptide sequence (nucleotides 299-322, primer I) and the repeat A8/. N9 (nucleotides ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 267, No. 33, Issue of November 25, pp. 2 4 ~ 2 - 2 4 ~ 3 , 1 3 9 2 Printed in U.S.A.

0 1992 by The American Soeiety for Biochemistry and Molecular Biology, Inc

The Human Type VI Collagen Gene mRNA AND PROTEIN VARIANTS OF THE a3 CHAIN GENERATED BY ALTERNATIVE SPLICING OF AN ADDITIONAL 5-END EXON* (Received for publication, June 17, 1992)

Stefania ZanussiS, Roberto DolianaS, Daniela SegatS, Paolo Bonaldos, and Alfonso ColombattiSll 11 From the SDivisione di Oncologia Sperimentale 2, Centro di Riferimento Oncologico, Auiano, the Slstituto di Istologia, o e Scienze Mediche, Universita’ di Udine, Universita’ diPadova, 35100 Padova, and the ~ D i ~ a r t i m edi~ t3io~ecno~gie 33100 Udine, Italy

The amino- and carboxyl-terminal globular domains the building blocks of the microfilament structures presentin of homologous several tissues (Colombatti et al., 1989; Kuo et al., 1989; Wu of type VI collagen are composedseveral modules similar to the type A collagen-binding modules et al., 1987). present in von Willebrand factor. The human a3(VI) The cDNAs for all three chainsof chicken and human type chain that contributes most of the amino-terminal VIglobcollagen have been cloned and sequenced (Bonaldo et al., ule appears heterogeneous insize as a result of alter1989, 1990; Chu et al., 1989, 1990; Koller et al., 1989). The native splicing of two exons (Stokes D. G., Saitta, B., cul(V1) and a2(VI) chains are very similar in structure and Timpl, R., and Chu, M.-L. (1991) J. Biol. Chern. 266, contain a short triple helix flanked by amino- and carboxyl8626-8633). In the present study, we report a further terminal globular domains, which consist of one and two characterizationof the 5’-end of the gene of the human a3(VI) chain and show that tran~riptioninitiates at repeated modules, respectively, of about 200 amino acid resimultiple sites. Southern blotting andDNA sequencing dues. The larger molecular mass of the a3(VI)chain is partly indicate that there is an additional type A exon (A91 due to additional amino acids in the carboxyl-terminal end N10) at about 1.8 kilobase pairs downstream of tbe (Bonaldo and Colombatti, 1989; Chu et al., 1990) and mostly exon coding for the signal peptide. The open reading due to thepresence of a very extended amino-terminal region, frame of this additional exon reveals 1 cysteine and of about 1600 residues, consisting of at least eight repetitive three potential N-glycosylation sites. Polymerase 200-amino-acid long modules. These modules share strong chain reaction, Northern blotting, and RNase protection assays demonstrate thatexon AQ/N10is subject to similarity with analogous segments present in several, apparalternative splicing in normal and tumor cell lines and ently unrelated proteins, which identify the superfamily of that this generates more protein variantsof thea3(VI) proteins embodying von Willibrand factor type A-like dochain thanexpectedbefore. A comparison with the mains (Colombatti and Bonaldo, 1991). Both 3’-end (Saitta corresponding amino-terminal globule of the chicken et al., 1990) and 5’-end (Doliana et al., 1990; Stokes et al., a3(VI) chain shows the presence of 1 additional cys- 1991; Saitta et al., 1992) variability has been reported for the teine in this portion of the molecule and suggests that a2(VI) and a3(VI) chains of type VI collagen indicating a human type VI collagenhasmore possibilities for potential for generation of alternative forms of the protein. structural and functional variations compared to In fact, inprevious studies it has been found that the a3(VI) chicken typeVI collagen. chain shows a size heterogeneity both at the mRNA and protein level (Engvall et al., 1986; Chu et al., 1987; Colombatti and Bonaldo, 1987; Wu et al., 1987; Ayad et ai, 1989; Colombatti et aL, 1989; Kielty et aL, 1990). Isolation of cDNA Type VI collagen is a component of an extensive network clones lacking about 600 base pairs, largely corresponding to of microfilaments interwoven with large collagen fibers and, the size of a type A module, suggests that alternative splicing in some tissues, in close contact with cell surfaces (von der could participate in modulating the organization and assembly Mark et al., 1984; Bruns et al., 1986; Keene et al., 1988). Type of type VI microfilaments (Doliana et al., 1990). VI collagen monomers contain three genetically distinct conHere the structureof 5‘-end of the human a3(VI)collagen stituent subunits of about h4, = 150,000,140,000, and 300,000 gene wasfurther investigated. We demonstrate that there are named al(VI), &(VI), and a3(VI) (Janderet al., 1983; Trueb two major and one minor transcription initiation sites in the and Winterhalter, 1986, Colombatti et al., 1987). The type VI human cu3(VI) gene and that thereis an additional exon (A9/ monomers form disulfide-bonded dimers andtetramers N10)’ coding for a von Willebrand factor type A-like module (Engvall et al., 1986; Colombatti et al., 1987), the latter being located in the large intron between the exons coding for the * This work wassupported by grants from the Associazione Italiana signal peptide and the previously identified type A module per la Ricerca sui Cancro, Minister0 della Universitae Ricerca (N9) at the extreme 5‘-end (Stokes et al., 1991). Moreover, Scientifica e Technologica SO%, and Fondo Sanitario Nazionale Progetti Finalizzati. The costs of publication of this article were defrayed in part by 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. I( To whom correspondence should be addressed Div. di Oncologia Sperimentale 2, Centro.di Riferimento Oncologico, ViaPedemontana Occidentale, 12, 33081 Aviano, Italy. Tel.: 434-659301; Fax: 434659428.

’ In accord with the identification of a Superfamily of proteins containing von Willebrand factor type A-like modules (Coiombatti and Bonaldo, 1991), of which type VI collagen is the main representative, we designate the amino-terminal modules of the a3(VI)chain as A8-A1. Since in the human sequences the same type of modules were named N9-N2 (Chu et al., 1990) for the sake of clarity we will use the double definition (Le. A8/N9-A1/N2) throughout. Positions in the a3(VI)chain sequence are given according to Chu et al., 1990.

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Human Type VI Collagen a3 Chain Variants the A9/N10 exon is expressed in normal and transformed cell lines and is subject to alternative splicing. MATERIALS AND METHODS

Isolation and Characterization of GenomicClones-A human genomic library from the glioblastoma 203 cell line cloned in EMBL-3 (Clontech Laboratories, Inc., Palo Alto, CA) vector was subdivided in 15 subpools. DNA was extracted from individual pools by standard procedures (Sambrook et al., 1989)and amplified by polymerase chain reaction (PCR)' using synthetic oligonucleotides prepared in a Synthesizer (Applied Biosystem, Foster City, CA). Nucleotides 438-467 (primer 111) and 800-822 (primer IV) (see Fig. 1) were derived from the cDNA sequences encoding the human a3(VI) module A8/N9 (Chu et al., 1990). Three subpools that showed an amplification signal of the expected size wereplated and theplaques transferred to nitrocellulose filters. Filters were hybridized to synthetic oligonucleotide probes derived from the cDNA sequences encoding the a3(VI)signal peptide sequence (nucleotides 299-322, primer I) and therepeat A8/ N9 (nucleotides 387-411, primer 11) and labeled with 13'P]ATP (Amersham International, Amersham, United Kingdom) and T4polynucleotide kinase (Boehringer Mannheim, GmbH, Federal Republic of Germany). Two clones that hybridized with both types of probe were isolated and one of these clones (Lh-1) is described here. Southern Blotting-Phage DNA was isolated by standard procedures. Digestions with appropriate restriction enzymes were performed as described by the manufacturers. Phage DNA fragments were separated by electrophoresis on 0.8% agarose gels, transferred t o nylon filters, and hybridized with synthetic oligonucleotides specific for the human a3(VI) sequence (primers I and 11) and with a cDNA probe derived from the chicken A9 module (nucleotides 355972) (Doliana et al., 1990). Nucleotide Sequence Analysis-Nucleotide sequences were obtained directly from the phage DNA by the dideoxy chain termination method (Sanger et al.,1977;Biggin et al., 1983) according to the procedure used by Manfioletti and Schneider (1988). Some sequences were determined using Taq DNA polymerase (Innis et al., 1988). Exon-coding sequences were independently confirmed by sequencing cDNAs which were amplified from total RNA using the RT/PCR method. RNA Extraction-Total RNA was prepared by acid guanidinium thiocyanate-phenol- chloroform extraction (Chomczynski and Sacchi, 1987) from the normal human fibroblastic cell lines, Flow 6000 and WI38, and from several human tumor cell lines including MG-63 osteosarcoma, A204 and RD-KD rhabdomyosarcoma, A172, T98, U87, U138 and U373 glioblastoma, and the SV40-transformed fibroblastic cell line W138VA13.All cell lines were obtained from the American Type Culture Collection (Rockville, MD.). Northern Blotting-Poly(A)+-enriched RNA was prepared from Flow 6000 and MG-63 cells by affinity chromatography (Sambrook et al., 1989). Electrophoresis of the RNA was performed on 1.1% (w/ v) agarose gel containing 2.3 M formaldehyde in MOPS buffer for 8 h at 150 V using 20 cm-long plates. RNA was then transferred onto nylon filters and hybridized with [a-"PI dCTP-labeled cDNA probes derived from the A9/N10 (nucleotides 1-618) and A7/N8 (nucleotides 944-1489) modules which were obtained by amplification of MG-63 RNA with the RT/PCR method. The filters were washed and exposed t o 0-max Hyperfilm (Amersham). RTIPCR-RTIPCR was performed according to the procedures described by Doliana et al. (1990),with the exception that thereaction was carried out through 25 cycles of amplification. Aliquots of the PCR mixture were electrophoretically, separated in agarose gel, and visualized by ethidium bromide staining. The oligonucleotides used and their position within the sequence are the following: sense primers A (nucleotides 266-290), D (295-314), E (439-468), and G (14981519), antisenseprimers B (605-625), C (1170-llgl), F (1588-1611), and H (2509-2531) (Fig. 6). Rapid Amplificationof cDNA Ends (RACE)-The RACE technique (Frohman etal., 1988) was applied to isolate the 5'-end of the human ru3(VI) transcripts. Ten ng of an antisense oligonucleotide (nucleotides 298-320) were used to prime first strand cDNA synthesis from MG-63 RNA. cDNAs were then purified by centrifugation using centricon 100 (Amicon Division, Beverly, MA) and polyadenylated

* The abbreviations used are: PCR, polymerase chain reaction; RT, reverse transcriptase; RACE, rapid amplification of cDNA ends; bp, base pair(s); MOPS, 4-morpholinepropanesulfonicacid.

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using 15 unitsof TdT (Promega Corporation, Madison, WI) and0.2 mM dATP. The cDNAs were amplified using a 5'-poly(dT) containingprimer and a 3' reverse nested primer (nucleotides 227-248). During the 40 amplification cycles three different profiles were applied I cycle, denaturation for 3 min at 96 "C, annealing for 2 min at 37 "C, and extension for 30 min at 72 "C; 11-VI cycles, denaturation for 1 min and 20 s at 95 "C, annealing for 2 min at 68 "C, and extension for 3 min at 72 'C; VII-XI cycles, denaturation for 1 min and 10 s, annealing for 1min and 30 s a t 62 "C, and extension for 2 min at 72 "C. A second and third round of amplifications were performed with nested 3"antisense primers (nucleotides 186-210 and 160-180, respectively) to augment the specificity of the amplification steps. RNase Protection-cDNA fragments were amplified by PCR using the following primers: nucleotides 187-211 and 176-200 and the largest fragment obtained by RACE was cloned into a pGEM 3zf(+) vector (Promega). After linearization with EcoRI, ~x-~'P-labeledantisense RNA probes were synthesized from the SP6 promoter with 25 units of SP6 RNA polymerase (Promega) (Melton et al., 1984) and annealed with total RNA (10 fig) extracted from normal and tumor human cell lines. Non-hybridized RNA was digested with 40 pg/ml of RNase A (Promega) and 2 rg/ml of RNase T1 (Promega) at 30 "C for 1h. After treatment with 0.1 gg/ml of proteinase K in 0.5% (w/v) SDS at 37 "C for 15 min, the undigested fragments were precipitated with ethanol and separated by electrophoresis on a 6% (w/v) polyacrylamide gel containing 6 M urea. Under these conditions no protected bands were detected when Escherichia coli tRNA instead of human RNA was added to the hybridization mixture. Expression of A9/NlO Fusion Proteins and Immunological Detection of A9/N10c Protein Variants-The A9/N10 module was amplified by PCR, inserted at the EcoRI-BglI sites of a pAX4b vector (Markmeyer et al., 1990) and expressed as a P-galactosidase fusion protein. The fusion protein was affinity purified on a column of p aminobenzyl-1-thio-8-D-galactopyranoside and used to produce a polyclonal antiserum. Specificity of the antiserum was assessed by immunoblotting on fusion proteinscontaining different human ot3(VI)modules. Indirect immunofluorescence of cells grown on tissue culture glass chamber slides (Nunc Inc., Naperville, IL) was carried out on cells fixed in 4% (v/v) paraformaldehyde in phosphate-buffered saline for 30 min before incubation with the antiserum. The slides were then incubated with anti-rabbit IgG fluorescein-conjugated goat antibodies. Stained fibrils were examined using a Leitz microscope and photographed with Kodak films. RESULTS AND DISCUSSION

The Structure of the 5'-End of the Human a3(VI) GeneSubpools of a human genomic library were screened by PCR amplification with oligonucleotide primers specific for the A8/N9 exon. One clone (Lh-1) of about 18 kilobases, which hybridized with oligonucleotide probes corresponding to sequences of both the presumed signal peptide and the A8/N9 module, was isolated and further studied by a combination of restriction mapping, Southern analysis, and selective sequencing. The physical map and a partial restriction map of the Lh-1 clone and the oligonucleotides used for the screening of the library are shown in Fig. 1. Identification of an Additional Exon Coding for a Type A Module-Phage DNA was digested with combinations of Sal1 (a restriction sitewithin the polylinker of EMBL-3) and either SmaI, or EcoRI, or AuaI. Fragments were separated on 0.8% (w/v) agarose gel, transferred onto nylon membranes, and hybridized with oligonucleotides derived from the presumed human signal peptide and the A8/N9 module. The two oligonucleotides hybridized with fragments of different size (Fig. 2). Since inthe chicken a3(VI) gene, an additional exon coding fora full type A module(A9) has recently been identified (Doliana et al., 1990), the filters were hybridized at lower stringency to a cDNA probe encoding the chicken A9 module, after stripping of the above probes with a solution of 0.5% (w/v) boiling SDS. Under these conditions, the chicken A9 probe recognized the same fragments asthe signal peptide oligonucleotide probe. The absence of additional bands on the

Lh-1

zzzzz

7

+ 1

9

A

A

'i

A

""-

""

0.

11y

p

= 0.6 kb

FIG. 1. Diagram of the physical map and partial restriction sites of clone Lh- 1. Exons are indicated hy npcn rrctnng1r.s and introns by thick 1inc.s. h'UT and KSI' represent noncoding sequences and sequencescoding for the presumed signal peptide. respectively. Restriction enzyme sites: A , Aunl; E , EcoRI; S , Smal. The positions of the oligonucleotides used to screen the human genomic litwary ( I - I V ) also are depicted.

P l r n F ]

A9 modules is about 77% at the nucleotide level and about 70% at the aminoacid level. kb oligonu. oligonu. cDNA In our analysis of the Lh-1 clonewe also sequenced part of the exon coding for the A8/N9 module. Upon closer exami";! E 6.5 . nation,thissequence did notmatch completelywith that reported by Chu et al. (1990) as i t contained six additional 1.3 . I, nucleotides. A cloningartifactseems unlikely sincedirect sequencing of a cDNA fragment amplified from human skin 2.3 . RNA by RT/PCR using primersspecific for the AR/N9 mod2.0 . ule confirmed the sequence obtainedfrom the genomic clone. In addition, the present nucleotides and deduced amino acids of thehuman A8/N9moduleperfectly match with those 1.3 . reported for the chicken A 8 module (Ronaldo et al., 1990). 1.0 . The additional nucleotides areall found in correspondence of gel doublets or tripletsand it seems likely that,dueto I123 compression, these nucleotides were overlooked in previous analyses (Chu etal., 1990). As a consequence of the inclusion FIG. 2. Southern blot analysis. DNA of clone Lh-1 was digested of these nucleotides, the cysteine at position 7 3 (Chu et al., with Sal1 together with one of three restriction enzymes asindicated, 1990) is substituted by another amino acid (Fig. 4). resolved on 0.8% (w/v) agarose gel, transferred to a nylon filter, and Determination of theTranscriptionInitiationSite-The hyhridized to radioactively labeled human oligonucleotides (SF' and A8/N!?) and a chicken cDNA ( A ! ? )probe. Restriction enzymes used: transcription initiation site of the human n W I ) gene was SmaI (lane I ) , IkoRI (lane 2 ) , and AuaI ( h a 3). SP, signal peptide. identified by RACE and by RNase protection studies. With On the k / t , the migration of standard restriction fragments is indi- RACE a number of cDNAs of different size were obtained cated in kilobases. after cloning the amplified fragments. These fragments extend to different 5'-ends (arrows in Fig. X ) , and the largest filter hybridized with the chicken A9 cDNA probe suggests one starts with the nucleotide corresponding to position +1 that the hybridization conditions were sufficiently stringent as reported by Chu et al. (1990). Since no larger fragments t o avoid any cross-hybridization of the chicken A9 probe to were detected by Southern blotting (Fig.S A ) , it is likely that non-homologous human type A modules. Since the genomic no other upstream initiation sites are present. The largest fragment obtained by RACE was inserted into clone Lh-1 does not contain any other downstream type A a pGEM 3Zf(+)vector and used to generateRNA probes that exon beside A8/N9, the positive hybridizationsignalsobtained with the chickenA9 cDNA probeimplied the existence were annealed to total RNA from the MG-63 cell line. Two of an additional type A exonin the proximity of the exon for major and one minor bandwere found after RNase digestion the signal peptide. DNA sequencing revealed an open reading of the protected RNA (Fig. 5 R ) . The same probe was also frame of 618 bp (Fig. 3) that matched very well with that of used to protect RNA from other cell lines and bands were of t h e chicken module A9. This open reading frame was inde- similar size (data not shown). The resulting bands were specific for the 210-base pairs probe since another probe propendently confirmed by direct sequencing of a cDNA fragtected bands of different size (Fig. 8). Rands of a size comment amplified from human skin RNA by RT/PCR (data not parable to the lowest RACE fragment (Fig. 5A ) were never shown).Thisexon is separated from the presumed signal obtained by RNaseprotection:thus, it is likely thatthis peptide exon by about 1.8 kilobase of intronic sequences. As shorterfragmentcorrespondstoatruncatedtranscription suggested from the deduced amino acid sequences (Fig. 3), the product. That transcription of the human n3VI) collagen A9/N10 module contains three potential N-glycosylation sites gene starts atmore than one siteis in line with what is known and 1 cysteine. Therefore, the human A9/N10 module differs for the chicken and human &!(VI) (Koller et al., 1991; Saitta from the corresponding chicken module that lacks cysteines et al.,1992) and for the murine nl(V1):' collagen genes. Howand only has one glycosylation site (Doliana etal., 1990). The ' P. Ronaldo, unpuhlished results. overall identity between the human A9/N10 and the chicken

-

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1 11231 11231

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H u m a n T y p e VCollagen I a3 Chain Variants

- +++ ++Human n u c l . Chicken nucl Human a a . Chicken aa

.

aaa

A

aaagca atg tct tttttt aaa gAT GTC

tct

c.c

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.

......... .c. ... V -

6 360 2 7

AAA AAT GGT GCG GCT GCT GAT ATA ATA TTP CTA GTG GAT TCC

48 402 16 21

TCT TGG ACC ATT GGA GAG GAA CAT TTC CAR CTT GTT CGA GAG .G. ..GA....G..C ..C S W T I G E E H F Q L V R E S " K - - -

90 444 30 35

TTT CTA TAT GAT Gl'T GTA AAA TCC TTA ..G ..G G.T F L Y D V V K S L A -

132 486 44 49

GAT TTC CAT TTT GCT CTG GTC CAG TTC AAC GGA AAC .G. ..A .G. D F H F A L V Q F N G N

174 528 58 63

ACC GAG TTC CTG TTA AAT ACG TAT CGT ACT AAA CAA GAA GTC ..A .A. ..C .CC T....C ..T..G T E F L L N T Y R T K Q E V Q - " P S N - D -

21 6 570 72 77

CTT TCT CAT ATT TCC AAC ATG TCTTAT ATP GGG GGA ACC AAT ..C..C ..CG.G..T C....C..G GG. .GC L S H I S N M S Y I G G T K - A - - P - M - - G S

258 612 86 91

........................... I F L V D

.G...C.T...C.TG K N G A A A D I R - V - V - " " " - -

......

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GCT GTG GGA GAA AAT

... .A. ...... .G. ...

............

A D

............

-

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S

"

-

...

G -

E G

N -

CAT ..C P H

CCA

.........

B N9h A8h

A8c

FIG. 4. Partial nucleotide sequence of repeat AS/N9 of human a3(VI) collagen. A , nucleotide sequence; B, deduced amino acid sequence. N9h, sequence derived from human cDNAs (Chu et al., 1990);A8h, sequence obtained from genomic clone Lh-1 and from RT/PCR amplified human RNA (this report);A&, sequence derived et al.,1990). The two regions in which from chicken cDNAs (Bonaldo the previous (Chu et al., 1990) deduced amino acid sequences differ from the present deduced sequences are boxed.

-

.........

" -

V -

R8C

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AARGCCCTCGG T T GGTGGTGGG AGTTGGCCAATATCG C TCGCC TTGAT AAAGCCCTCGG~TTIGCTGGTGGG~AGTTGGCCAATATCG~C€TCGCC€TTGRT AAGGCACTCRGTTTCCGCGGTGGCAAGGARGCTAACACTGGTGCAGCACTGGAG

N9h ABh

...

...

-

u-

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111

207 194

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172

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TCRTTCTCCTGGCCTCCRGCTCCTGCRAGG---

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CAG ACT GGA AAA GGA TTA GAA TAC ATA ATG GCA AAG CAC CTC ..G C.. ..C .AG ..C ..T Q T G K G L E Y I M A K H L K - - - - L I E N - -

...............

A..

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300 654 100 105

ACC AAG GCT GCT GGA AGC CTG GCC GGT GAC GGA GTC CCT CAG ..T..A AGA ..GA....A..C ..C T K A A G S L A G D G V P Q R - S E " - -

342 696 114 119

GTT ATC GTA GTG TTA ACT GAT GGA CAC TCG AAGGAT ..TA.. ..G..C ..A..CC.. V I V V L T D G H S K D 1""Q - Q -

384 738 128 133

GCT CTG CCC TCA GCCGAA CTT AAG TCT GCT GAT GTT AAC GTG ..A T.T .TC ..A..G..CC....A A.. A L P S A E L K S A D V N V S V " " H - - M

426 780 142 147

AAA TTT GCA ATT GGA GTT GAG GAT GCA GAT GAA GGA GCG TTA A....GG.C..C..GC.. ..G .TG ..G.A. ..G F A I G V E D A D E G A L K I - V - - Q - - V - - E - -

4 68 822 156 158

GAA ATA

............

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...

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CTT ... GGC .AT GTG G L

...

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GCAAGT GAA CCG CTC CG. ..CT..G.. A S E P L R - F

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D

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B C FIG. 5. Analysis of exon EUT for transcription start sites.

A

A , autoradiography of the RACE products run on a 2% (w/v) agarose gel, blotted, and hybridized with a ["'PIdATP-labeled internal oligonucleotide. B, autoradiography of an RNase protection assay. The probe depicted at thebotton extends from position 1to position 210. Lane I contains probe, RNase, and 10 pg of total RNA of MG-63 cells; lane 2 contains probe alone. Double-stranded RNA fragments were separated on a 6% polyacrylamide sequencing gel. C, nucleotide sequence around the transcription initiationsites. The transcription initiation sites identified by RACE are indicated by arrows and those determined by RNase protection by brackets. The discrepancy between the results of the two methods is most likely due to intrinsic differences of the twoprocedures.

AATATG CAT ATGTTC ARC CTA .CC ..CC.T N M H M F N L D T - L - - -

.........

ever, the pattern of transcription sites for the a3(VI)chain is much simpler than the pattern for both al(V1) and a2(VI) chains that have six to eight start sites each. It is known that GAG AAT TTT ACC TCA CTT CAT GAC ATA GTA GGA AAC TTA GTG the choice of mRNA species is regulated in a tissue-specific 51 0 864 G.T..C ... .G. ... ..T ... G.. manner or during development and is related to translation E N F T S L H D I V G N L V 170 efficiency or mRNA stability. Whether the tissue variability " - - A - - G " D - 161 reported for the a3(VI) chain may be explained in terms of TCC TGT GTG CAT TCA TCC GTG AGT CCA 352 GAA AGG GCT GGG GAC mRNA selection by the use of different transcription initiaG.AA....C.G.A.C 90 6 ... A...C. ...... C A . ..A.C. tion sites remains to be determined. S @ V H S S V S P E R A G D 184 A S - R T - M T - Q " A 175 Expression and Splicing Pattern of the A9/NlO Exon-In the study of Chu et al. (1990), as well as in our preliminary ACG GAA ACC CTP AAA GAC A X ACA Ggt aat gg 594 948 .AA .G. CTG G.. screening of a human placenta cDNA library, clones that 198 T E T L K D I T contained the newly discovered A9/N10 module were never K G L V 189 identified. This suggests that the exon coding for the A9/N10 FIG. 3. Comparison of the nucleotide sequences of the hu- module is not expressed constitutively but is present only in man A9/N10and chicken A9 a3(VI) chain modules. First line human andsecond line chicken nucleotide sequence; third line human rare mRNA species. Inorder to ascertain the presence of and fourth line chicken deducedamino acid sequence(one-letter code). RNAs containing the A9/N10 exon, we analyzed its expres..G E

I

"

"

............

......

...

....................

Nucleotides and amino acids of the human A9/N10 sequence are numbered on the left-hand side. The numbers of the human A9/N10 sequence are given starting with the firstnucleotide and amino acid. In the human A9/N10 sequence three potential N-attachment sites

for oligosaccharides are underlined and a cysteine residue is circled. Dots and hyphens indicate nucleotides and aminoacids identical in the chicken A9 (u3(VI) chain module, respectively.

Human Type V I Collagen (u3Variants Chain

24086

sion by RT/PCR in normal fibroblasts (Flow 6000) and in tumor cells (MG-63osteosarcoma). A series of amplified fragments corresponding to the expected bands were generated. Evidence for spliced and unspliced A9/N10 transcripts was obtained in both cell lines (Fig. 6, lanes 1-4). Furthermore, in view of the fact that the chicken A9, A8, and A6 modules undergo alternative splicing (Doliana et al., 1990), we opted to also investigate the probable variations in the region comprised between the signal peptide and the A5/N6 module. Splicing of A9/N10, A8/N9, or of both exons from the same transcript, and splicing of A6/N7 were detected in a pattern similar to that observed for chicken RNA (Fig. 6). If amplification was carried out for 40 cycles, splicing of a portion of A2/N3 exon also was obtained as reported previously by Stokes et al. (1991). This is at variance with the situation in chick tissues where we were unable to demonstrate anysplicing of A2 exon (data not shown). Similarly,in accordance with findings in the chick (Doliana et al., 1990), splicing of the human A7/N8 exon could never be demonstrated (Fig. 6). That the different fragments amplified by the RT/PCR assay were genuine was confirmed by hybridization with labeled oligonucleotides specific for the different exons (data not shown). Expression of a3(VI) transcripts was further examined by Northern blotting of poly(A)+-enriched RNA isolated from the two cell lines. A probe which recognizes the constitutive A7/N8 exon showed a strong hybridization signal with MG63 cells and a weaker signalwith RNAfrom normal fibroblasts

(Fig. 7). However, when an AS/NlO-specific probe was used, a much greater difference between the tumor and thenormal cell line was detected. Scanning densitometry of the autoradiogram indicated that the intensityof the A9/N10 signals in MG-63 cells uersus Flow 6000 fibroblasts was nearly 10-fold higher whereas the difference in the intensity of the constitutive A7/N8 signal was only obout 2.5-fold suggesting that splicing of A9/N10exonseemsmoreefficientinnormal fibroblasts. RNase protection analysis was used in order to ascertain the extent of the differential splicing of the A9/N10 exon in normal fibroblast cell lines and ina panel of tumor cell lines. RNAs were hybridizedwitha-"P-labeled antisensetranscripts designed to overlap a segment of the A9/N10 exon and a segment of the adjoining untranslated 5'-end and signal peptide region. The predicted fragment size for the included form (A9/N10+)is 360 nucleotides, whereas the fully excluded form protects a 160-nucleotide fragment (Fig. 8). Both normal fibroblast cell lines and three out of six tumor cell lines express a3(VI) mRNA in comparable quantity;all these five cell lines have A9/N10+ and A9/N10- mRNAs (Fig. 8 A ) . In addition to thesemajor bands, there are a few minor unpredicted bands suggesting that the labeled transcript is unable to be fully protected by the A9/N10+ mRNA. Densitometric measurements of the protected fragments reveal that the ratios of included to excluded A9/N10 exon is lower in the two normal fibroblasts compared to the tumor cell lines. Accordingly, in Flow 6000 fibroblasts the A9/N10+ band represents 5.4% of

A __rsFl

a3CVI)mRNA template oligonucleotide primers

A -P

I

A9/N10 AWN9 '.._,.........'.: ..'............ ..... ..._.. .

D

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A6/N7

I

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I

........

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E B

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c

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-+e

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c

B 953 bp

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.

.... ,, '. ....... 939 bP -. . . . .. + .. + 4A B C pY49/NlqA8/N9 [A7M8]

D

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590 bp

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-. .. . .. 353 bp .._....,..' e -+ A B ~1~9lNlqA8M I A97 M 8 A C -b c . . ., .. '..,. . ...'.339 hp .... ...

I

-

expected

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observed

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1035 bp 435 bp

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FIG.6. PCR analysis of the a3(VI) mRNA transcripts. PCR was performed after first strand cDNA synthesis using total RNA isolated from Flow 6000 fibroblasts (lanes 1, 3, 5 , 7, 9, 11 ) or MG-63 cells (lanes 2, 4 , 6, 8, 10, 12). Amplified products were separated on a 1.0% (w/v) agarosegel, and the DNA hands were visualized with ethidium bromide and U V light. The primer used are: lanes 1 and 2, D and H ; lanes 3 and 4 , A and R (the 953 transcript is clearly visible only after40 PCR cycles); lanes .5 and 6, A and C; lanes 7 and 8, E and F; lanes 9-12, G and H . In negative control reaction mixtures the reverse transcriptase (lane 1 1 ) or the template RNA (lane 12) were omitted. A HaeIII digest of 1'74 DNA was used as standard marker.

HumanVIType

Collagen a3Variants Chain

1.4 -

5.3-

2.8 -

1

2

1 2

FIG. 7. Northern blot analysis of human cell lines. Each lane was loaded with 10 pg of poly(A)+-enriched RNA. Filters were hybridized to [a-'"PIdCTP-labeled probesderived from A7/N8 and A9/ N10 human-specific sequences. Exposures times were one (A7/N8) or four (A9/N10) davs. On the left the migrationof molecular markers is indicated in kilobases. 1 2

3

4

5

6

7

8

9 1 0 1 1

12 13

14 15

360 (AS/NlO+)+

-a A

B UT l S P l

A9/N 10

-Probe 360 n t FIG. 8. RNase protection assay of A9/N10 exon. The uniformly labeled single-stranded RNA probe of 360 nucleotides is depicted at the bottom. Splicing out of A9/N10 exon is expected to yield a protected fragment of 160 nucleotides. Full protection yields a protected fragmentsof 360 nucleotides. A, in vitrocell lines: 1, Flow 6000; 2, RD-KD; 3, MG-63; 4, T98; 5,A172; 6, VA13; 7, U87; 8, A204; 9, WI38; IO, U373; 11, U138.B. Fresh tumor tissues: 12, histiocytic sarcoma; 13, gastric lymphoma; 14, follicular lymphoma; 15, ovarian carcinoma. Each lane contains the probe plus 10 pg of total RNA. T h e products were run on 6% (w/v) sequencing gels and exposed for 2 days a t room temperature.

24087

normal and tumor cells are the findings with another extracellular matrix glycoprotein that has been extensively investigated. The splicing pattern of fibronectin was found to be deregulated in transformed cells and in malignancies (Borsi et al., 1987; Vartio et al., 1987; Barone et al., 1989; Oyama et al., 1990). In fact, fibronectin variants containing the I11 CS, ED-A, and in particular the ED-B exons areexpressed to a greater extent in human transformed cells and freshly isolated tumors than in their normal counterparts. There are also a few reports on type VI collagen expression in transformed cells and tumor tissues, inwhich the expression of this collagen has been found dramatically reduced (Carter, 1982; Trueb et al., 1985; Schreier et al., 1988), or increased compared to normal tissues (Jakkola et al., 1989). The polymorphism of the a3(VI) primary transcript both in chicken (Doliana et al., 1990) and human (Stokes etdl., 1991; present data) cells and tissues suggests that, inanalogy with the optionalskipping of fibronectin ED-B exon, the a3(VI) chain variants may be expressed in response to different environmental stimuli related to the transformation process. Whether this depends on the presence of different, independently regulated factors, or whether the same trans-acting factor(s) areinvolved in the regulation of alternative splicing of different exons is stilla matter of investigation. Expression of the A9/NlO aS(VI) Chain Variants-In order to determine whether the alternativelyspliced A9/N10 exon resulted in theexpression of a type VI collagen with a variant a3 chain, we produced an antiserum against a recombinant bacterial fusion protein that expressed the A9/N10 module. Fig. 9 shows the pattern of reactivity of this polyclonal antiserum which in Western blottingrecognizes only the A9/ N10+ fusion protein, whereas no reactivity is detected with the A6/N7+ fusion protein. The antiserum also was assayed by indirect immunofluorescence onculturedMG-63 cells. This analysis demonstrated that extracellular proteins reacting with the anti- A9/N10 antiserum are secreted andincorporated into a fibrillar meshwork in the extracellular matrix deposited by the cells in uitro (Fig. 10B). Forcomparison the staining obtained witha polyclonal antibody against thepepsin-resistant form of type VI collagen is shown (Fig. 1OA). It will be interesting to investigate whether A9/N10+ the variant a3(VI) chains are detected only in the extracellular matrixof certain tissues and/orspecific developmental stages. CONCLUSIONS

In this study we report some structural characteristics of the 5'-end of the human a3(VI) gene and provide information on additional mRNA variation in both 5"untranslated and 5"translated regions. The primarysequence of an open readMr

I

2

3

4

the total specific signal whereas in the MG-63 osteosarcoma 205 cells the same band represents 19%. We extended our observations also to a few freshly isolated human tumortissues. In each case (Fig. 8 B ) , the upper band corresponding to protection of exon A9/N10 is much less intense than in the tumor cell lines. It is likely that the discrepancy seen with fresh 116tumor tissuesreflects the presenceof a mixture of tumor and normal cellswith different capacities to generate splicing. 97 This is consistent with recent observations by Stokes et al. (1991) who demonstrated by a sensitive S1 nuclease protecFIG. 9. Immunoblotting of recombinant fusion proteins. A tion analysis that splicing of A8/N9 exon was much less polyclonal antiserum against the fusion protein of @-galactosidase efficientinthreedifferenthumantumor cell lines when and A9/N10 module was reacted with 20 pg of bacterial lysate of A9/ compared to normal skinfibroblasts. N10 (lanes 1 and 3) or A6/N7 (lanes 2 and 4 ) before (lanes I and 2) Relevant to the discussion of the alternative splicing in or after (lanes 3 and 4 ) removal of 0-galactosidase reacting antibodies. 4

24088

Human Type V I Collagen (u3Variants Chain

A "

Chicken a3 (VI)

.

.. T

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AT

,

A6

A5

A4

A3

A2 "

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B Chicken

Human

a1 (VI)

a2 (VI)

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D A 3/N1

FIG. 11. Schematic model of t h e a m i n o - t e r m i n a l g l o b u l a r d o m a i n s of h u m a n a n d c h i c k e n t y pVI e collagen. A, diagram of the amino-terminal domainsof (r3(VI) collagen chains. The alterna-

FIG. 10. Antigenlocalization by indirectimmunofluoresc e n c e o n M G - 6 3 cells. A , fixed cells were reacted with: A, polyclonal antiserum to the pepsin-form of human type VI collagen; H , polyclonal antiserum against the recombinant fusion protein of B-galactosidase and A9/N10 module; C, preimmune serum.

tively expressed domains are shaded.C, cysteine; ?, potential glycosylation site. H , comparison between the human and chicken aminoterminalglobulardomains. A wavy lineindicatesthecollagenous sequences.In thehuman (rB(VI), dependingonthepresenceor absence of both or of either one of A9/N10 and A6/N7 alternatively spliced modules, there could be a free cysteine.

given molecule following a different splicing pattern. If both or neither A9/N10 nor A6/N7 exons are expressed then the number of cysteine residues at the amino-globular domain would be odd leaving 1cysteine free to make anothercovalent ing frame of 618 bp coding for a previously undetected type bond. At present the prevalent notionis that disulfide linked A repeat (A9/N10)is presented along with its splicing pattern. basic type VI collagen units that I t is already known that the overall modularstructure of dimers and tetramers are the are dissociable from microfilaments by strongdenaturing human and chicken (u3(VI) chain is similar and hence the present identification of the A9/N10 exon in a human ge- agents (Engel et al., 1985). Thissuggests that disulfide bondnomic clone extends the structural similarity of the a3(VI) ing is not one of the major mechanisms that participate in chain between these two species. This finding suggests that thestabilization of microfilaments formed by endtoend the functionof type VI collagen variants that express the A9/ association of tetramers. Nevertheless, the potential for covalent bonding between two tetramers or between a type VI N10 exon might be similar in the two species. Within this general conservation there arefew a differences that are worth tetramerandadifferentextracellularmatrixconstituent type VI noting. The amino-terminal end of the human (u3(VI) chain should be keptinmind.Forinstance,branched collagen microfilaments have been identified in different tishas several potential glycosylation sites that are absent (exsues by electron microscopy (Engvall et al., 1986; Keene et al., cept for one) in the chicken molecule(Fig. 11A) and that potentially could generate additional molecular heterogeneity 1988; Fleischmajer et al., 1991) and disulfide bonding might by attachment of branched oligosaccharides. Furthermore, be involved in this type of assembly. apart from the different positions of the cysteine residues in Acknou:ledgments-We thank Dr. A. Ruhlmann for providing us the two species, the human A9/N10 repeat has an additional with thePAX 4b vector. We are grateful to Antonella Moro for typing cysteine residue that brings the total number of predicted the manuscript. cysteines in thisregion of the human molecule to two instead REFERENCES of only one of the chicken molecule. This difference is more striking in view of the fact that the positioning of the other Ayad, S.,Marriott, A,, Morgan, K., and Grant, M. E. (1989) Biochem. J . 2 6 2 , X3-761 cysteine residues in the amino-globular domainof human and Barone, M. V., Henchcliffe, C., Baralle, F. E., and Paolella, G . (1989) EMRO J. 8, 1079-1085 chicken type VI collagen is similar (Fig. 11B). The fact that Biggin, M. D., Gibson, T. ,J., and Hong, G . F. (1983) Proc. Natl. Acad. Sci. in the human(u3(VI) chain both cysteineresidues are present L. S. A. 80,3963-3965 in alternatively spliced exons, while in the chicken (u3(VI) Bonaldo, P., and Colomhatti, A. (1989) J. Riol. Chem. 2 6 4 , 20235-20239 P., Kusso, V., Bucciotti, F., Bressan, G. M., and Colomhatti, A. (1989) chain the only cysteine is contained in the constitutive A7 Honaldo, J . H i d . Chem. 264,557.5-5580 exon, augments thepossibilities for structural/functional var- Bonaldo, P., Russo, V., Bucciotti, F., Doliana, R., and Colombatti, A. (1990) Biochemistry 2 9 , 1245-1254 iations of the human typeVI collagen molecules. For instance, Bruns, K., Press, W., Engvall, E., Timpl, R. and Gross, J. (1986) J. Cell Riol. both cysteines, either one, or none could be expressed in a 103,3393-404

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