Ž .Biochimica et Biophysica Acta 1354 1997 183–188

Short sequence-paper

ž / 1Complete primary structure of human collagen type XIV Undulin

Michael Bauer, Walburga Dieterich, Tobias Ehnis, Detlef Schuppan )

Free UniÕersity of Berlin, Klinikum Benjamin Franklin, Department of Gastroenterology, Hindenburgdamm 30, D-12200 Berlin,Germany

Received 29 May 1997; accepted 14 July 1997

Abstract

A partial cDNA sequence coding for the human extracellular matrix protein undulin has been completed. The completedsequence provides conclusive evidence for the suggested identity of undulin and collagen type XIV. Two differently sizedpolyproteins of 1780 and 1796 amino acids, with an overall amino acid sequence identity of 75% compared to chicken

X Ž .CXIV, emerge from variant 3 sequence ends encoding the C-terminal non-collagenous NC NC1 domain of humancollagen type XIV. q 1997 Elsevier Science B.V.

Keywords: Undulin; Collagen type XIV; Extracellular matrix; FACIT; von Willebrand factor; Fibronectin

Ž .Collagen type XIV CXIV , a member of theŽFACIT fibril associated collagens with interrupted

. w xtriple helices family of collagens 1,2 , as well as therelated matrix protein undulin, is found in associationwith collagen fibrils in differentiated mesenchymal

w xtissues 3–5 . The deduced protein sequence of cDNAclones covering approximately 40% of human un-dulin showed a modular arrangement of domains

Ž .with homology to fibronectin type III FN III repeatsŽ . w xor von Willebrand factor A vWF A domains 6 .

Comparison of the partial amino acid sequence of

Abbreviations: RACE, rapid amplification of cDNA ends;PCR, polymerase chain reaction; bp, base pairs; FACIT, fibrilassociated collagens with interrupted triple helices; CXIV, colla-gen type XIV

) Corresponding author. Fax: q49 30 8445 4017; E-mail:schuppan@is.in-berlin.de

1 DNA-sequences reported in this paper have been submitted tothe EMBLrGenBank Data Libraries with accession numbersY11709, Y11710 and Y11711.

w xundulin with that of chicken CXIV 7,8 , and with theamino acid sequence of proteolytic human CXIV

w xfragments 9 , strongly suggested that undulin andCXIV are identical proteins. Chicken CXIV is com-

Ž .posed of a large N-terminal non-collagenous NCdomain NC3, followed by two short collagenoussegments, COL2 and COL1, that are separated by ashort NC2 domain, and the NC1 domain at the

w xC-terminus 8,10 . CXIV may modulate the supra-molecular organization of collagen fibrils, similar tothe function assigned to the FACIT collagen type IX,

w xwhich is found in cartilage 2 . Recently, a proteogly-can variant of CD44 was identified as a cellular

w xreceptor for human CXIVrundulin 11 , suggesting arole of CXIVrundulin in cell adhesion and possiblysignal transduction. In order to clarify the relationshipof CXIV and undulin we completed the cDNA anddeduced protein sequence of human undulin.

X Žw x5 RACE-PCR 12,13 Marathon Kit Manual,.Clontech was used to extend the partial human

X Ž .undulin sequence at the 5 end Fig. 1 . The first

0167-4781r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved.Ž .PII S0167-4781 97 00131-0

( )M. Bauer et al.rBiochimica et Biophysica Acta 1354 1997 183–188184

Fig. 1. Overlapping cDNA clones completing the humanCXIVrundulin sequence were obtained by repeated library

Ž X . Ž X Xscreening l3 1–3 and RACE-PCR 5 RACE a–c, 3 RACE.d–f . Alternative poly-adenylation signals created two different

X Ž .sized 3 RACE-PCR products d,e encoding an identical NC1Ž . Xdomain. f shows a third 3 RACE-PCR product encoding a

Žvariant NC1 domain black colour represents cDNA sequenceŽ . Ž ..different from d and e . UN1 is a previously described partialw xundulin clone 6 .

Ž .ATG codon Fig. 2, nucleotides 244–246 is immedi-Ž .ately followed by a stop codon nucleotides 247–249 .

Therefore, translation is expected to start at the sec-Ž .ond ATG codon nucleotides 265–267 , which is in a

favorable sequence context for translation initiationw x14 . A putative signal peptide of 28 amino acids witha characteristically high content of hydrophobicresidues is encoded by the following nucleotide se-

Ž .quence nucleotides 265–348 . Similar to one re-w xported chicken CXIV sequence 8 the mature human

ŽCXIVrundulin starts with a FN III repeat nucleo-.tides 349–618 . Interestingly, the chicken CXIV se-

w xquence of a second report 10 lacks this N-terminalFN III domain. Studies with recombinant fragmentsof human CXIVrundulin showed that the N-terminalFN III repeat mediates binding of CXIVrundulin todecorin, a small chrondroitinrdermatan sulfate pro-

w x Žteoglycan 22 . A linker sequence nucleotides 619–.708 connects the N-terminal FN III repeat to a vWF

Ž .A domain nucleotides 709–1245 and to a cluster ofseven FN III repeats already covered by the previ-

w xously described undulin clones UN1 and UN2 6 . Inw xcontrast to chicken CXIV 10 no evidence for alter-

native splicing at the 5X end was found for humanCXIVrundulin.

Ž .Screening of a placental cDNA library StratageneŽ .recovered overlapping phage clones Fig. 1 that

further extended the partial human undulin sequence

X X Ž .Fig. 2. 5 RACE-PCR was used to complete the human CXIVrundulin sequence at the 5 end. Placental or RD rhabdomyosarcoma cellmRNA was reverse transcribed using a gene specific primer located ) 500 bp downstream of the 5X ends of the previously characterized

Ž . Ž .partial undulin clones UN1 v and UN2 ) . Subcloned RACE-PCR products were sequenced using the dideoxy chain terminationŽ .method TaqTrack or Silver Sequence, Promega . Amino acids of the N-terminal FN III repeat are in italics. A putative heparin binding

w x X Ž . Žsite is boxed 20 . Sequences corresponding to gene specific primers used in 5 RACE- underlined or long-distance PCR thick.underlined are indicated.

( )M. Bauer et al.rBiochimica et Biophysica Acta 1354 1997 183–188 185

X Ž .at the 3 end. Conceptional translation Fig. 3 re-vealed a second vWF A domain C-terminal to thecluster of FN III repeats, and a domain with homol-ogy to the globular N-terminal NC4 domain of the a1

chain of collagen type IX. C-terminal to the thuscompleted large N-terminal NC3 domain, additionaldomains with high homology to the COL2, NC2, and

Ž .COL1 domains of chicken CXIV Table 1 werefound. Only the C-terminal NC1 domain displayed areduced homology between human and chicken. Thecompleted cDNA sequence for human undulin pro-vides conclusive evidence for the identity of humanCXIV and undulin. To verify our 3X sequence we

X Žw xperformed 3 RACE-PCR 13 , and Marathon Kit.Manual, Clontech . One of the resulting differently

sized 3XRACE-PCR products confirmed the 3X endŽ Ž ..Fig. 3 A identified in phage screening. A secondproduct was apparently generated by usage of an

Ž Ž .alternative poly-adenylation signal Fig. 3 A , nu-.cleotides 5928–5933 . Surprisingly a third product

Ž Ž ..encoded a variant NC1 domain Fig. 3 B , whichŽ .shared only the first 11 amino acids boxed in Fig. 3

with the prior NC1 domain. The two divergent 3X

sequences were confirmed by Northern analysis with3X end specific probes. All probes hybridized tomRNA’s of about 6.5 kb in line with previous resultsw x w x6–8,10 . In addition, long-distance PCR 15 with

X Ž .the 5 primer Fig. 2, nucleotides 265–291 located atthe translation start site and 3X variant specific primersŽ Ž .Fig. 3 A , nucleotides 6001–6022, respectively,

Ž ..5751–5772 in Fig. 3 B , spanning the completecoding region of human CXIV, produced the ex-

Ž .pected PCR products not shown .Analogous to human CXIV there are also two NC1

w xvariants of chicken CXIV 8 . Alignment of the corre-sponding human and chicken NC1 domains demon-

Žstrates that the human domains are much shorter Fig..4 . Seven amino acids C-terminal to the conserved

CXXXXC sequence motif at the COL1–NC1 junc-tion, which is thought to be characteristic for FACIT

Table 1Similarity of corresponding human CXIVrundulin and chicken CXIV domains deduced from the cDNA sequences. For further details

Ž .refer to the text. Note the lower percentage of identical amino acids with chicken CXIV identity in the putative signal peptide, linkers1–3 of the NC3 domain, and the C-terminal NC1 domains

Ž . Ž .Domain Length residues Position Identity with chicken CXIV % Length differences

Put. Signal 28 1– 28 21FN type III-1 90 29– 118 82Linker-1 29 119– 147 39vWF A-1 189 148– 336 83Linker-2 15 337– 351 53FN type III-2 90 352– 441 77FN type III-3 92 442– 533 80FN type III-4 89 534– 622 67FN type III-5 90 623– 712 61Linker-3 20 713– 732 20 Human 7 residues shorterFN type III-6 95 733– 827 81 Human 3 residues longerFN type III-7 91 828– 918 73FN type III-8 88 919–1006 69Linker-4 15 1007–1021 73 Human 6 residues shortervWF A-2 190 1022–1211 87

Ž .NC4 CIX like 250 1212–1461 81 Human 3 residues longerŽ .NC3 complete 1433 29–1461 76 Human 7 residues shorter

COL2 149 1462–1610 81 Human 3 residues shorterNC2 43 1611–1653 79COL1 106 1654–1759 78NC1 short variant 21 1760–1780 52 Human 67 residues shorterNC1 long variant 37 1760–1796 62 Human 82 residues shorter

( )M. Bauer et al.rBiochimica et Biophysica Acta 1354 1997 183–188186

( )M. Bauer et al.rBiochimica et Biophysica Acta 1354 1997 183–188 187

Fig. 4. Comparison of the N-terminal sequences of bovine, human, and chicken CXIV. Identical amino acids of the human sequences areŽ . w xin bold letters. A : Alignment of the short NC1 variants and a partial bovine NC1 sequence 21 . The CXXXXC motif at the COL1–NC1

Ž .junction, thought to be characteristic for the FACIT collagen family, is well conserved. B : Alignment of the larger NC1 variants. Aminoacids of the larger chicken NC1 not included in the smaller chicken variant are in italics.

collagens, the NC1 sequences become dissimilar.However, following a gap both human NC1 variantscontain amino acid stretches with homology to thecorresponding chicken NC1 domains. These se-quences which have been conserved between humanand chicken may be required for yet unknown func-tions of the NC1 domain. Also, between chickencollagen types XII and XIV, the C-terminal NC1domain displayed the highest sequence dissimilarity.Thus, it was speculated that the NC1 domain mayplay a role in the selection of the correct type ofFACIT collagen for association with a given collagen

w xfibril 8 . Furthermore, the NC1 domain may assist inthe assembly of appropriate chains during triple-helix

w xformation 16–18 . The two variant NC1 domainsobserved in CXIV might differently affect these pro-cesses. In addition to the suspected functional role ofthe variant NC1 domains, the 3X ends might contain

elements that are important for the regulation ofCXIV gene expression. The optional usage of analternative poly-adenylation signal within one 3X se-quence variant results in a mRNA with a shortened 3X

Ž X . Xuntranslated region 3 UTR . Variations in the 3 UTRhave been shown to modulate mRNA stability and

w xthus to modify gene expression 19 . Switching be-tween alternative poly-adenylation sites could be oneregulatory tool of cells to achieve and maintain atissue- and development-specific CXIV gene expres-sion. However, till today there is no experimentalproof for this in case of CXIV.

Supported by Grants Schu 646r1–4 and SFB 366C5 from the Deutsche Forschungsgemeinschaft. D.Schuppan is recipient of a Hermann-and-Lilly-Schill-ing-professorship. This publication contains parts ofthe Ph.D. thesis of M. Bauer. We thank Dr. H.-D.Orzechowski for critically reading the manuscript.

Fig. 3. The alternative 3X sequences of human CXIVrundulin. Amino acid residues of collagen triple helical sequences are in italics.Identical residues of the variant C-terminal NC1 domains are boxed and poly-adenylation signals are in lower case letters. Sequences

X Ž . Ž . Ž . Xcorresponding to gene specific primers used in 3 RACE- underlined or long-distance PCR thick underlined are indicated. A : The 3Ž .sequence of the shorter CXIVrundulin variant. v marks the first nucleotide not included in the previously described undulin clone

w x Ž . Ž . XUN1 6 , ) the alternative polyadenylation site. B : 3 cDNA sequence of the long C-terminal NC1 variant.

( )M. Bauer et al.rBiochimica et Biophysica Acta 1354 1997 183–188188

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