Honing and carboxyl-propeptide analysis of human type III procollagen

Two human cDNA l i b r a r i e s prepared from normal f i b r o b l a s t (GM3348) and rhabdomyosarcoma (CCL136) mRNAs were screened under cross h y b r i d i z a t i o n cond i t i ons w i th a genomic fragment coding f o r exons 2 and 3 o f the avian type I I I procol lagen C00H-propept1de (Yamada, Y . , Mudryj , M., S u l l i v a n , M. and deCrombrugghe, B. (1983) J . B1o l . Chem. 258, 2758-2761). One cDNA clone con ta in ing a 1.12 kb I n s e r t was I s o l a t e d from the CCL136 l i b r a r y and l a t e r used to I d e n t i f y a GM3348 der ived clone w i t h a 2.4 kb I n s e r t . Comparison o f the human and avian type I I I C-term1nal propept ides revealed much more divergence 1n the f i r s t 54 amino a d d s f o l l ow ing the terminal cys te ine o f the t r i p l e h e l i c a l region than 1s present 1n the a l ( i ) and a 2 ( I ) proco l lagen chains of these spec ies. Ana lys is of poly (A) RNA from normal human f i b r o b l a s t and tumor c e l l l i n e s showed tha t they d i f f e r e d g r e a t l y 1n the r e l a t i v e amounts of a l ( l ) , a 2 ( l ) , and a l ( l l l ) mRNAs. Furthermore, as we p rev ious ly repor ted f o r the a l ( i ) and a 2(1) t ranscr ip ts , mul t ip le mRNAs also hybridize to the cloned a l ( I I I ) DNA. INTRODUCTION Five major and at least f i ve minor types of collagen comprise the most abundant protein family found 1n vertebrates (reviews 1,2; and ref. 3-7). In general, collagen 1s synthesized in the form of procollagen molecules composed of three polypeptide chains consist ing of a central t r i p l e hel ical domain with the repeating Gly-X-Y sequence, and shorter carboxy and amino propeptides. The d i f fe rent chains are the products of probably more than f i f teen genes which have recently begun to be Isolated from several species (reviews 8,9; and ref . 10-17). Determination of the DNA sequences of the genes and thei r encoded amino add sequences are fundamental to understanding the function of the various types of collagen and the factors cont ro l l ing the i r synthesis. Previously, human cDNA clones coding for the al and a2 chains of the type I procollagen heterotrimer (pro al >2 (pro a2) were Ident i f ied (18,19) and used to Isolate overlapping genomic fragments (20,21). Analysis of these two genes, which are located on d i f fe ren t chromosomes (22), showed that the coding region 1s frequently Interrupted © IRL Press Limited, Oxford, England. 9383 Nucleic Acids Research by about 50 sometimes very la rge Int rons (20,21) . Although the mRNA t ransc r i p t s of these genes are normally found at a two to one r a t i o r e f l e c t i n g the type I collagen subunit composit ion, the leve ls can vary 1n disease states (23) . To study the coordinate and d i f f e r e n t i a l expression of several procol l agens, we constructed two cDNA l i b r a r i e s contain ing sequences corresponding to d i f f e r e n t types of procollagen 1n add i t ion to type I . Here we report the I s o l a t i o n and charac te r i za t ion of cDNA clones coding for about ha l f of the human a l ( m ) procollagen cha in . From the DNA sequence of these c lones, part of the t r i p l e he l i ca l and 3' untranslated region and the en t i re C-term1nal propeptide was der ived. While the te lopept lde and adjacent 5 par t of the C00H-propept1de of avian (24,25) and human type I I I are more divergent than seen 1n the ccl(I) and a2 ( I ) chains of these species (26-28) , the major par t of the C00H-propept1de 1s conserved. Comparison of t h i s domain 1n three human procollagen chains also shows s i g n i f i c a n t homology In the regions where the cysteine residues and g lycos lya t ion attachment s i te are loca ted . Together with e luc ida t ing the s t i l l undetermined ro le o f type I I I , these probes w i l l be valuable 1n def in ing mutations and regulatory abnormal i t ies 1n genetic and acquired diseases of connective t i s sue . MATERIALS AND METHODS Enzymes and Isotopes Res t r i c t i on endonucleases were purchased from New England Bio labs, DNA polymerase I and proteinase K from Boehringer Mannheim, T4 DNA Ugase and rabb i t r e t i cu locy te lysate from Bethesda Research Laborator ies. A l l enzymes were used according to manufacturers suggestions. Formamide was obtained from Fluka, o l i go dT ce l l u l ose and primers for DNA sequencing from Col laborat ive Research, and n i t r o c e l l u l o s e f i l t e r s from Schleicher S Schuei l . Labeled Isotopes were purchased from Amersham. Cell l i n e 013348 was obtained from the Human Genetic Mutant Cell Repository, Camden, New Jersey, and CCL136 and HT1080 from the American Type Culture C o l l e c t i o n , Rockv i l le , Maryland. RNA P u r i f i c a t i o n and cONA Cloning Poly (A) RNA was Iso lated by o l i go dT ce l lu lose chromatography and size f rac t iona ted on a sucrose-SDS gradient (19). F i r s t strand DNA synthesis was performed 1n a 150 ul react ion contain ing 5ug of mRNA, 0.7ug o l i go dT 12-18 primer, 1.30mH dGTP, . 53CTM TTP, . 32 mM dCTP, . 27mM dATP and 55 uni ts /ml of AMV reverse t ranscr ip tase p u r i f i e d by DEAE and CM sepharose