Role of the pro-alpha2(I) COOH-terminal region in assembly of type I collagen: truncation of the last 10 amino acid residues of pro-alpha2(I) chain prevents assembly of type I collagen heterotrimer.
Procollagen (Type I) contains a noncollagenous COOH-terminal propeptide (C-propeptide) hypothesized to be important in directing chain association and alignment during assembly. We previously expressed human pro-alpha2(I) cDNA in rat liver epithelial cells, W8, that produce only pro-alpha1(I) trimer collagen (Lim et al. [1994] Matrix Biol. 14: 21-30). In the resulting cell lines, alpha2(I) assembled with alpha1(I) forming heterotrimers. Using this cell system, we investigated the importance of the COOH-terminal propeptide sequence of the pro-alpha2(I) chain for normal assembly of type I collagen. Full-length human pro-alpha2(I) cDNA was cloned into expression vectors with a premature stop signal eliminating the final 10 amino acids. No triple-helical molecules containing alpha2(I) were detected in transfected W8 cells, although pro-alpha2(I) mRNA was detected. Additional protein analysis demonstrated that these cells synthesize small amounts of truncated pro-alpha2(I) chains detected by immunoprecipitation with a pro-alpha2(I) antibody. In addition, since the human-rat collagen was less thermostable than normal intraspecies collagen, wild-type and C-terminal truncated mouse cDNAs were expressed in mouse D2 cells, which produced only type I trimers. Results from both systems were consistent, suggesting that the last 10 amino acid residues of the pro-alpha2(I) chain are important for formation of stable type I collagen.
Lim
A L
AL
Department of Biochemistry, Boston University School of Medicine, Massachusetts 02118, USA.
Doyle
S A
SA
Balian
G
G
Smith
B D
BD
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003094:Collagen / Q000737:chemistry / Q000378:metabolism*; D017931:DNA Primers; D018076:DNA, Complementary; D019281:Dimerization; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D051381:Rats
D017931:DNA Primers; D018076:DNA, Complementary; D011498:Protein Precursors; D003094:Collagen