Deletion of the pro-alpha 1(I) N-propeptide affects secretion of type I collagen in Chinese hamster lung cells but not in Mov-13 mouse cells.
We have introduced two mutations into a full-length human pro-alpha 1(I) cDNA that delete 114 amino acids or the entire 139 amino acids of the N-propeptide domain. Wild-type and mutated versions of the cDNA were introduced into cultured Chinese hamster lung (CHL) cells, which do not produce endogenous type I collagen, and into Mov-13 mouse cells, which produce endogenous pro-alpha 2(I) chains but not pro-alpha 1(I) chains. As judged by resistance to proteases, neither mutation impaired intracellular triple helical assembly of human alpha 1(I) homotrimers in CHL cells, or of chimeric type I collagen comprised of human alpha 1(I) and mouse alpha 2(I) chains in Mov-13 cells. Thus, the N-propeptide is not necessary for intracellular assembly of the main helical collagen domain of type I collagen. In CHL cells the rate of secretion of the mutant homotrimers was greatly reduced as compared to wild type homotrimers, and by immunofluorescence and immunoelectron microscopy, the mutant chains were shown to be accumulated in large vesicular expansions of the rough endoplasmic reticulum. When such cells were retransfected with cDNA encoding wild-type human alpha 2(I) chains, mutant alpha 1(I) chains were not rescued and heterotrimers containing the mutant chains were also retained in the intracellular vesicles. By contrast, deletion of the N-propeptide did not affect secretion of heterotrimers containing mutant chains from Mov-13 cells. Thus, an intact N-propeptide appears necessary for efficient secretion of type I collagen from some but not all cell types.
Lee
S T
ST
Department of Pathology and Laboratory Medicine, University of Wisconsin, Madison 53706.
Lee
S
S
Peters
D P
DP
Hoffman
G G
GG
Stacey
A
A
Greenspan
D S
DS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003094:Collagen / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics*; D000090602:Collagen Type I, alpha 1 Chain; D006224:Cricetinae; D003412:Cricetulus; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D006801:Humans; D008168:Lung; D046911:Macromolecular Substances; D051379:Mice; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009838:Oligodeoxyribonucleotides; D011347:Procollagen / Q000235:genetics*; D015183:Restriction Mapping; D017384:Sequence Deletion; D014162:Transfection
C000716128:Col1a1 protein, mouse; D000090602:Collagen Type I, alpha 1 Chain; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011347:Procollagen; D003094:Collagen