The type I collagen pro alpha 1(I) COOH-terminal propeptide N-linked oligosaccharide. Functional analysis by site-directed mutagenesis.
The C-propeptides of the pro alpha 1(I) and pro alpha 2(I) chains of type I collagen are each substituted with a single high-mannose N-linked oligosaccharide. Conservation of this motif among the fibrillar collagens has led to the proposal that the oligosaccharide has structural or functional importance, but a role in collagen biosynthesis has not been unambiguously defined. To examine directly the function of the pro alpha 1(I) C-propeptide N-linked oligosaccharide, the acceptor Asn residue was changed to Gln by site-directed mutagenesis. In transfected mouse Mov13 and 3T6 cells, unglycosylated mutant pro alpha 1(I) folded and assembled normally into trimeric molecules with pro alpha 2(I). In biosynthetic pulse-chase experiments mutant pro alpha 1(I) were secreted at the same rate as wild-type chains; however, following secretion, the chains were partitioned differently between the cell layer and medium, with a greater proportion of the mutant pro alpha 1(I) being released into the medium. This distribution difference was not eliminated by the inclusion of yeast mannan indicating that the high-mannose oligosaccharide itself was not binding to the matrix or the fibroblast surface after secretion. Subtle alterations in the tertiary structure of unglycosylated C-propeptides may have decreased their affinity for a cell-surface component. Further support for a small conformational change in the mutant C-propeptides came from experiments suggesting that unglycosylated pro alpha 1(I) chains were cleaved in vitro by the purified C-proteinase slightly less efficiently than wild-type chains. Mutant and normal pro alpha 1(I) were deposited with equal efficiency into the 3T6 cell accumulated matrix, thus the reduced cleavage by C-proteinase and altered distribution in the short pulse-chase experiments were not functionally significant in this in vitro extracellular matrix model system.
Lamandé
S R
SR
Department of Paediatrics, University of Melbourne, Royal Children's Hospital, Parkville, Victoria, Australia.
Bateman
J F
JF
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D055395:Bone Morphogenetic Protein 1; D019485:Bone Morphogenetic Proteins; D002240:Carbohydrate Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D008666:Metalloendopeptidases; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000378:metabolism*; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D011347:Procollagen / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational
D019485:Bone Morphogenetic Proteins; D009844:Oligosaccharides; D010455:Peptides; D011347:Procollagen; D010450:Endopeptidases; D008666:Metalloendopeptidases; C525707:Bmp1 protein, mouse; D055395:Bone Morphogenetic Protein 1