Analysis of glycosaminoglycan substitution in decorin by site-directed mutagenesis.
Posttranslational glycosaminoglycan attachment to decorin, a chondroitin/dermatan sulfate proteoglycan, was studied by expression of a wild-type decorin cDNA and several mutagenized forms in two types of mammalian cells. Transfection of the wild-type cDNA resulted in the synthesis of an authentic chondroitin/dermatan sulfate proteoglycan similar to the decorin molecule synthesized by cultured human fibroblasts. Conversion of the serine residue that serves as the attachment site for the sole glycosaminoglycan chain in decorin to a threonine residue greatly reduced the efficiency of the glycosaminoglycan substitution. Less than 10% of the threonine-mutated core protein acquired a glycosaminoglycan chain, whereas most of the core protein was secreted without such substitution. Expression of cDNA in which an alanine residue had been introduced into the substituted serine position resulted in the secretion of core protein with no detectable glycosaminoglycan. Conversion to alanine of either one of the glycine residues that are adjacent to the substituted serine yielded the proteoglycan form of decorin. These results show that the xylosyltransferase responsible for the initiation of the glycosaminoglycan chain on the core protein can use a threonine residue for this substitution instead of a serine residue, but that such substitution is only partial, creating a "part-time" proteoglycan. Moreover, variations are possible in the sequence context of a glycosaminoglycan-substituted serine residue without loss of glycosaminoglycan substitution. The conformation of the substitution site may therefore be important for xylosyltransferase recognition.
Mann
D M
DM
La Jolla Cancer Research Foundation, California 92037.
Yamaguchi
Y
Y
Bourdon
M A
MA
Ruoslahti
E
E
D000818:Animals; D002460:Cell Line; D002852:Chromatography, Ion Exchange; D004247:DNA / Q000235:genetics*; D058575:Decorin; D016326:Extracellular Matrix Proteins; D006025:Glycosaminoglycans / Q000235:genetics*; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D015345:Oligonucleotide Probes; D010957:Plasmids; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D011509:Proteoglycans / Q000235:genetics* / Q000302:isolation & purification; D012984:Software; D013912:Threonine; D014162:Transfection
C546878:DCN protein, human; D058575:Decorin; D016326:Extracellular Matrix Proteins; D006025:Glycosaminoglycans; D015345:Oligonucleotide Probes; D011509:Proteoglycans; D013912:Threonine; D004247:DNA