Different usage of the glycosaminoglycan attachment sites of biglycan

Biglycan is a member of the small leucine-rich proteoglycan family. Its cor e protein comprises two chondroitin/dermatan sulfate attachment sites on se rine 42 and serine 47, respectively, which are the fifth and tenth amino ad d residues, respectively, after removal of the prepro peptide. Because the regulation of glycosaminoglycan chain assembly is not fully understood and because of the in vivo existence of monoglycanated biglycan, mutant core pr oteins were stably expressed in human 293 and Chinese hamster ovary cells i n which i) either one or both serine residues were converted into alanine o r threonine residues, ii) the number of acidic amino acids N-terminal of th e respective serine residues was altered, and iii) a hexapeptide was insert ed between the mutated site 1 and the unaltered site 2, Labeling experiment s with [S-35]sulfate and [S-35]methionine indicated that serine 42 was almo st fully used as the glycosaminoglycan attachment site regardless of whethe r site 2 was available or not for chain assembly. In contrast, substitution of site 2 was greatly influenced by the presence or absence of serine 42, although additional mutations demonstrated a direct influence of the amino acid sequence between the two sites. When site 2 was not substituted with a glycosaminoglycan chain, there was also no assembly of the linkage region. These results indicate that xylosyltransferase is the rate-limiting enzyme in glycosaminoglycan chain assembly and implicate a cooperative effect on the xylosyl transfer to site 2 by xylosylation of site 1, which probably be comes manifest before the removal of the propeptide. It is shown additional ly that biglycan expressed in 293 cells may still contain the propeptide se quence and may carry heparan sulfate chains as well as sulfated N-linked ol igosaccharides.