title: Different usage of the glycosaminoglycan attachment sites of biglycan.
abstract: Biglycan is a member of the small leucine-rich proteoglycan family. Its core protein comprises two chondroitin/dermatan sulfate attachment sites on serine 42 and serine 47, respectively, which are the fifth and tenth amino acid 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 proteins were stably expressed in human 293 and Chinese hamster ovary cells in which i) either one or both serine residues were converted into alanine or threonine residues, ii) the number of acidic amino acids N-terminal of the respective serine residues was altered, and iii) a hexapeptide was inserted between the mutated site 1 and the unaltered site 2. Labeling experiments with [(35)S]sulfate and [(35)S]methionine indicated that serine 42 was almost fully used as the glycosaminoglycan attachment site regardless of whether 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 becomes manifest before the removal of the propeptide. It is shown additionally that biglycan expressed in 293 cells may still contain the propeptide sequence and may carry heparan sulfate chains as well as sulfated N-linked oligosaccharides.
authors.affiliation: Institute of Physiological Chemistry and Pathobiochemistry, University of Münster, D-48149 Münster, Germany. kresse@uni-muenster.de
authors.forename: H
authors.initials: H
authors.lastname: Kresse
authors.forename: D G
authors.initials: DG
authors.lastname: Seidler
authors.forename: M
authors.initials: M
authors.lastname: Muller
authors.forename: E
authors.initials: E
authors.lastname: Breuer
authors.forename: H
authors.initials: H
authors.lastname: Hausser
authors.forename: P J
authors.initials: PJ
authors.lastname: Roughley
authors.forename: E
authors.initials: E
authors.lastname: Schonherr
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D058578:Biglycan; D001665:Binding Sites; D016466:CHO Cells; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D016384:Consensus Sequence; D006224:Cricetinae; D016326:Extracellular Matrix Proteins; D006025:Glycosaminoglycans / Q000737:chemistry* / Q000378:metabolism*; D019763:Heparin Lyase / Q000378:metabolism; D006801:Humans; D007930:Leucine; D008715:Methionine / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010768:Phosphoserine; D011509:Proteoglycans / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D012694:Serine; D013431:Sulfates / Q000378:metabolism; D013462:Sulfur Radioisotopes; D014162:Transfection
chemical_list: C546884:BGN protein, human; C546885:Bgn protein, mouse; D058578:Biglycan; D016326:Extracellular Matrix Proteins; D006025:Glycosaminoglycans; D011509:Proteoglycans; D011994:Recombinant Proteins; D013431:Sulfates; D013462:Sulfur Radioisotopes; D010768:Phosphoserine; D012694:Serine; D008715:Methionine; D019763:Heparin Lyase; D007930:Leucine