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.

Biglycan, a Danger Signal That Activates the NLRP3 Inflammasome via Toll-like and P2X Receptors

“…All animal work was done in accordance with the German Animal Protection Law and was approved by the Ethics Review Committee for laboratory animals of the District Government of Muenster and Darmstadt, Germany. Purification of Human Biglycan-Expression of human biglycan in 293 HEK cells has been described previously (31). For purification of the native proteoglycan, containing two chondroitin/dermatan sulfate chains (31), the conditioned medium was supplemented with proteinase inhibitors (0.1 M ⑀-aminon-caproic acid, 10 mM EDTA, 5 mM benzamidine, 10 mM N-ethylmaleimide, 1 mM phenylmethylsulfonyl fluoride) and passed over a DEAE-Trisacryl-M (Pall) column, followed by elution with 20 mM Tris/HCl, pH 7.4, containing 1 M NaCl.…”

Characterization of ADAMTS-9 and ADAMTS-20 as a Distinct ADAMTS Subfamily Related to Caenorhabditis elegans GON-1

“…Ͼ185,000). Although there are a number of SerGly or Gly-Ser motifs in both ADAMTS-9 and ADAMTS-20, most are within presumed disulfide-bonded domains and lack the expected sequence context for xylosyltransferase recognition (25,26). However, one motif in the middle of the AD-AMTS-9 spacer domain with the sequence Glu-Tyr-Ser 830 -GlySer 832 -Glu-Thr-Ala-Val-Glu lies within a sequence context that is compatible with GAG attachment to Ser 830 or Ser 832 .…”

Deficiency of Biglycan Causes Cardiac Fibroblasts to Differentiate into a Myofibroblast Phenotype

“…Purified Biglycan-To reconstitute the bgn content, human biglycan was purified as described previously (35)(36)(37) and added to collagen type 1 (PureCol Collagen, Nutacon) at 0.5 g/ml prior to coating of cell culture plates. Subsequently, cell proliferation was determined by cell counting.…”