Identification of the predominant glycosaminoglycan-attachment site in soluble recombinant human thrombomodulin: potential regulation of functionality by glycosyltransferase competition for serine474

Abstract

Thrombomodulin (TM) is an endothelial cell thrombin receptor that converts thrombin from a procoagulant to an anticoagulant enzyme. It has previously been shown that TM is expressed in both a high-M(r) form containing chondroitin sulphate and a low-M(r) form lacking this modification. Site-directed mutagenesis of a soluble human TM derivative (TMD1) was employed to determine the attachment site(s) of this functionally important oligosaccharide on the core protein. Although there are four serine residues within the Ser/Thr-rich domain of TMD1 that might support glycosaminoglycan assembly, our analysis demonstrates that the primary site of attachment is at Ser474, and evidence is presented for low levels of attachment at Ser472. It was possible to improve the overall degree of attachment by mutating Ser472 to glutamic acid (so as to conform Ser474 to the xylosyltransferase acceptor consensus acidic-Gly-Ser-Gly-acidic); however, a significant proportion (approx. 35%) of the total TM still lacked a glycosaminoglycan moiety. Mutants that possess a substitution for Ser474 show an increased mobility of their low-M(r) form on SDS/PAGE compared with native TMD1. Isolation and sequencing of a C-terminal peptide demonstrated that this serine is modified in the low-M(r) form of native TMD1. An apparent 'acceptor consensus overlap' at Ser474 suggests that the mechanism behind the glycosaminoglycan split of TM may involve a competition for substrate between xylosyltransferase and N-acetylgalactosaminyltransferase.

Novel Proteoglycan Linkage Tetrasaccharides of Human Urinary Soluble Thrombomodulin, SO4-3GlcAβ1–3Galβ1–3(±Siaα2–6)Galβ1–4Xyl

“…Hence, the binding of chondroitin sulfate seems to be important for several of the versatile functions of TM and to affect its cell-surface anticoagulant potential. Chondroitin sulfate is linked to Ser-474 in the high molecular weight TM of recombinant TM, but Ser-474 is also modified with a small substituent in the low molecular weight TM (9,10). By way of explanation of the fact that TM is expressed in two distinct forms, Gerlitz et al (9) postulated a model involving glycosyltransferase competition between xylosyltransferase and N-acetylgalactosaminyltransferase for Ser-474, whereas Lin et al (10) suggested that occupation of the adjacent O-linked glycosylation may be important because of the steric hindrance for xylosyltransferase of the cell line.…”

“…The protein possesses five potential N-linked glycosylation sites (7) as deduced from its amino acid sequence, whereas the detection of GalNAc suggests the presence of Olinked sugar chains (8). Although uTM does not contain a glycosaminoglycan, recombinant TM and some TMs obtained from cultured human endothelial cells are expressed in both a high molecular weight TM containing chondroitin sulfate and a low molecular weight TM lacking this modification (9,10). Recombinant TM expressed in Chinese hamster ovary cells contains chondroitin 4-sulfate (11).…”

Distinct Functions of Activated Protein C Differentially Attenuate Acute Kidney Injury

“…52 In brief, the APCs were serially diluted in buffer consisting of 150 mM NaCl, 20 mM Tris-HCl pH 7.40, and 2 mg/ml BSA. APTTs were then determined by preincubating 20 l of the APC dilution (or buffer alone, baseline control) with 50 l of citrated plasma and 50 l of APTT reagent (Helena Laboratories 5385) for 5 min at 37°C in a 96-well plate (CoStar 3596).…”

Different Usage of the Glycosaminoglycan Attachment Sites of Biglycan

“…The situation is additionally complicated by the fact that established attachment sites are not necessarily used regularly, giving rise to so-called part-time proteoglycans. This has been considered as a result of the possibility that the attachment site is not always recognized by xylosyltransferase and that there may be competition for substrate between xylosyltransferase and glycoprotein N-acetylgalactosaminyltransferase (8). Furthermore, in the glycosaminoglycan-free form of the part-time proteoglycan thrombomodulin, the respective serine residue is substituted with the linkage region tetrasaccharide GlcA␤1-3Gal␤1-3Gal-␤1-4Xyl (9), raising the possibility that it is the transfer of the fifth monosaccharide (either GalNAc or GlcNAc) that represents the most critical step in glycosaminoglycan chain biosynthesis.…”