Title : 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
Output (sent_index, trigger,
protein,
sugar,
site):
- 0. site, , thrombomodulin, site, -
Output(Part-Of) (sent_index,
protein,
site):
*Output_Site_Fusion* (sent_index,
protein,
sugar,
site):