title: ADAMTS-13 glycans and conformation-dependent activity.
abstract: Essentials The impact of N-linked glycosylation on ADAMTS-13 function has not been fully explored. The activity of glycan modified ADAMTS-13 was investigated under static and shear stress conditions. Terminal sialic acid on the metalloprotease domain glycans are important for ADAMTS-13 activity. The CUB domain glycans modulate ADAMTS-13 activity.
  
  
  
  Background ADAMTS-13 activity can be regulated by its conformation, whereby interactions between the C-terminal CUB domains and the spacer domain maintain ADAMTS-13 in a closed conformation. ADAMTS-13 contains 10 N-linked glycans, with four sites present in theTSP2 through to CUB domains that may contribute to its conformation. Objectives/Methods We hypothesized that glycosylation contributes to ADAMTS-13 conformation and function. The proteolytic activity of glycan-modified ADAMTS-13 was assessed under static and shear stress conditions. Results Enzymatic removal of terminal silaic acid or entire N-linked glycan chains decreased activity against FRETS-VWF73 at pH 7.4 and against full-length von Willebrand factor (VWF) under shear stress. Using truncated ADAMTS-13, we demonstrated that this was attributable to loss of sialic acid from the glycans in the metalloprotease domain and an effect of N-linked glycosylation in the TSP2 through to CUB domains. Mutation of the N-linked glycan sites in the MDTCS domains reduced or abolished protein expression. However, the N707Q, N828Q, N1235Q and N1354Q (TSP2, TSP4, CUB1, and CUB2 domains, respectively) variants were expressed normally. Interestingly, the N707Q and N828Q variants showed reduced activity against FRETS-VWF73, but normal activity under flow conditions. In contrast, the N1235Q and N1354Q variants had enhanced activity against FRETS-VWF73 and VWF under shear stress. Immunoprecipitation experiments confirmed that loss of N-linked glycans in the CUB domains significantly reduced the interaction with the spacer domain and enhanced binding to the 6A6 anti-ADAMTS-13 antibody, which recognizes a cryptic epitope in the metalloprotease domain. Conclusions Together, these data demonstrate that the N-linked glycans of ADAMTS-13 play a crucial role in regulating ADAMTS-13 activity.
authors.affiliation: Department of Haematology, Faculty of Medicine, Hammersmith Hospital Campus, Imperial College London, London, UK.
authors.forename: A A
authors.initials: AA
authors.lastname: Nowak
authors.affiliation: Department of Haematology, Faculty of Medicine, Hammersmith Hospital Campus, Imperial College London, London, UK.
authors.forename: H E R
authors.initials: HER
authors.lastname: O'Brien
authors.affiliation: Department of Haematology, Faculty of Medicine, Hammersmith Hospital Campus, Imperial College London, London, UK.
authors.forename: P
authors.initials: P
authors.lastname: Henne
authors.affiliation: Department of Haematology, Faculty of Medicine, Hammersmith Hospital Campus, Imperial College London, London, UK.
authors.forename: A
authors.initials: A
authors.lastname: Doerr
authors.affiliation: Laboratory for Thrombosis Research, Catholic University Leuven, Leuven, Belgium.
authors.forename: K
authors.initials: K
authors.lastname: Vanhoorelbeke
authors.affiliation: Department of Haematology, Faculty of Medicine, Hammersmith Hospital Campus, Imperial College London, London, UK.
authors.forename: M A
authors.initials: MA
authors.lastname: Laffan
authors.affiliation: Department of Haematology, Faculty of Medicine, Hammersmith Hospital Campus, Imperial College London, London, UK.
authors.forename: T A J
authors.initials: TAJ
authors.lastname: McKinnon
mesh_terms: D000071120:ADAMTS13 Protein / Q000737:chemistry*; D001665:Binding Sites; D000939:Epitopes / Q000737:chemistry; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D011134:Polysaccharides / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D000072417:Protein Domains; D011499:Protein Processing, Post-Translational; D059748:Proteolysis; D011994:Recombinant Proteins / Q000737:chemistry; D012794:Sialic Acids / Q000737:chemistry; D014841:von Willebrand Factor / Q000737:chemistry
chemical_list: D000939:Epitopes; D011134:Polysaccharides; D011994:Recombinant Proteins; D012794:Sialic Acids; D014841:von Willebrand Factor; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human
keywords: ADAMTS-13; glycosylation; proteolysis; thrombosis; von Willebrand factor