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ADAMTS-13 glycans and conformation-dependent activity.

A A Nowak1, H E R O'Brien1, P Henne1

  • 1Department of Haematology, Faculty of Medicine, Hammersmith Hospital Campus, Imperial College London, London, UK.

Journal of Thrombosis and Haemostasis : JTH
|April 4, 2017
PubMed
Summary
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N-linked glycosylation significantly impacts ADAMTS-13 activity. Terminal sialic acid on metalloprotease domain glycans and CUB domain glycans are crucial for regulating ADAMTS-13 function under various conditions.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Proteomics

Background:

  • ADAMTS-13 (A Disintegrin and Metalloproteinase with Thrombospondin type 1 repeats, member 13) activity is modulated by its conformation, influenced by interactions between C-terminal CUB domains and the spacer domain.
  • ADAMTS-13 possesses 10 N-linked glycosylation sites, with four located in the TSP2 to CUB domains, potentially affecting its conformation and function.

Purpose of the Study:

  • To investigate the impact of N-linked glycosylation on ADAMTS-13 conformation and proteolytic activity.
  • To determine the role of specific glycosylation sites and terminal sialic acid in ADAMTS-13 function under static and shear stress conditions.

Main Methods:

  • Assessed proteolytic activity of glycan-modified ADAMTS-13 against FRETS-VWF73 and full-length von Willebrand factor (VWF) under static and shear stress.
Keywords:
ADAMTS-13glycosylationproteolysisthrombosisvon Willebrand factor

Related Experiment Videos

  • Utilized enzymatic deglycosylation and site-directed mutagenesis (N-linked glycan site variants) to study the effects of glycosylation.
  • Employed immunoprecipitation to analyze protein interactions and antibody binding.

    • Main Results:

    • Enzymatic removal of terminal sialic acid or N-linked glycans reduced ADAMTS-13 activity under static and shear stress conditions.
    • N-linked glycans in the metalloprotease domain (via sialic acid) and CUB domains are critical for optimal ADAMTS-13 function.
    • Mutations in CUB domain glycans (N1235Q, N1354Q) enhanced activity under shear stress and reduced interaction with the spacer domain, while mutations in TSP2/TSP4 domains (N707Q, N828Q) reduced activity under static conditions.

    Conclusions:

    • N-linked glycosylation is a key regulator of ADAMTS-13 activity, influencing its conformation and substrate interaction.
    • Specific glycosylation sites, particularly those in the metalloprotease and CUB domains, play distinct roles in modulating ADAMTS-13 function.
    • Understanding these glycosylation impacts is crucial for comprehending ADAMTS-13's role in hemostasis and thrombosis.