N-Glycans of ADAMTS13 modulate its secretion and von Willebrand factor cleaving activity (original) (raw)

Severe deficiency of ADAMTS13, a plasma metalloprotease, leads to thrombotic thrombocytopenic purpura. ADAMTS13 contains 10 putative N-glycosylation sites in or near its metalloprotease sequence, spacer region, thrombospondin type 1 repeat no. 4 (TSR no. 4), and CUB domains. Tunicamycin treatment markedly decreased the secretion of ADAMTS13 into the culture medium of transfected cells. Nevertheless, the protease was efficiently secreted from N-acetylglucosaminyltransferase I–deficient Lec1 Chinese hamster ovary cells, indicating that N-glycosylation in the endoplasmic reticulum, but not the conversion of oligomannose to complex N-glycans in the Golgi complex, is important for secretion. However, ADAMTS13 with oligomannose N-glycans cleaved its substrate, von Willebrand factor (VWF) multimers, less effectively, with a higher Km but similar kcat value. In mutagenesis analysis, decreased secretion and VWF cleaving activity was observed with the N146Q and N828Q mutants, while decreased ...