N-Glycans of ADAMTS13 modulate its secretion and von Willebrand factor cleaving activity.
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 K(m) but similar k(cat) value. In mutagenesis analysis, decreased secretion and VWF cleaving activity was observed with the N146Q and N828Q mutants, while decreased secretion only was observed with the N552Q mutant of ADAMTS13. Enzymatic removal of N-glycans from ADAMTS13 did not affect its VWF cleaving activity. Thus, N-glycosylation is necessary for efficient secretion of ADAMTS13, while conversion of the N-glycans from oligomannose to complex type in the Golgi complex enhances the proteolytic activity of the protease toward VWF multimers. After its secretion, ADAMTS13 does not require N-glycans for its VWF cleaving activity.
Zhou
Wenhua
W
Section of Hemostasis and Thrombosis, Division of Hematology/Oncology, Pennsylvania State University College of Medicine-Milton S. Hershey Medical Center, Hershey, PA 17033, USA.
Tsai
Han-Mou
HM
D051722:ADAM Proteins / Q000235:genetics / Q000378:metabolism*; D000071120:ADAMTS13 Protein; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D006031:Glycosylation; D006801:Humans; D008358:Mannose / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D011134:Polysaccharides / Q000378:metabolism*; D014841:von Willebrand Factor / Q000378:metabolism*
D011134:Polysaccharides; D014841:von Willebrand Factor; D051722:ADAM Proteins; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human; D008358:Mannose