O-Fucosylation Is Required for ADAMTS13 Secretion

Lindsay M. Ricketts, Malgosia Dlugosz, Kelvin B. Luther, Robert S. Haltiwanger, Elaine M. Majerus
2007 Journal of Biological Chemistry  
ADAMTS13 is a plasma metalloproteinase that cleaves von Willebrand factor to smaller, less thrombogenic forms. Deficiency of ADAMTS13 activity in plasma leads to thrombotic thrombocytopenic purpura. ADAMTS13 contains eight thrombospondin type 1 repeats (TSR), seven of which contain a consensus sequence for the direct addition of fucose to the hydroxyl group of serine or threonine. Mass spectral analysis of tryptic peptides derived from human ADAMTS13 indicate that at least six of the TSRs are
more » ... x of the TSRs are modified with an O-fucose disaccharide. Analysis of [ 3 H]fucose metabolically incorporated into ADAMTS13 demonstrated that the disaccharide has the structure glucose-␤1,3-fucose. Mutation of the modified serine to alanine in TSR2, TSR5, TSR7, and TSR8 reduced the secretion of ADAMTS13. Mutation of more than one site dramatically reduced secretion regardless of the sites mutated. When the expression of protein O-fucosyltransferase 2 (POFUT2), the enzyme that transfers fucose to serines in TSRs, was reduced using siRNA, the secretion of ADAMTS13 decreased. A similar outcome was observed when ADAMTS13 was expressed in a cell line unable to synthesize the donor for fucose addition, GDPfucose. Although overexpression of POFUT2 did not affect the secretion of wild-type ADAMTS13, it did increase the secretion of the ADAMTS13 TSR1,2 double mutant but not that of ADAMTS13 TSR1-8 mutant. Together these findings indicate that O-fucosylation is functionally significant for secretion of ADAMTS13. ADAMTS13 is a plasma metalloprotease that cleaves von Willebrand factor to smaller, less thrombogenic fragments. ADAMTS13 is a member of the ADAMTS family of metalloproteases that are characterized by a conserved domain structure. These include a metalloprotease domain, a disintegrin domain, a thrombospondin type 1 repeat (TSR), 2 a cysteine-. 2 The abbreviations used are: TSR, thrombospondin type I repeat; POFUT2, protein O-fucosyltransferase 2; POFUT1, protein O-fucosyltransferase 1; ER, endoplasmic reticulum; EGF, epidermal growth factor; CHO, Chinese hamster ovary; Hex, hexose, dHex, deoxyhexose; VWF, von Willebrand factor; siRNA, small interfering RNA; HPLC, high pressure liquid chromatography; dT, deoxy thymidine; MS, mass spectrometry; MS/MS, tandem mass spectrometry; LC-MS/MS, laser chromatography-MS/MS.
doi:10.1074/jbc.m700317200 pmid:17395589 fatcat:em73nzzbv5hjtfr4egazato2fi