O-Fucosylation Is Required for ADAMTS13 Secretion

Abstract

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 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 cysteinerich domain, and a spacer domain and conclude with a variable number of additional TSRs (1). ADAMTS13 uniquely contains seven additional TSRs and two CUB1 domains at its carboxyl end (2-4) (Fig. 1A). TSRs contain ϳ60 amino acids with conserved tryptophans and cysteines. They were first described in thrombospondin type 1 and are homologous to the properdin repeat found in many components of the complement system. Thrombospondin type 1 is a protein that is thought to play a role in angiogenesis, cell adhesion, and motility (5, 6). Many interactions of thrombospondin-1 are thought to be mediated through amino acids within the TSRs (7). For example, binding of thrombospondin-1 to the endothelial cell protein, CD36, can be inhibited by the peptide CSVTCG, which is found in the TSRs of thrombospondin-1 (8). The three TSRs of thrombospondin-1 have been shown to contain a fucose directly linked to a serine or threonine within a putative consensus sequence of C 1 XX(S/T)C 2 G (where C 1 and C 2 are the 1st and 2nd conserved cysteines in the TSR (9)), which is the putative CD36 binding region. The fucose on the TSRs was further modified with a glucose in ␤1-3 linkage to form a disaccharide (9, 10). Subsequent analysis ...

The D173G mutation in ADAMTS-13 causes a severe form of congenital thrombotic thrombocytopenic purpura

“…In particular, the O-fucosylation occurring at 6 TSP1 repeats of ADAMTS13 and the N-glycosylation process appear to be critical for folding, secretion and proteolytic activity of ADAMTS-13 (45). The protein folding machinery of the ER consists of three major classes of proteins: (i) foldases, (ii) molecular chaperones, and (iii) the oligosaccharide processing enzymes as well as lectin chaperones calnexin and calreticulin, which all are involved in protein quality control (46). Among the foldases, the peptidyl-prolyl…”

stall Encodes an ADAMTS Metalloprotease and Interacts Genetically With Delta in Drosophila Ovarian Follicle Formation

“…Since N-glycosylation can affect protein folding and secretion efficiency [e.g., synaptotagmin 1 and inhibin/activin (Han et al 2004;Antenos et al 2007)], it is possible that these sites in Stl either facilitate secretion in the absence of a strong signal peptide (Stl-B) or boost a weak signal sequence (Stl-A). Other ADAMTS oligosaccharide modifications, C-mannosylation and O-fucosylation, typically occur within the TSRs and can be important for function (e.g., ADAMTS13) (Gonzalez De Peredo et al 2002;Paakkonen et al 2006;Ricketts et al 2007). Capacity for C-mannosylation is noteworthy in the context of Stl, because the solitary central TSR of Stl (with no C-terminal TSRs) is one of the major structural differences that marginalize Stl within the ADAMTS family and because C-mannosylation occurs only on secreted proteins.…”

Thrombospondins: from structure to therapeutics

“…O-fucosylation occurs within the CSX(S/T)C sequence of TSRs, where the underlined Ser or Thr is fucosylated. This is a post-translational modification that requires that the module be folded and is mediated by O-fucosyltransferase 2 [22]. Studies of ADAMTS13 and ADAMTS-like-1/Punctin-1 have demonstrated that O-fucosylation of TSRs is critical for protein maturation and secretion [22,23].…”

“…This is a post-translational modification that requires that the module be folded and is mediated by O-fucosyltransferase 2 [22]. Studies of ADAMTS13 and ADAMTS-like-1/Punctin-1 have demonstrated that O-fucosylation of TSRs is critical for protein maturation and secretion [22,23]. Interestingly, this modification is so far unique for TSRs and is, for instance, distinct from O-fucosylation of EGF-like repeats, which is catalyzed by O-fucosyltransferase 1 [22,23].…”

“…The modifications dot the surface of THBSs and likely act as beacons to guide chaperones to particularly hard-to-fold structures in order to ensure the proper maturation of this complicated molecule. For example, the O-fucosylation sites in the TSRs lie at the interface between modules, where it may be critical to have the quality control that has been attributed to the glycosylation machinery [22,23].…”