C-Mannosylation and O-Fucosylation of the Thrombospondin Type 1 Module

Abstract

Thrombospondin-1 (TSP-1) is a multidomain protein that has been implicated in cell adhesion, motility, and growth. Some of these functions have been localized to the three thrombospondin type 1 repeats (TSRs), modules of ϳ60 amino acids in length with conserved Cys and Trp residues. The Trp residues occur in WXXW patterns, which are the recognition motifs for protein C-mannosylation. This modification involves the attachment of an ␣-mannosyl residue to the C-2 atom of the first tryptophan. Analysis of human platelet TSP-1 revealed that Trp-368, -420, -423, and -480 are C-mannosylated. Mannosylation also occurred in recombinant, baculovirally expressed TSR modules from Sf9 and "High Five" cells, contradictory to earlier reports that such cells do not carry out this reaction. In the course of these studies it was appreciated that the TSRs in TSP-1 undergo a second form of unusual glycosylation. By using a novel mass spectrometric approach, it was found that Ser-377, Thr-432, and Thr-489 in the motif CSX(S/T)CG carry the O-linked disaccharide Glc-Fuc-O-Ser/Thr. This is the first protein in which such a disaccharide has been identified, although protein O-fucosylation is well described in epidermal growth factor-like modules. Both C-and O-glycosylations take place on residues that have been implicated in the interaction of TSP-1 with glycosaminoglycans or other cellular receptors.

Modification of Epidermal Growth Factor-like Repeats withO-Fucose

“…Recently the presence of O-fucose in a different protein context was reported (27). Hofsteenge and co-workers showed the presence of the disaccharide Glc-Fuc O-linked to serines and threonines within the three thrombospondin type 1 repeats (TSR) of human thrombospondin-1.…”

Thrombospondins: from structure to therapeutics

“…Functions associated with the TSRs found in THBS-1 include cell attachment, angiogenesis inhibition, protein-protein interactions, and protein-glycosaminoglycan interactions [12]. The module is subject to two unusual carbohydrate modifications, introduction of a mannose onto a conserved tryptophan, and of a fucose-glucose onto a conserved serine or threonine [21] (Fig. 2D).…”

“…Two fucose moieties were bound to the complex at Thr432 and Thr489. Predicted C-mannosylation was not observed, as the S2 cell expression system is incapable of this modification [19,21]. However, it was observed that the Cδ1 atoms of the predicted tryptophan modification sites are exposed in the structure.…”

“…C-mannosylation is co-translational and is mediated by dolichol-P-mannose. It occurs within the TSRWXXW sequence, and specifically Trp368, Trp420, Trp423, and Trp480 of THBS-1 each form a C-mannose [21]. Other proteins with a WXXW sequence are also subject to C-mannosylation [21].…”

“…It occurs within the TSRWXXW sequence, and specifically Trp368, Trp420, Trp423, and Trp480 of THBS-1 each form a C-mannose [21]. Other proteins with a WXXW sequence are also subject to C-mannosylation [21]. O-fucosylation occurs within the CSX(S/T)C sequence of TSRs, where the underlined Ser or Thr is fucosylated.…”