C-mannosylation and O-fucosylation of the thrombospondin type 1 module

Thrombospondin-1 (TSP-1) is a multidomain protein that has been implicated in cell adhesion, motility, and growth. Some of these functions have been l ocalized to the three thrombospondin type 1 repeats (TSRs), modules of simi lar to 60 amino acids in length with conserved Cys and Trp residues. The Tr p residues occur in WXXW patterns, which are the recognition motifs for pro tein C-mannosylation. This modification involves the attachment of an cu-ma nnosyl residue to the C-2 atom of the first tryptophan. Analysis of human p latelet TSP-1 revealed that Trp-368, -420, -423, and -480 are C-mannosylate d. Mannosylation also occurred in recombinant, baculovirally expressed TSR modules from Sf9 and "High Five" cells, contradictory to earlier reports th at such cells do not carry out this reaction. In the course of these studie s it was appreciated that the TSRs in TSP-1 undergo a second form of unusua l 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-Lin ked disaccharide Glc-Fuc-O-Ser-Thr. This is the first protein in which such a disaccharide has been identified, although protein O-fucosylation is wel l described in epidermal growth factor-like modules. Both C- and O-glycosyl ations take place on residues that have been implicated in the interaction of TSP-1 with glycosaminoglycans or other cellular receptors.