Glycosylation of integrins has been implicated in the modulation of their function. Characterisation of carbohydrate moieties of alpha(3) and beta(1) subunits from non-metastatic (WM35) and metastatic (A375) human melanoma cell lines was carried out on peptide-N-glycosidase F-released glycans using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). beta(1) integrin subunit from both cell lines displayed tri- and tetraantennary oligosaccharides complex type glycans, but only in A375 cell line was the sialylated tetraantennary complex type glycan (Hex(7)HexNAc(6)FucSia(4)) present. In contrast, only alpha(3) subunit from metastatic cells possessed beta1-6 branched structures. Our data indicate that the beta(1) and alpha(3) subunits expressed by the metastatic A375 cell line carry beta1-6 branched structures, suggesting that these cancer-associated glycan chains may modulate tumor cell adhesion by affecting the ligand binding properties of alpha(3)beta(1) integrin. In direct ligand binding assays, alpha(3)beta(1) integrin from both cell lines binds strongly to fibronectin and to much lesser degree to placental laminin. No binding to collagen IV was observed. Enzymatic removal of sialic acid residues from purified alpha(3)beta(1) integrin stimulates its adhesion to all examined ECM proteins. Our data suggest that the glycosylation profile of alpha(3)beta(1) integrin in human melanoma cells correlates with the acquisition of invasive capacity during melanoma progression.

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The tetraspanin CD151 forms a stoichiometric complex with integrin ␣3␤1 and regulates its endocytosis. We observed that down-regulation of CD151 in various epithelial cell lines changed glycosylation of ␣3␤1. In contrast, glycosylation of other transmembrane proteins, including those associated with CD151 (e.g. ␣6␤1, CD82, CD63, and emmprin/CD147) was not affected. The detailed analysis has shown that depletion of CD151 resulted in the reduction of Fuc␣1-2Gal and bisecting GlcNAc-␤(134) linkage on N-glycans of the ␣3 integrin subunit. The modulatory activity of CD151 toward ␣3␤1 was specific, because stable knockdown of three other tetraspanins (i.e. CD9, CD63, and CD81) did not affect glycosylation of the integrin. Analysis of ␣3 glycosylation in CD151-depleted breast cancer cells with reconstituted expression of various CD151 mutants has shown that a direct contact with integrin is required but not sufficient for the modulatory activity of the tetraspanin toward ␣3␤1. We also found that glycosylation of CD151 is also critical; Asn 159 3 Gln mutation in the large extracellular loop did not affect interactions of CD151 with other tetraspanins or ␣3␤1 but negated its modulatory function. Changes in the glycosylation pattern of ␣3␤1 observed in CD151-depleted cells correlated with a dramatic decrease in cell migration toward laminin-332. Migration toward fibronectin or static adhesion of cells to extracellular matrix ligands was not affected. Importantly, reconstituted expression of the wild-type CD151 but not glycosylation-deficient mutant restored the migratory potential of the cells. These results demonstrate that CD151 plays an important role in post-translation modification of ␣3␤1 integrin and strongly suggest that changes in integrin glycosylation are critical for the promigratory activity of this tetraspanin.

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