β1-6 branching of cell surface glycoproteins may contribute to uveal melanoma progression by up-regulating cell motility

PURPOSE: This study investigated the influence of integrin expression as well as the oligosaccharide structure of surface N-glycoproteins on cell behavior of two primary uveal (92–1 and Mel202) and two primary cutaneous (FM55P and IGR-39) melanoma cell lines. METHODS: Cell adhesion to fibronectin and cell migration on fibronectin (wound healing) were selected as the studied cell behavior parameters. The percentage of cells positive for expression of selected integrins was estimated by flow cytometric analysis. The influence of β1–6 branched complex-type N-oligosaccharides on wound healing on fibronectin was investigated. Cell surface β1–6 branched N-oligosaccharides were measured by their specific binding to PHA-L followed by flow cytometry, and the fibronectin receptors bearing β1–6 GlcNAc branched N-linked glycans were identified. In addition, the transcript of GnT-V (the enzyme that catalyzes the addition of N-acetylglucosamine to the core mannose of di- and tri-antennary N-glycans through a β1–6 linkage) was analyzed by semiquantitative RT–PCR. RESULTS: Unlike the two examined cutaneous melanoma cell lines, neither of the uveal melanoma cells adhered to fibronectin. The adhesion efficiency of IGR-39 cells was twice that of FM55P cells. In contrast, uveal melanoma cells repaired scratch wounds on fibronectin-coated surfaces twice as fast as cutaneous melanoma cells did. The expression of α(3)β(1), α(4)β(1), α(5)β(1,) and α(v)β(3) integrins, acting as fibronectin receptors, differed between the tested cell lines, and no distinct pattern distinguished uveal melanoma from cutaneous melanoma except for high expression of α(4)β(1) integrin on both FM55P and IGR-39 cells. The results also demonstrated that the high levels of α(3)β(1), α(4)β(1), and α(5)β(1) integrin expression on IGR-39 cells promoted their strong attachment to fibronectin-coated surfaces. In addition, 92–1, Mel202, and FM55P cells showed no or low adhesion to fibronectin, perhaps the result of low expression of fibronectin receptors excluding high expression of α(4)β(1) integrin in FM55P cells. Cell migration was significantly decreased in three out of four PHA-L-treated cell lines, suggesting that β1–6 branched complex type N-oligosaccharides are critical for 92–1, Mel202, and FM55P cell motility. Semiquantitative RT–PCR analysis showed that the tested cells did not differ in mRNA levels of β1–6 –N-acetylglucosaminyltransferase V. However, FACS analysis showed that 92–1, Mel202 and IGR-39 cells expressed significantly higher amounts of β1–6 branched N-oligosaccharides on the cell surface than FM55P cells did. All examined α(3), α(5), α(v), and β(1) integrin subunits were shown to bear β1–6 branched N-linked glycans. CONCLUSIONS: The role of integrins and their N-glycosylation in the regulation of uveal melanoma growth and progression is largely unknown. These results reveal that cell surface complex-type N-glycans with GlcNAc β1–6 branches are important factors determining the migration of primary uveal melanoma cells on fibronectin.