Beta1-6 branching of cell surface glycoproteins may contribute to uveal melanoma progression by up-regulating cell motility.

Molecular vision (2008-04-04)
Małgorzata Przybyło, Ewa Pocheć, Paweł Link-Lenczowski, Anna Lityńska

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. 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 beta1-6 branched complex-type N-oligosaccharides on wound healing on fibronectin was investigated. Cell surface beta1-6 branched N-oligosaccharides were measured by their specific binding to PHA-L followed by flow cytometry, and the fibronectin receptors bearing beta1-6 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 beta1-6 linkage) was analyzed by semiquantitative RT-PCR. 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 alpha(3)beta(1), alpha(4)beta(1), alpha(5)beta(1), and alpha(v)beta(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 alpha(4)beta(1) integrin on both FM55P and IGR-39 cells. The results also demonstrated that the high levels of alpha(3)beta(1), alpha(4)beta(1), and alpha(5)beta(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 alpha(4)beta(1) integrin in FM55P cells. Cell migration was significantly decreased in three out of four PHA-L-treated cell lines, suggesting that beta1-6 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 beta1-6 -N-acetylglucosaminyltransferase V. However, FACS analysis showed that 92-1, Mel202 and IGR-39 cells expressed significantly higher amounts of beta1-6 branched N-oligosaccharides on the cell surface than FM55P cells did. All examined alpha(3), alpha(5), alpha(v), and beta(1) integrin subunits were shown to bear beta1-6 branched N-linked glycans. 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 beta1-6 branches are important factors determining the migration of primary uveal melanoma cells on fibronectin.

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Trypsin-EDTA solution, 0.25%, sterile-filtered, BioReagent, suitable for cell culture, 2.5 g porcine trypsin and 0.2 g EDTA • 4Na per liter of Hanks′ Balanced Salt Solution with phenol red
Trypsin-EDTA solution, 1 ×, sterile; sterile-filtered, BioReagent, suitable for cell culture, 0.5 g porcine trypsin and 0.2 g EDTA • 4Na per liter of Hanks′ Balanced Salt Solution with phenol red
Poly-L-lysine solution, 0.01%, sterile-filtered, BioReagent, suitable for cell culture
Poly-L-lysine solution, 0.1 % (w/v) in H2O
Poly-L-lysine solution, mol wt 150,000-300,000, 0.01%, sterile-filtered, BioReagent, suitable for cell culture
Trypsin-EDTA solution, 10 ×, sterile-filtered, BioReagent, suitable for cell culture, 5.0 g porcine trypsin and 2 g EDTA • 4Na per liter of 0.9% sodium chloride
Trypsin-EDTA solution, 1 ×, sterile-filtered, BioReagent, suitable for cell culture, 500 BAEE units porcine trypsin and 180 μg EDTA • 4Na per ml in Dulbecco′s PBS without calcium and magnesium
Anti-Mouse IgG (Fab specific)–Alkaline Phosphatase antibody produced in goat, affinity isolated antibody, buffered aqueous solution

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