Characterisation of α3β1 and αvβ3 integrin N-oligosaccharides in metastatic melanoma WM9 and WM239 cell lines

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Abstract

It is well documented that glycan synthesis is altered in some pathological processes, including cancer. The most frequently observed alterations during tumourigenesis are extensive expression of β1,6-branched complex type N-glycans, the presence of poly-N-acetyllactosamine structures, and high sialylation of cell surface glycoproteins. This study investigated two integrins, α3β1 and αvβ3, whose expression is closely related to cancer progression. Their oligosaccharide structures in two metastatic melanoma cell lines (WM9, WM239) were analysed with the use of matrix-assisted laser desorption ionisation mass spectrometry. Both examined integrins possessed heavily sialylated and fucosylated glycans, with β1,6-branches and short polylactosamine chains. In WM9 cells, α3β1 integrin was more variously glycosylated than αvβ3; in WM239 cells the situation was the reverse. Functional studies (wound healing and ELISA integrin binding assays) revealed that the N-oligosaccharide component of the tested integrins influenced melanoma cell migration on vitronectin and α3β1 integrin binding to laminin-5. Additionally, more variously glycosylated integrins exerted a stronger influence on these parameters. To the best of our knowledge, this is the first report concerning structural characterisation of αvβ3 integrin glycans in melanoma or in any cancer cells.

Introduction

Over half of the proteins present in living organisms are glycosylated [1], and many of them are expressed in cell membranes. For all glycoproteins this modification is a major determinant of their activity, folding and/or stability [2]. It is well documented in the literature that glycan synthesis is altered in some pathological processes, including cancer [3], [4], [5]; the most frequently observed alterations during tumourigenesis are extensive expression of β1,6-branched tri- and tetraantennary complex type N-glycans [6], [7], [8], [9], the presence of poly-N-acetyllactosamine structures, and high sialylation of cell surface glycoproteins. In the case of the cell surface receptors, the changed glycosylation profile may affect their presence in the cell membrane [10]. Carbohydrates have been shown to interact with proteins during recognition events, so any changes in the structure of the sugar component may influence the glycoprotein's ligand binding ability [11]. Hence the common observation is that altered glycosylation modifies migration and adhesive properties and supports the metastatic potential of cancer cells.

Integrins, a large family of cell membrane receptors, are involved in important processes such as cell–cell and cell–extracellular matrix (ECM) adhesion. They are built of two subunits: α and β. Both subunits are glycosylated and possess multiple potential N-glycosylation sites [12]. It is reported that an increase of the metastatic potential of tumour cells is accompanied by changes in the expression level of several adhesive molecules, including integrins. Altered integrin expression is associated with tissue invasion and metastasis in many types of cancer, but there is no universal pattern of integrin expression in cancer cells [13], [14]. In melanoma, α3β1 and αvβ3 integrins are recognised as specific markers of tumour progression, and αvβ3 in particular is used as a marker to distinguish the radial growth phase (RGP) from the vertical growth phase (VGP) [15], [16], [17].

The aim of this study was to analyse glycan pools of α3β1 and αvβ3 integrins purified from two human melanoma cell lines of different origin: WM9 from lymph node metastasis, and WM239 from skin metastasis. Moreover, the biological relevance of these findings was examined by ELISA integrin binding and wound healing assays to determine whether the oligosaccharides attached to these integrins modified their interaction with primary ligands — laminin-5 and vitronectin. To the best of our knowledge this is the first report of a structural analysis of αvβ3 integrin oligosaccharides in melanoma or in any cancer cells.

Section snippets

Chemicals

The Immobilon P membrane was from Milipore (Bedford, MA, USA). Rabbit polyclonal antisera against α3, αv, β3 integrin subunits, mouse monoclonal antiserum against β1 integrin subunit, affi-gel coupled with anti-αvβ3 antibody and vitronectin (VN) were purchased from Chemicon International (Temecula, CA). RPMIG 1640 medium, bovine serum albumin (BSA), trypsin/EDTA solution, protease inhibitor cocktail, octyl-β-d-glucopyranoside, pNpp (p-nitrophenyl phosphate disodium), Phaseolus vulgaris

Results

In order to purify the integrins from human melanoma WM9 and WM239 cell lines, affinity chromatography on GD-6 peptide (for α3β1) and immunoaffinity chromatography with immobilised antibodies against αvβ3 integrin were performed. The presence of isolated proteins in the collected material was confirmed by SDS-PAGE under non-reducing conditions followed by Western blotting with specific antibodies (Fig. 1). The molecular weights of integrin subunits in WM9 and WM239, respectively, were as

Discussion

In the present study we showed that α3β1 and αvβ3 integrins purified from two human melanoma cell lines derived from particular metastasis sites differ in their glycosylation profiles. Both subunits of α3β1 integrin in WM9 cells originating from lymph node metastasis showed more diverse glycan types than did WM239 cells originating from skin metastasis. In the case of αvβ3 integrin it was just the reverse: the glycan pool from WM239 cells was much more diverse than in WM9 cells. Nevertheless,

Acknowledgments

This work was supported by the Polish State Committee for Scientific Research (PB/0939/P05/2004/26) and by Jagiellonian University (BW/12a/IZ/2003).

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