Biochimica et Biophysica Acta (BBA) - General Subjects
Characterisation of α3β1 and αvβ3 integrin N-oligosaccharides in metastatic melanoma WM9 and WM239 cell lines
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).
References (53)
- et al.
On the frequency of protein glycosylation deduced from analysis of the SWISS-PROT database
Biochim. Biophys. Acta
(1999) - et al.
Glycoprotein glycosylation and cancer progression
Biochim. Biophys. Acta
(1999) - et al.
Expression of the vacuolar H+-ATPase 16-kDa subunit result in the Triton X-100-insoluble aggregation of β1 integrin and reduction of its cell surface expression
J. Biol. Chem.
(2004) - et al.
The α3β1 integrin is involved in melanoma cell migration and invasion
Exp. Cell Res.
(1995) A symplification of the protein assay method of Lowry et al. which is more generally applicable
Anal. Biochem.
(1977)- et al.
The Arg-Gly-Asp binding domain of the vitronectin receptor
J. Biol. Chem.
(1988) - et al.
Characterization of the oligosaccharide component of α3β1 integrin from human bladder carcinoma cell line T24 and its role in adhesion and migration
Eur. J. Cell. Biol.
(2006) - et al.
Identification of proteins bearing β1-6 branched N-glycans in human melanoma cell lines from different progression stages by tandem mass spectrometry analysis
Biochim. Biophis. Acta
(2007) - et al.
Sialilated β1,6 branched N-oligosaccharides modulated adhesion, chemotaxis and motility of melanoma cells: effect on invasion and spontaneous metastasis properties
Biochim. Biophys. Acta
(2006) - et al.
Cell sialilation and tumor metastasis: metastatic potential of B16 melanoma variants correlates with their relative numbers of specific penultimate oligosaccharide structures
J. Biol. Chem.
(1988)
Variant glycosylation: an underappreciated regulatory mechanism for β1 integrins
Biochim. Biophys. Acta
Overexpression of the cell adhesion molecule L1 is associated with metastasis in cutaneous malignant melanoma
Eur. J. Cancer
Expression of a carbohydrate signal, sialyl-dimeric Lex antigen, is associated with metastatic potential of transitional cell carcinoma of the human urinary bladder
Biochem. Biophys. Res. Commun.
Deletion of core fucosylation on α3β1 integrin down-regulates its functions
J. Biol. Chem.
The α1-6-fucosyltransferase gene and its biological significance
Biochim. Biophys. Acta
Increased UDP-GclNAc:Gal β1-3GalNAc-R (GlcNAc to GalNAc) β1-6-N-acetylglucosaminyltransferase activity in metastatic murine tumor cell lines. Control of polylactosamine synthesis
J. Biol. Chem.
Inhibition of a specific N-glycosylation activity results in attenuation of breast carcinoma cell invasiveness-related phenotypes: inhibition of epidermal growth factor-induced dephosphorylation of focal adhesion kinase
J. Biol. Chem.
Effects of glycosylation on peptide conformation: a synergistic experimental and computational study
J. Am. Chem. Soc.
Protein glycosylation in development and disease
Bioessays
Regulation of integrin functions by N-glycans
Glycoconj. J.
Protein glycosylation in cancer biology: an overview
J. Clin. Pathol. Mol. Pathol.
Aberrant N-glycosylation of β1 integrin causes reduced α5β1 integrin clustering and stimulates cell migration
Cancer Res.
Glycosylation defining cancer malignancy: new wine in an old bottle
Proc. Natl. Acad. Sci. U. S. A.
Characterization of glycosylation and adherent properties of melanoma cell lines
Cancer Immun. Immunother.
Adhesion properties of human bladder cell lines with extracellular matrix components: the role of integrins and glycosylation
Acta Biochim. Pol.
Functionally distinct roles for glycosylation of α and β integrin chains in cell–matrix interactions
Proc. Natl. Acad. Sci. U. S. A.
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