Identification of proteins bearing β1–6 branched N-glycans in human melanoma cell lines from different progression stages by tandem mass spectrometry analysis Małgorzata Przybyło, Danuta Martuszewska, Ewa Pocheć, Dorota Hoja-Łukowicz, Anna Lityńska ⁎ Department of Glycoconjugate Biochemistry, Institute of Zoology, Jagiellonian University, Ingardena 6, 30-060 Kraków, Poland Received 3 January 2007; received in revised form 24 April 2007; accepted 15 May 2007 Available online 31 May 2007 Abstract The common structural alterations in the cell-surface glycoproteins concern the highly elevated expression of tri- and tetra-antennary β1–6-N- acetylglucosamine (β1–6 GlcNAc) bearing N-glycans, which are recognised by Phaseolus vulgaris agglutinin (PHA-L). In this report we identified proteins bearing β1–6 GlcNAc branched N-glycans in three human melanoma cell lines: WM35 — from the primary tumour site, as well as WM239 and WM9 from different metastatic sites: the skin and the lymph node, respectively, by tandem mass spectrometry (MS/MS) on PHA-L agarose bound material, followed by immunochemical identification. Our results show that melanoma cell lines differ from each other in the number of N-glycoproteins bearing β1–6 GlcNAc branched oligosaccharides. Among identified proteins the largest group consists of integrin subunits. In addition, L1-CAM, Mac-2 binding protein, melanoma cell adhesion molecule, intercellular adhesion molecule, melanoma associated antigen, tumour rejection antigen-1, melanoma-associated chondroitin sulfate proteoglycan 4 and lysosome-associated membrane protein (LAMP- 1) were found. It was indicated that WM35 cell line showed the lowest number of proteins possessing β1–6 GlcNAc branched N-glycans in comparison to metastatic WM9 and WM239 cell lines. Our data suggest that changes in the number of proteins being a substrate for GlcNAc-TV are better correlated with melanoma development and progression than with expression of cell adhesion molecules. © 2007 Elsevier B.V. All rights reserved. Keywords: β1–6 branching; Glycosylation; Integrins; Melanoma; MS/MS analysis; PHA-L 1. Introduction A number of studies have suggested that cancer cells differ from their normal counterparts in a wide variety of features, including glycosylation. Covalently linked sugar chains are thought to play an important role in the control of cellular function and cellular recognition in multi-cellular organisms. Therefore, alteration in the carbohydrate structures of glycoproteins found in various tumours are considered to be the basis of abnormal social behaviour of tumour cells, such as invasion into the surrounding tissue and metastasis. One of the common structural alterations in the cell-surface glycans observed in various human and rodent tumours is highly elevated expression of β1–6-N-acetylglucosamine (β1–6 GlcNAc) branched tri- and tetra-antennary complex type N- glycans [1]. The formation of β1–6-GlcNAc branches is controlled via the activity of N-acetylglucosaminyltransferase V (GlcNAc- TV), which catalyses the addition of GlcNAc to the mannose of tri- and tetra-antennary N-glycans through a β1–6 linkage. It has been well documented that GlcNAc-TV level is elevated as a result of transformation by viruses, several oncogenes, including members of the ras family and v- src [2–4]. Furthermore, enhanced activity of GlcNAc-TV and larger number of glycoproteins detected by Phaseolus vulgaris agglutinin (PHA-L), which preferred ligands are β1–6 branched N-glycans [5], correlates with higher invasive potential, metastasis, reconstruction of the vascular system, and growth of tumour cells [1,6–11]. Interestingly, GlcNAc-TV activity has recently been reported to be elevated also during Biochimica et Biophysica Acta 1770 (2007) 1427 – 1435 www.elsevier.com/locate/bbagen ⁎ Corresponding author. Tel.: +48 12 633 63 77x2405; fax: +48 12 634 37 16. E-mail address: lita@zuk.iz.uj.edu.pl (A. Lityńska). 0304-4165/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2007.05.006