Original article 479 N-glycoproteins bearing b1–6 branched oligosaccharides from the A375 human melanoma cell line analysed by tandem mass spectrometry Danuta Ochwat, Dorota Hoja-Lukowicz and Anna Lityn ´ ska Tumour-associated alterations of cell surface glycosylation play a crucial role in the adhesion and metastasis of cancer cells. It is well known that the metastatic potential is associated with increased GlcNAc b1–6 branching in N- glycans of tumour cells specifically recognized by a lectin from Phaseolus vulgaris leukoagglutinin (PHA-L). We identified proteins bearing GlcNAc b1–6 branched N- glycans in the A375 human melanoma cell line by affinity chromatography separation on a PHA-L agarose column, followed by immunoidentification and tandem mass spec- trometry (MS/MS) analysis. Amongst the proteins identi- fied were integrin subunits a 2 , a 3 , a 5 and b 1 , as well as N- cadherin and lysosome-associated membrane proteins (LAMP-1 and LAMP-2). In addition, L1, Mac-2 binding protein (Mac-2-BP), activated leukocyte cell adhesion molecule/CD166 (ALCAM) and melanotransferrin were shown to react with PHA-L. Some of these proteins are connected mainly with nervous tissues or the immune system and play a crucial role in cell adhesion processes. The presence of GlcNAc b1–6 branched oligosaccharides in these proteins may influence their adhesion properties, reducing adhesion of the cells to the extracellular matrix (ECM) and thus facilitating tumour cell invasion. Melanoma Res 14:479–485  c 2004 Lippincott Williams & Wilkins. Melanoma Research 2004, 14:479–485 Keywords: b1–6 branching, glycosylation, human melanoma, MS/MS Laboratory for Glycobiology, Institute of Zoology, Department of Animal Physiology, Jagiellonian University, Ingardena 6, 30-060 Krako ´ w, Poland. Sponsorship: This work was supported by the State Committee for Scientific Research, Poland. PB/0287/P04/2001/21. Correspondence and requests for reprints to Anna Lityn ´ ska, Laboratory for Glycobiology, Institute of Zoology, Department of Animal Physiology, Jagiellonian University, Ingardena 6, 30-060 Krako ´ w, Poland. Tel: (48-12) 633-63-77, ext 2405; fax: (48-12) 634-37-16; e-mail: Lita@zuk.iz.uj.edu.pl Received 9 September 2003 Accepted (after revision) 6 September 2004 Introduction The metastasis of tumour cells is associated with the aberrant glycosylation profile of cell surface glycoproteins [1–3], in particular terminal sialylation [4] and the presence of b1–6 branched N-linked oligosaccharides containing poly-N-acetyllactosamine structures [5]. There is increasing evidence that progression of cancer from a tumorigenic to a metastatic phenotype is directly associated with increased levels of b1–6 branched N- linked oligosaccharides, as the result of hyperactivity of N-acetylglucosaminyltransferase V (GlcNAc TnV) [6–8]. These oligosaccharides, which have the sequence GlcNAc b(1–6) Mana 1–6Man –R, can be characterized by specific binding to the Phaseolus vulgaris leukoaggluti- nin (PHA-L) lectin [9]. PHA-L is the sole reagent exhibiting reactivity towards b1–6 branches, and its binding is not influenced either by the addition of N- acetyllactosamine repeats or sialic acid residues [9,10]. Interestingly, only a small number of glycoproteins are suitable substrates for GlcNAc TnV activity. Those identified so far include lysosome-associated membrane proteins (LAMP-1, LAMP-2) [11], carcinoembryonic antigen (CEA) from colon tumours [12,13] and b 1 integrins. The altered glycosylation of b 1 integrins contributes to the abnormal behaviour of cells, resulting in reduced adhesion and increased invasion into the surrounding tissues, as well as metastasis to distant regions [1,8,14,15]. Experimental findings have indicated an association between b1–6 branches and the metastatic formation of melanoma cells [16]. PHA-L-positive coarse vesicles rich in b1–6 branched oligosaccharides have also been documented in melanomas [17]. In a previous study, we demonstrated that melanoma cells from metastatic sites contained more glycoproteins with b1–6 branches than those from melanoma in situ [18]. Of the PHA-L-positive proteins in the A375 cell line, only N-cadherin has been identified with specific antibodies [18]. Although it is well known that alterations in N-linked b1–6 branched oligosaccharides accompany the malignant transformation of tumour cells, it is not clearly established which of the glycoproteins undergoes altered glycosylation. Therefore, the present investigation was performed to further identify N-glycoproteins bearing b1–6 branched oligosac- charides in the highly metastatic A375 human melanoma cell line. Materials and methods Chemicals Fetal bovine serum (FBS) was obtained from GibcoBRLt (Paisley, UK). RPMI-1640 medium, PHA-L immobilized 0960-8931  c 2004 Lippincott Williams & Wilkins Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.