Glycoconjugate Journal 20, 483–492, 2004 C  2004 Kluwer Academic Publishers. Manufactured in The Netherlands. The structure of the oligosaccharides of N-cadherin from human melanoma cell lines * Dorota Cio lczyk-Wierzbicka 1 , Angela Amoresano 3 , Annarita Casbarra 3 , Dorota Hoja- Lukowicz 2 , Anna Lity ´ nska 2 and Piotr Laidler 1 1 Intitute of Medical Biochemistry, Medical College, Jagiellonian University, Krak´ ow, Poland, 2 Institute of Zoology, Department of Animal Physiology, Jagiellonian University, Krak´ ow, Poland, 3 Dipartimento di Chimica Organica e Biochimica, Universit ` a degli Studi di Napoli “Federico II”, Napoli, Italy N-cadherin is calcium-dependent cell adhesion molecule that mediates cell-cell adhesion and also modulates cell migra- tion and tumor invasion. N-cadherin is a heavily glycosylated protein. Many studies have demonstrated that malignant transformation of a number of cell types correlates with changes of cell surface N-linked oligosacharides. We have studied the carbohydrate profile of N-cadherin synthesized in human melanoma cell lines and the effect of this protein and complex N-glycans on in vitro migration of melanoma cells from the primary tumor site—WM35 and from different metastatic sites WM239 (skin), WM9 (lymph node), and A375 (solid tumor). N-cadherin was immunoprecipitated with anti-human N-cadherin polyclonal antibodies. Characterization of its carbohydrate moieties was carried out by SDS-PAGE electrophoresis and blotting, followed by immunochemical identification of the N-cadherin polypeptides and on-blot deglycosylation using PNGase F for glycan release. N-glycans were separated by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and their structures identified by the computer matching of the resulting masses with those derived from a sequence database. The assay of in vitro chemotaxic cell migration was performed using QCM TM Cell Invasion Assay (Chemicon). N-cadherin from WM35 (primary tumor site) possessed high-mannose and biantennary complex type glycans with α2–6 linked sialic acid. N-cadherin from WM239, WM9, and A375 cell lines possessed mostly tri- or tetra-antennary complex type glycans. In addition, N-cadherin from WM9 (lymph node metastatic site) and A375 (solid tumor metastatic site) contained heavily α-fucosylated complex type chains with α2,3 linked sialic acid. Blocking of N-cadherin-mediated intercellular interaction by N-cadherin-specific antibodies significantly (of about 40%) inhibited migration of melanoma cells. Inhibition of synthesis of complex type N-glycans by swainsonine (mannosidase II inhibitor) led to 50% decrease of cell migration. The results indicated differences between N-cadherin glycans from primary and metastatic sites and confirmed influence of N-cadherin and complex -type N-glycans on in vitro migration of melanoma cells. Published in 2004. Keywords: N-cadherin, N-glycans, melanoma, MALDI MS, migration Abbreviations: HexNAc: N-acetyl-hexosamine; Fuc: fucose; Sia: sialic acid; MALDI MS: matrix-assisted laser desorption ionisation mass spectrometry; PNGase: F peptide N-glycosidase F. Introduction The pattern of expression of cell adhesion molecules and their properties play a pivotal role in controlling processes of cell division, migration, differentiation and death. ∗ This work was supported by the State Committee for Scientific Research KBN, Poland (W L/133/P/L Medical College, Jagiellonian University). To whom correspondence should be addressed: Dorota Cio lczyk- Wierzbicka, Institute of Medical Biochemistry, Medical College, Jagiel- lonian University, M. Kopernika 7, 31-034 Krak´ ow. Tel/Fax: (48 12) 422 32 72; E-mail: mbciolcz@cyf-kr.edu.pl Cadherins are transmembrane glycoproteins, which provide strong intercellular adhesion in a Ca 2+ dependent manner. Clas- sic cadherins are the transmembrane components of cellular junctions. They are composed of three segments: an large extra- cellular domain, which mediates homophilic type cell adhesion, a transmembrane domain, and a highly conserved cytoplasmic domain, that interacts with catenins to link cadherins to the actin cytoskeleton. Cadherins play a major role in epithelial cell-cell adhesion [1,2]. Moreover, their role in cell differentiation, trans- formation, and invasion has been also documented [3–5] with some of these functions most probably depending upon the ac- tivaction of intracellular signal transduction cascades [6].