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The structure of the oligosaccharides of N-cadherin from human melanoma cell lines

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Abstract

N-cadherin is calcium-dependent cell adhesion molecule that mediates cell-cell adhesion and also modulates cell migration 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™ 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.

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Authors and Affiliations

  1. Intitute of Medical Biochemistry, Medical College, Jagiellonian University, Kraków, Poland

    Dorota Ciołczyk-Wierzbicka & Piotr Laidler

  2. Dipartimento di Chimica Organica e Biochimica, Università degli Studi di Napoli “Federico II”, Napoli, Italy

    Angela Amoresano & Annarita Casbarra

  3. Institute of Zoology, Department of Animal Physiology, Jagiellonian University, Kraków, Poland

    Dorota Hoja-Łukowicz & Anna Lityńska

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  1. Dorota Ciołczyk-Wierzbicka
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  2. Angela Amoresano
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  3. Annarita Casbarra
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Correspondence to Dorota Ciołczyk-Wierzbicka.

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Ciołczyk-Wierzbicka, D., Amoresano, A., Casbarra, A. et al. The structure of the oligosaccharides of N-cadherin from human melanoma cell lines. Glycoconj J 20, 483–492 (2003). https://doi.org/10.1023/B:GLYC.0000038294.72088.b0

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