Expression of PNA-binding sites on specific glycoproteins by human melanoma cells is associated with a high metastatic potential

Lectin-binding patterns of seven human melanoma clones and variants selected from the same parental cell line and differing in their spontaneous metastatic potential in an animal model were compared by flow cytometry and Scatchard analysis. Human melanoma clones and variants with high and low metastatic potential could be distinguished by their peanut agglutinin (PNA)–binding patterns, but not by their wheat germ agglutinin (WGA)–, Ulex europaeus agglutinin I (UEA I)–, and soybean agglutinin (SBA)–binding patterns. Low metastatatic clones and variants proved to be made up of a single poorly peanut agglutinin–binding cell population (2.20–3.52 × 10⁶ sites/cell, Ka = 2.48–2.75 × 10⁶ M^?1). By contrast, highly metastatic variants were found to be constituted by two cellular subpopulations, exhibiting respectively a moderate (2.62–3.72 × 10⁶ sites/cell) and a high peanut agglutinin staining (17.68–18.76 × 10⁶ sites/cell). One highly metastatic clone was found to be homogeneously constituted by a single population of cells strongly binding this lectin (18.86 × 10⁶ sites/cell) with an association constant of 4.06 × 10⁶ M^?1. Using an EPICS V cytometer, these two subpopulations were sorted from a highly metastatic variant and tested for their metastatic abilities: cells with high PNA binding generated a higher frequency of metastases than did moderately PNA-binding cells. Following treatment with Vibrio cholerae neuraminidase, all cells from all variants and clones were brightly labeled by PNA, collecting in a single peak with similar fluorescence intensities. Electrophoresis of total cellular proteins and subsequent detection with labeled PNA on Western blots show two major PNA-reactive glycoproteins with apparent molecular weights of 140 and 110 kDa (MAGP1 and MAGP2), expressed only in highly metastatic cells, but which can be strongly labeled by PNA in slightly metastatic cells following a treatment with neuraminidase. These results provide evidence that the expression of terminal galactose (β1–3)N-acetyl galactosamine structures, positioned on MAGP1 and MAGP2 glycoproteins, is associated with the metastatic potential of human melanoma cells.