Metastasis-associated murine melanoma cell surface galactosyltransferase: characterization of enzyme activity and identification of the major surface substrates.
Surface galactosyltransferase (GT) has been described on a variety of cells where it is believed to be involved in cell-cell and cell-substratum adhesion. Here we show that B16 metastatic murine melanoma cells exhibit a 5-fold higher cell surface GT activity than their nonmetastatic counterparts, although total GT activity in NP-40 solubilized cells is similar for both melanoma variants. Interestingly, on living cells, this cell surface GT almost exclusively galactosylates an endogenous glycoprotein (Mr = 110,000). This metastasis-associated GT is specific for terminal D-N-acetylglucosamine, catalyzes the formation of a beta 1-4 linkage, does not recognize polylactosaminoglycans, and has its specificity altered from D-N-acetylglucosamine to D-glucose by alpha-lactalbumin; yet the Mr = 110,000 protein is not a major substrate when exogenous bovine GT is used on the outside of living cells. In addition to this protein-specific endogenous GT activity, another cell surface GT activity that selectively galactosylates glucosylceramide is also prominent. Endogenous galactosylation of both protein and glycolipid substrates is reduced when the membrane is solubilized by the detergent NP-40 but remains unaltered in the presence of digitonin, which permeabilizes but does not dissolve the membrane. These data suggest that the GTs and their substrates are associated on the cell surface. Chloroquine treatment of intact cells leads to a 4-fold and a 3-fold increase in galactosylation of the Mr = 110,000 protein and glucosylceramide, respectively, suggesting that these two substrates normally reside mostly in the lysosomal or Golgi compartments. The increased expression of lysosomal membrane proteins on the surfaces of highly metastatic cells may, in part, also explain the galactosylation differences observed. These studies further suggest that increased surface localization of certain glycosyltransferases with highly restricted in situ substrate specificities may be a common feature of highly metastatic tumor cells.
Passaniti
A
A
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Hart
G W
GW
D000818:Animals; D015703:Antigens, CD; D002462:Cell Membrane / Q000378:metabolism; D002738:Chloroquine / Q000494:pharmacology; D002855:Chromatography, Thin Layer; D005677:G(M1) Ganglioside; D005690:Galactose / Q000378:metabolism; D005700:Galactosyltransferases / Q000378:metabolism*; D006017:Glycolipids / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism; D006028:Glycosphingolipids / Q000378:metabolism; D006031:Glycosylation; D007536:Isomerism; D007768:Lactalbumin / Q000494:pharmacology; D007790:Lactosylceramides; D008545:Melanoma / Q000201:enzymology* / Q000473:pathology; D051379:Mice; D008970:Molecular Weight; D009362:Neoplasm Metastasis; D011499:Protein Processing, Post-Translational; D014407:Tumor Cells, Cultured
D015703:Antigens, CD; D006017:Glycolipids; D006023:Glycoproteins; D006028:Glycosphingolipids; D007790:Lactosylceramides; D005677:G(M1) Ganglioside; C009744:CDw17 antigen; C018835:asialo GM1 ganglioside; D002738:Chloroquine; D007768:Lactalbumin; D005700:Galactosyltransferases; D005690:Galactose