Decrease in polylactosaminoglycans associated with lysosomal membrane glycoproteins during differentiation of CaCo-2 human colonic adenocarcinoma cells.
The proportion of labeled polylactosaminoglycans found in glycoproteins decreases during spontaneous differentiation of CaCo-2 human colonic adenocarcinoma cells to enterocytes in culture (A. Youakim and A. Herscovics, Biochem. J., 247: 299-306, 1987). To identify polylactosaminoglycan-containing glycoproteins, CaCo-2 cells were incubated with [3H]glucosamine or [3H]fucose, for 24 h, and membrane glycoproteins solubilized with 0.5% Nonidet P-40 were fractionated by affinity chromatography on Datura stramonium (DSA)-agarose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography showed that a restricted set of glycoproteins with a molecular weight of about 100,000 bound to DSA-agarose. These labeled glycoproteins were shown to contain polylactosaminoglycans by DSA-agarose chromatography and endo-beta-galactosidase digestion of Pronase-derived glycopeptides. Immunoprecipitation of the [3H]glucosamine-labeled Nonidet P-40 extract with polyclonal antibodies to the lysosomal membrane proteins h-lamp-1 and h-lamp-2 followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography also revealed a band with a molecular weight of about 100,000. The immunoprecipitates were digested with Pronase, and the resulting glycopeptides were first fractionated on Bio-Gel P-6 into excluded (Fraction I) and included (Fraction II) glycopeptides, and then by DSA-agarose affinity chromatography. A much greater proportion of labeled glycopeptides in undifferentiated cells (3 to 5 days in culture) than in differentiated cells (19 to 27 days in culture) was recovered in Fraction I; these glycopeptides were bound to DSA-agarose and were sensitive to endo-beta-galactosidase. This decrease in polylactosaminoglycans was associated primarily with h-lamp-1. These results indicate that h-lamp-1 of CaCo-2 cells contains polylactosaminoglycans and that it undergoes a change in glycosylation with differentiation.
Youakim
A
A
McGill Cancer Center, McGill University, Montreal, Quebec, Canada.
Romero
P A
PA
Yee
K
K
Carlsson
S R
SR
Fukuda
M
M
Herscovics
A
A
D000230:Adenocarcinoma / Q000378:metabolism* / Q000473:pathology; D000606:Amino Sugars / Q000378:metabolism*; D002454:Cell Differentiation; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003110:Colonic Neoplasms / Q000378:metabolism* / Q000473:pathology; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D000606:Amino Sugars; D008562:Membrane Glycoproteins; D011134:Polysaccharides