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Decrease in Polylactosaminoglycans Associated with Lysosomal Membrane Glycoproteins during Differentiation of CaCo-2 Human Colonic Adenocarcinoma Cells¹

McGill Cancer Center, McGill University, Montreal, Quebec, Canada H3G 1Y6 [A. Y., P. A. R., K. Y., A. H.] and La Jolla Cancer Research Foundation, Cancer Research Center, La Jolla, California 92037 [S. R. C., M. F.]

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-polacrylamide 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-ß-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-ß-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.

¹ This work was supported by the Medical Research Council of Canada, by the Cancer Research Society, and by Grant CA-48737 to M. K. from the NIH.

² Supported by a Studentship from the Fonds de la Recherche en Santé du Québec. Present address: Department of Biochemistry and Molecular Biology, M. D. Anderson Hospital and Tumor Institute, 1515 Holcombe Boulevard, Box 117, Houston, TX 77030.

³ To whom requests for reprints should be addressed, at the McGill Cancer Center, 3655 Drummond St., Montreal, Quebec, Canada H3G 1Y6.

Received 4/11/89. Revised 8/21/89. Accepted 9/18/89.

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