Biomedical subjects

A Youakim

Publications and source records attributed to A Youakim.

3 recordsLinked to original sources

PubMed1991

Evidence for a molecular distinction between Golgi and cell surface forms of beta 1,4-galactosyltransferase.

beta 1,4-Galactosyltransferase (GalTase) is present on the plasma membrane of many cell types in addition to its traditional location within the Golgi compartment. Recently, the GalTase gene has been shown to encode two proteins that are identical throughout their length except that one has an additional 13-amino acid extension in its amino-terminal cytoplasmic domain. We present evidence here suggesting that the longer GalTase protein, containing this unique 13-amino acid peptide, is preferentially targeted to the plasma membrane, and the shorter GalTase protein resides primarily within the Golgi compartment. S1 nuclease protection assays of RNA from a variety of cells and tissues show that the relative abundance of the short and long GalTase mRNAs correlates with GalTase-specific activities in the Golgi and plasma membranes, respectively. Furthermore, transfection of cDNAs encoding either the long or short GalTase protein into F9 embryonal carcinoma cells suggests that the long GalTase protein is preferentially expressed on the cell surface. These results propose a molecular distinction between the Golgi and cell surface forms of GalTase as well as a novel mechanism for targeting glycoproteins to the cell surface.

Amino Acid Sequence↗

PubMed1989

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.

Adenocarcinoma↗

PubMed1987

Differentiation-associated decrease in the proportion of fucosylated polylactosaminoglycans of CaCo-2 human colonic adenocarcinoma cells.

CaCo-2 cells are human colonic adenocarcinoma cells which can differentiate spontaneously into enterocytes when maintained confluent for extended periods of time. Cells kept in culture for 4 days (rapidly growing), 7-9 days (early confluence) and 19-22 days (late confluence) were incubated for 24 h with L-[5,6-3H]fucose or D-[6-3H]glucosamine in order to examine the changes in glycoprotein carbohydrate structure that occur during this differentiation. Labelled glycopeptides obtained by exhaustive Pronase digestion of the cell-surface and cell-pellet fractions were fractionated on Bio-Gel P-6. A high-Mr glycopeptide fraction which was excluded from Bio-Gel P-6 was present in all cases. These glycopeptides were then fractionated by affinity chromatography on Datura stramonium agglutinin-agarose. The glycopeptides which were specifically bound to the lectin column were largely degraded by endo-beta-galactosidase, thereby indicating that they consisted of fucosylated polylactosaminoglycans. The proportion of labelled polylactosaminoglycans decreased with increasing time in culture, whereas sucrase activity, which is characteristic of differentiated enterocytes, increased. These results demonstrate that a relatively large decrease in the proportion of fucosylated polylactosaminoglycans occurs with differentiation of CaCo-2 cells.

Adenocarcinoma↗