Abstract
We have metabolically labeled the human pancreatic tumor cell line PANC-1 with high specific activity tritiated sugar precursors to study the expression of glycosphingolipids by this cell type. We have used a combination of detergent solubilization, exhaustive protease digestion, ceramide glycanase digestion, and reverse-phase chromatography to isolate glycosphingolipid-derived oligosaccharides specifically labeled in their component sugars. A significant proportion of the oligosaccharides derived from polar glycosphingolipids were of high molecular mass (greater than 2000 Da). The results of compositional studies, lectin affinity chromatography, and methylation analysis suggested that this high molecular weight fraction consists of lactosaminoglycan type oligosaccharides derived from polyglycosylceramides. There are on average three beta 1-6 linked N-acetyllactosamine branches attached to the polylactosamine backbone in this type of glycosphingolipid-derived oligosaccharide. The majority of the oligosaccharides also contain 1-2 mol of sialic acid that are linked alpha 2-3 to penultimate galactose. The results indicate that PANC-1 cells, like human colorectal tumor cells, express highly extended neolacto type glycosphingolipids. However, the lactosaminoglycan sequences are highly branched, unlike those associated with colorectal tumor cells.
Highlights
We have metabolically labeled the human pancreatic tumor celllinePANC-1withhighspecificactivity tritiated sugar precursors to study the expression of glycosphingolipids by this cell type
Increase the sensitivity of the analyses, we have incubated cells with high specific activity [3H]sugar precursors to label the glycosphingolipids in their oligosaccharide component
Analysis of the glycosphingolipid-derived oligosaccharides obtained from the nativ[3eH]glycolipids suggests thatPANC-1 cells synthesize a population of very large, highly branched lactosaminoglycan type glycosphingolipids that were originally designated polyglycosylceramides [11]
Summary
[6-3H]Gal (25 Ci/mmol), [6-3H]GlcNH2(30 Ci/mmol), [6-3H]Fuc’ (25 Ci/mmol) and Pronasewere purchased from ICN Radiochemicals (Costa Mesa, CA). Sep-Pak CIS cartridges were purchased from Waters tal tumor cells, express highly extended neolacto type glycosphingolipids. The lactosaminoglycan sequences are highlybranched, unlike those associated with colorectal tumor cells. Glycosphingolipids are ubiquitous mammalian cell membrane constituents that arceomposed of a monosaccharide or oligosaccharidelinked to ceramide,a membrane anchoring component. Glycosphingolipids have been shownto represent receptors for bacterial toxins, adhesion sites for pathogenic bacteria and cell matrix proteins, blood group antigens, and tylphenoxypolyethoxyethanol),PWM coupled to agarose (1.5 mg/ml) and dextran (Mr523,000) were purchased from Sigma. RCA,,, immobilized on agarose (4 mg/ml), WFAagarose (4 mg/ml gel), GS-11-agarose(4 mg/ml),LTA-agarose (4 mg/ ml), GS-I lectin, and WGA were purchased from E-Y Laboratories (San Mateo, CA). WGA was immobilized on Affi-Gel 10 as described by the manufacturer a t a final concentration of 30 mg/ml gel. This type of glycolipid has alsobeen implicated in cell-cell recognition and growth inhibition (reviewed in Ref. 3)
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