Our research for the next year involves the completion of our analysis of the asparagine-linked oligosaccharides of BHK and polyoma-transformed BHK cells by sequential lectin-affinity chromatography (SLAC) and methylation analyses. Two lectins, E-phytohemagglutinin and one from Datura stamonium, will be added to the analysis of the glycopeptides. Next, each of the 29 L-PHA-resistant PyBHK clones will be screened for its ability not to bind labeled L-PHA using the procedure we have developed for the parental PyBHK and the BHK cells. Any clone which can be isolated that is L-PHA?-? will be radiolabeled with [2-?3?H]-mannose, and its Asnlinked glycopeptides analyzed by SLAC and compared to those from the parental PyBHK cells. The next series of experiments involves the production of tumors in hamsters by injection of PyBHK cells and their lectin-resistant variants. We will be able to compare the Asn-linked glycopeptides of the tumors by radiolabeling in organ culture and then SLAC analysis. We wish to know if a particular oligosaccharide phenotype is selected for in vivo. We will finish the analysis of the glycosyltransferases that participate in the synthesis of the Asn-linked oligosaccharides. We will determine if the specific activities of these enzymes change upon transformation and will attempt to explain the differences in the oligosaccharide structures between the two cell types in terms of enzyme activity differences.
The final aim will be to finish the examination of the various glycoproteins on the surfaces of the BHK, PyBHK, and variant cells which express the highly branched structures and determine if the increase in branched structures occurs on all or only a few of these glycoproteins. This analysis, coupled with the results of the glycosyltransferase activity comparisons, should allow us to focus on the immediate cause in the changes in oligosaccharide branching patterns. (A)
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