Previous studies have demonstrated the existence of a class of sulfated N-linked oligosaccharides in eukaryotic glycoproteins. However, little is currently known about their detailed structure, biosynthesis, regulation, or biological significance. In this proposal, we describe the establishment of an enzymatic method for quantitative and nondegradative release of intact sulfated N-linked oligosaccharides directly from whole proteins, and their complete separation from all other sulfated macromolecules. This method will be used to look for differences in the expression of sulfated N-linked oligosaccharides in total cell homogenates and on specific glycoproteins in the following continuous tissue culture systems: (1) CHO and mouse lymphoma cells with specific defects in N-linked glycosylation and/or treated with inhibitors of processing glycosidases; (2) NIH 3T3 cells before and after transformation by specific classes of oncogenes; (3) metastatic and nonmetastatic tumors cells; and (4) leukemia cells in various stages of induced differentiation. On the basis of the results of this screening, one or two of these systems will be chosen for further study. The individual oligosaccharides will be fractionated and the types of sulfated monosaccharides identified. This will be followed by a detailed structural analysis of the sulfated oligosaccharides, with regard to the location, linkages, and types of component sugars, using conventional and newly developed methods. This will define any differences in structure found within the systems chosen for study. The information obtained from the structural analysis will be used to predict the biosynthetic pathways for the generation of the sulfated oligosaccharides in these cells, and to predict possible levels of modulation that could explain any differences found. Finally, we will design assays that will identify the enzyme and substrates involved in the sulfation reactions in question.
The specific aim of this work will be to see if differences in the expression of the enzymes involved could explain differences in sulfation of N-linked oligosaccharides in these systems. In the long run, we hope to extend these studies to attempt to understand potential biological roles of the sulfation of N-linked oligosaccharides in processes such as tumorigenesis and metastasis formation. (B)
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