Many plasma membrane receptors, such as those for insulin, for LDL, for acetylcholine, etc., are glycoproteins. All of these receptors contain N-linked oligosaccharides. Although many of the steps are known that lead to the assembly of the lipid-linked oligosaccharide donor, few of the enzymes have been purified or characterized, and little is known about how the pathway is regulated. Such information is of considerable importance since alterations in carbohydrate structure may be involved is such disease conditions as atherosclerosis, diabetes, cancer, etc. We propose to purify and study several enzymes that appear likely to be regulatory sites in the pathway. One of these enzymes is the GlcNAc-1-P transferase that catalyzes the first step in the pathway; another is the mannosyl transferase that adds the sixth mannose to the lipid- linked oligosaccharides. In this latter reaction, there is a change in mannosyl donor from GDP-mannose to dolichyl-P-mannose. We think we have some good assays for these enzymes and some reasonable affinity steps for purification. We propose to do these studies in several plant systems for a number of reasons including the fact that soybean or sycamore cells are very inexpensive to grow in culture and it is quite reasonable to grow large amounts of cells (i.e., up to a Kg of cells). In addition, the enzymes of the lipid-linked saccharide pathway in plants appear to be much more stable after solubilization than those of animal cells. Thus purification should be more feasible in the plant system. We also plan to purify several of the processing enzymes to homogeneity in order to prepare polyclonal antibodies. Such antibodies will be used for enzyme localization as well as for studies on biosynthesis and targeting of these enzymes. We now have antibody against glucosidase II, an early processing enzyme, and will finish the purification of mannosidase II, a mid stage enzyme, and GlcNAc transferase II, a late-stage processing enzyme. Among the questions to be addressed is whether plants have cis, medial and trans Golgi, and whether the various activities are in different areas of the Golgi. Other questions deal with the signals that direct these proteins to the specific sites. Since we have many of these processing enzymes purified, we will attempt to set up an in vitro processing system. As glycoproteins become cloned in bacteria, it will become important to be able to glycosylate and process these proteins in cell-free systems.
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