A group of post-translational modification mutants in Dictyostelium discoideum appear to have decreased amounts of sulfate and/or mannose-6-phosphate on the oligosaccharides of their lysosomal enzymes. Phenotypically, some of these mutants are deficient in either the coordinate secretion of several lysosomal enzymes or in the accumulation of at least one of these developmentally regulated enzymes. Still others appear to have repackaged one lysosomal enzyme into a different vesicle. All of these mutants may be blocked at different steps of normal oligosaccharide processing of lysosomal enzymes which may, in turn, lead to these various abnormal phenotypes. To address this question the project will examine the detailed structure of 3H-labeled oligosaccharide of lysosomal enzymes in each of these mutants in terms of size, charge, location of Man-6-P and sulfate groups and the linkage relationships of each of the various saccharides. The results of these analyses will be compared to those previously obtained from analysis of similar oligosaccarides isolated from the lysosomal enzymes of normal cells. The nature of the structural alterations found in each mutant will serve as a guide to identifying its primary defect. The results of the analysis of the altered oligosaccharide structures in the mutants can be used to dissect the various steps n the biosynthesis and processing of these complex oligosaccharides. In some instances we may be able to establish a causal relationship between an altered oligosaccharide and proper cellular localization or secretion of lysosomal enzymes. These results may be useful in determining whether slime molds like mammalian fibroblasts target their lysosomal enzymes via a Man-6-P specific intracellular receptor or whether another pathway is used. This could be significant since other mammalian cell types such as liver and spleen do not use the Man-6-P receptor system to target their lysosomal enzymes. The mechanism of packaging in the other system is unknown.
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