I-CELL DISEASE AND MENTAL RETARDATION

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Lysosomal enzymes share a unique pathway of biosynthesis and posttranslational modification. The long-term objectives of this grant involve studies of structural information on acid hydrolases which is necessary for their intracellular sorting and subsequent targeting to lysosomes or extracellular fluids. I-cell disease (ICD) and pseudoHurler polydystrophy (PHP), two inherited childhood disorders, will be used as model systems. The primary defect responsible for these disorders is an absence or severe reduction of glcNac phosphotransferase, a key enzyme involved in the biosynthesis and targeting of acid hydrolases to lysosomes. Evidence exists for genetic and biochemical heterogeneity in ICD and PHP. The existence of complementation groups within and between the two disorders suggest the involvement of more than one gene mutation which affects the structure and function of the transferase. One experimental approach will examine selected properties of the enzyme from the different complementation groups, such as the enzyme's stability under various conditions including temperature, addition of protease inhibitors, sucrose loading, and the enzyme's interaction with various lectins. The purification of the transferase from autopsied human liver will be carried out using a combination of conventional and affinity column methods. The characterization of kinetic, chemical, and physical properties of the enzyme from both the normal and mutant sources will be useful in understanding the functions of the different gene product(s) required for enzyme activity. The production of polyclonal antisera will allow us to probe the basis for each mutation by comparing the biosynthesis and processing of immunoprecipitable material in normal and mutant cell lines after adding labeled amino acids or 3H mannose. Lymphoblasts prepared from normal controls, ICD and PHP patients will also be examined as an alternate source to study the glcNac phosphotransferase protein from the various complementation groups. A second approach in studying the structural requirements for intracellular segregation of acid hydrolases will employ cultured normal, ICD, and PHP skin fibroblasts and lymphoblasts that have been previously pulsed with 3H-mannose and grown in the presence or absence of various agents known to interfere with normal glycosylation (tunicamycin), posttranslational processing (swainsonine) and intracellular transport and secretion (monensin). Subcellular fractionation by density gradient centrifugation on percoll and/or free flow electrophoresis will allow separation of the fractions containing labeled acid hydrolases and subsequent analysis of the types of oligosaccharides present.