STRUCTURE, BIOSYNTHESIS, AND FUNCTION OF GLYCOPROTEINS

我们的研究有两个基本的研究。 第一个是要了解 糖蛋白的寡糖单元是如何合成的， 在生物过程中作为识别标记发挥作用。 二是 在分子水平上理解新合成的溶酶体酶 被导向溶酶体。 对于第一个目标，我们计划使用凝集素- 在寡糖生物合成中具有特异性阻断的抗性细胞系 以发现寡糖合成的新途径。 几 将进行方法以理解溶酶体酶靶向。 第一、 我们计划识别溶酶体的共同蛋白质识别标志物， 产生磷酸甘露糖基残基所需的酶， 用作将蛋白质靶向溶酶体的识别信号。 到 这样做，我们将构建嵌合分子之间的分泌 蛋白质胃蛋白酶原和溶酶体酶组织蛋白酶D来定义 磷酸转移酶识别所需的组织蛋白酶D区域， 随后磷酸化。 我们计划克隆磷酸转移酶， 使用该克隆研究粘脂沉积症II型患者的缺陷， 两种常染色体隐性遗传病的磷酸转移酶。 我们 还计划克隆α-N-乙酰葡糖胺-1-磷酸二酯N- 乙酰氨基葡萄糖苷酶，生成所需的第二种酶， 磷酸甘露糖残基。 此外，我们计划对cDNA进行诱变， 对于阳离子非依赖性和阳离子依赖性甘露糖6-磷酸 受体，以产生具有突变和缺失的受体分子， 它们的胞质结构域。 这些cDNA将被转染到受体中 阴性小鼠L细胞，以确定对受体功能的影响， 高尔基体的溶酶体酶分选和细胞表面的内吞作用。

Our studies have two basic studies. The first is to understand how the oligosaccharide units of glycoproteins are synthesized and how they function as recognition markers in biologic processes. The second is to understand at the molecular level how newly synthesized lysosomal enzymes are directed to lysosomes. For the first goal, we plan to use lectin- resistant cell lines with specific blocks in oligosaccharide biosynthesis to uncover new pathways for the synthesis of oligosaccharides. Several approaches will be made to understand lysosomal enzyme targeting. First, we plan to identify the common protein recognition marker for lysosomal enzymes required for the generation of phosphomannosyl residues which serve as the recognition signal for targeting proteins to lysosomes. To do this, we will construct chimeric molecules between the secretory protein pepsinogen and the lysosomal enzyme cathepsin D to define the regions on cathepsin D needed for recognition by phosphotransferase and subsequence phosphorylation. We plan to clone phosphotransferase and to use this clone to study the defects in patients with mucolipidosis II and III, two autosomal recessive genetic disorders of phosphotransferase. We also plan to clone alpha N-acetylglucosamine-1-phosphodiester N- acetylglucosaminidase, the second enzyme required for the generation of phosphomannosyl residues. In addition, we plan to mutagenize the cDNA's for the cation-independent and the cation-dependent mannose 6-phosphate receptors to generate receptor molecules with mutations and deletions in their cytoplasmic domains. These cDNA's will be transfected into receptor negative mouse L cells to determine the effects on receptor function in lysosomal enzyme sorting at the Golgi and endocytosis at the cell surface.