GLYCOPROTEIN PROCESSING GLUCOSIDASES

我们研究的最终目的是了解蛋白质糖基化生化机制各组成部分的结构/功能关系，以及它们在动物生长发育过程中基因表达的分子基础。研究的重点是乳腺个体发育过程中N-连接糖蛋白的生物合成和调控。这些蛋白质的前体碳水化合物单位的生物合成是由三个分支的Glc3Man9GlcNAC2-P-P-Dolicol逐步地、Dolicol连接的组装启动的，然后将其整体转移到RER中的新生多肽。随后，对寡糖部分进行广泛的重新建模，以产生完整的糖蛋白。糖苷酶I和II在N-连锁糖蛋白合成的翻译后成熟阶段是并列的，因为这两种酶的顺序作用触发了细胞分泌途径中糖蛋白成熟的寡糖加工和多肽折叠机制。抑制糖苷酶I和II可以干扰内质网糖蛋白的正常折叠、转运和排出，并导致错误折叠的糖蛋白积聚和降解。寡糖加工的损伤已被证明影响许多糖蛋白的生物活性、细胞表面受体的运输、成肌细胞融合、病毒组装和感染性、体外逆转细胞转化的表型以及抑制体内肿瘤细胞的转移。在前期研究的基础上，我们提出了以下具体目标：1和2.通过新型光亲和探针的标记、与自杀底物的接合以及对进化保守的亲核性和酶中酸性残基的诱变相结合，确定糖苷酶I和II的氨基酸残基和催化亲核体；3，表达具有催化活性的重组形式的葡萄糖苷酶I和II，并确定这些酶的晶体结构；4.研究亚基相互作用在乳腺发育和产乳分化过程中对葡萄糖苷酶II调控的意义。N-连接糖蛋白是糖蛋白中最大的一类，参与多种生物学现象，参与多种病理过程，包括恶性肿瘤、动脉粥样硬化、多种遗传性疾病以及导致艾滋病的宿主病毒相互作用等。转基因生物技术有可能为糖蛋白药物的“分子制药”提供一个极好的生物反应器。

The ultimate objective of our research is to understand the structure/function relationships of the components of the biochemical machinery for protein glycosylation and the molecular basis of their gene expression during animal growth and development. The focus of investigation is on the biosynthesis and regulation of N-linked glycoproteins in the mammary gland during its ontogeny. The biosynthesis of the precursor carbohydrate unit of these proteins is initiated by a stepwise, dolichol-linked assembly of the tri-branched Glc3Man9GlcNAC2-P-P-dolichol followed by its transfer en bloc to the nascent polypeptide in the RER. Subsequently, an extensive re-modeling of the oligosaccharide moiety occurs to give rise to completed glycoproteins. Glucosidases I and II are critically juxtapositioned in the post-translational maturation phase of N-linked glycoprotein synthesis since the sequential action of these two enzymes serves as a trigger for the oligosaccharide processing and polypeptide folding machinery for glycoprotein maturation in the secretory pathway of the cell. Inhibition of glucosidases I and II has been shown to interfere with normal folding, transport and egress of glycoproteins from the ER and cause accumulation and degradation of the malfolded glycoproteins. The impairment of oligosaccharide processing has been shown to affect the biological activity of many glycoproteins, transport of receptors of the cell surface, myoblast fusion, virus assembly and infectivity, reversal of the transformed phenotype of cells in vitro, and inhibition of tumor cell metastasis in vivo. Based on preliminary studies, we propose to pursue the following specific aims: 1 and 2. Identify the active site of amino acid residues and the catalytic nucleophile of glucosidases I and II by a combination of labeling with novel photoaffinity probes, conjugation with a suicide substrate and mutagenesis of selected evolutionary conserved nucleophiles and acidic residues in the enzymes; 3, Express catalytically active recombinant forms of Glucosidases I and II, and determine the crystal structure of the enzymes; 4. Investigate the significance of subunit interaction in the regulation of glucosidase II during the development and lactogenic differentiation of the mammary gland. N-linked glycoproteins, with diverse and versatile sugar moieties, represent the largest class of glycoproteins, participate in myriad biological phenomena, and are implicated in numerous pathologies, including malignancy, atherosclerosis, many genetic disorders and host-viral interaction leading to AIDS. Transgenic biotechnology can potentially provide the mammary gland as an excellent bioreactor for a 'molecular pharming' of glycoprotein pharmaceuticals.