GLYCOPROTEIN PROCESSING GLUCOSIDASES

我们研究的最终目标是了解蛋白质糖基化生化机制的组成部分的结构/功能关系及其在动物生长发育过程中基因表达的分子基础。研究的重点是在其个体发育过程中的乳腺N-连接糖蛋白的生物合成和调节。这些蛋白质的前体碳水化合物单元的生物合成起始于三分支Glc 3 Man 9 GlcNAC 2-P-P-dolichol的逐步的、dolichol连接的组装，随后将其整体转移到RER中的新生多肽。随后，寡糖部分发生广泛的重新建模以产生完整的糖蛋白。葡萄糖醛酸酶I和II在N-连接糖蛋白合成的翻译后成熟阶段是关键性并置的，因为这两种酶的顺序作用作为细胞分泌途径中糖蛋白成熟的寡糖加工和多肽折叠机制的触发剂。已显示抑制葡糖醛酸酶I和II干扰糖蛋白从ER的正常折叠、转运和外出，并引起错误折叠的糖蛋白的积累和降解。寡糖加工的损伤已被证明影响许多糖蛋白的生物活性、细胞表面受体的转运、成肌细胞融合、病毒组装和感染性、体外细胞转化表型的逆转以及体内肿瘤细胞转移的抑制。在初步研究的基础上，我们建议实现以下具体目标：1和2。通过结合新型光亲和探针标记、与自杀底物缀合以及选择的进化保守的亲核试剂和酶中的酸性残基的诱变，鉴定葡糖醛酸酶I和II的氨基酸残基和催化亲核试剂的活性位点; 3、表达葡糖醛酸酶I和II的催化活性重组形式，并确定酶的晶体结构; 4.研究亚基相互作用在乳腺发育和泌乳分化过程中葡萄糖苷酶II调节中的意义。N-连接的糖蛋白，具有不同的和通用的糖部分，代表了最大类别的糖蛋白，参与无数的生物学现象，并涉及许多病理学，包括恶性肿瘤，动脉粥样硬化，许多遗传疾病和宿主-病毒相互作用导致艾滋病。转基因生物技术可以潜在地为糖蛋白药物的“分子嫁接”提供优良的乳腺生物反应器。

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.