GLYCOPROTEIN PROCESSING GLUCOSIDASES

糖蛋白加工葡萄糖苷酶

基本信息

批准号：
6182286
负责人：
INDER K VIJAY
金额：
$ 35.99万
依托单位：
UNIV OF MARYLAND, COLLEGE PARK
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-09-01 至 2003-08-31
项目状态：
已结题

项目摘要

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.

我们研究的最终目的是了解蛋白质糖基化生化机制成分的结构/功能关系，以及在动物生长和发育过程中其基因表达的分子基础。研究的重点放在乳腺中N连接糖蛋白的生物合成和调节过程中。这些蛋白质的前体碳水化合物单位的生物合成是由三支三支球菌的GLC3MAN9GLCNAC2-P-P-P-P-DOLICHOL的逐步的，多甲基连接的组件启动的，然后是其在RER中向新生的多肽转移的。随后，发生寡糖部分的广泛重新建模，从而产生完整的糖蛋白。葡萄糖酶I和II在N连接糖蛋白合成的翻译后成熟阶段进行了严格并置，因为这两种酶的顺序作用是触发寡糖处理和多肽折叠机器的寡糖处理和分泌途径的多肽折叠机器。抑制葡萄糖酶I和II的抑制已显示出干扰正常的折叠，糖蛋白从ER中的折叠，转运和出口，并导致碎糖蛋白的积累和降解。已显示寡糖加工的损害会影响许多糖蛋白的生物学活性，细胞表面受体的转运，成肌细胞融合，病毒组装和感染性，体外细胞转化的表型的反转以及肿瘤细胞转移的抑制作用。基于初步研究，我们建议追求以下特定目的：1和2。确定氨基酸残基的活性位点以及葡萄糖酶I和II的催化核定型，通过将标记与新型光接触探针结合结合，结合了与所选核化核量和酸性残基的抗性底物的结合和酸性的诱导底物和酸性突变; 3，葡萄糖酶I和II的表达催化活性重组形式，并确定酶的晶体结构； 4。研究亚基相互作用在乳腺发育和乳酸分化过程中葡萄糖酶II的调节中的重要性。具有多种多样和多功能糖部分的N连接糖蛋白代表了最大的糖蛋白类，它参与了无数的生物学现象，并且与许多病理相关，包括恶性，动脉粥样硬化，许多遗传疾病和宿主相互作用，导致艾滋病。转基因生物技术可以潜在地提供乳腺作为糖蛋白药物“分子药物”的出色生物反应器。