GLYCOPROTEIN PROCESSING GLUCOSIDASES

我们研究的最终目的是了解动物生长发育过程中蛋白质糖基化生化机制组成部分的结构/功能关系及其基因表达的分子基础。研究的重点是n -连接糖蛋白在乳腺个体发育过程中的生物合成和调控。这些蛋白质的前体碳水化合物单位的生物合成是由三支链glc3man9glcnac2 - p - p -多醇的一步式多醇连接组装开始的，然后将其整体转移到内质网中的新生多肽。随后，低聚糖部分的广泛重新建模产生完整的糖蛋白。糖苷酶I和糖苷酶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是触发细胞分泌途径中糖蛋白成熟的低聚糖加工和多肽折叠机制。葡萄糖苷酶I和II的抑制已被证明会干扰糖蛋白从内质网的正常折叠、运输和输出，并导致折叠不良的糖蛋白的积累和降解。低聚糖加工的损伤已被证明会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、与自杀底物偶联和诱变选择进化保守的亲核试剂和酶中的酸性残基，鉴定葡萄糖苷酶I和II的氨基酸残基和催化亲核试剂的活性位点；3、表达具有催化活性的葡萄糖苷酶I和葡萄糖苷酶II的重组形式，并确定酶的晶体结构；4. 探讨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调控中的意义。n -连锁糖蛋白是糖蛋白中最大的一类，具有多样化和多用途的糖段，参与无数的生物现象，并涉及许多病理，包括恶性肿瘤、动脉粥样硬化、许多遗传疾病和导致艾滋病的宿主-病毒相互作用。转基因生物技术可以为乳腺提供一个极好的生物反应器，用于糖蛋白药物的“分子制药”。

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.