FUNCTIONS OF HUMAN MILK FAT GLOBULE GLYCOPROTEINS

本项目的长期目标是界定 人乳脂肪球（HMFG）膜的糖蛋白成分， 抑制病毒受体结合和感染性。 由于轮状病毒是 是婴儿和儿童严重胃肠炎的主要原因， 体内和体外感染性测定目前可用于 首先，他们将被强调。 接下来我们将研究人类 免疫缺陷病毒（艾滋病毒），因为艾滋病毒的围产期传播是 母乳喂养的作用 传播仍有争议。 我们已经证明牛奶 粘蛋白（MM）复合物，其含有MM和相关的70 KDa， 可能是46 KDa的糖蛋白，与轮状病毒结合并抑制其 在体外和体内的感染性，并可能抑制HIV结合其CD 4 受体的 因此，具体目标是：1）生产成熟，不成熟， 和主要HMFG糖蛋白的重组片段，以鉴定 病毒抑制中的功能域及其结构。 成熟 糖蛋白将从HMFG中单独纯化。 不成熟的形式将 在乳腺癌细胞系中生物合成制备， 分子不同，然后以类似的方式纯化。 部分 具有抗病毒活性的HMFG组分的完整cDNA克隆 将从乳腺cDNA文库中分离并用于制备， 未成熟和选择的原核和真核细胞的转染 这些分子的片段具有不同程度的糖基化。 （二） 利用这些分子探针， 将首先通过特异性受体结合试验进行测试。 那么传染性 对轮状病毒的抑制将通过体外和体内试验进行测试 和HIV的体外试验。 3)单克隆抗体将是 对MM成分，干扰其抑制 病毒-受体相互作用和使用表位作图确定的表位 和寡糖的分析。 该计划结合了我们的专业知识， Yolken博士在儿科感染性疾病中的HMFG糖蛋白 以及Newburg博士在牛奶低聚糖化学分析方面的贡献。

The long-term objective of this project is to delineate the role of glycoprotein components of the human milk fat globule (HMFG) membrane in inhibiting viral receptor binding and infectivity. Since rotaviruses are a major cause of severe gastroenteritis in infants and children, and since both in vivo and in vitro infectivity assays are currently available for them, they will be initially emphasized. Next we will study human immunodeficiency virus (HIV), since perinatal transmission of HIV is becoming a major health concern and the role of breast feeding in transmission is still controversial. We have already shown that the milk mucin (MM) complex, which contains the MM and associated 70 KDa and possibly the 46 KDa glycoproteins, binds to rotaviruses and inhibits their infectivity in vitro and in vivo, and may inhibit HIV binding to its CD4 receptor. Therefore, the Specific Aims are: 1) Produce mature, immature, and recombinant fragments of the major HMFG glycoproteins to identify the functional domain and its structure in virus inhibition. Mature glycoproteins will be purified individually from HMFG. Immature forms will be prepared biosynthetically in breast carcinoma cell lines that process the molecules differently and then purified in a similar manner. Partial and complete cDNA clones of the HMFG components with antiviral activity will be isolated from breast cDNA libraries and used to prepare, by transfection of procaryotic and eucaryotic cells, immature and selected fragments of these molecules with different degrees of glycosylation. 2) With these molecular probes the functional domains for antiviral activity will be first tested by specific receptor binding assay. Then infectivity inhibition for rotavirus will be tested with in vitro and in vivo assays and for HIV, with in vitro assays. 3) Monoclonal antibodies will be developed against the MM component that interfere with its inhibition of virus-receptor interaction and the epitopes defined, using epitope mapping and analysis of oligosaccharides. This program combines our expertise on the HMFG glycoproteins, that of Dr. Yolken in pediatric infectious disease and of Dr. Newburg in chemical analysis of milk oligosaccharides.