FUNCTIONS OF HUMAN MILK FAT GLOBULE GLYCOPROTEINS

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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.