Characterization of Spider Mucin & Associated Compounds

Tillinghast, Edward

Abstract

Mucins are essential to the function of the respiratory, gastrointestinal, and reproductive systems of all vertebrates. Yet, in spite of malfunctions associated with certain diseases (e.g. cystic fibrosis), our knowledge of the structure and hydration of mucins remains uncertain. Much of this uncertainty results from the difficulty in separating mucins from other tissue components and artifacts resulting from tissue proteases. The webs of orb-weaving spiders present a very accessible system with which to seek answers to the deficiencies in our knowledge. The adhesive droplets of the web consist of a mucin-like molecule (Glycoprotein A), four major middle molecular weight proteins (MMW), and six low molecular weight, water-soluble compounds (LMW). Because the web is extracorporeal, neither the MMW nor LMW would be artifacts resulting from tissue proteases. Moreover evidence suggests that Glycoprotein A depends upon these compounds to achieve its required conformation and hydration state. The adhesive droplets, therefore, present a unique opportunity to explore the structure of mucins and the roles of its associated compounds. It is proposed here to (1) purify Glycoprotein A from the web of A. aurantia and to characterize it according to molecular weight (ultracentrifugation, gel filtration, electron microscopy), general structural features (distribution of oligosaccharides, sulfur, phosphate), using PAGE and radiolabelled precursors, glycan structure by gas-liquid chromatography, and specific glycosidases. (2) To examine the hypothesis that the MMW are a family of polypeptides that link Glycoprotein A into higher aggregates using PAGE and peptide mapping. Finally, we will initiate electron microscopic studies designed to visualize Glycoprotein A in the presence of MMW and LMW so as to deduce their contributions to mucin structure.