We plan to establish the location and structure of carbohydrates on human Fibronectins (Fn) as produced by cells in different tissues, and as produced at stages of differentiation. Fibronectins, a class of cellular and secreted glycoproteins, are important in wound healing, and are therefor significant in the survival of mammalian species, including man. As a first approach we will study the chemical structures and locations on the polypeptide chain, of oligosaccharides derived from human placental (fetal cellular) Fibronectin. For this purpose we have isolated 500mg qquantities of human placental cellular fibronectin, and have preliminary evidence of extensive differences in the quantity, structures and location of complex sugar chains when compared with the adult plasma form. The cellular form is more protease resistant. We have purified the 43K protease-resistant chymotryptic fragment (which contains 1/4 to 1/3 of the carbohydrate in human fibronectins) in quantities large enough to perform both mass spectral and proton-NMR studies, and are localizing and sequencing glycopeptides of unusual structure on the remaining polypeptide. Our interest is in the larger questions of the function, specificity and control of glycosylation of proteins. Chemical strcutura information gained in this project will suggest the enzymes which are controlled for specific glycocylation sites. The protection of proteins against proteases by oligosaccharides is another important aspect of this work, since the more highly glocosylated cellular fibronectin (6 chains vs 4 in plasma Fn) is much less susceptible to the enzymes we use for fragmentation. We intend to study protease-resistance in relation to the location of the oligosaccharide chains in these fibronectins. As sequencing of this significant human protein proceeds in other laboratories, our chemical studies on the carbohydrates will help complete the picture of its biochemical properties and lead to a chemical understanding of Fibronectins' many forms and biological effects. Development of sensitive methods for precise structure determination in complex sugars: We are planning in the first year to begin studies in utilization of revolutionary technological advances in mass spectrometry, including fast atom bombardment ionization, high field magnets for high mass, link scanning to scrutinize daughter ions, and collision activation prior to a third mass filtration stage (MS-MS). These new techniques will provide the next generation of methodology for carbohydrate sequencing.
| Yoon, E; Laine, R A (1992) Linkage position determination in a novel set of permethylated neutral trisaccharides by collisional-induced dissociation and tandem mass spectrometry. Biol Mass Spectrom 21:479-85 |
| Yoon, E; Laine, R A (1992) Synthesis of four novel trisaccharides by induction of loose acceptor specificity in Gal beta 1----4 transferase (EC 2.4.1.22): Galp(beta 1----4)Glcp(X)Glc where X = beta 1----3: beta 1----4: beta 1----6: alpha 1----4. Glycobiology 2:161-8 |
