The long term goal of this project is to understand the function of complex carbohydrates on secreted and cell-surface glycoproteins through structural chemistry. The proposal for the enxt budget period comprises an investigation on the effect of glycosylation of fibronectin on its intracellular matrix binding properties. Fibronectin (Fn) from human placental tissue and plasma will be used in this study, since the protein is readily availabale. Fn is important in medicine because of its probable role as the cell-binging component in the extracellular matrix and in its crucial participation in wound healing. We plan the following experiments: 1) Mapping of the location of specific types of oligosaccharide chains on the Fn 44Kda chymotryptic collagen-binding domain: In its most highly glycosylated form Fn contains polylactosamine structures, and has a lower binding affinity for denatured collagen (gelatin) than its less glycosylated counterparts. We plan to map the location of N-linked polylactosamine structures, which occur on the most highly glycosylated, weaker-binding forma. 2) Determination of carbohydrate structural parameters resulting in weakened collagen binding: This second part of our plan is to partially degrade the oligosaccharide chains on the more weakly binding, highly glycosylated Fn and its fragments, using the partially degraded fragments for comparative binding studies with gelatin and a 21Kda col-lagen fragment to find the molecular limits of the effects on collagen-binding by certain saccharides. 3) Use of physical probes to examine the extent of the effect of glycosylation on the protein conformation of the gelatin-binding domain of fibronectin: Tryptophan fluorescence will be used to probe the proximity and effect of the polylactosamine chain on the Fn-gelatin binding site. We will examine whether tighter binding resulting from removal of carbohydrate arises from protein conformation alterations, as judged by changes in tryptophan fluorescence, and whether the protein conformation is altered by the presence of either complex or polylactosamine chains on the polypeptide.
| 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 |
