The goal of this project is to map the carbohydrate binding site of a beta-galactoside specific lectin. Several members of this unique class of endogenous lectins are expressed on the surface of tumorigenic cells and have been implicated in the promotion of cell-cell adhesion; one of the necessary steps in tumor metastasis.
The specific aim of this proposal is to probe the molecular features of the saccharide binding site so that those moieties on the beta- galactoside and the lectin which impart specificity to this lectin; carbohydrate interaction can be identified. This type of detailed knowledge is required in order to determine precisely which glycoconjugates can serve as functional ligands in vivo. Based on this information, they, altered ligands capable of specifically inhibiting unwanted cell-cell interactions could potentially be engineered. For this study, the beta-galactoside specific lectin present in human placenta will be used. Sufficient quantities of purified human placental lectin (HPL) can be readily isolated from a reasonable amount of starting material. """"""""Isotope-directed"""""""" nuclear Overhauser effect (IDNOE) NMR experiments will be used to locate those amino acids contained in the binding site. These experiments require carbon-13 labeled saccharides. Various carbon-13 containing lactoses and N-acetyllactosamines, isotopically labeled at one or several positions on the pyranose rings, will be enzymatically synthesized. A combination of two-dimensional NMR techniques will be used to assign the protein proton resonances. Those amino acid protons in close proximity to the protons on the bound 13C labeled lactoses will be identified from the results of the IDNOE experiments. Each selectively labeled disaccharide will give a set of interproton NOE's which will serve as a probe on one specific area of the binding site.
