CLONING, EXPRESSION, PURIFICATION AND CHARACTERIZATION OF DOMAIN B OF WHEAT GERM AGGLUTININ

• Main Author: Rice, Ann C.
• Format: Others
• Published: VCU Scholars Compass 1994
• Subjects: Biochemistry
• Online Access: https://scholarscompass.vcu.edu/etd/5270; https://scholarscompass.vcu.edu/cgi/viewcontent.cgi?article=6362&context=etd

Wheat germ agglutinin (WGA) belongs to a family of dimeric chitin binding lectins specific for N-acetyl-D-glucosamine (GlcNAc) and N-acetyl-D-neuraminic acid (NeuNAc). The polypeptide chain consists of a tandem repeat of four conserved 4.3 kDa domains (A, B, C, D) stabilized by four disulfide bonds. Saccharide binding occurs in the dimer interface where domains of different subunits are in contact (A with D, B with C). An aromatic amino acid rich pocket on one domain (sufficient for saccharide binding) and a polar region on the contacting domain constitute a complete binding site. Saccharide binding affinities may differ among the four unique sites (eight/dimer) due to sequence divergence. Two equivalent sites/monomer were detected in solution. However, conclusive evidence is lacking as to their locations on the dimer. To delineate individual specificities and dimerization requirements, it was desirable to isolate and characterize each site independently. This thesis describes an expression system by which single WGA domains can be efficiently generated as functional proteins. The B-domain was cloned first, because binding was observed at this site in several WGA-oligosaccharide crystal complexes. Two B-domain sequences were cloned varying at residue 28 (Ala→Ser). In a putative domain dimer the polar Ser28 would mimic an H-bond observed to stabilize NeuNAc in the WGA B-site from the contacting C-domain. The domains were expressed as fusion proteins from which they were proteolytically separated and isolated in high yields. The recombinant domains were shown to associate with chitin (poly-GlcNAc). The correct tertiary structure was inferred by saccharide binding ability and antibody recognition. All cysteines were found to be in disulfide linkages. Isothermal titration calorimetry showed that (GlcNAc)3&4, binding to both B-domain mutants is seven-fold weaker than to WGA (Kd3.5x 10-4M versus 0.54x 10-4M). Binding of N-acetylneuraminyl-lactose was undetectable. Gel filtration, Mass spectral and NMR analysis indicated that the recombinant domains exist as monomers in solution. Thus, the complete WGA binding site was not reproduced and the low affinity reflects only the interactions of the saccharide with the aromatic pocket.