Role of conserved and nonconserved residues in the Ca(2+)-dependent carbohydrate-recognition domain of a rat mannose-binding protein. Analysis by random cassette mutagenesis.
The carbohydrate-recognition domain of rat serum mannose-binding protein A has been subjected to random cassette mutagenesis. Mutant domains, expressed in bacteria, were initially screened for binding to invertase-coated nitrocellulose and then analyzed further for Ca2+ affinity, saccharide binding, resistance to proteolysis, and oligomerization. The results are consistent with previous evolutionary and structural studies. Six out of seven completely inactive mutants have changes in residues directly involved in ligating Ca2+. Most changes in conserved residues which form part of the hydrophobic core characteristic of Ca(2+)-dependent (C-type) animal lectins result in decreased affinity for Ca2+, even though these residues are distant from the Ca2+ sites. Changes can be made in large portions of the surface without affecting saccharide binding. The results indicate that the precise arrangement of the regular portion of the domain containing the hydrophobic core is necessary for formation of a stable Ca(2+)-ligated structure under physiological conditions. The data also suggest that the saccharide-binding site is likely to be in close proximity to the bound Ca2+.
Quesenberry
M S
MS
Department of Biochemistry and Molecular Biophysics, Columbia University, New York, New York 10032.
Drickamer
K
K
D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D050260:Carbohydrate Metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002413:Cations, Divalent; D003432:Cross-Linking Reagents; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D008358:Mannose / Q000378:metabolism*; D037601:Mannose-Binding Lectin; D016296:Mutagenesis; D010641:Phenotype; D010957:Plasmids; D011487:Protein Conformation; D051381:Rats
D000596:Amino Acids; D002352:Carrier Proteins; D002413:Cations, Divalent; D003432:Cross-Linking Reagents; D037601:Mannose-Binding Lectin; C069858:mannose binding protein A; D004247:DNA; D008358:Mannose; D002118:Calcium