Effects of substitution of conserved amino acid residues on the sugar-binding property of the tandem-repeat 32-kDa galectin of the nematode Caenorhabditis elegans.
The 32-kDa galectin (LEC-1) of the nematode Caenorhabditis elegans (C elegans) is composed of two tandemly repeated homologous sequences, each containing a carbohydrate-recognition domain (CRD). Using the polymerase chain reaction (PCR) with LEC-1 cDNA as a template and "megaprimers", we performed site-directed mutagenesis to substitute conserved amino acid residues in these domains. The resultant mutated LEC-1s were produced in E. coli, and their binding abilities were estimated by affinity chromatography. When one of the conserved amino acid residues in the first lectin domain was substituted, the binding ability of the mutant protein to asialofetuin-agarose was reduced but still remained. The binding ability of such mutants was similar to that of the recombinant half molecule containing the second lectin domain (Ch). However, when mutations were introduced into the second lectin domain, the binding ability of these mutant lectins to asialofetuin-agarose was significantly reduced just like the half recombinant molecule containing the first lectin domain (Nh). The different effects of the substitution of amino acid residues on the two lectin domains suggest that the binding properties of the two sites are different and that LEC-1 acts as a "heterobifunctional crosslinker."
Arata
A
A
Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, Teikyo University, Kanagawa, Japan. y-arata@pharm.teikyo-u.ac.jp
Sekiguchi
M
M
Hirabayashi
J
J
Kasai
K
K
D000818:Animals; D001483:Base Sequence; D017173:Caenorhabditis elegans / Q000378:metabolism*; D050260:Carbohydrate Metabolism; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D037161:Galectins; D006388:Hemagglutinins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D017931:DNA Primers; D037161:Galectins; D006388:Hemagglutinins; D011994:Recombinant Proteins