| Budget Amount *help |
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1997: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1996: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Research Abstract |
Legume lectins are one of the largest lectin families and they resemble each other in their physico-chemical properties although they differ in their carbohydrate specificities. The carbohydrate-binding sites of these lectins consist of 2 conserved amino acids on beta pleated sheets and 2 loops. One of these loops contains transition metals, calcium and manganese, and keep the amino acid residues of the sugar-binding site at the required positions. Amino acid sequences of this loop play an important role in the carbohydrate-binding specificities of these lectins. Random mutation was introduced in a part of cDNA coding Bauhinia purpurea lectin (BPA) which correspond to this carbohydrate-biniding loop. Mutated lectin library expressed on the surface of lambda foo phages were screened by the panning method. Several clones having the affinity for mannose, N-acetylgalactosamine, or N-acetylglucosamine were isolated, respectively. Random mutation was also introduced into the cDNA encoding Maackia amurensis lectin (MAH) and the mutated cDNAs were introduced into plasmid of pGEX-2T.Sixteen clones of recombinant mutated lectins were rondomlly chosen to investigate their specificities. All of these were found to contain different residues in sugar-binding domain, and reacted strongly with anti-MAH polyclonal antibody. Hemagglutination of the purified mutated lectins expressed in E.coli cells were tested against human, bovine, pocine, equine and chicken erythrocytes, as compared to wild type MAH.Variations in carbohydrate-binding specificities aong sixteen colnes were revealed though sialidase treatment of cells did not cause a significant decrease in hemagglutinating acitivity conparde to MAH.
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