Identification of functional site of AFP by recombinant molecule.
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概要
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We would like to focus on two topics which were recently made in our laboratoty on structure/function relationship of alpha-fetoprotein (AFP) by recombinant DNA technology: identification of estrogen binding site of rat AFP, and evaluation of glycosylation of two possible N-glycosylation sites in rat AFP. In order to identify estrogen binding site of rat AFP, a series of rat-human chimeric AFPs was designed, since rat AFP binds to estrogen while human AFP does not. Out of 7 rat-human chimeric AFPs, only those which carry a rat segment 426-467 in human back ground sequence conserved the estrogen binding activity, indicating the segment 426-467, where 15 amino acid substitutions were observed, contained the residues responsible for estrogen binding. A variety of human mutant AFPs in which some human-specific amino acid residues were substituted with those of rat AFP was obtained by site-directed mutagenesis employing polymerase chain reaction. Among 17 human mutant AFPs carrying different combinations of substitutions, those carrying 436 A→V, 437A→S, 438 T→I, 450 L→R, 451 L→S, 457 A→L, 461 I→Y conserved sufficient binding activities, strongly suggesting that these amino acid residues were responsible for the estrogen binding activity in rat AFP. The rat AFP resolves on polyacrylamide gel electrophoreses (PAGE) into two discrete bands, "Slow"and"Fast", and this electrophoretic pattern was shown by us to depend on the heterogeneity of N-glycosylation. For evaluation of glycosylation at two possible N-glycosylation sites in rat AFP, we designed three mutant rat AFPs, which had either substitution of 93Asn→Gln, 229Asn→Gln, or 93Asn→Gln+229Asn→Gln, as well as recombinant rat AFP without mutation. SDS-PAGE before and after glycopeptidase F treatment of the mutant AFPs indicated that the"Fast"had one carbohydrate moiety at 229Asn and the"Slow"had two carbohydrate moieties at 93Asn and 229Asn. It was therefore indicated that 229Asn was fully glycosylated while 93Asn was partially.
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