Abstract
The substrate specificity of rat brain neurolysin was rapidly modified by semirational mutagenesis coupled with a yeast molecular display system. Neurolysin mainly recognizes substrates with sequential six residues close to the scissile bond in polypeptides, cleaving a peptide bond in the center position of the six residues. To alter the recognition of the P2' amino acid of substrates by neurolysin, six residues of neurolysin, Asp467, Arg470, Glu510, Tyr606, Tyr610 and Tyr611, which might be involved in the formation of the neurolysin S2' subsite, were individually and comprehensively substituted. The protein libraries of mutant neurolysins comprising 120 species were displayed on the yeast cell surface and screening was carried out using two fluorescence-quenching peptides, the matrix metalloproteinase-2/9- (MMPs-2/9-) and MMP-3-specific substrates, which consisted of similar amino acids, except for alanine (for MMPs-2/9) or glutamic acid (for MMP-3) at the P2' amino acid position. Among mutant neurolysins, the Y610L mutant neurolysin exhibited a marked change in substrate specificity. Steady-state kinetic analysis of the purified Y610L mutant neurolysin revealed that the binding efficiency toward the MMP-3-specific substrate was about 3-fold higher than that toward the MMP-2/9-specific substrate. These results indicate that Tyr610 of neurolysin is the important residue to recognize the P2' amino acid of substrates.
Highlights
Peptidases catalyze the hydrolysis of peptide bonds, which includes processing of bioactive peptides to regulate signal transduction, length regulation of antigenic peptides for the antigen presentation of major histocompatibility molecules and cleavage of peptides for the recycling system of amino acids
The protein libraries of mutant neurolysins comprising 120 species were displayed on the yeast cell surface and screening was carried out using two fluorescence-quenching peptides, the matrix metalloproteinase-2/9- (MMPs-2/9-) and MMP-3-specific substrates, which consisted of similar amino acids, except for alanine or glutamic acid at the P20 amino acid position
A number of neurolysin residues were comprehensively substituted to enhance its binding ability toward the MMP-3-specific peptide, which contains charged glutamic acid at the P20 amino acid position, the substitution of Tyr610 with hydrophobic leucine was most effective, suggesting that P20 amino acids of substrates bind to the neurolysin S20 subsite by hydrophobic interaction, and not by charge interaction of both side chains
Summary
Peptidases catalyze the hydrolysis of peptide bonds, which includes processing of bioactive peptides to regulate signal transduction, length regulation of antigenic peptides for the antigen presentation of major histocompatibility molecules and cleavage of peptides for the recycling system of amino acids. Modification of the substrate specificity of peptidases using protein engineering technology is a powerful strategy to gain the basic knowledge of peptide recognition. The construction of a gene library and its conversion to a protein library following screening for function has progressed with the development of several protein-display systems in protein engineering (Boder and Wittrup, 1997; Ueda and Tanaka, 2000; Benhar, 2001; Frankel et al, 2003; Daugherty, 2007; Seeling and Szostak, 2007). The protein library is constructed on the basis of a semirational strategy as follows (Antikainen et al, 2003; Geddie and Matsumura, 2004; Rui et al, 2004; Schmitzer et al, 2004; Shiraga et al, 2005; Fukuda et al, 2007). Multiple and specific residues are selected on the basis of structural and functional knowledge, DNA codons encoding the selected residues are comprehensively changed using mixed primers (Chica et al, 2005). The focused library designed by the semirational approach has decreased library size and provided a higher possibility for acquiring positive clones than in the case of a freely randomized library constructed by the directed evolution approach including the error-prone PCRand DNA-shuffling methods
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