Addition of Phagocytosis Stimulating Activity

Recently, Yoshikawa et al. isolated a phagocytosis stimulating peptide from trypsin digests of soybean proteins (72). This peptide, named soymetide, is derived from the a' subunit of P-conglycinin, and its primary sequence is MITLAIPVNKPGR. Met at its N-terminus is essential for the activity and MITL, the four residues from N-terminus, is the shortest peptide exhibiting phagocytosis stimulation. When the Thr, third residue from the N-terminus of the soymetide, was replaced by Phe or Trp, the activities of the modified peptides greatly increased (Thr < Phe < Trp) (72). Although the regions of the a and P subunits corresponding to the soymetide are highly conserved among the three subunits, and the cleavage sites for trypsin exist at both sides of the regions, their digests by trypsin do not exhibit the activity. The reason is that the residues of the a and P subunits corresponding to the N-terminal Met in the soymetide, which is essential for the activity, are Leu and Ile, respectively. It was also found that Lys124 in the P subunit was not favorable for phagocytosis stimulation (72).

We attempted to introduce the phagocytosis stimulating peptide sequence into the P subunit by replacing Ile122 and Lys124 of the wild type with Met and Thr/Phe/Trp (I122M/K124T, I122M/K124F and I122M/K124W), respectively (73). These residues are located in the P barrel of the core region and their side chains are inside the molecule. Therefore, there is a possibility that the introduction of the phagocytosis stimulating peptide sequence into the P subunit might induce the loss of the folding ability by causing unfavorable contacts, especially in the case of the replacement of Lys with Phe or Trp, which have side chains bigger than that of Lys.

At first, we simulated the models of the three mutants using the three dimensional structure of the P subunit by the programs Insight II and Discover to confirm whether these mutants could fold correctly or not. The RMSD for the entire Ca atoms between the starting and simulated monomer models was around 0.67 A for all the mutants. The distance of Ca atoms between the wild type and the simulated models at the positions 122 and 124 were 0.47-0.49 and 0.29-0.50 A in all the mutants, respectively. These values mean that the position of the backbone scarcely moved. Furthermore, no unfavorable van der Waal's interactions between the side chains of the replaced residues and those of their neighboring residues were observed. Therefore, we concluded that all the mutants could fold correctly. To confirm our assumption that all the mutants could fold correctly based on the molecular modeling, we characterized the structural features of the mutants expressed in E. coli by circular dichroism measurement, differential scanning calorimetry, and gel filtration column chromatography. No significant difference in circular dichroism spectra was observed between the wild type and the mutants. This result indicates that the mutations for introducing the phagocytosis stimulating peptide sequence into the P subunit have little effect on the secondary structure. Tm values of all the mutants measured by differential scanning calorimetry were 1.9-3.1°C lower than that of the wild type. Although there is a hydrogen bond between Lys124 and Tyr109 in the P barrel of the wild type, all the mutants lost this hydrogen bond by the replacement of Lys124. Therefore, the loss of the hydrogen bond of the mutants might induce a slight decrease in Tm values. In gel filtration chromatography, all the mutants eluted similarly to the wild type. The results of circular dichroism measurement, differential scanning calorimetry, and gel filtration chromatography indicate that all the mutants folded correctly similar to the wild type. Further, we determined the crystal structure of I122M/K124W to investigate the effect of mutations in detail, because Trp was biggest among the introduced residues (Figure 2.12). The distances of Ca between Ile and Met (residue number 122) and between Lys and Trp (residue number 124) were 0.48 A and 0.17 A, respectively. Furthermore, no unfavorable van der Waal's interactions between the side chains of the replaced residues and those of their neighboring residues were observed. These results indicate that the replacement has little influence on the backbone structures and that the assumption about the conformation of I122M/K124W from the simulated model is correct. Further, all the mutants exhibited phagocytosis stimulating activity in the order of I122M/K124T < I122M/K124F < I122M/K124W as expected, and the wild type did not (Figure 2.13). The results confirm that we could introduce the phagocytosis stimulating peptide sequence into the P subunit with the correct folding.

When bioactive peptides are introduced into food proteins, a simulation based on three dimensional structures is a powerful tool to estimate the folding ability of the mutants. In the future, the accumulation of data on three dimensional structures of higher resolutions and on the folding abilities of mutants will make it possible to introduce bioac-tive peptide sequences exactly into food proteins by protein engineering.

Figure 2.12 Structural comparison of the mutation site of I122M/K124W with corresponding site of the wild type. The Ca traces of wild type and I122M/K124W are represented by lines, respectively. The view in panel (b) is related to that depicted in panel (a) by a rotation of 90°. Dotted line indicates a hydrogen bond. (From: Maruyama, N., Y. Maruyama, T. Tsuruki, E. Okuda, M. Yoshikawa, S. Utsumi, Biochim. Biophys. Acta 1648:99-104, 2003.)

Figure 2.12 Structural comparison of the mutation site of I122M/K124W with corresponding site of the wild type. The Ca traces of wild type and I122M/K124W are represented by lines, respectively. The view in panel (b) is related to that depicted in panel (a) by a rotation of 90°. Dotted line indicates a hydrogen bond. (From: Maruyama, N., Y. Maruyama, T. Tsuruki, E. Okuda, M. Yoshikawa, S. Utsumi, Biochim. Biophys. Acta 1648:99-104, 2003.)

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