Creation of soybean beta-conglycinin beta with strong phagocytosis-stimulating activity.
beta-Conglycinin is composed of three kinds of subunit: alpha, alpha' and beta. A phagocytosis-stimulating peptide sequence (MITLAIPVNKPGR), soymetide, exists in the alpha' subunit of beta-conglycinin. Met at N terminus of the soymetide is essential for the activity. When Thr at the third residue from N terminus of the soymetide is replaced by Phe or Trp, the phagocytosis-stimulating activity greatly increases (Thr<Phe<Trp). The beta subunit does not exhibit the phagocytosis-stimulating activity because the residues corresponding to the first and third residues in the soymetide are Ile and Lys, respectively. In this study, we introduced the phagocytosis-stimulating peptide sequence (Ile-->Met, Lys-->Thr, Phe, or Trp) into the beta subunit after confirmation of the effects of residue replacements by molecular modeling, suggesting that the introduced mutations might not prevent the correct folding. The studies of circular dichroism (CD), gel filtration and differential scanning calorimetry (DSC) of the mutants (I122M/K124T, I122M/K124F, I122M/K124W) expressed in E. coli demonstrated that they folded and self-assembled similarly to the wild type. This was confirmed by X-ray analysis of I122M/K124W crystal where the biggest residue tryptophane was introduced. The three mutants exhibited phagocytosis activities after digestion by trypsin, and the order was the wild type<I122M/K124T<I122M/K124F<I122M/K124W as expected.
Maruyama
Nobuyuki
N
Laboratory of Food Quality Design and Development, Graduate School of Agriculture, Kyoto University, Uji, Japan.
Maruyama
Yukie
Y
Tsuruki
Takahiro
T
Okuda
Eiko
E
Yoshikawa
Masaaki
M
Utsumi
Shigeru
S
D052179:Antigens, Plant; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D005916:Globulins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D006358:Hot Temperature; D008958:Models, Molecular; D010587:Phagocytosis / Q000502:physiology; D017434:Protein Structure, Tertiary; D055314:Seed Storage Proteins; D030262:Soybean Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D013025:Glycine max / Q000737:chemistry* / Q000235:genetics
D052179:Antigens, Plant; D005916:Globulins; D055314:Seed Storage Proteins; D030262:Soybean Proteins; C013894:beta-conglycinin protein, Glycine max