Studies on purification, structures and characterization of protease inhibitors from Apios americana tubers

概要

Apios americana Medikus is a perennial leguminous plant native to North America, which can produce edible tubers formed from protuberances on stolons. Apios tubers have higher nutritional value. They are rich in protein, carbohydrate, dietary fiber, calcium and iron. In addition, Apios tubers also contain some bioactive components, such as isoflavones with antioxidant activity and ACE inhibitors that can decrease the blood pressure. Apios tubers contain higher contents of protein, iron, and dietary fiber, and are considered as health food materials. In order to enhance the application value of Apios as a functional food, our research group had focused on bioactive proteins existing in the Apios tubers and purified β-amylase, polygalacturonase inhibitor, lectin and Bowman-Birk trypsin inhibitor so far. Among these proteins, AATI showed an inhibitory effect on proliferation of the U937, K563, J774.1 and HeLa cell lines.

Interestingly, during purifying the AATI, another type of protease inhibitor proteins with molecular masses of 6 kDa and 13 kDa were found in the crude proteins of Apios tubers.

The purpose of this study was to purify the newly-found protease inhibitor and characterize its bioactivity by the cDNA cloning technique and functional expression in Escherichia coli cells.

In the Chapter 2, the Kunitz-type protease inhibitors were purified by the DEAE-Cellufine A-500 column, Butyl-Cellufine column, Sephadex G-50 column, and RP-HPLC. N-terminal amino acid sequences of purified proteins showed that two kinds of Kunitz-type protease inhibitors (AKPI1: 20 kDa and AKPI2: 19 kDa) existed in Apios tubers. Besides the presence of 19 kDa AKPI2 protein, two-chain form of AKPI2 protein fragments [13/12 kDa (chain A), and 6 kDa (chain B)] were observed and AKPI1, AKPI2, and its fragments were able to be separated by RP-HPLC. Although the separated peptides had trypsin and chymotrypsin inhibitory activities, the inhibition constants (Ki) were not able to be determined, because of the low yield and the effect of reagent in the purification process.

To determine the amino acid sequences and characterize the targeting protease from the amino acid sequence, the cDNA cloning of AKPI1 and AKPI2 was performed in the chapter 3 and chapter 4.

The specific primers of AKPI1 used for degenerated PCR were designed based on the amino acid sequences of the digested peptides by lysyl-endopeptidase and the N-terminal amino acid sequence of AKPI1. From degenerated PCR, 5ʹ-RACE and 3ʹ-RACE, 348, 242 and 448 bp AKPI1 cDNA fragments were obtained, respectively.

These fragments were combined by the full-length PCR with the full-length primers designed from 5′-and 3′-UTR. The AKPI1 cDNA consisted of 809 nucleotides with a 624 bp open reading frame which encoded a polypeptide of 208 amino acids, a 46 bp 5′-UTR, and a 139 bp 3′-UTR (DDBJ accession no. LC504755). The mature protein had a polypeptide of 190 amino acids with a molecular mass of 20,594 Da. From BLAST search, the AKPI1 was relatively similar to the Kunitz-type inhibitors in other legumes such as Gm2, Glycine max Kunitz-type trypsin inhibitor (ACA23207.1), Ca, Cicer arietinum Kunitz-type trypsin inhibitor (NP_001266050.1), and Ps, Pisum sativum Kunitz-type protease inhibitor (O82711.1), which shared 65-75% amino acid residues with AKPI1 at the identical position. The AKPI1 had five cysteine residues located at the positions of 62, 107, 125, 158 and 165. Unlike these general KPIs, the reactive site P1 residue of AKPI1 was speculated to be Gly85-Ile86-Ser87, suggested that AKPI1 would belong to a new sub-family of the Kunitz-type protease inhibitors.

The cDNA cloning of AKPI2 was performed by the same methods for AKPI1. The degenerated PCR of AKPI2 was performed using the specific degenerated PCR primers designed based on the N-terminal amino acid sequences of AKPI2 (19 kDa) and chain B (6 kDa) protein. The degenerated PCR, 5′-RACE and 3′-RACE gave 315, 254 and 450 bp PCR products. The AKPI2 cDNA consisted of 794 nucleotides with a 603 bp open reading frame encoding a polypeptide of 201 amino acid residues, a 49 bp 5′-UTR and a 142 bp 3′-UTR (DDBJ accession no. LC504756). The mature AKPI2 protein consisted of 177 amino acid residues with a molecular mass of 19,336 Da. N-terminal amino acid sequence of 6 kDa AKPI2 fragment suggested that the AKPI2 was cleaved at the position of Ser149-Asp150 to generate two-chain form (chain A and B consisted of at least 125 and 52 amino acid residues, respectively). Four cysteine residues were located at the positions of 64, 108, 159 and 166 that could form two disulfide bridges. P1 residue of the reactive site of AKPI2 was speculated to be Leu88, suggesting that AKPI2 preferred to bind to chymotrypsin rather than trypsin. The short chain (chain B) of AKPI2 contained two cysteine residues, and the two polypeptide chains (chain A and chain B) were detected by the SDS-PAGE in the absence of β-mercaptoethanol. This indicated that the two polypeptide chains were not held together by disulfide bonds, implying that AKPI2 might represent an unreported type of Kunitz-type protease inhibitor. The AKPI1 and AKPI2 only shared 30% amino acid residues at the identical position.

In order to study whether the single-chain form of AKPI2 (19 kDa) had protease inhibitory activity, the recombinant AKPI2 protein (rAKPI2) was produced in E. coli BL21 CodonPlus (DE3) RIL cells with the expression vector pET-22b and purified by the Sephadex G-75 column chromatography, as described in the chapter 5. The yield of purified rAKPI2 was 16 mg/ L medium.

In the chapter 6, the purified rAKPI2 were used for inhibition assay. The inhibition constants (Ki) of rAKPI2 toward trypsin and chymotrypsin were calculated to be 6.1×10-6 M and 4.4×10-7 M, respectively. The result of heat-stability measurement of rAKPI2 showed that it was stable from 37℃ to 60℃ regarding to the chymotrypsin and trypsin inhibitory activity. Besides, rAKPI2 had weak inhibitory activity against elastase but not against papain and subtilisin. Furthermore, rAKPI2 showed the inhibitory activity of proliferation of U937 in a dose and time dependent manner but it was not caused by apoptosis. rAKPI2 also inhibited the growth of yeast Candida buinensis. If the inhibitory activity of rAKPI2 against more kinds of cancer cells and pathogen bacteria are measured in future, the application prospect of rAKPI2 as antimicrobial and anticancer agent will be broadened.