Kenji Takahashi

Widespread tissue expression of nepenthesin-like aspartic protease genes in Arabidopsis thaliana

There are approximately 69 genes encoding aspartyl protease homologues in Arabidopsis thaliana, and most of the gene products constitute a novel subfamily of aspartic proteases. However, their physiological roles are largely unknown. As an initial step to shed light on the roles of these nepenthesin-like aspartic proteases (NAPs), a phylogenetic tree was constructed, which indicated that these proteases are classified into several distinct sub-sub-groups. Based on these results, specific primers were designed for genes selected from several of these groups and their tissue expression was investigated using RT-PCR. The results indicated that these genes are widely expressed in several tissues, such as leaves, stems, seeds and pods, suggesting ubiquitous occurrence and multiple functions of the corresponding proteases in the tissues of A. thaliana.

A Cysteine Endopeptidase (“Dionain”) Is Involved in the Digestive Fluid of Dionaea muscipula (Venus’s Fly-trap)

The carnivorous plant Dionaea muscipula (Venus’s flytrap) secretes proteinases into the digestive fluid to digest prey proteins. In this study, we obtained evidence that the digestive fluid contains a cysteine endopeptidase, presumably belonging to the papain family, through inhibitor studies and partial amino acid sequencing of the major SDS–PAGE band protein. The name “dionain” is proposed for the enzyme.

Stability Profiles of Nepenthesin in Urea and Guanidine Hydrochloride: Comparison with Porcine Pepsin A

Nepenthesin, an aspartic endopeptidase from the pitcher fluid of Nepenthes, was found to be markedly less stable than porcine pepsin A when treated with urea or guanidine hydrochloride. This is in sharp contrast with its remarkably high pH/temperature stability as compared with porcine pepsin A. No protein with such a stability profile has been reported to date.

Inhibition of rat liver cathepsins B and L by the peptide aldehyde benzyloxycarbonyl-leucyl-leucyl-leucinal and its analogues

Cathepsins B and L belong to the papain superfamily of cysteine proteases and play important roles in various physiological and pathological processes. In the course of studies on their inhibitors, we examined the inhibitory effects of the peptide aldehyde benzyloxycarbonyl-leucyl-leucyl-leucinal (ZLLLal) and its analogues. As a result, rat liver cathepsins B and L were shown to be strongly inhibited by them. The concentration required for 50% inhibition (IC50) by ZLLLal was 88 nM for cathepsin B and 163 nM for cathepsin L. Moreover, various analogues of ZLLLal, including 2-furancarbonyl-, nicotinyl-, isonicotinyl- and 4-morpholinylsuccinyl-LLLals, and some acetyl-Pro (AcP)-containing analogues, AcPLLLal and AcPPLLLal, were shown to inhibit both enzymes more strongly than ZLLLal. Among them, isonicotinyl-LLLal was most inhibitory against both cathepsins B (IC50, 12 nM) and L (IC50, 20 nM). Several of these inhibitors were indicated to be somewhat more soluble in aqueous media than ZLLLal.

Substrate Specificities of Porcine and Bovine Enteropeptidases toward the Peptide Val-(Asp)4-Lys-Ile-Val-Gly and Its Analogs

The substrate specificities of porcine and bovine enteropeptidases were investigated using the peptide Val-(Asp)4-Lys-Ile-Val-Gly and its various analogs with mutations in the (Asp)4-Lys sequence as substrates. The results indicated that in addition to P1 Lys, P2 Asp in the substrates is most important, that P3 Asp is additionally important, and that P5 Asp contributes somewhat to the susceptibility, and that P4 Asp is the least important. These results were essentially identical as between porcine and bovine enteropeptidases.

Urea-dependent unfolding of HIV-1 protease studied by circular dichroism and small-angle X-ray scattering

HIV-1 protease is responsible for the maturation of infective virions, and is one of the targets of drugs against AIDS. It is an aspartic protease with a 99-resiude polypeptide dimerized. Previous study with fluorescence and sedimentation measurements revealed that the protein was unfolded with concomitant dissociation of the subunits. In the present study, we investigated urea-dependent unfolding of HIV-1 protease with CD and SAXS in order to monitor the secondary structure and the global size and shape of the molecule, respectively. The unfolding parameters estimated by both methods were almost the same, indicating that the dissociation of the subunits accompanied the disruption of their internal structures. This is in line with the previous results, and moreover some residual structures were suggested to be present in the unfolded state. The distinct difference, as compared with the unfolding of pepsin, was interpreted from the point of their molecular architectures.

The P1 and P1′ Residue Specificities of Physarolisin I, a Serine-Carboxyl Peptidase from the True Slime Mold Physarum polycephalum

The P1 and P1′ residue specificities of physarolisin I were investigated using combinatorial peptide substrates. The results indicated that certain hydrophobic residues and acidic residues are preferred at the P1 position and some hydrophobic residues at the P1′ position. This P1 specificity, different from other serine-carboxyl peptidases, appears to be explained partially by the nature of the S1 subsite residues.

Structure and function studies on enzymes with a catalytic carboxyl group(s): from ribonuclease T1 to carboxyl peptidases

A group of enzymes, mostly hydrolases or certain transferases, utilize one or a few side-chain carboxyl groups of Asp and/or Glu as part of the catalytic machinery at their active sites. This review follows mainly the trail of studies performed by the author and his colleagues on the structure and function of such enzymes, starting from ribonuclease T1, then extending to three major types of carboxyl peptidases including aspartic peptidases, glutamic peptidases and serine-carboxyl peptidases.

Bromein, a Bromelain Inhibitor from the Pineapple Stem: Structural and Functional Characteristics

Bromelain inhibitor, bromein, is a proteinase-inhibitor specific to the cysteine proteinase bromelain from pineapple stem. In the stem, eight bromein isoforms are known to exist, and each isoform has a short peptide (light chain) and a long one (heavy chain) with five disulfide bonds. The three-dimensional structure of the sixth isoform (bromein-6) is composed of inhibitory and stabilizing domains, and each domain contains a three-stranded antiparallel β-sheet. The genomic sequence of a bromein precursor encodes three homologous bromein isoform domains, and each isoform domain has a signal peptide, three interchain peptides between the light chain and heavy chain, two interdomain peptides and a propeptide. Interestingly, at the protein level, bromein- 6 appears to share a similar folding and disulfide-bonding connectivity with Bowman-Birk serine proteinase inhibitors and shows weak inhibition toward chymotrypsin and trypsin. However, no significant similarity was found between them at the genomic level. This indicates that they have evolved convergently to possess such a structural similarity. To identify the essential reactive site(s) with bromelain, we investigated the inhibitory activity of 44 kinds of the single/double and insertion/ deletion mutants of bromein-6 towards stem bromelain. As a result, it was shown that both the appropriate positioning and the complete side-chain structure of Leu10 in the light chain are absolutely crucial for the inhibition, with an additional measure of importance for the preceding Pro9. Bromein and stem bromelain coexist in the acidic vacuoles of the stem tissue, and one of the key role of bromein appears to be the regulation of the bromelain activity.