Takeshi Kobayakawa

Characterization of the glutamyl endopeptidase from Staphylococcus aureus expressed in Escherichia coli

V8 protease, a member of the glutamyl endopeptidase I family, of Staphylococcus aureus V8 strain (GluV8) is widely used for proteome analysis because of its unique substrate specificity and resistance to detergents. In this study, an Escherichia coli expression system for GluV8, as well as its homologue from Staphylococcus epidermidis (GluSE), was developed, and the roles of the prosegments and two specific amino acid residues, Val69 and Ser237, were investigated. C‐terminal His6‐tagged proGluSE was successfully expressed from the full‐length sequence as a soluble form. By contrast, GluV8 was poorly expressed by the system as a result of autodegradation; however, it was efficiently obtained by swapping its preprosegment with that of GluSE, or by the substitution of four residues in the GluV8 prosequence with those of GluSE. The purified proGluV8 was converted to the mature form in vitro by thermolysin treatment. The prosegment was essential for the suppression of proteolytic activity, as well as for the correct folding of GluV8, indicating its role as an intramolecular chaperone. Furthermore, the four amino acid residues at the C‐terminus of the prosegment were sufficient for both of these roles. In vitro mutagenesis revealed that Ser237 was essential for proteolytic activity, and that Val69 was indispensable for the precise cleavage by thermolysin and was involved in the proteolytic reaction itself. This is the first study to express quantitatively GluV8 in E. coli, and to demonstrate explicitly the intramolecular chaperone activity of the prosegment of glutamyl endopeptidase I.

Interaction between the N-terminal and Middle Regions Is Essential for the in Vivo Function of HSP90 Molecular Chaperone

At the primary structure level, the 90-kDa heat shock protein (HSP90) is composed of three regions: the N-terminal (Met1–Arg400), middle (Glu401–Lys615), and C-terminal (Asp621–Asp732) regions. In the present study, we investigated potential subregion structures of these three regions and their roles. Limited proteolysis revealed that the N-terminal region could be split into two fragments carrying residues Met1 to Lys281 (or Lys283) and Glu282 (or Tyr284) to Arg400. The former is known to carry the ATP-binding domain. The fragments carrying the N-terminal two-thirds (Glu401–Lys546) and C-terminal one-third of the middle region were sufficient for the interactions with the N- and C-terminal regions, respectively. Yeast HSC82 that carried point mutations in the middle region causing deficient binding to the N-terminal region could not support the growth of HSP82-depleted cells at an elevated temperature. Taken together, our data show that the N-terminal and middle regions of the HSP90 family protein are structurally divided into two respective subregions. Moreover, the interaction between the N-terminal and middle regions is essential for the in vivo function of HSP90 in yeast.

Identification and Characterization of Prokaryotic Dipeptidyl-peptidase 5 from Porphyromonas gingivalis

Background: Dipeptidyl-peptidases (DPPs) are key factors for amino acid metabolism and bacterial growth of asaccharolytic Porphyromonas gingivalis. Results: DPP5, which is specific for Ala and hydrophobic residues, is expressed in the periplasmic space of P. gingivalis. Conclusion: DPP5 was discovered in prokaryotes for the first time. Significance: The discovery of DPP5 expands understanding of amino acid and energy metabolism in prokaryotes. Porphyromonas gingivalis, a Gram-negative asaccharolytic anaerobe, is a major causative organism of chronic periodontitis. Because the bacterium utilizes amino acids as energy and carbon sources and incorporates them mainly as dipeptides, a wide variety of dipeptide production processes mediated by dipeptidyl-peptidases (DPPs) should be beneficial for the organism. In the present study, we identified the fourth P. gingivalis enzyme, DPP5. In a dpp4-7-11-disrupted P. gingivalis ATCC 33277, a DPP7-like activity still remained. PGN_0756 possessed an activity indistinguishable from that of the mutant, and was identified as a bacterial orthologue of fungal DPP5, because of its substrate specificity and 28.5% amino acid sequence identity with an Aspergillus fumigatus entity. P. gingivalis DPP5 was composed of 684 amino acids with a molecular mass of 77,453, and existed as a dimer while migrating at 66 kDa on SDS-PAGE. It preferred Ala and hydrophobic residues, had no activity toward Pro at the P1 position, and no preference for hydrophobic P2 residues, showed an optimal pH of 6.7 in the presence of NaCl, demonstrated Km and kcat/Km values for Lys-Ala-MCA of 688 μm and 11.02 μm−1 s−1, respectively, and was localized in the periplasm. DPP5 elaborately complemented DPP7 in liberation of dipeptides with hydrophobic P1 residues. Examinations of DPP- and gingipain gene-disrupted mutants indicated that DPP4, DPP5, DPP7, and DPP11 together with Arg- and Lys-gingipains cooperatively liberate most dipeptides from nutrient oligopeptides. This is the first study to report that DPP5 is expressed not only in eukaryotes, but also widely distributed in bacteria and archaea.

Substitution of only two residues of human Hsp90α causes impeded dimerization of Hsp90β

Two isoforms of the 90-kDa heat-shock protein (Hsp90), i.e., Hsp90α and Hsp90β, are expressed in the cytosol of mammalian cells. Although Hsp90 predominantly exists as a dimer, the dimer-forming potential of the β isoform of human and mouse Hsp90 is less than that of the α isoform. The 16 amino acid substitutions located in the 561–685 amino acid region of the C-terminal dimerization domain should be responsible for this impeded dimerization of Hsp90β (Nemoto T, Ohara-Nemoto Y, Ota M, Takagi T, Yokoyama K. Eur J Biochem 233: 1–8, 1995). The present study was performed to define the amino acid substitutions that cause the impeded dimerization of Hsp90β. Bacterial two-hybrid analysis revealed that among the 16 amino acids, the conversion from Ala558 of Hsp90β to Thr566 of Hsp90α and that from Met621 of Hsp90β to Ala629 of Hsp90α most efficiently reversed the dimeric interaction, and that the inverse changes from those of Hsp90α to Hsp90β primarily explained the impeded dimerization of Hsp90β We conclude that taken together, the conversion of Thr566 and Ala629 of Hsp90α to Ala558 and Met621 is primarily responsible for impeded dimerization of Hsp90β.

Amino acid residues modulating the activities of staphylococcal glutamyl endopeptidases.

Abstract The glutamyl endopeptidase family of enzymes from staphylococci has been shown to be important virulence determinants of pathogenic family members, such as Staphylococcus aureus. Previous studies have identified the N-terminus and residues from positions 185–195 as potentially important regions that determine the activity of three members of the family. Cloning and sequencing of the new family members from Staphylococcus caprae (GluScpr) and Staphylococcus cohnii (GluScoh) revealed that the N-terminal Val residue is maintained in all family members. Mutants of the GluV8 enzyme from S. aureus with altered N-terminal residues, including amino acids with similar properties, were inactive, indicating that the Val residue is specifically required at the N-terminus of this enzyme family in order for them to function correctly. Recombinant GluScpr was found to have peptidase activity intermediate between GluV8 and GluSE from Staphylococcus epidermis and to be somewhat less specific in its substrate requirements than other family members. The 185–195 region was found to contribute to the activity of GluScpr, although other regions of the enzyme must also play a role in defining the activity. Our results strongly indicate the importance of the N-terminal and the 185–195 region in the activity of the glutamyl endopeptidases of staphylococci.

Single nucleotide polymorphism that accompanies a missense mutation (Gln488His) impedes the dimerization of Hsp90.

A single nucleotide polymorphism (SNP) that causes a missense mutation of highly conserved Gln488 to His of the α isoform of the 90-kDa heat shock protein (Hsp90α) molecular chaperone is observed in Caucasians. The mutated Hsp90α severely reduced the growth of yeast cells. To investigate this molecular mechanism, we examined the domain–domain interactions of human Hsp90α by using bacterial 2-hybrid system. Hsp90α was expressed as a full-length form, N-terminal domain (residues 1–400), or middle (residues 401–617) plus C-terminal (residues 618–732) domains (MC domain/amino acids 401–732). The Gln488His substitution in MC domain did not affect the intra-molecular interaction with N-terminal domain, whereas the dimeric interaction-mediated by the inter-molecular interaction between MC domains was decreased to 32%. Gln488Ala caused a similar change, whereas Gln488Thr, which exceptionally occurs in mitochondrial Hsp90 paralog, fully maintained the dimeric interaction. Therefore, the SNP causing Gln488His mutation could abrogate the Hsp90 function due to reduced dimerization.

Degradation of Incretins and Modulation of Blood Glucose Levels by Periodontopathic Bacterial Dipeptidyl Peptidase 4

ABSTRACT Severe periodontitis is known to aggravate diabetes mellitus, though molecular events related to that link have not been fully elucidated. Porphyromonas gingivalis, a major pathogen of periodontitis, expresses dipeptidyl peptidase 4 (DPP4), which is involved in regulation of blood glucose levels by cleaving incretins in humans. We examined the enzymatic characteristics of DPP4 from P. gingivalis as well as two other periodontopathic bacteria, Tannerella forsythia and Prevotella intermedia, and determined whether it is capable of regulating blood glucose levels. Cell-associated DPP4 activity was found in those microorganisms, which was effectively suppressed by inhibitors of human DPP4, and molecules sized 73 kDa in P. gingivalis, and 71 kDa in T. forsythia and P. intermedia were immunologically detected. The kcat/Km values of recombinant DPP4s ranged from 721 ± 55 to 1,283 ± 23 μM−1s−1 toward Gly-Pro-4-methylcoumaryl-7-amide (MCA), while those were much lower for His-Ala-MCA. Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) analysis showed His/Tyr-Ala dipeptide release from the N termini of incretins, glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide, respectively, with the action of microbial DPP4. Moreover, intravenous injection of DPP4 into mice decreased plasma active GLP-1 and insulin levels, accompanied by a substantial elevation in blood glucose over the control after oral glucose administration. These results are the first to show that periodontopathic bacterial DPP4 is capable of modulating blood glucose levels the same as mammalian DPP4; thus, the incidence of periodontopathic bacteremia may exacerbate diabetes mellitus via molecular events of bacterial DPP4 activities.