Treatment resistance of rheumatoid arthritis relates to infection of periodontal pathogenic bacteria: a case-control cross-sectional study

Recent studies have shown that periodontitis is associated with rheumatoid arthritis (RA) and periodontal bacteria, such as Aggregatibacter actinomycetemcomitans (Aa) and Porphyromonas gingivalis (Pg) are involved in the pathogenesis of RA via citrullinated proteins. Smoking has also been shown to be involved in the pathogenesis of RA; however, the extent of this involvement is still poorly understood. In addition, RA and polymyalgia rheumatica (PMR) are sometimes difficult to differentiate; however, the relationship between PMR and the factors from smoking and periodontal bacteria is unclear. The aim of this study was to clarify the relationship between periodontal pathogenic bacterial infections and smoking in patients with RA or PMR. This case-control study included 142 patients with untreated RA or PMR. This study evaluated the serum antibody titers against periodontal pathogenic bacterial antigens and an anti-citrullinated peptide antibody (ACPA). In patients with RA, the relationship between antibody titers and disease activity of RA and response after 3 months of treatment was also investigated. Additionally, the effects of smoking were evaluated. Although there was no significant difference in serum antibody titer against periodontal pathogenic bacteria between the ACPA-positive RA group and the ACPA-negative PMR group, we found an association between the elevated antibody titer against Pg and the degree of ACPA value, especially between negative group and high-value positive group (>= 100 U/mL). The antibody titers against Aa and Pg did not differ depending on disease activity score 28 (DAS28) at baseline; however, patients with high antibody titers had poor RA therapeutic response as judged by DAS28 after 3 months. We could not find any association between smoking and any of these parameters. Periodontal pathogenic bacteria, especially Pg, are associated with elevated ACPA levels. Our findings suggest that Pg and Aa infections interfere with the therapeutic response of RA

Novel activity of Streptomyces aminopeptidase P

Streptomyces aminopeptidase P enzymes are proline-specific peptidases that belong to the peptidase M24 family. To evaluate the activity of a commercial Streptomyces aminopeptidase P, named 'XPO DUET', we performed three experiments involving degradation of tryptic casein, production of free amino acids from casein hydrolysate, and hydrolysis of synthetic peptides. Using an ion-trap liquid chromatography-mass spectrometry (LC-MS) apparatus, we demonstrate that XPO DUET could degrade FFVAPFPEVFGK, an allergic and bitter peptide, VAPFPEVFGK, and PEVFGK from tryptic casein. All amino acids, except Ala, Asp, Glu, and Tyr, were released in an XPO DUET activity-dependent manner during the hydrolysis of casein hydrolysate. LC-MS analysis also revealed the ability of XPO DUET to completely hydrolyze Phe-Phe-Phe into free Phe. Thus, we confirm that XPO DUET possesses broader specificity than its known activity toward Xaa-Pro peptides. Because XPO DUET is a food-grade peptidase, it is useful in the bioprocessing of protein hydrolysates through its combination with other food-grade peptidases

Alteration of Leucine Aminopeptidase from Streptomyces septatus TH-2 to Phenylalanine Aminopeptidase by Site-Directed Mutagenesis

To tailor leucine aminopeptidase from Streptomyces septatus TH-2 (SSAP) to become a convenient biocatalyst, we are interested in Phe221 of SSAP, which is thought to interact with the side chain of the N-terminal residue of the substrate. By using saturation mutagenesis, the feasibility of altering the performance of SSAP was evaluated. The hydrolytic activities of 19 mutants were investigated using aminoacyl p-nitroanilide (pNA) derivatives as substrates. Replacement of Phe221 resulted in changes in the activities of all the mutants. Three of these mutants, F221G, F221A, and F221S, specifically hydrolyzed l-Phe-pNA, and F221I SSAP exhibited hydrolytic activity with l-Leu-pNA exceeding that of the wild type. Although the hydrolytic activities with peptide substrates decreased, the hydrolytic activities with amide and methyl ester substrates were proportional to the changes in the hydrolytic activities with pNA derivatives. Furthermore, based on a comparative kinetic study, the mechanism underlying the alteration in the preference of SSAP from leucine to phenylalanine is discussed

Change in Substrate Preference of Streptomyces Aminopeptidase through Modification of the Environment around the Substrate Binding Site

We attempted to alter the substrate preference of aminopeptidase from Streptomyces septatus TH-2 (SSAP). Because Asp198 and Phe221 of SSAP are located in the substrate binding site, we screened 2,000 mutant enzymes with D198X/F221X mutations. By carrying out this examination, we obtained two enzymes; one specifically hydrolyzed an arginyl derivative, and the other specifically hydrolyzed a cystinyl derivative (65- and 12.5-fold higher k(cat) values for hydrolysis of p-nitroanilide derivatives than those of the wild type, respectively)

C-terminal loop of Streptomyces phospholipase D has multiple functional roles

We have recently shown that two flexible loops of Streptomyces phospholipase D (PLD) affect the catalytic reaction of the enzyme by a comparative study of chimeric PLDs. Gly188 and Asp191 of PLD from Streptomyces septatus TH-2 (TH-2PLD) were identified as the key amino acid residues involved in the recognition of phospholipids. In the present study, we further investigated the relationship between a C-terminal loop of TH-2PLD and PLD activities to elucidate the reaction mechanism and the recognition of the substrate. By analyzing chimeras and mutants in terms of hydrolytic and transphosphatidylation activities, Ala426 and Lys438 of TH-2PLD were identified as the residues associated with the activities. We found that Gly188 and Asp191 recognized substrate forms, whereas residues Ala426 and Lys438 enhanced transphosphatidylation and hydrolysis activities regardless of the substrate form. By substituting Ala426 and Lys438 with Phe and His, respectively, the mutant showed not only higher activities but also higher thermostability and tolerance against organic solvents. Furthermore, the mutant also improved the selectivity of the transphosphatidylation activity. The residues Ala426 and Lys438 were located in the C-terminal flexible loop of Streptomyces PLD separate from the highly conserved catalytic HxKxxxxD motifs. We demonstrated that this C-terminal loop, which formed the entrance of the active well, has multiple functional roles in Streptomyces PLD

Repeat-Length-Independent Broad-Spectrum Shuffling, a Novel Method of Generating a Random Chimera Library In Vivo

We describe a novel method of random chimeragenesis based on highly frequent deletion formation in the Escherichia coli ssb-3 strain and a deletion-directed chimera selection system that uses the rpsL(+) gene as a reporter. It enables the selection of chimeras without target gene expression and can therefore be applied to cytotoxic targets. When this system was applied to phospholipase D genes from Streptomyces septatus TH-2 and Streptomyces halstedii subsp. scabies K6 (examples of cytotoxic targets), chimeragenesis occurred between short identical sequences at the corresponding position of the parental genes with large variations. Chimeragenesis was >1,000 times more frequent in the ssb-3 background than in the ssb(+) background. We called this system repeat-length-independent broad-spectrum shuffling. It enables the convenient chimeragenesis and functional study of chimeric proteins. In fact, we found two amino acid residues related to the thermostability of phospholipase D (Phe426 and Thr433) by comparing thermostability among the chimeric enzymes obtained

Understanding Antidiabetic Potential of Oligosaccharides from Red Alga Dulse <i>Devaleraea inkyuleei</i> Xylan by Investigating α-Amylase and α-Glucosidase Inhibition

In this study, the α-glucosidase (maltase-glucoamylase: MGAM) and α-amylase inhibitory properties elicited by xylooligosaccharides (XOSs) prepared from dulse xylan were analysed as a potential mechanism to control postprandial hyperglycaemia for type-2 diabetes prevention and treatment. Xylan was purified from red alga dulse powder and used for enzymatic hydrolysis using Sucrase X to produce XOSs. Fractionation of XOSs produced xylobiose (X2), β-(1→3)-xylosyl xylobiose (DX3), xylotriose (X3), β-(1→3)-xylosyl-xylotriose (DX4), and a dulse XOS mixture with n ≥ 4 xylose units (DXM). The different fractions exhibited moderate MGAM (IC50 = 11.41–23.44 mg/mL) and α-amylase (IC50 = 18.07–53.04 mg/mL) inhibitory activity, which was lower than that of acarbose. Kinetics studies revealed that XOSs bound to the active site of carbohydrate digestive enzymes, limiting access to the substrate by competitive inhibition. A molecular docking analysis of XOSs with MGAM and α-amylase clearly showed moderate strength of interactions, both hydrogen bonds and non-bonded contacts, at the active site of the enzymes. Overall, XOSs from dulse could prevent postprandial hyperglycaemia as functional food by a usual and continuous consumption