Pyroglutamyl leucine, a peptide in fermented foods, attenuates dysbiosis by increasing host antimicrobial peptide

PyroGlu-Leu is present in certain food protein hydrolysates and traditional Japanese fermented foods. Our previous study demonstrated that the oral administration of pyroGlu-Leu (0.1 mg/kg body weight) attenuates dysbiosis in mice with experimental colitis. The objective of this study was to elucidate why such a low dose of pyroGlu-Leu attenuates dysbiosis in different animal models. High fat diet extensively increased the ratio of Firmicutes/Bacteroidetes in feces of rats compared to control diet. Oral administration of pyroGlu-Leu (1 mg/kg body weight) significantly attenuated high fat diet-induced dysbiosis. By focusing on the production of intestinal antimicrobial peptides, we found that pyroGlu-Leu significantly increased the level of 4962 Da peptides, which identified as the propeptide of rattusin or defensin alpha 9, in ileum. We also observed increased tryptic fragment peptides from rattusin in the lumen. Here, we report that orally administered pyroGlu-Leu attenuates dysbiosis by increasing in the host antimicrobial peptide, rattusin.

Aggregates with lysozyme and ovalbumin show features of amyloid-like fibrils

The interaction of egg-white lysozyme with N-ovalbumin, the native form of egg-white ovalbumin with the denaturation temperature, Tm, of 78 °C, was investigated by the inhibition of lysozyme muramidase activity, differential scanning calorimetry, and circular dichroism assay as indicators. Signals for the interaction were the most prominent when the mixture of lysozyme and N-ovalbumin was co-heated at 72 °C, slightly lower than the Tm of N-ovalbumin. The interaction was also marked when unheated lysozyme was mixed with N-ovalbumin preheated at 72 °C. Moreover, the mixture rapidly formed fibrous precipitates, which were positive for thioflavin T fluorescent emission, a marker for the amyloid fibril formation. Also electron microscopic observation exhibited features of fibrils. The interaction potency of ovalbumin was ascribed to the tryptic fragment ILELPFASGT MSMLVLLPDE VSGLEQLESIINFEK (residues 229–263), derived from the 2B strands 2 and 3 of ovalbumin. From lysozyme, on the other hand, the chymotryptic peptide RNRCKGTDVQAW (residues 112–123), including cluster 6, and the chymotryptic/tryptic peptide GILQINSRW (residues 54–62), including cluster 3, were responsible for the interaction with N-ovalbumin. Interestingly, this nonamer peptide was found to have the ability to self-aggregate. To the authors knowledge, this may be the first report to document the possible involvement of dual proteins in the formation of amyloid-like fibrils.

N-linked glycosylation of VWF modulates its interaction with ADAMTS13

We examined the role of N-linked glycan structures of VWF on its interaction with ADAMTS13. PNGase F digestion followed by lectin analysis demonstrated that more than 90% of VWF N-linked glycan chains could be removed from the molecule (PNG-VWF) without disruption of its multimeric structure or its ability to bind to collagen. PNG-VWF had an approximately 4-fold increased affinity for ADAMTS13 compared with control VWF. PNG-VWF was cleaved by ADAMTS13 faster than control VWF and was also proteolysed in the absence of urea. Occupancy of the N-linked glycan sites at N1515 and N1574 and their presentation of ABO(H) blood group sugars were confirmed with an isolated tryptic fragment. Recombinant VWF was mutated to prevent glycosylation at these sites. Mutation of N1515 did not alter ADAMTS13 binding or increase rate of ADAMTS13 proteolysis. Mutation of N1574 increased the susceptibility of VWF to ADAMTS13 proteolysis and allowed cleavage in the absence of urea. Mutation of N1574 in the isolated recombinant VWF-A2 domain also increased binding and ADAMTS13 proteolysis. These data demonstrate that the N-linked glycans of VWF have a modulatory effect on the interaction with ADAMTS13. At least part of this effect is conformational, but steric hindrance may also be important.

Characterization of a Bifidobacterium longum BORI Dipeptidase Belonging to the U34 Family

ABSTRACT A dipeptidase was purified from a cell extract of Bifidobacterium longum BORI by ammonium sulfate precipitation and chromatography on DEAE-cellulose and Q-Sepharose columns. The purified dipeptidase had a molecular mass of about 49 kDa and was optimally active at pH 8.0 and 50°C. The enzyme was a strict dipeptidase, being capable of hydrolyzing a range of dipeptides but not tri- and tetrapeptides, p-nitroanilide derivatives of amino acids, or N- or C-terminus-blocked dipeptides. A search of the amino acid sequence of an internal tryptic fragment against protein sequences deduced from the total genome sequence of B. longum NCC2705 revealed that it was identical to an internal sequence of the dipeptidase gene (pepD), which comprised 1,602 nucleotides encoding 533 amino acids with a molecular mass of 60 kDa, and thereby differed considerably from the 49-kDa mass of the purified dipeptidase. To understand this discrepancy, pepD was cloned into an Escherichia coli expression vector (pBAD-TOPO derivative) to generate the recombinant plasmids pBAD-pepD and pBAD-pepD-His (note that His in the plasmid designation stands for a polyhistidine coding region). Both plasmids were successfully expressed in E. coli, and the recombinant protein PepD-His was purified using nickel-chelating affinity chromatography and reconfirmed by internal amino acid sequencing. The PepD sequence was highly homologous to those of the U34 family of peptidases, suggesting that the B. longum BORI dipeptidase is a type of cysteine-type N-terminal nucleophile hydrolase and has a β-hairpin motif similar to that of penicillin V acylase, which is activated by autoproteolytic processing.

Reactivity of free thiol groups in type-I inositol trisphosphate receptors

The IP3R (inositol 1,4,5-trisphosphate receptor) Ca2+-release channel is known to be sensitive to thiol redox state. The present study was undertaken to characterize the number and location of reactive thiol groups in the type-I IP3R. Using the fluorescent thiol-reactive compound monobromobimane we found that approx. 70% of the 60 cysteine residues in the type-I IP3R are maintained in the reduced state. The accessibility of these residues was assessed by covalently tagging the IP3R in membranes with a 5 kDa or 20 kDa MPEG [methoxypoly(ethylene glycol) maleimide]. MPEG reaction caused a shift in the mobility of IP3R on SDS/PAGE that was blocked by pretreatment of the membranes with dithiothreitol, N-ethylmaleimide, mersalyl or thimerosal, indicating that MPEG reactivity was specific to thiol groups on the IP3R. Trypsin cleavage of the type-I IP3R generates five defined domains. In cerebellum membranes, MPEG reacted over a 5 min interval with tryptic fragment I and fragment III, but not fragments II, IV or V. Fragment I appears as a doublet in cerebellum membranes, corresponding to the presence and absence of the SI splice site in this region (SI is a spliced domain corresponding to amino acids 318–332). Only the fragment I band corresponding to the SI(+) splice form shifted after reaction with MPEG. Expression of SI(+) and SI(−) spliced forms in COS cell microsomes confirmed this result. The MPEG-induced shift was not prevented when the cysteine residue present in the SI splice domain (C326A) or the remaining seven cysteine residues in fragment I were individually mutated. Of the combination mutations screened, only the mutation of C206/214/326A blocked MPEG reactivity in fragment I. We conclude that a set of highly reactive cysteine residues in fragment I are differentially accessible in the SI(+) and SI(−) splice variants of the type-I IP3R.

COOH-terminal 26-amino acid residues of progastrin are sufficient for stimulation of mitosis in murine colonic epithelium in vivo

Transgenic mice (hGAS) that overexpress human progastrin are more susceptible than wild-type mice (FVB/N) to the induction of colonic aberrant crypt foci (ACF) and adenomas by the chemical carcinogen azoxymethane. We have previously shown significantly increased levels of colonic mitosis in hGAS compared with FVB/N mice after γ-radiation. To investigate whether the effects of progastrin observed in hGAS colon require the presence of other forms of circulating gastrin, we have crossed hGAS (hg+/+) with gastrin knockout (G−/−) mice to generate mice that express progastrin and no murine gastrin (G−/−hg+/+). After azoxymethane, G−/−hg+/+ mice developed significantly more ACF than control G−/−hg−/− mice (which do not express any forms of gastrin). G−/−hg+/+ mice also exhibited significantly increased colonic mitosis both before and after exposure to 8 Gray Gy γ-radiation or 50 mg/kg azoxymethane compared with G−/−hg−/−. Treatment of G−/−hg−/− mice with synthetic progastrin (residues 21–101 of human preprogastrin) or G17 extended at its COOH terminus corresponding to the COOH-terminal 26-amino-acid residues of human preprogastrin (residues 76–101, G17-CFP) resulted in continued colonic epithelial mitosis after γ-radiation, whereas glycine-extended gastrin-17 and the COOH-terminal tryptic fragment of progastrin [human preprogastrin-(96–101)] had no effect. Immunoneutralization with an antibody against G17-CFP before γ-radiation significantly decreased colonic mitosis in G−/−hg+/+ mice to levels similar to G−/−hg−/−. We conclude that progastrin does not require the presence of other forms of gastrin to exert proliferative effects on colonic epithelia and that the portion of the peptide responsible for these effects is contained within amino acid residues 76–101 of human preprogastrin.

The N5-Glutamine S-Adenosyl-l-Methionine-Dependent Methyltransferase PrmC/HemK in Chlamydia trachomatis Methylates Class 1 Release Factors

ABSTRACT The gene prmC, encoding the putative S-adenosyl-l-methionine (AdoMet)-dependent methyltransferase (MTase) of release factors (RFs) of the obligate intracellular pathogen Chlamydia trachomatis, was functionally analyzed. Chlamydial PrmC expression suppresses the growth defect of a prmC knockout strain of Escherichia coli K-12, suggesting an interaction of chlamydial PrmC with E. coli RFs in vivo. In vivo methylation assays carried out with recombinant PrmC and RFs of chlamydial origin demonstrated that PrmC methylates RFs within the tryptic fragment containing the universally conserved sequence motif Gly-Gly-Gln. This is consistent with the enzymatic properties of PrmC of E. coli origin. We conclude that C. trachomatis PrmC functions as an N 5-glutamine AdoMet-dependent MTase, involved in methylation of RFs.