Josette Perrier

Deoxynivalenol inhibits the expression by goblet cells of intestinal mucins through a PKR and MAP kinase dependent repression of the resistin‐like molecule β

SCOPE The food-associated mycotoxin deoxynivalenol (DON) is known to affect intestinal functions. However, its effect on intestinal mucus is poorly characterized. METHODS AND RESULTS We analyzed the effects of DON on human goblet cells (HT29-16E cells) and porcine intestinal explants. Results showed that subtoxic doses of DON (as low as 1 μM) decreased mucin (MUC) production. qPCR analysis demonstrated that this inhibition was due to a specific decrease in the level of mRNA encoding for the intestinal membrane-associated (MUC1) and the secreted MUCs (MUC2, MUC3). Mechanistic studies demonstrated that DON effect relied on the activation of the protein kinase R and the mitogen-activated protein kinase p38 ultimately leading to the inhibition of the expression of resistin-like molecule beta, a known positive regulator of MUC expression. CONCLUSION Taken together, our results show that at low doses found in food and feed, DON is able to affect the expression and production of MUCs by human and animal goblet cells. Due to the important role of MUCs in the barrier function and in the interaction of commensal bacteria with the host, such effect could explain the observed modifications in the microbial diversity and the increased susceptibility to enteric infection following exposure to DON.

The Food-Associated Ribotoxin Deoxynivalenol Modulates Inducible NO Synthase in Human Intestinal Cell Model

The intestinal epithelium possesses active immune functions including the production of proinflammatory cytokines and antimicrobial molecules such as nitric oxide (NO). As observed with immune cells, the production of NO by the intestinal epithelium is mainly due to the expression of the inducible NO synthase (iNOS or NOS2). Epithelial immune functions could be affected by many factors including pathogenic microorganisms and food-associated toxins (bacterial and fungal). Among the various mycotoxins, deoxynivalenol (DON) is known to alter the systemic and intestinal immunity. However, little is known about the effect of DON on the production of NO by the intestinal epithelium. We studied the impact of DON on the intestinal expression of iNOS using the Caco-2 cell model. In line with its proinflammatory activity, we observed that DON dose-dependently up-regulates the expression of iNOS mRNA. Surprisingly, DON failed to increase the expression of iNOS protein. When testing the effects of DON on cytokine-mediated induction of iNOS, we found that very low concentrations of DON (ie, 1 µM) decrease the amount of iNOS protein but not of iNOS mRNA. We demonstrated that DONs effect on iNOS protein relies on its ability to activate signal pathways and to increase iNOS ubiquitinylation and degradation through the proteasome pathway. Taken together, our results demonstrate that although DON causes intestinal inflammation, it suppresses the ability of the gut epithelium to express iNOS and to produce NO, potentially explaining the increased susceptibility of animals to intestinal infection following exposure to low doses of DON.

α-Galactosidase/sucrose kinase (AgaSK), a novel bifunctional enzyme from the human microbiome coupling galactosidase and kinase activities.

Background: Raffinose, an abundant carbohydrate in plants, is degraded into galactose and sucrose by intestinal microbial enzymes. Results: AgaSK is a protein coupling galactosidase and sucrose kinase activity. The structure of the galactosidase domain sheds light onto substrate recognition. Conclusion: AgaSK produces sucrose-6-phosphate directly from raffinose. Significance: Production of sucrose-6-phosphate directly from raffinose points toward a novel glycolytic pathway in bacteria. α-Galactosides are non-digestible carbohydrates widely distributed in plants. They are a potential source of energy in our daily food, and their assimilation by microbiota may play a role in obesity. In the intestinal tract, they are degraded by microbial glycosidases, which are often modular enzymes with catalytic domains linked to carbohydrate-binding modules. Here we introduce a bifunctional enzyme from the human intestinal bacterium Ruminococcus gnavus E1, α-galactosidase/sucrose kinase (AgaSK). Sequence analysis showed that AgaSK is composed of two domains: one closely related to α-galactosidases from glycoside hydrolase family GH36 and the other containing a nucleotide-binding motif. Its biochemical characterization showed that AgaSK is able to hydrolyze melibiose and raffinose to galactose and either glucose or sucrose, respectively, and to specifically phosphorylate sucrose on the C6 position of glucose in the presence of ATP. The production of sucrose-6-P directly from raffinose points toward a glycolytic pathway in bacteria, not described so far. The crystal structures of the galactosidase domain in the apo form and in complex with the product shed light onto the reaction and substrate recognition mechanisms and highlight an oligomeric state necessary for efficient substrate binding and suggesting a cross-talk between the galactose and kinase domains.

Functional characterization of a vacuolar invertase from Solanum lycopersicum: Post-translational regulation by N-glycosylation and a proteinaceous inhibitor

Plant vacuolar invertases, which belong to family 32 of glycoside hydrolases (GH32), are key enzymes in sugar metabolism. They hydrolyse sucrose into glucose and fructose. The cDNA encoding a vacuolar invertase from Solanum lycopersicum (TIV-1) was cloned and heterologously expressed in Pichia pastoris. The functional role of four N-glycosylation sites in TIV-1 has been investigated by site-directed mutagenesis. Single mutations to Asp of residues Asn52, Asn119 and Asn184, as well as the triple mutant (Asn52, Asn119 and Asn184), lead to enzymes with reduced specific invertase activity and thermostability. Expression of the N516D mutant, as well as of the quadruple mutant (N52D, N119D, N184D and N516D) could not be detected, indicating that these mutations dramatically affected the folding of the protein. Our data indicate that N-glycosylation is important for TIV-1 activity and that glycosylation of N516 is crucial for recombinant enzyme stability. Using a functional genomics approach a new vacuolar invertase inhibitor of S. lycopersicum (SolyVIF) has been identified. SolyVIF cDNA was cloned and heterologously expressed in Escherichia coli. Specific interactions between SolyVIF and TIV-1 were investigated by an enzymatic approach and surface plasmon resonance (SPR). Finally, qRT-PCR analysis of TIV-1 and SolyVIF transcript levels showed a specific tissue and developmental expression. TIV-1 was mainly expressed in flowers and both genes were expressed in senescent leaves.

Ruminococcus gnavus E1 modulates mucin expression and intestinal glycosylation

The molecular cross‐talk between commensal bacteria and the gut play an important role in the maintenance of the intestinal homeostasis and general health. Here, we studied the impact of a major Gram‐positive anaerobic bacterium of the human gut microbiota, that is, Ruminococcus gnavus on the glycosylation pattern and the production of intestinal mucus by the goblet cells.

Hydrolytic Fate of 3/15-Acetyldeoxynivalenol in Humans: Specific Deacetylation by the Small Intestine and Liver Revealed Using in Vitro and ex Vivo Approaches

In addition to deoxynivalenol (DON), acetylated derivatives, i.e., 3-acetyl and 15-acetyldexynivalenol (or 3/15ADON), are present in cereals leading to exposure to these mycotoxins. Animal and human studies suggest that 3/15ADON are converted into DON after their ingestion through hydrolysis of the acetyl moiety, the site(s) of such deacetylation being still uncharacterized. We used in vitro and ex vivo approaches to study the deacetylation of 3/15ADON by enzymes and cells/tissues present on their way from the food matrix to the blood in humans. We found that luminal deacetylation by digestive enzymes and bacteria is limited. Using human cells, tissues and S9 fractions, we were able to demonstrate that small intestine and liver possess strong deacetylation capacity compared to colon and kidneys. Interestingly, in most cases, deacetylation was more efficient for 3ADON than 15ADON. Although we initially thought that carboxylesterases (CES) could be responsible for the deacetylation of 3/15ADON, the use of pure human CES1/2 and of CES inhibitor demonstrated that CES are not involved. Taken together, our original model system allowed us to identify the small intestine and the liver as the main site of deacetylation of ingested 3/15ADON in humans.

Identification of the zinc binding ligands and the catalytic residue in human aspartoacylase, an enzyme involved in Canavan disease.

Canavan disease is an autosomal‐recessive neurodegenerative disorder caused by a lack of aspartoacylase, the enzyme that degrades N‐acetylaspartate (NAA) into acetate and aspartate. With a view to studying the mechanisms underlying the action of human aspartoacylase (hASP), this enzyme was expressed in a heterologous Escherichia coli system and characterized. The recombinant protein was found to have a molecular weight of 36 kDa and kinetic constants K m and k cat of 0.20 ± 0.03 mM and 14.22 ± 0.48 s−1, respectively. Sequence alignment showed that this enzyme belongs to the carboxypeptidase metalloprotein family having the conserved motif H21xxE24(91aa)H116. We further investigated the active site of hASP by performing modelling studies and site‐directed mutagenesis. His21, Glu24 and His116 were identified here for the first time as the residues involved in the zinc‐binding process. In addition, mutations involving the Glu178Gln and Glu178Asp residues resulted in the loss of enzyme activity. The finding that wild‐type and Glu178Asp have the same K m but different k cat values confirms the idea that the carboxylate group contributes importantly to the enzymatic activity of aspartoacylase.

Specific enhancement of acylase I and acylpeptide hydrolase activities by the corresponding N-acetylated substrates in primary rat hepatocyte cultures

The specific effects of N‐acetyl‐L‐methionine on acylase I activity and of both N‐acetyl‐L‐alanyl‐L‐alanine and N‐acetyl‐L‐methionyl‐L‐alanine on N‐acylpeptide hydrolase activity were investigated in primary rat hepatocyte cultures. Each of the above two substrates is known to be much more rapidly hydrolyzed than other derivatives of the same type under optimum enzyme assay conditions. After a two‐day incubation of the substrates in the presence of primary rat hepatocyte cultures, the N‐acetylaminoacid was found to specifically induce an increase in the acylase I activity, whereas the two N‐acetylated peptides increased the acylpeptide hydrolase activity in the soluble 100000 × g fraction from the culture medium. No change in any of the enzyme activities could be detected during the same period of time when the medium was not supplemented with N‐acetylated substrates. In addition, the acylase I activity showed a dose dependent response when the N‐acetyl‐L‐methionine concentration increased from 10 fold to 50 fold. It is therefore suggested that the efficient hydrolysis of each type of substrate that occured in the 48 h hepatocyte cell cultures was due to the increase observed in the overall activity of the corresponding enzymes. The ratio of acylpeptide hydrolase to that of acylase I increased considerably when the medium was supplemented with N‐acetylpeptides, and decreased in the presence of the N‐acetylaminoacid.

Radical Copolymerization of Vinyl Ethers and Cyclic Ketene Acetals as a Versatile Platform to Design Functional Polyesters

Free-radical copolymerization of cyclic ketene acetals (CKAs) and vinyl ethers (VEs) was investigated as an efficient yet simple approach for the preparation of functional aliphatic polyesters. The copolymerization of CKA and VE was first predicted to be quasi-ideal by DFT calculations. The theoretical prediction was experimentally confirmed by the copolymerization of 2-methylene-1,3-dioxepane (MDO) and butyl vinyl ether (BVE), leading to rMDO =0.73 and rBVE =1.61. We then illustrated the versatility of this approach by preparing different functional polyesters: 1) copolymers functionalized by fluorescent probes; 2) amphiphilic copolymers grafted with poly(ethylene glycol) (PEG) side chains able to self-assemble into PEGylated nanoparticles; 3) antibacterial films active against Gram-positive and Gram-negative bacteria (including a multiresistant strain); and 4) cross-linked bioelastomers with suitable properties for tissue engineering applications.

Acylase 1 expression in rat intestinal crypt–villus axis

Acylase 1 was investigated at the cellular level in the rat small intestine along the enterocyte—differentiation axis. As confirmed by microscopic analysis, villus tip cells and crypt cells of rat jejunal mucosa were successfully separated using the Weiser method. The proliferating undifferentiated crypt cells showed much higher ACY 1 activity levels than the villus cells, with a 6.4‐fold decrease as the cells migrated and differentiated along the crypt—villus axis. RT‐PCR studies on mRNA extracted from isolated cells showed that ACY 1 mRNA was mainly expressed in crypt cells, reaching levels that were 12‐fold higher than those recorded in other cell types along the whole enterocyte differentiation axis. It was concluded that the expression of ACY 1 in the intestinal crypt cells is regulated at the mRNA level. Immunohistochemistry revealed the expression of ACY 1 in the absorbing lineage cells from the ileal and colonic crypts and the absence of ACY 1 in the mucus producing goblet cells. These findings proposed ACY 1 as a new marker transcript for absorbing cells of intestinal crypt, which can be used to monitor the process of intestinal N‐α‐acetylated protein metabolism.