Ruminococcus gnavus E1 modulates mucin expression and intestinal glycosylation.

AIMS: 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. METHODS AND RESULTS: Our results showed that R. gnavus E1 specifically increases the expression and the glycosylation level of the intestinal glyco-conjugates by goblet cells in the colonic mucosa of mono-associated mice with R. gnavus E1 as well as in human HT29-MTX cells. Such an effect was mediated through induction of the level of mRNA encoding for the major intestinal gel-forming mucin such as MUC2 and various glycosyltransferase enzymes. CONCLUSIONS: This study demonstrates for the first time that R. gnavus E1 possess the ability to modulate the glycosylation profile of the glyco-conjugate molecules and mucus in goblet cells. SIGNIFICANCE AND IMPACT OF THE STUDY: Furthermore, we demonstrated that R. gnavus E1 modified specifically the glycosylation pattern and MUC2 expression by means of a small soluble factor of peptidic nature (<3 kDa) and heat stable in the HT29-MTX cell.

Functional analysis of family GH36 α-galactosidases from Ruminococcus gnavus E1: insights into the metabolism of a plant oligosaccharide by a human gut symbiont.

Ruminococcus gnavus belongs to the 57 most common species present in 90% of individuals. Previously, we identified an α-galactosidase (Aga1) belonging to glycoside hydrolase (GH) family 36 from R. gnavus E1 (M. Aguilera, H. Rakotoarivonina, A. Brutus, T. Giardina, G. Simon, and M. Fons, Res. Microbiol. 163:14-21, 2012). Here, we identified a novel GH36-encoding gene from the same strain and termed it aga2. Although aga1 showed a very simple genetic organization, aga2 is part of an operon of unique structure, including genes putatively encoding a regulator, a GH13, two phosphotransferase system (PTS) sequences, and a GH32, probably involved in extracellular and intracellular sucrose assimilation. The 727-amino-acid (aa) deduced Aga2 protein shares approximately 45% identity with Aga1. Both Aga1 and Aga2 expressed in Escherichia coli showed strict specificity for α-linked galactose. Both enzymes were active on natural substrates such as melibiose, raffinose, and stachyose. Aga1 and Aga2 occurred as homotetramers in solution, as shown by analytical ultracentrifugation. Modeling of Aga1 and Aga2 identified key amino acids which may be involved in substrate specificity and stabilization of the α-linked galactoside substrates within the active site. Furthermore, Aga1 and Aga2 were both able to perform transglycosylation reactions with α-(1,6) regioselectivity, leading to the formation of product structures up to [Hex](12) and [Hex](8), respectively. We suggest that Aga1 and Aga2 play essential roles in the metabolism of dietary oligosaccharides and could be used for the design of galacto-oligosaccharide (GOS) prebiotics, known to selectively modulate the beneficial gut microbiota.

Intron retention regulates the expression of pectin methyl esterase inhibitor (Pmei) genes during wheat growth and development.

Pectin is an important component of the plant cell wall and its remodelling occurs during normal plant growth or following stress responses. Pectin is secreted into the cell wall in a highly methyl-esterified form and subsequently de-methyl-esterified by pectin methyl esterase (PME), whose activity is controlled by the pectin methyl esterase inhibitor protein (PMEI). Cereal cell wall contains a low amount of pectin; nonetheless the level and pattern of pectin methyl esterification play a primary role during development or pathogen infection. Since few data are available on the role of PMEI in plant development and defence of cereal species, we isolated and characterised three Pmei genes (Tdpmei2.1, Tdpmei2.2 and Tdpmei3) and their encoded products in wheat. Sequence comparisons showed a low level of intra- and inter-specific sequence conservation of PMEIs. Tdpmei2.1 and Tdpmei2.2 share 94% identity at protein level, but only 20% identity with the product of Tdpmei3. All three Tdpmei genes code for functional inhibitors of plant PMEs and do not inhibit microbial PMEs or a plant invertase. RT-PCR analyses demonstrated, for the first time to our knowledge, that Pmei genes are regulated by intron retention. Processed and unprocessed transcripts of Tdpmei2.1 and Tdpmei2.2 accumulated in several organs, but anthers contained only mature transcripts. Tdpmei3 lacks introns and its transcript accumulated mainly in stem internodes. These findings suggest that products encoded by these Tdpmei genes control organ- or tissue-specific activity of specific PME isoforms in wheat.

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 H(21)xxE(24)(91aa)H(116). 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.

Aminoacylase I deficiency: a novel inborn error of metabolism.

This is the first report of a patient with aminoacylase I deficiency. High amounts of N-acetylated amino acids were detected by gas chromatography-mass spectrometry in the urine, including the derivatives of serine, glutamic acid, alanine, methionine, glycine, and smaller amounts of threonine, leucine, valine, and isoleucine. NMR spectroscopy confirmed these findings and, in addition, showed the presence of N-acetylglutamine and N-acetylasparagine. In EBV transformed lymphoblasts, aminoacylase I activity was deficient. Loss of activity was due to decreased amounts of aminoacylase I protein. The amount of mRNA for the aminoacylase I was decreased. DNA sequencing of the encoding ACY1 gene showed a homozygous c.1057 C>T transition, predicting a p.Arg353Cys substitution. Both parents were heterozygous for the mutation. The mutation was also detected in 5/161 controls. To exclude the possibility of a genetic polymorphism, protein expression studies were performed showing that the mutant protein had lost catalytic activity.

Both binding sites of the starch-binding domain of Aspergillus niger glucoamylase are essential for inducing a conformational change in amylose.

The interaction of the two binding sites of the starch-binding domain (SBD) of Aspergillus niger glucoamylase 1 (GA-I) with substrate has been investigated by using atomic force microscopy (AFM) and UV difference spectroscopy in combination with site-specific mutants of both SBD and GA-I. The SBD possesses two binding sites with distinct affinities towards the soluble linear substrate maltoheptaose; dissociation constants (K(d)) of 17 and 0.95 microM were obtained for W563 K (binding site 2 mutant) and W590 K (binding site 1 mutant), respectively, compared to an apparent K(d) of 23 microM for the wild-type SBD. Further, the two sites are almost but not totally independent of each other for binding, since abolishing one site does not prevent the amylose chain binding to the other site. Using AFM, we show that the amylose chains undergo a conformational change to form loops upon binding to the SBD, using either the recombinant wild-type SBD or a catalytically inactive mutant of GA-I. This characteristic conformation of amylose is lost when one of the SBD binding sites is eliminated by site-directed mutagenesis, as seen with the mutants W563 K or W590 K. Therefore, although each binding site is capable of simple binding to a ligand, both sites must be functional in order to induce a gross conformational change of the amylose molecules. Taken together these data suggest that for the complex with soluble amylose, SBD binds to a single amylose chain, site 1 being responsible for the initial recognition of the chain and site 2 being involved in tighter binding, leading to the circularisation of the amylose chain observed by AFM. Binding of the SBD to the amylose chain results in a novel two-turn helical amylose complex structure. The binding of parallel amylosic chains to the SBD may provide a basis for understanding the role of the SBD in facilitating enzymatic degradation of crystalline starches by glucoamylase 1.

The rat kidney acylase I, characterization and molecular cloning. Differences with other acylases I.

The soluble acylase I from rat kidney was purified to homogeneity using a five-step procedure. As the resulting protein was found to have a relative molecular mass of 125 kDa based on size-exclusion chromatography and 44 kDa based on SDS/PAGE, the native protein was taken to consist of three subunits. The amino-acid sequence of a peptide resulting from limited proteolysis of the polypeptide chain with proteinase K, which was determined by microsequencing (RHEFHALRAGFALDEGLA), was found to be very similar to the corresponding sequence of porcine kidney acylase I. However, as N-furyl-acryloyl-L-methionine, a synthetic substrate for porcine acylases, was not hydrolyzed by the rat enzyme, it was suggested that the polypeptide chain might differ in other respects from those of the other acylases I. A full length cDNA coding for the rat kidney acylase I was therefore isolated and found to contain a 1224-bp open reading frame encoding a protein consisting of 408 amino-acid residues, which corresponded to a calculated molecular mass of 45 847 Da per subunit. The deduced amino-acid sequence showed 93.6% and 87.2% identity with that of the human liver and porcine kidney, respectively.

Distribution and subcellular localization of acylpeptide hydrolase and acylase I along the hog gastro-intestinal tract.

The distribution of acylase I and acylpeptide hydrolase along the hog small intestine was investigated. No significant changes in their respective specific activity was found when the intestine was cut off and divided into eight segments (taken every 200 cm) so as to specifically study the duodenum, jejunum and ileum. Upon performing subcellular fractionation of hog enterocytes, it was observed that acylpeptide hydrolase is a soluble enzyme, while acylase I is essentially a soluble protein accounting for only 5% of the activity associated with the whole membrane fraction. The membrane-bound acylase I was neither solubilized by phosphatidylinositol-specific phospholipase C from Bacillus cereus nor by detergents which are commonly used to solubilize alkaline phosphatase, a glycosylphosphatidylinositol-anchored protein. When a phase separation was carried out in Triton X-114, all the anchored-membrane proteins of the intestinal membranes were located in the detergent-rich phase, while acylase I was present in the detergent-poor phase. Finally, the immunolabeling of intestinal cells with specific antibodies definitively established the cytoplasmic localization of acylase I. Acylpeptide hydrolase and acylase I therefore both are located in the enterocyte cytoplasm.

The membrane-bound basic carboxypeptidase from hog intestinal mucosa(1).

The carboxypeptidase activity occurring in hog intestinal mucosa is apparently due to two distinct enzymes which may be responsible for the release of basic COOH-terminal amino acids from short peptides. The plasma membrane-bound carboxypeptidase activity which occurs at neutral optimum pH levels was found to be enhanced by CoCl(2) and inhibited by guanidinoethylmercaptosuccinic acid, o-phenanthroline, ethylenediamine tetraacetic acid and cadmium acetate; whereas the soluble carboxypeptidase activity which occurs at an optimum pH level of 5.0 was not activated by CoCl(2) and only slightly inhibited by o-phenanthroline, ethylenediamine tetraacetic acid, NiCl(2) and CdCl(2). The latter activity was presumably due to lysosomal cathepsin B, which is known to be present in the soluble fraction of hog intestinal mucosa. Although the membrane-bound enzyme was evenly distributed along the small intestine, it was not anchored in the phospholipidic bilayer via a glycosyl-phosphatidylinositol moiety, as carboxypeptidase M from human placenta is. The enzyme was not solubilized by phosphatidylinositol-specific phospholipase C, but was solubilized to practically the same extent by several detergents. The purified trypsin-solubilized form is a glycoprotein with a molecular mass of 200 kDa, as determined by performing SDS-PAGE and gel filtration, which differs considerably from the molecular mass of human placental carboxypeptidase M (62 kDa). It was found to cleave lysyl bonds more rapidly than arginyl bonds, which is not so in the case of carboxypeptidase M, and immunoblotting analysis provided further evidence that hog intestinal and human placental membrane-bound carboxypeptidases do not bear much resemblance to each other. Since the latter enzyme has been called carboxypeptidase M, it is suggested that the former might be carboxypeptidase D, the recently described new member of the carboxypeptide B-type family.

Cloning, sequencing and further characterization of acylpeptide hydrolase from porcine intestinal mucosa.

Acylpeptide hydrolase was purified to homogeneity from porcine intestinal mucosa using a seven-step procedure including ammonium sulfate precipitation, gel filtration as well as anion exchange and affinity chromatography. The specific activity of the enzyme reached 105000 nmol/mg protein per min and the purification was as high as 5500-fold. This tetrameric enzyme is composed of four apparently identical subunits, the molecular mass of which was estimated to be 75 kDa, based on the results of amino acid analysis and gel electrophoresis performed under denaturing conditions. It is likely that the NH(2)-terminal residue may be acetylated, while serine was found to be the COOH-terminal residue. The hydrolytic activity of the enzyme toward N-acetyl-L-alanine p-nitroanilide at the optimum pH value was increased twofold in the presence of the chloride anion. The K(m) value calculated from the kinetics of the hydrolysis of acetylalanyl peptides was found to be 0.7+/-0.1 mM, whereas the V(max) values decreased from 200 to 50 nmol/min per microgram of enzyme, depending on the peptidic chain lengths. The V(max) value of the synthetic substrate (250 nmol/min per microgram of enzyme) was 25-500% higher than those of the acetylalanyl peptides, depending on the peptide chain length, although the enzyme affinity was slightly lower (1.8 mM as compared with 0.7 mM). In line with data on other animal species and on various tissues, the enzyme seemed likely to be a serine protease, since it was readily inhibited by diisopropyl fluorophosphate and diethyl pyrocarbonate. A 2377-nucleotide long cDNA coding for the enzyme was isolated from pig small intestine. The deduced amino acid sequence consisted of 731 residues and showed a single different amino acid with that of the porcine liver APH, except the N-terminal amino acid which is still probably lacking.

The hog intestinal mucosa acylase I: subcellular localization, isolation, kinetic studies and biological function.

The soluble acylase I (N-acylamino acid amidohydrolase, EC 3.5.1.14) from hog intestinal mucosa was 11,000-fold purified for the first time using a new four-step procedure involving an immunoaffinity chromatography. The resulting protein, which had an isoelectric point of 5.2 and a M(r) of 90,000 was composed of two apparently identical N-acylated polypeptide chains. Its amino acid composition was comparable to that of hog kidney acylase I. The enzyme had a pH optimum at 8.0 and required Zn2+ or Co2+. The optimal temperature for the acylase reaction was 40 degrees C and the activation energy of thermodenaturation was estimated at 260 kJ mol-1. The enzyme was strongly inhibited when preincubated with chelating agents, by diethyl pyrocarbonate under histidine-modifying conditions as well as by sulfhydryl compounds. The reaction of the purified enzyme with the synthetic substrate furylacryloyl-L-methionine was partly characterized as follows: Km = 0.22 +/- 0.03 mM, kcat = 128.0 +/- 17.8 s-1 and kcat/Km = 5.8 +/- 1.6 x 10(5) M-1 s-1. The L-stereoisomer of methionine competitively inhibited the enzyme reaction with a Ki of 3.4 +/- 0.2 mM. It is suggested that acylase I might not only be involved in the catabolism of intracellular N-acylated protein but also be responsible for the biological utilization of N-acylated food proteins.