Function of xyloglucan endotransglucosylase/hydrolases in rice

Background and Aims

Although xyloglucans are ubiquitous in land plants, they are less abundant in Poales species than in eudicotyledons. Poales cell walls contain higher levels of β-1,3/1,4 mixed-linked glucans and arabinoxylans than xyloglucans. Despite the relatively low level of xyloglucans in Poales, the xyloglucan endotransglucosylase/hydrolase (XTH) gene family in rice (Oryza sativa) is comparable in size to that of the eudicotyledon Arabidopsis thaliana. This raises the question of whether xyloglucan is a substrate for rice XTH gene products, whose enzyme activity remains largely uncharacterized.

Methods

This study focused on OsXTH19 (which belongs to Group IIIA of the XTH family and is specifically expressed in growing tissues of rice shoots), and two other XTHs, OsXTH11 (Group I/II) and OsXTH20 (Group IIIA), for reference, and measurements were made of the enzymatic activities of three recombinant rice XTHs, i.e. OsXTH11, OsXTH20 and OsXTH19.

Key Results

All three OsXTH gene products have xyloglucan endohydrolase (XEH, EC 3·2·1·151) activity, and OsXTH11 has both XEH and xyloglucan endotransglycosylase (XET, EC 2·4·1207) activities. However, these proteins had neither hydrolase nor transglucosylase activity when glucuronoarabinoxylan or mixed-linkage glucan was used as the substrate. These results are consistent with histological observations demonstrating that pOsXTH19::GUS is expressed specifically in the vicinity of tissues where xyloglucan immunoreactivity is present. Transgenic rice lines over-expressing OsXTH19 (harbouring a Cauliflower Mosaic Virus 35S promoter::OsXTH19 cDNA construct) or with suppressed OsXTH19 expression (harbouring a pOsXTH19 RNAi construct) did not show dramatic phenotypic changes, suggesting functional redundancy and collaboration among XTH family members, as was observed in A. thaliana.

Conclusions

OsXTH20 and OsXTH19 act as hydrolases exclusively on xyloglucan, while OsXTH11 exhibits both hydrolase and XET activities exclusively on xyloglucans. Phenotypic analysis of transgenic lines with altered expression of OsXTH19 suggests that OsXTH19 and related XTH(s) play redundant roles in rice growth.     

Barley xyloglucan xyloglucosyl transferases bind xyloglucan-derived oligosaccharides in their acceptor-binding regions in multiple conformational states

Three barley xyloglucan endotransglycosylases (HvXETs), known as xyloglucan xyloglucosyl transferases (EC 2.4.1.207), were subjected to kinetic and computational docking studies. The k_cat·K_m⁻¹ values with the reduced [³H]-labelled XXXG, XXLG/XLXG and XLLG acceptor substrates were 0.02 × 10⁻², 0.1 × 10⁻² and 3.2 × 10⁻² s⁻¹ μM⁻¹, while the K_m constants were 10.6, 8.6 and 5.3 mM, obtained for HvXET3, HvXET4 and HvXET6, respectively. Docking of XLLG in acceptor-binding regions revealed that at least two conformational states were likely to participate in all isoforms. The assessments of kinetic and computational data indicated that the disposition of aromatic residues at the entrance to the active sites and the flexibility of proximal COOH-terminal loops could orient acceptors more or less favourably during binding, thus leading to tighter or weaker K_m constants. The data suggested that binding of acceptors in HvXETs is guided by contributions from the conserved residues in the active sites and by the of neighbouring loops.     

Positional specifity of acetylxylan esterases on natural polysaccharide: An NMR study

Background

Microbial degradation of acetylated plant hemicelluloses involves besides enzymes cleaving the glycosidic linkages also deacetylating enzymes. A detailed knowledge of the mode of action of these enzymes is important in view of the development of efficient bioconversion of plant materials that did not undergo alkaline pretreatment leading to hydrolysis of ester linkages.

Methods

In this work deacetylation of hardwood acetylglucuronoxylan by acetylxylan esterases from Streptomyces lividans (carbohydrate esterase family 4) and Orpinomyces sp. (carbohydrate esterase family 6) was monitored by ¹H-NMR spectroscopy.

Results

The ¹H-NMR resonances of all acetyl groups in the polysaccharide were fully assigned. The targets of both enzymes are 2- and 3-monoacetylated xylopyranosyl residues and, in the case of the Orpinomyces sp. enzyme, also the 2,3-di-O-acetylated xylopyranosyl residues. Both enzymes do not recognize as a substrate the 3-O-acetyl group on xylopyranosyl residues α-1,2-substituted with 4-O-methyl-d-glucuronic acid.

Conclusions

The ¹H-NMR spectroscopy approach to study positional and substrate specificity of AcXEs outlined in this work appears to be a simple way to characterize catalytic properties of enzymes belonging to various CE families.

Significance

The results contribute to development of efficient and environmentally friendly procedures for enzymatic degradation of plant biomass.     

A novel dienelactone hydrolase from the thermoacidophilic archaeon Sulfolobus solfataricus P1: Purification,characterization, and expression

Background

Dienelactone hydrolases catalyze the hydrolysis of dienelactone to maleylacetate, which play a key role for the microbial degradation of chloroaromatics via chlorocatechols. Here, a thermostable dienelactone hydrolase from thermoacidophilic archaeon Sulfolobus solfataricus P1 was the first purified and characterized and then expressed in Escherichia coli.

Methods

The enzyme was purified by using several column chromatographys and characterized by determining the enzyme activity using p-nitrophenyl caprylate and dienelactones. In addition, the amino acids related to the catalytic mechanism were examined by site-directed mutagenesis using the identified gene.

Results

The enzyme, approximately 29 kDa monomeric, showed the maximal activity at 74 °C and pH 5.0, respectively. The enzyme displayed remarkable thermostability: it retained approximately 50% of its activity after 50 h of incubation at 90 °C, and showed high stability against denaturing agents, including various detergents, urea, and organic solvents. The enzyme displayed substrate specificities toward trans-dienelactone, not cis-isomer, and also carboxylesterase activity toward p-nitrophenyl esters ranging from butyrate (C₄) to laurate (C₁₂). The k_cat/K_m ratios for trans-dienelactone and p-nitrophenyl caprylate (C₈), the best substrate, were 92.5 and 54.7 s⁻¹ μM⁻¹, respectively.

Conclusions

The enzyme is a typical dienelactone hydrolase belonging to α/β hydrolase family and containing a catalytic triad composed of Cys151, Asp198, and His229 in the active site.

General significance

The enzyme is the first characterized archaeal dienelactone hydrolase.     

Edible blue-green algae reduce the production of pro-inflammatory cytokines by inhibiting NF-κB pathway in macrophages and splenocytes

Background

Chronic inflammation contributes to the development of pathological disorders including insulin resistance and atherosclerosis. Identification of anti-inflammatory natural products can prevent the inflammatory diseases.

Methods

Anti-inflammatory effects of blue-green algae (BGA), i.e., Nostoc commune var. sphaeroides Kützing (NO) and Spirulina platensis (SP), were compared in RAW 264.7 and mouse bone marrow-derived macrophages (BMM) as well as splenocytes from apolipoprotein E knockout (apoE^−/−) mice fed BGA.

Results

When macrophages pretreated with 100 μg/ml NO lipid extract (NOE) or SP lipid extract (SPE) were activated by lipopolysaccharide (LPS), expression and secretion of pro-inflammatory cytokines, such as tumor necrosis factor α (TNFα), interleukin 1β (IL-1β), and IL-6, were significantly repressed. NOE and SPE also significantly repressed the expression of TNFα and IL-1β in BMM. LPS-induced secretion of IL-6 was lower in splenocytes from apoE^−/− fed an atherogenic diet containing 5% NO or SP for 12 weeks. In RAW 264.7 macrophages, NOE and SPE markedly decreased nuclear translocation of NF-κB. The degree of repression of pro-inflammatory gene expression by algal extracts was much stronger than that of SN50, an inhibitor of NF-κB nuclear translocation. Trichostatin A, a pan histone deacetylase inhibitor, increased basal expression of IL-1β and attenuated the repression of the gene expression by SPE. SPE significantly down-regulated mRNA abundance of 11 HDAC isoforms, consequently increasing acetylated histone 3 levels.

Conclusion

NOE and SPE repress pro-inflammatory cytokine expression and secretion in macrophages and splenocytes via inhibition of NF-κB pathway. Histone acetylation state is likely involved in the inhibition.

General significance

This study underscores natural products can exert anti-inflammatory effects by epigenetic modifications such as histone acetylation.     

Structural and catalytic roles of residues located in β13 strand and the following β-turn loop in Fibrobacter succinogenes 1,3-1,4-β-d-glucanase

Background

Fibrobacter succinogenes 1,3-1,4-β-d-glucanase (Fsβ-glucanase) is the only naturally occurring circularly permuted β-glucanase among bacterial glucanases with reverse protein domains. We characterized the functional and structural significance of residues 200–209 located in the domain B of Fsβ-glucanase, corresponding to the major surface loop in the domain A region of Bacillus licheniformis glucanase.

Methods

Rational design approaches including site-directed mutagenesis, initial-rate kinetics, and structural modeling analysis were used in this study.

Results

Our kinetic data showed that D202N and D206N exhibited a 1.8- and 1.5-fold increase but G207N, G207−, F205L, N208G and T204F showed a 7.0- to 2.2-fold decrease, in catalytic efficiency (k_cat/K_M) compared to the wild-type enzyme. The comparative energy ΔΔG_b value in individual mutant enzymes was well correlated to their catalytic efficiency. D206R mutant enzyme exhibited the highest relative activity at 50 °C over 10 min, whereas K200F was the most heat-sensitive enzyme.

Conclusions

This study demonstrates that Phe205, Gly207, and Asn208 in the Type II turn of the connecting loop may play a role in the catalytic function of Fsβ-glucanase.

General significance

Residues 200–209 in Fsβ-glucanase resided at the similar structural topology to that of Bacillus enzyme were found to play some similar catalytic function in glucanase.     

Evolution of mixed-linkage (1 -> 3, 1 -> 4)-β-D-glucan (MLG) and xyloglucan in Equisetum (horsetails) and other monilophytes

Background and Aims

Horsetails (Equisetopsida) diverged from other extant eusporangiate monilophytes in the Upper Palaeozoic. They are the only monilophytes known to contain the hemicellulose mixed-linkage (1 → 3, 1 → 4)-β-d-glucan (MLG), whereas all land plants possess xyloglucan. It has been reported that changes in cell-wall chemistry often accompanied major evolutionary steps. We explored changes in hemicelluloses occurring during Equisetum evolution.

Methods

Hemicellulose from numerous monilophytes was treated with lichenase and xyloglucan endoglucanase. Lichenase digests MLG to di-, tri- and tetrasaccharide repeat-units, resolvable by thin-layer chromatography.

Key Results

Among monilophytes, MLG was confined to horsetails. Our analyses support a basal trichotomy of extant horsetails: MLG was more abundant in subgenus Equisetum than in subgenus Hippochaete, and uniquely the sister group E. bogotense yielded almost solely the tetrasaccharide repeat-unit (G4G4G3G). Other species also gave the disaccharide, whereas the trisaccharide was consistently very scarce. Tetrasaccharide : disaccharide ratios varied interspecifically, but with no consistent difference between subgenera. Xyloglucan was scarce in Psilotum and subgenus Equisetum, but abundant in subgenus Hippochaete and in the eusporangiate ferns Marattia and Angiopteris; leptosporangiate ferns varied widely. All monilophytes shared a core pattern of xyloglucan repeat-units, major XEG products co-chromatographing on thin-layer chromatography with non-fucosylated hepta-, octa- and nonasaccharides and fucose-containing nona- and decasaccharides.

Conclusions

G4G4G3G is the ancestral repeat-unit of horsetail MLG. Horsetail evolution was accompanied by quantitative and qualitative modification of MLG; variation within subgenus Hippochaete suggests that the structure and biosynthesis of MLG is evolutionarily plastic. Xyloglucan quantity correlates negatively with abundance of other hemicelluloses; but qualitatively, all monilophyte xyloglucans conform to a core pattern of repeat-unit sizes.     

Characterization of ecto-ATPase activity in the surface of LLC-PK1 cells and its modulation by ischemic conditions

Background

The concentration of extracellular nucleotides is regulated by enzymes that have their catalytic site facing the extracellular space, the so-called ecto-enzymes.

Methods

We used LLC-PK1 cells, a well-characterized porcine renal proximal tubule cell line, to biochemically characterize ecto-ATPase activity in the luminal surface. The [γ-³²P]Pi released after reaction was measured in aliquots of the supernatant by liquid scintillation.

Results

This activity was linear with time up to 20 min of reaction and stimulated by divalent metals. The ecto-ATPase activity measured in the presence of 5 mM MgCl₂ was (1) optimum at pH 8, (2) insensitive to different inhibitors of intracellular ATPases, (3) inhibited by 1 mM suramin, an inhibitor of ecto-ATPases, (4) sensitive to high concentrations of sodium azide (NaN₃) and (5) also able to hydrolyze ADP in the extracellular medium. The ATP:ADP hydrolysis ratio calculated was 4:1. The ecto-ADPase activity was also inhibited by suramin and NaN₃. The dose–response of ATP revealed a hyperbolic profile with maximal velocity of 25.2 ± 1.2 nmol Pi x mg^− 1 x min^− 1 and K_0.5 of 0.07 ± 0.01 mM. When cells were submitted to ischemia, the E-NTPDase activity was reduced with time, achieving 71% inhibition at 60 min of ischemia.

Conclusion

Our results suggest that the ecto-ATPase activity of LLC-PK1 cells has the characteristics of a type 3 E-NTPDase which is inhibited by ischemia.

General Significance

This could represent an important pathophysiologic mechanism that explains the increase in ATP concentration in the extracellular milieu in the proximal tubule during ischemia.     

Mode of action of acetylxylan esterases on acetyl glucuronoxylan and acetylated oligosaccharides generated by a GH10 endoxylanase

Background

Substitutions on the xylan main chain are widely accepted to limit plant cell wall degradability and acetylations are considered as one of the most important obstacles. Hence, understanding the modes of action of a range of acetylxylan esterases (AcXEs) is of ample importance not only to increase the understanding of the enzymology of plant decay/bioremediation but also to enable efficient bioconversion of plant biomass.

Methods

In this study, the modes of action of acetylxylan esterases (AcXEs) belonging to carbohydrate esterase (CE) families 1, 4, 5 and 6 on xylooligosaccharides generated from hardwood acetyl glucuronoxylan were compared using MALDI ToF MS. Supporting data were obtained by following enzymatic deacetylation by ¹H NMR spectroscopy.

Conclusions

None of the used enzymes were capable of complete deacetylation, except from linear xylooligosaccharides which were completely deacetylated by some of the esterases in the presence of endoxylanase. A clear difference was observed between the performance of the serine-type esterases of CE families 1, 5 and 6, and the aspartate-metalloesterases of family CE4. The difference is mainly due to the inability of CE4 AcXEs to catalyze deacetylation of 2,3-di-O-acetylated xylopyranosyl residues. Complete deacetylation of a hardwood acetyl glucuronoxylan requires additional deacetylating enzyme(s).

General significance

The results contribute to the understanding of microbial degradation of plant biomass and outline the way to achieve complete saccharification of plant hemicelluloses which did not undergo alkaline pretreatment.     

Trichoderma reesei CE16 acetyl esterase and its role in enzymatic degradation of acetylated hemicellulose

Background

Trichoderma reesei CE16 acetyl esterase (AcE) is a component of the plant cell wall degrading system of the fungus. The enzyme behaves as an exo-acting deacetylase removing acetyl groups from non-reducing end sugar residues.

Methods

In this work we demonstrate this exo-deacetylating activity on natural acetylated xylooligosaccharides using MALDI ToF MS.

Results

The combined action of GH10 xylanase and acetylxylan esterases (AcXEs) leads to formation of neutral and acidic xylooligosaccharides with a few resistant acetyl groups mainly at their non-reducing ends. We show here that these acetyl groups serve as targets for TrCE16 AcE. The most prominent target is the 3-O-acetyl group at the non-reducing terminal Xylp residues of linear neutral xylooligosaccharides or on aldouronic acids carrying MeGlcA at the non-reducing terminus. Deacetylation of the non-reducing end sugar may involve migration of acetyl groups to position 4, which also serves as substrate of the TrCE16 esterase.

Conclusion

Concerted action of CtGH10 xylanase, an AcXE and TrCE16 AcE resulted in close to complete deacetylation of neutral xylooligosaccharides, whereas substitution with MeGlcA prevents removal of acetyl groups from only a small fraction of the aldouronic acids. Experiments with diacetyl derivatives of methyl β-d-xylopyranoside confirmed that the best substrate of TrCE16 AcE is 3-O-acetylated Xylp residue followed by 4-O-acetylated Xylp residue with a free vicinal hydroxyl group.

General significance

This study shows that CE16 acetyl esterases are crucial enzymes to achieve complete deacetylation and, consequently, complete the saccharification of acetylated xylans by xylanases, which is an important task of current biotechnology.     

A novel bifunctional peptidic aspartic protease inhibitor inhibits chitinase A from Serratia marcescens: Kinetic analysis of inhibition and binding affinity

Background

Chitinase inhibitors have chemotherapeutic potential as fungicides, pesticides and antiasthmatics. The majority of chitinase inhibitors reported are natural products like argifin, argifin linear fragments, argadin, allosamidin and disulfide-cyclized peptides. Here, we report a novel peptidic inhibitor API (Aspartic Protease Inhibitor), isolated from Bacillus licheniformis that inhibits chitinase A (ChiA) from Serratia marcescens.

Methods

The binding affinity of API with ChiA and type of inhibition was determined by the inhibition kinetics assays. Fluorescence and CD spectroscopic analysis and chemical modification of API with different affinity reagents elucidated the mechanism of binding of API with ChiA.

Results and conclusions

The peptide has an amino acid sequence N-Ile¹-Cys²-Glu³-Ala⁴-Glu⁵-His⁶-Lys⁷-Trp⁸-Gly⁹-Asp¹⁰-Tyr¹¹-Leu¹²-Asp¹³-C. The ChiA–API kinetic interactions reveal noncompetitive, irreversible and tight binding nature of API with I₅₀ = 600 nM and K_i = 510 nM in the presence of chromogenic substrate p-nitrophenyl-N,N′-diacetyl-β-chitobioside[p-NP-(GlcNAc)₂]. The inhibition progress curves show a two-step slow tight binding inhibition mechanism with the rate constant k₅ = 8.7 ± 1 × 10^− 3 s^− 1 and k₆ = 7.3 ± 0.6 × 10^− 5 s^− 1. CD-spectra and tryptophanyl fluorescence analysis of ChiA incubated with increasing API concentrations confirms conformational changes in enzyme structure which may be due to irreversible denaturation of enzyme upon binding of API. Chemical modifications by WRK abolished the anti-chitinase activity of API and revealed the involvement of carboxyl groups in the enzyme inactivation. Abolished isoindole fluorescence of OPTA-labeled ChiA demonstrates the irreversible denaturation of ChiA upon incubation with API for prolonged time and distortion of active site of the enzyme.

General significance

The data provide useful information that could lead to the generation of drug-like, natural product-based chitinase inhibitors.     

Selenium-binding protein 1: Its physiological function,dependence on aryl hydrocarbon receptors,and role in wasting syndrome by 2,3,7,8-tetrachlorodibenzo-p-dioxin

Background

Selenium-binding protein 1 (Selenbp1) is suggested to play a role in tumor suppression, and may be involved in the toxicity produced by dioxin, an activator of aryl hydrocarbon receptors (AhR). However, the mechanism or likelihood is largely unknown because of the limited information available about the physiological role of Selenbp1.

Methods

To address this issue, we generated Selenbp1-null [Selenbp1 (−/−)] mice, and examined the toxic effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in this mouse model.

Results

Selenbp1 (−/−) mice exhibited only a few differences from wild-type mice in their apparent phenotypes. However, a DNA microarray experiment showed that many genes including Notch1 and Cdk1, which are known to be enhanced in ovarian carcinoma, are also increased in the ovaries of Selenbp1 (−/−) mice. Based on the different responses to TCDD between C57BL/6J and DBA/2J strains of mice, the expression of Selenbp1 is suggested to be under the control of AhR. However, wasting syndrome by TCDD occurred equally in Selenbp1 (−/−) and (+/+) mice.

Conclusions

The above pieces of evidence suggest that 1) Selenbp1 suppresses the expression of tumor-promoting genes although a reduction in Selenbp1 alone is not very serious as far as the animals are concerned; and 2) Selenbp1 induction by TCDD is neither a pre-requisite for toxicity nor a protective response for combating TCDD toxicity.

General significance

Selenbp1 (−/−) mice exhibit little difference in their apparent phenotype and responsiveness to dioxin compared with the wild-type. This may be due to the compensation of Selenbp1 function by a closely-related protein, Selenbp2.     

Inorganic phosphate uptake in Trypanosoma cruzi is coupled to K cycling and to active Na extrusion

Background

Orthophosphate (P_i) is a central compound in the metabolism of all organisms, including parasites. There are no reports regarding the mechanisms of P_i acquisition by Trypanosoma cruzi.

Methods

³²P_i influx was measured in T. cruzi epimastigotes. The expression of P_i transporter genes and the coupling of the uptake to Na⁺, H⁺ and K⁺ fluxes were also investigated. The transport capacities of different evolutive forms were compared.

Results

Epimastigotes grew significantly more slowly in 2 mM than in 50 mM P_i. Influx of P_i into parasites grown under low P_i conditions took place in the absence and presence of Na⁺. We found that the parasites express TcPho84, a H⁺:P_i-symporter, and TcPho89, a Na⁺:P_i-symporter. Both P_i influx mechanisms showed Michaelis–Menten kinetics, with a one-order of magnitude higher affinity for the Na⁺-dependent system. Collapsing the membrane potential with carbonylcyanide-p-trifluoromethoxyphenylhydrazone strongly impaired the influx of P_i. Valinomycin (K⁺ ionophore) or SCH28028 (inhibitor of (H⁺ + K⁺)ATPase) significantly inhibited P_i uptake, indicating that an inwardly-directed H⁺ gradient energizes uphill P_i entry and that K⁺ recycling plays a key role in P_i influx. Furosemide, an inhibitor of the ouabain-insensitive Na⁺-ATPase, decreased only the Na⁺-dependent P_i uptake, indicating that this Na⁺ pump generates the Na⁺ gradient utilized by the symporter. Trypomastigote forms take up P_i inefficiently.

Conclusions

P_i starvation stimulates membrane potential-sensitive P_i uptake through different pathways coupled to Na⁺ or H⁺/K⁺ fluxes.

General significance

This study unravels the mechanisms of P_i acquisition by T. cruzi, a key process in epimastigote development and differentiation to trypomastigote forms.     

Choline kinase beta is required for normal endochondral bone formation

Background

Choline kinase has three isoforms encoded by the genes Chka and Chkb. Inactivation of Chka in mice results in embryonic lethality, whereas Chkb^−/− mice display neonatal forelimb bone deformations.

Methods

To understand the mechanisms underlying the bone deformations, we compared the biology and biochemistry of bone formation from embryonic to young adult wild-type (WT) and Chkb^−/− mice.

Results

The deformations are specific to the radius and ulna during the late embryonic stage. The radius and ulna of Chkb^−/− mice display expanded hypertrophic zones, unorganized proliferative columns in their growth plates, and delayed formation of primary ossification centers. The differentiation of chondrocytes of Chkb^−/− mice was impaired, as was chondrocyte proliferation and expression of matrix metalloproteinases 9 and 13. In chondrocytes from Chkb^−/− mice, phosphatidylcholine was slightly lower than in WT mice whereas the amount of phosphocholine was decreased by approximately 75%. In addition, the radius and ulna from Chkb^−/− mice contained fewer osteoclasts along the cartilage/bone interface.

Conclusions

Chkb has a critical role in the normal embryogenic formation of the radius and ulna in mice.

General Significance

Our data indicate that choline kinase beta plays an important role in endochondral bone formation by modulating growth plate physiology.     

An ectotherm homologue of human predicted gene NAT16 encodes histidine N-acetyltransferase responsible for Nα-acetylhistidine synthesis

Background

Nα-Acetylhistidine (NAH) is present in very high concentrations exclusively in the brain and lens of ectothermic vertebrates, including ray-finned fishes, amphibians and reptiles, and not in those of endothermic birds and mammals. Although NAH is known to be synthesized from l-His and acetyl-CoA by histidine N-acetyltransferase (HISAT; EC 2.3.1.33), the gene encoding HISAT has remained unknown for any organism.

Methods

HISAT was purified from the blue mackerel brain, and its partial amino acid sequences were analyzed using mass spectrometry and Edman degradation. Using the sequence information, the corresponding gene was cloned and sequenced. Recombinant proteins encoded by the fish gene and its human homologue were expressed in a cell-free translation system.

Results

HISAT was identified to be a protein encoded by a fish homologue of the human predicted gene NAT16 (N-acetyltransferase 16). HISAT is an unstable enzyme that is rapidly and irreversibly inactivated during preincubation at 37 °C in the absence of acetyl-CoA. In fish brain, the HISAT gene is expressed as two splice variants containing an identical ORF but differing lengths of 5′-UTR. Both variants are expressed exclusively in the fish brain and lens. Interestingly, the recombinant human NAT16 protein, unlike the recombinant fish HISAT, has only trace enzyme activity for NAH synthesis.

Conclusions

These results propose that the function of mammalian NAT16 has been altered from l-His acetylation (NAH synthesis) to another different biological role.

General significance

The molecular identification of HISAT will allow progress in the understanding of the physiological function of NAH in ectothermic vertebrates.     

Cholesterol reduction ameliorates glucose-induced calcium handling and insulin secretion in islets from low-density lipoprotein receptor knockout mice

Aims/hypothesis

Changes in cellular cholesterol level may contribute to beta cell dysfunction. Islets from low density lipoprotein receptor knockout (LDLR^−/−) mice have higher cholesterol content and secrete less insulin than wild-type (WT) mice. Here, we investigated the association between cholesterol content, insulin secretion and Ca^2 + handling in these islets.

Methods

Isolated islets from both LDLR^−/− and WT mice were used for measurements of insulin secretion (radioimmunoassay), cholesterol content (fluorimetric assay), cytosolic Ca^2 + level (fura-2AM) and SNARE protein expression (VAMP-2, SNAP-25 and syntaxin-1A). Cholesterol was depleted by incubating the islets with increasing concentrations (0–10 mmol/l) of methyl-beta-cyclodextrin (MβCD).

Results

The first and second phases of glucose-stimulated insulin secretion (GSIS) were lower in LDLR^−/− than in WT islets, paralleled by an impairment of Ca^2 + handling in the former. SNAP-25 and VAMP-2, but not syntaxin-1A, were reduced in LDLR^−/− compared with WT islets. Removal of excess cholesterol from LDLR^−/− islets normalized glucose- and tolbutamide-induced insulin release. Glucose-stimulated Ca^2 + handling was also normalized in cholesterol-depleted LDLR^−/− islets. Cholesterol removal from WT islets by 0.1 and 1.0 mmol/l MβCD impaired both GSIS and Ca^2 + handling. In addition, at 10 mmol/l MβCD WT islet showed a loss of membrane integrity and higher DNA fragmentation.

Conclusion

Abnormally high (LDLR^−/− islets) or low cholesterol content (WT islets treated with MβCD) alters both GSIS and Ca^2 + handling. Normalization of cholesterol improves Ca^2 + handling and insulin secretion in LDLR^−/− islets.     

Increased renal semicarbazide-sensitive amine oxidase activity and methylglyoxal levels in aristolochic acid-induced nephrotoxicity

Aims

Aristolochic acid (AA) nephrotoxicity is related to accumulation of methylglyoxal (MGO) and N^ε-(carboxymethyl)lysine (CML) in the mouse kidney. We studied the activity of renal semicarbazide-sensitive amine oxidase (SSAO), a key enzyme involved in MGO generation, in AA-treated mice, and investigated nephroprotective effects produced by metformin, a MGO scavenger.

Methods

Mice were orally administered water or metformin for 15 days (12 or 24 mg kg^− 1 day^− 1), and injected AA (5 mg kg^− 1 day^− 1) intraperitoneally for 8 days starting on day 8. Renal function was studied, and histopathological examination, determination of renal SSAO activity, and measurement of MGO levels were performed.

Key findings

Compared to control mice, AA-injected mice showed significant renal damage and approximately 2.7-fold greater renal SSAO activity (p < 0.05). Further, compared to control treatment, administration of 12 mg/kg metformin inhibited formation of renal lesions, and significantly decreased renal MGO levels (37.33 ± 9.78 vs. 5.89 ± 2.64 μg/mg of protein, respectively, p < 0.01). In the AA-treated mice, metformin also inhibited the accumulation of CML in renal tubules, but did not affect SSAO activity.

Significance

This study is the first to show elevated renal SSAO activity in AA-treated mice, which could be involved in MGO accumulation. Moreover, MGO scavenging by metformin reduces AA nephrotoxicity. These findings suggest that reducing MGO accumulation produces nephroprotection, revealing new therapeutic strategies for the management. SSAO is a key enzyme involved in MGO generation, and consequently, inhibition of renal SSAO activity is worth investigating in AA nephrotoxicity and other renal pathologies further.     

Efficient transfer of sialo-oligosaccharide onto proteins by combined use of a glycosynthase-like mutant of Mucor hiemalis endoglycosidase and synthetic sialo-complex-type sugar oxazoline

Background

An efficient method for synthesizing homogenous glycoproteins is essential for elucidating the structural and functional roles of glycans of glycoproteins. We have focused on the transglycosylation activity of endo-β-N-acetylglucosaminidase from Mucor hiemalis (Endo-M) as a tool for glycoconjugate syntheses, since it can transfer en bloc the oligosaccharide of not only high-mannose type but also complex-type N-glycan onto various acceptors having an N-acetylglucosamine residue. However, there are two major bottlenecks for its practical application: the low yield of the transglycosylation product and the difficulty to obtain the activated sugar oxazoline substrate, especially the sialo-complex type one.

Methods

We carried out the transglycosylation using a glycosynthase-like N175Q mutant of Endo-M, which was found to possess enhanced transglycosylation activity with sugar oxazoline as a donor substrate, in combination with an easy preparation of the sialo-complex-type sugar oxazoline from natural sialoglycopeptide in egg yolk.

Results

Endo-M-N175Q showed efficient transglycosylation toward sialo-complex-type sugar oxazoline onto bioactive peptides and bovine ribonuclease B, and each sialylated compound was obtained in significantly high yield.

Conclusions

Highly efficient and simple chemo-enzymatic syntheses of various sialylated compounds were enabled, by a combination of a simple synthesis of sialo-complex-type sugar oxazoline and the Endo-M-N175Q catalyzed transglycosylation.

General significance

Our method would be very useful for a practical synthesis of biologically important glycopeptides and glycoproteins.     

Heme-iron utilization by Leptospira interrogans requires a heme oxygenase and a plastidic-type ferredoxin-NADP reductase

Background

Heme oxygenase catalyzes the conversion of heme to iron, carbon monoxide and biliverdin employing oxygen and reducing equivalents. This enzyme is essential for heme-iron utilization and contributes to virulence in Leptospira interrogans.

Methods

A phylogenetic analysis was performed using heme oxygenases sequences from different organisms including saprophytic and pathogenic Leptospira species. L. interrogans heme oxygenase (LepHO) was cloned, overexpressed and purified. The structural and enzymatic properties of LepHO were analyzed by UV–vis spectrophotometry and ¹H NMR. Heme-degrading activity, ferrous iron release and biliverdin production were studied with different redox partners.

Results

A plastidic type, high efficiently ferredoxin-NADP⁺ reductase (LepFNR) provides the electrons for heme turnover by heme oxygenase in L. interrogans. This catalytic reaction does not require a ferredoxin. Moreover, LepFNR drives the heme degradation to completeness producing free iron and α-biliverdin as the final products. The phylogenetic divergence between heme oxygenases from saprophytic and pathogenic species supports the functional role of this enzyme in L. interrogans pathogenesis.

Conclusions

Heme-iron scavenging by LepHO in L. interrogans requires only LepFNR as redox partner. Thus, we report a new substrate of ferredoxin-NADP⁺ reductases different to ferredoxin and flavodoxin, the only recognized protein substrates of this flavoenzyme to date. The results presented here uncover a fundamental step of heme degradation in L. interrogans.

General significance

Our findings contribute to understand the heme-iron utilization pathway in Leptospira. Since iron is required for pathogen survival and infectivity, heme degradation pathway may be relevant for therapeutic applications.