Biomarkers for subclinical bovine mastitis: a high throughput TMT-based proteomic investigation.

Mastitis represents the biggest threat to the health and productivity of dairy cows, leading to substantial economic losses in milk production. It manifests in two forms: clinical mastitis, easily diagnosed by visible symptoms, and subclinical mastitis (SCM), which lacks overt clinical signs. SCM's elusive nature often results in it going undetected, thus facilitating the spread of the disease-causing agent due to lack of treatment. Finding a reliable biomarker for early SCM would reduce the possibility of mastitis spreading in the herd, reduce the need for antibiotic use and ultimately reduce milk losses for producers. Utilizing state-of-the-art proteomics techniques, 138 milk samples from dairy cows in continental Croatia underwent analysis. These samples were categorized into four groups based on the Zagreb Mastitis Test (ZMT) and microbiological analysis: lowSCC- (n = 20), lowSCC + (n = 20), medSCC + (n = 79), and highSCC + (n = 19). A total of 386 proteins were identified and quantified, with 76 proteins showing significant differential abundances among the groups. Many of these proteins are linked to the innate immune system, as well as neutrophil and platelet degranulation processes. Through fold changes observed between groups, 15 proteins exhibiting biomarker characteristics for subclinical mastitis (SCM) were identified. Among these, five proteins-cathelicidins (-1, -4, and -7), lactoferrin, and haptoglobin-showed particular promise.

Broad protection against invasive fungal disease from a nanobody targeting the active site of fungal b-1,3-glucanosyltransferases.

Invasive fungal disease accounts for ~3.8 million deaths annually, an unacceptable rate that urgently prompts the discovery of new knowledge-driven treatments. We report the use of camelid single-domain nanobodies (Nbs) against fungal ß-1,3-glucanosyltransferases (Gel) involved in ß-1,3-glucan transglycosylation. Crystal structures of two Nbs with Gel4 from Aspergillus fumigatus revealed binding to a dissimilar CBM43 domain and a highly conserved catalytic domain across fungal species, respectively. Anti-Gel4 active site Nb3 showed significant antifungal efficacy in vitro and in vivo prophylactically and therapeutically against different A. fumigatus and Cryptococcus neoformans isolates, reducing the fungal burden and disease severity, thus significantly improving immunocompromised animal survival. Notably, C. deneoformans (serotype D) strains were more susceptible to Nb3 and genetic Gel deletion than C. neoformans (serotype A) strains, indicating a key role for ß-1,3-glucan remodelling in C. deneoformans survival. These findings add new insights about the role of b-1,3-glucan in fungal biology and demonstrate the potential of nanobodies in targeting fungal enzymes to combat invasive fungal diseases.

Characterization and LC-MS/MS based proteomic analysis of extracellular vesicles separated from blood serum of healthy and dogs naturally infected by Babesia canis. A preliminary study.

Canine babesiosis is a rapidly spreading tick-borne disease in Europe, which entails protozoan parasites invading red blood cells. Small extracellular vesicles (EVs) (< 200 nm) were isolated from the serum of 15 healthy and 15 by Babesia canis naturally infected dogs aimed to distinguish EV characteristics and protein profiles. There were no significant differences (P = 0.05) observed in the mean sizes and concentrations of serum EVs between the healthy and canine babesiosis groups. Despite a higher number of Canis lupus proteins detected in EVs from serum of diseased dogs, there were no statistically significant differences (P < 0.05) in the number of protein IDs between the experimental groups. We successfully identified 211 Canis lupus proteins across both experimental groups, of which 147 Canis lupus proteins were validated as being EV-associated. This data set is accessible via the ProteomeXchange PXD047647. EVs isolated from serum of B. canis infected dogs were Cd9+, Cd63+, Cd81+, and Cd82+. Furthermore, 73 Canis lupus proteins were validated as EV-associated and specific for EVs isolated from serum of B. canis-infected dogs. These were predominantly membrane and cytosolic proteins, and innate and adaptive immune system-related proteins, especially those involved in adhesion and proteoglycan mechanisms like integrins. Enrichment was also observed for proteins involved in vascular and cellular responses, including signalling pathways such as VEGF, VEGFR, and the LKB1 network. When only blood-related sites of EV expression were evaluated, the origins of EV proteins were mostly cells of immune system. These were dendritic cells, neutrophils, B cells, monocytes and platelets. In general, proteins were enriched in pathways that collectively regulate various cellular processes, including immune responses, communication, signal transduction, membrane trafficking, and apoptosis. Serum EVs and their protein cargo may have an important role in both the invasion of B. canis and the host's response to the parasitic infection, nevertheless, additional experimental research is warranted. The overall count of identified EV proteins of parasitic origin, meeting cut off criteria of two peptides and 1 % FDR, was relatively low.

Quantitative serum proteome analysis using tandem mass tags in dogs with epilepsy.

This study included four groups of dogs (group A: healthy controls, group B: idiopathic epilepsy receiving antiepileptic medication (AEM), group C: idiopathic epilepsy without AEM, group D: structural epilepsy). Comparative quantitative proteomic analysis of serum samples among the groups was the main target of the study. Samples were analyzed by a quantitative Tandem-Mass-Tags approach on the Q-Exactive-Plus Hybrid Quadrupole-Orbitrap mass-spectrometer. Identification and relative quantification were performed in Proteome Discoverer. Data were analyzed using R. Gene ontology terms were analyzed based on Canis lupus familiaris database. Data are available via ProteomeXchange with identifier PXD041129. Eighty-one proteins with different relative adundance were identified in the four groups and 25 were master proteins (p < 0.05). Clusterin (CLU), and apolipoprotein A1 (APOA1) had higher abundance in the three groups of dogs (groups B, C, D) compared to controls. Amine oxidase (AOC3) was higher in abundance in group B compared to groups C and D, and lower in group A. Adiponectin (ADIPOQ) had higher abundance in groups C compared to group A. ADIPOQ and fibronectin (FN1) had higher abundance in group B compared to group C and D. Peroxidase activity assay was used to quantify HP abundance change, validating and correlating with proteomic analysis (r = 0.8796). SIGNIFICANCE: The proteomic analysis of serum samples from epileptic dogs indicated potential markers of epilepsy (CLU), proteins that may contribute to nerve tissue regeneration (APOA1), and contributing factors to epileptogenesis (AOC3). AEM could alter extracellular matrix proteins (FN1). Illness (epilepsy) severity could influence ADIPOQ abundance.

Pharmacogenomics of Dementia: Personalizing the Treatment of Cognitive and Neuropsychiatric Symptoms.

Dementia is a syndrome of global and progressive deterioration of cognitive skills, especially memory, learning, abstract thinking, and orientation, usually affecting the elderly. The most common forms are Alzheimer's disease, vascular dementia, and other (frontotemporal, Lewy body disease) dementias. The etiology of these multifactorial disorders involves complex interactions of various environmental and (epi)genetic factors and requires multiple forms of pharmacological intervention, including anti-dementia drugs for cognitive impairment, antidepressants, antipsychotics, anxiolytics and sedatives for behavioral and psychological symptoms of dementia, and other drugs for comorbid disorders. The pharmacotherapy of dementia patients has been characterized by a significant interindividual variability in drug response and the development of adverse drug effects. The therapeutic response to currently available drugs is partially effective in only some individuals, with side effects, drug interactions, intolerance, and non-compliance occurring in the majority of dementia patients. Therefore, understanding the genetic basis of a patient's response to pharmacotherapy might help clinicians select the most effective treatment for dementia while minimizing the likelihood of adverse reactions and drug interactions. Recent advances in pharmacogenomics may contribute to the individualization and optimization of dementia pharmacotherapy by increasing its efficacy and safety via a prediction of clinical outcomes. Thus, it can significantly improve the quality of life in dementia patients.

Distinguishing Natural Infections of the Bovine Mammary Gland by Staphylococcus from Streptococcus spp. Using Quantitative Milk Proteomics.

Bovine mastitis is the most frequent disease on dairy farms, which leads to a decrease in the health welfare of the animals and great economic losses. This study was aimed at determining the quantitative variations in the milk proteome caused by natural infection by Staphylococcus and Streptococcus species in order to gain further understanding of any discrepancies in pathophysiology and host immune responses, independent of the mastitis level. After identification of Staphylococcus (N = 51) and Streptococcus (N = 67) spp., tandem mass tag (TMT)-labeled quantitative proteomic and liquid chromatography-mass spectrometry (LC-MS/MS) techniques on a modular Ultimate 3000 RSLCnano system coupled to a Q Exactive Plus was applied on aseptically sampled milk from Holstein cows. Proteome Discoverer was used for protein identification and quantitation through the SEQUEST algorithm. Statistical analysis employing R was used to identify differentially abundant proteins between the groups. Protein classes, functions and functional-association networks were determined using the PANTHER and STRING tools and pathway over-representation using the REACTOME. In total, 156 master bovine proteins were identified (two unique peptides, p < 0.05 and FDR < 0.001), and 20 proteins showed significantly discrepant abundance between the genera (p < 0.05 and FDR < 0.5). The most discriminatory proteins per group were odorant-binding protein (higher in staphylococci) and fibrinogen beta chain protein (higher in streptococci). The receiver operating characteristic (ROC) curve showed that protein kinase C-binding protein NELL2, thrombospondin-1, and complement factor I have diagnostic potential for differentiating staphylococci and streptococci intramammary infection and inflammation. Improved understanding of the host response mechanisms and recognition of potential biomarkers of specific-pathogen mastitis, which may aid prompt diagnosis for control implementation, are potential benefits of this study.

Constitutive Serotonin Tone as a Modulator of Brown Adipose Tissue Thermogenesis: A Rat Study.

Brown adipose tissue (BAT), an important regulator of thermogenic and metabolic processes, is considered a promising target to combat metabolic disorders. The neurotransmitter and hormone serotonin (5HT) is a major modulator of energy homeostasis, with its central and peripheral pools acting in opposing ways. To better understand how individual variations in 5HT homeostasis influence the thermogenic functionality of BAT, we used a rat model consisting of two sublines with constitutively increased (high-5HT) or decreased (low-5HT) whole-body 5HT tone, developed by selective breeding for platelet 5HT parameters. We have shown that animals with constitutively low 5HT activity maintained at a standard housing temperature (22 °C) have greater interscapular BAT (iBAT) mass and higher iBAT metabolic activity (as evidenced by measurements of iBAT temperature and glucose uptake), accompanied by increased iBAT mRNA expression of key thermogenic genes, compared to animals with high 5HT tone. In response to further thermogenic challenges-intermittent cold exposure or treatment with a ß3-adrenergic agonist-5HT sublines show several functional and molecular differences linking constitutively low endogenous 5HT tone to higher BAT activity/capacity. Overall, the results support a role of 5-HT in the control of BAT thermogenesis They also suggest that individuals with lower 5HT activity may be more sensitive to ß3-adrenergic drugs.

Serum proteome profiling of naturally acquired Babesia rossi infection in dogs.

Babesiosis is a disease of significant medically and veterinary importance with worldwide distribution. It is caused by intra-erythrocyte protozoal parasites, with Babesia rossi causing the most severe clinical signs of all the large Babesia parasites infecting dogs. The disease can be clinically classified into uncomplicated and complicated forms with a wide range of clinical presentations from a mild, subclinical illness to complicated forms and death. The aim of this study was to assess serum proteomic profiles from dogs with babesiosis and healthy dogs using a label-based proteomics approach. Altogether 32 dogs naturally infected with B. rossi (subdivided into 18 uncomplicated cases and 14 complicated cases of babesiosis) and 20 healthy dogs were included. There were 78 proteins with significantly different abundances between the three groups of dogs. Elucidation of proteins and pathways involved in canine babesiosis caused by B. rossi have revealed key differences associated with haemostasis, innate immune system, lipid metabolism and inflammation. Shotgun proteomic profiling allowed identification of potential serum biomarkers for differentiation of disease severity in canine babesiosis caused by B. rossi. These findings may be applicable to the study of host-parasite interactions and the development of novel therapeutic targets.

Serum Proteomic Profiles Reflect the Stages of Myxomatous Mitral Valve Disease in Dogs.

Canine myxomatous mitral valve disease (MMVD) is similar to Barlow's form of MMVD in humans. These valvulopathies are complex, with varying speeds of progression. We hypothesized that the relative abundances of serum proteins would help identify the consecutive MMVD stages and discover new disease pathways on a systemic level. To identify distinction-contributing protein panels for disease onset and progression, we compared the proteomic profiles of serum from healthy dogs and dogs with different stages of naturally occurring MMVD. Dogs were divided into experimental groups on the basis of the left-atrium-to-aorta ratio and normalized left ventricular internal dimension in diastole values. Serum was collected from healthy (N = 12) dogs, dogs diagnosed with MMVD in stages B1 (N = 13) and B2 (N = 12) (asymptomatic), and dogs diagnosed with MMVD in chronic stage C (N = 13) (symptomatic). Serum biochemistry and selected ELISAs (galectin-3, suppression of tumorigenicity, and asymmetric dimethylarginine) were performed. Liquid chromatography-mass spectrometry (LC-MS), tandem mass tag (TMT) quantitative proteomics, and statistical and bioinformatics analysis were employed. Most of the 21 serum proteins with significantly different abundances between experimental groups (p < 0.05, FDR ˂ 0.05) were classified as matrix metalloproteinases, protease inhibitors, scaffold/adaptor proteins, complement components, anticoagulants, cytokine, and chaperone. LC-MS TMT proteomics results obtained for haptoglobin, clusterin, and peptidase D were further validated analytically. Canine MMVD stages, including, for the first time, asymptomatic B1 and B2 stages, were successfully distinguished in dogs with the disease and healthy dogs on the basis of the relative abundances of a panel of specific serum proteins. Most proteins with significantly different abundances were involved in immune and inflammatory pathways. Their role in structural remodeling and progression of canine MMVD must be further investigated. Further research is needed to confirm the resemblance/difference with human MMVD. Proteomics data are available via ProteomeXchange with the unique dataset identifier PXD038475.

Antioxidant and Antiproliferative Activities of Kale (Brassica oleracea L. Var. acephala DC.) and Wild Cabbage (Brassica incana Ten.) Polyphenolic Extracts.

Brassicaceae are rich in healthy phytochemicals that have a positive impact on human health. The aim of this study was to analyze the phenolic compounds and antioxidant and anticancer potential of traditional Croatian kale (Brassica oleracea L. var. acephala DC.) and wild cabbage (Brassica incana Ten.) extracts. The phenolic groups and antioxidant activity were determined by spectrophotometry, selected phenolic compounds (ferulic acid, sinapic acid, salicylic acid, kaempferol, and quercetin) were analyzed by LC-MS/MS, and anticancer potential was evaluated in vitro using HeLa cells. The extracts of both plant species are rich in phenolic compounds and showed significant antioxidant activity at similar levels. LC-MS/MS detected sinapic acid as the most abundant phenolic acid, followed by ferulic acid, while salicylic acid was present at lower concentrations. A comparative analysis showed that wild cabbage contained significantly more sinapic acid, while kale contained more kaempferol and quercetin. Both Brassica extracts at a concentration of 50 µg mL-1 showed an antiproliferative effect on HeLa cells, while they did not affect the proliferation of normal human skin fibroblasts. Wild cabbage extract also showed an antiproliferative effect on HeLa cells at a lower applied concentration of 10 µg mL-1 of extracts. The clonogenic analysis also revealed the inhibitory effect of the extracts on HeLa colony growth.

Profiling the alterations of serum proteome in dairy cows with retained placenta using high-throughput tandem mass tags quantitative approach.

BACKGROUND: Retained placenta (RP), a quite common disorder in dairy cows, shows a high negative impact on their health status and milk production. AIM: To investigate the difference in the serum proteome between the cows with RP and the physiologic puerperium (PP). MATERIAL & METHODS: Analysis of serum samples from nine cows with RP and six with PP using high-resolution liquid chromatography-tandem mass spectrometry approach. The proteins differing in the relative abundance between the PP and RP groups were classified using the Protein Analysis Through Evolutionary Relationship tool. For the pathway enrichment analysis, the REACTOME tool, with the human genome as the background, was employed. The criterion for significance was the false discovery rate corrected P-value less than 0.05. RESULTS: In total 651 proteins were identified with altered relative abundance of ten proteins. Among them, seven had higher, and three showed lower relative abundance in RP than in the PP group. The differently abundant proteins participated in 15 pathways: six related to hemostasis, three involved in lipoprotein metabolism, and the remaining ones associated with for instance redox homeostasis, post-translational modification, and scavenging. Finally, the validation of the proteomic results showed that haptoglobin and lipopolysaccharide-binding protein levels reliably differentiated between the RP and PP groups. CONCLUSION: The pattern of serum proteome alterations in the cows with RP mirrored several interplaying mechanisms underlying the systematic response to the presence of RP, therefore representing a source to mine for predictive or prognostic biomarkers.

Liver Proteome Alterations in Red Deer (Cervus elaphus) Infected by the Giant Liver Fluke Fascioloides magna.

Liver fluke infections are recognised as diseases with worldwide distribution and considerable veterinary and public health importance. The giant liver fluke, Fascioloides magna, is an important non-native parasite which has been introduced to Europe, posing a threat to the survival of local wildlife populations such as red deer (Cervus elaphus). The aim of the study was to analyse differences in liver proteomes between F. magna-infected and control red deer groups using a label-based high-throughput quantitative proteomics approach. The proteomics analysis identified 234 proteins with differential abundance between the control and infected groups. Our findings showed that F. magna infection in this definitive host is associated with changes in the metabolism of proteins and fatty acids, oxidative stress, fibrosis, and signaling pathways. The identified proteins and associated biological pathways represent a valuable contribution to the understanding of host-parasite interactions and the pathogenesis of liver fluke infection.

Effect of N-acetyl chito-oligosaccharides on the biosynthesis and properties of chitin in Saccharomyces cerevisiae.

Chitin exists in yeast cells both as free and bound in a complex with ß-1,3/ß-1,6-glucan. The formation of covalent links between chitin and ß-glucans is catalyzed by the enzymes Crh1 and Crh2, acting as transglycosylases. We found that N-acetyl-chito-oligosaccharides, as well as laminarioligosaccharides, the respective products of partial hydrolysis of chitin, and ß-1,3-glucan, interfered with reactions catalyzed by Crh1p and Crh2p in vitro. However, the N-acetyl-chito-oligosaccharides did not influence the growth rate of the yeast, neither did they affect the yeast phenotype, but they prolonged the lag phase. Inhibition of Crh1 and Crh2 in vivo with oligosaccharides derived from chitin leads to an increase of alkali-soluble chitin and a decrease in the amount of chitin linked to ß-glucans. In addition, yeast cells growing in the presence of N-acetyl-D-chito-oligosaccharides accumulated more chitin than control cells.

Iron overload in aging Bmp6­/­ mice induces exocrine pancreatic injury and fibrosis due to acinar cell loss.

The relationship between hemochromatosis and diabetes has been well established, as excessive iron deposition has been reported to result in impaired function of the endocrine and exocrine pancreas. Therefore, the objective of the present study was to analyze the effects of iron accumulation on the pancreata and glucose homeostasis in a bone morphogenetic protein 6­knockout (Bmp6­/­) mouse model of hemochromatosis. The sera and pancreatic tissues of wild­type (WT) and Bmp6­/­ mice (age, 3 and 10 months) were subjected to biochemical and histological analyses. In addition, 18F­fluorodeoxyglucose biodistribution was evaluated in the liver, muscle, heart, kidney and adipose tissue of both animal groups. The results demonstrated that 3­month­old Bmp6­/­ mice exhibited iron accumulation preferentially in the exocrine pancreas, with no signs of pancreatic injury or fibrosis. No changes were observed in the glucose metabolism, as pancreatic islet diameter, insulin and glucagon secretion, blood glucose levels and glucose uptake in the liver, muscle and adipose tissue remained comparable with those in the WT mice. Aging Bmp6­/­ mice presented with progressive iron deposits in the exocrine pancreas, leading to pancreatic degeneration and injury that was characterized by acinar atrophy, fibrosis and the infiltration of inflammatory cells. However, the aging mice exhibited unaltered blood glucose levels and islet structure, normal insulin secretion and moderately increased α­cell mass compared with those in the age­matched WT mice. Additionally, iron overload and pancreatic damage were not observed in the aging WT mice. These results supported a pathogenic role of iron overload in aging Bmp6­/­ mice leading to iron­induced exocrine pancreatic deficiency, whereas the endocrine pancreas retained normal function.

Autologous blood coagulum containing rhBMP6 induces new bone formation to promote anterior lumbar interbody fusion (ALIF) and posterolateral lumbar fusion (PLF) of spine in sheep.

In the present study, we evaluated an autologous bone graft substitute (ABGS) composed of recombinant human BMP6 (rhBMP6) dispersed within autologous blood coagulum (ABC) used as a physiological carrier for new bone formation in spine fusion sheep models. The application of ABGS included cervical cage for use in the anterior lumbar interbody fusion (ALIF), while for the posterolateral lumbar fusion (PLF) sheep model allograft devitalized bone particles (ALLO) were applied with and without use of instrumentation. In the ALIF model, ABGS (rhBMP6/ABC/cage) implants fused significantly when placed in between the L4-L5 vertebrae as compared to control (ABC/cage) which appears to have a fibrocartilaginous gap, as examined by histology and micro CT analysis at 16 weeks following surgery. In the PLF model, ABGS implants with or without ALLO showed a complete fusion when placed ectopically in the gutter bilaterally between two decorticated L4-L5 transverse processes at a success rate of 88% without instrumentation and at 80% with instrumentation; however the bone volume was 50% lower in the instrumentation group than without, as examined by histology, radiographs, micro CT analyses and biomechanical testing at 27 weeks following surgery. The newly formed bone was uniform within ABGS implants resulting in a biomechanically competent and histologically qualified fusion with an optimum dose in the range of 100 µg rhBMP6 per mL ABC, while in the implants that contained ALLO, the mineralized bone particles were substituted by the newly formed remodeling bone via creeping substitution. These findings demonstrate for the first time that ABGS (rhBMP6/ABC) without and with ALLO particles induced a robust bone formation with a successful fusion in sheep models of ALIF and PLF, and that autologous blood coagulum (ABC) can serve as a preferred physiological native carrier to induce new bone at low doses of rhBMP6 and to achieve a successful spinal fusion.

Role of Sirt3 in Differential Sex-Related Responses to a High-Fat Diet in Mice.

Metabolic homeostasis is differently regulated in males and females. Little is known about the mitochondrial Sirtuin 3 (Sirt3) protein in the context of sex-related differences in the development of metabolic dysregulation. To test our hypothesis that the role of Sirt3 in response to a high-fat diet (HFD) is sex-related, we measured metabolic, antioxidative, and mitochondrial parameters in the liver of Sirt3 wild-type (WT) and knockout (KO) mice of both sexes fed with a standard or HFD for ten weeks. We found that the combined effect of Sirt3 and an HFD was evident in more parameters in males (lipid content, glucose uptake, pparγ, cyp2e1, cyp4a14, Nrf2, MnSOD activity) than in females (protein damage and mitochondrial respiration), pointing towards a higher reliance of males on the effect of Sirt3 against HFD-induced metabolic dysregulation. The male-specific effects of an HFD also include reduced Sirt3 expression in WT and alleviated lipid accumulation and reduced glucose uptake in KO mice. In females, with a generally higher expression of genes involved in lipid homeostasis, either the HFD or Sirt3 depletion compromised mitochondrial respiration and increased protein oxidative damage. This work presents new insights into sex-related differences in the various physiological parameters with respect to nutritive excess and Sirt3.

Activation of brown adipose tissue in diet-induced thermogenesis is GC-C dependent.

Uroguanylin (UGN) is released from the intestine after a meal. When applied in brain ventricles, UGN increases expression of markers of thermogenesis in brown adipose tissue (BAT). Therefore, we determine the effects of its receptor, guanylate cyclase C (GC-C), on mouse interscapular BAT (iBAT) activity during diet-induced thermogenesis (DIT). The activation of iBAT after a meal is diminished in GC-C KO mice, decreased in female wild type (WT) mice, and abolished in old WT animals. The activation of iBAT after a meal is the highest in male WT animals which leads to an increase in GC-C expression in the hypothalamus, an increase in iBAT volume by aging, and induction of iBAT markers of thermogenesis. In contrast to iBAT activation after a meal, iBAT activation after a cold exposure could still exist in GC-C KO mice and it is significantly higher in female WT mice. The expression of GC-C in the proopiomelanocortin neurons of the arcuate nucleus of the hypothalamus but not in iBAT suggests central regulation of iBAT function. The iBAT activity during DIT has significantly reduced in old mice but an intranasal application of UGN leads to an increase in iBAT activity in a dose-dependent manner which is in strong negative correlation to glucose concentration in blood. This activation was not present in GC-C KO mice. Our results suggest the physiological role of GC-C on the BAT regulation and its importance in the regulation of glucose homeostasis and the development of new therapy for obesity and insulin resistance.

Mechanisms of redundancy and specificity of the Aspergillus fumigatus Crh transglycosylases.

Fungal cell wall synthesis is achieved by a balance of glycosyltransferase, hydrolase and transglycosylase activities. Transglycosylases strengthen the cell wall by forming a rigid network of crosslinks through mechanisms that remain to be explored. Here we study the function of the Aspergillus fumigatus family of five Crh transglycosylases. Although crh genes are dispensable for cell viability, simultaneous deletion of all genes renders cells sensitive to cell wall interfering compounds. In vitro biochemical assays and localisation studies demonstrate that this family of enzymes functions redundantly as transglycosylases for both chitin-glucan and chitin-chitin cell wall crosslinks. To understand the molecular basis of this acceptor promiscuity, we solved the crystal structure of A. fumigatus Crh5 (AfCrh5) in complex with a chitooligosaccharide at the resolution of 2.8 Å, revealing an extensive elongated binding cleft for the donor (-4 to -1) substrate and a short acceptor (+1 to +2) binding site. Together with mutagenesis, the structure suggests a "hydrolysis product assisted" molecular mechanism favouring transglycosylation over hydrolysis.

Engineering the acceptor substrate specificity in the xyloglucan endotransglycosylase TmXET6.3 from nasturtium seeds (Tropaeolum majus L.).

KEY MESSAGE: The knowledge of substrate specificity of XET enzymes is important for the general understanding of metabolic pathways to challenge the established notion that these enzymes operate uniquely on cellulose-xyloglucan networks. Xyloglucan xyloglucosyl transferases (XETs) (EC 2.4.1.207) play a central role in loosening and re-arranging the cellulose-xyloglucan network, which is assumed to be the primary load-bearing structural component of plant cell walls. The sequence of mature TmXET6.3 from Tropaeolum majus (280 residues) was deduced by the nucleotide sequence analysis of complete cDNA by Rapid Amplification of cDNA Ends, based on tryptic and chymotryptic peptide sequences. Partly purified TmXET6.3, expressed in Pichia occurred in N-glycosylated and unglycosylated forms. The quantification of hetero-transglycosylation activities of TmXET6.3 revealed that (1,3;1,4)-, (1,6)- and (1,4)-ß-D-glucooligosaccharides were the preferred acceptor substrates, while (1,4)-ß-D-xylooligosaccharides, and arabinoxylo- and glucomanno-oligosaccharides were less preferred. The 3D model of TmXET6.3, and bioinformatics analyses of identified and putative plant xyloglucan endotransglycosylases (XETs)/hydrolases (XEHs) of the GH16 family revealed that H94, A104, Q108, K234 and K237 were the key residues that underpinned the acceptor substrate specificity of TmXET6.3. Compared to the wild-type enzyme, the single Q108R and K237T, and double-K234T/K237T and triple-H94Q/A104D/Q108R variants exhibited enhanced hetero-transglycosylation activities with xyloglucan and (1,4)-ß-D-glucooligosaccharides, while those with (1,3;1,4)- and (1,6)-ß-D-glucooligosaccharides were suppressed; the incorporation of xyloglucan to (1,4)-ß-D-glucooligosaccharides by the H94Q variant was influenced most extensively. Structural and biochemical data of non-specific TmXET6.3 presented here extend the classic XET reaction mechanism by which these enzymes operate in plant cell walls. The evaluations of TmXET6.3 transglycosylation activities and the incidence of investigated residues in other members of the GH16 family suggest that a broad acceptor substrate specificity in plant XET enzymes could be more widespread than previously anticipated.