We report on the homo-and hetero-transglycosylation activities of the HvXET3 and HvXET4 xyloglucan xyloglucosyl transferases (XET; EC 2.4.1.207) from barley (Hordeum vulgare L.), and the visualisation of these activities in young barley roots using Alexa Fluor 488-labelled oligosaccharides. We discover that these isozymes catalyse the transglycosylation reactions with the chemically defined donor and acceptor substrates, specifically with the xyloglucan donor and the penta-galacturonide [a(1-4)GalAp] 5 acceptorthe homogalacturonan (pectin) fragment. This activity is supported by 3D molecular models of HvXET3 and HvXET4 with the docked XXXG donor and [a(1-4)GalAp] 5 acceptor substrates at the À4 to +5 subsites in the active sites. Comparative sequence analyses of barley isoforms and seed-localised TmXET6.3 from nasturtium (Tropaeolum majus L.) permitted the engineering of mutants of TmXET6.3 that could catalyse the hetero-transglycosylation reaction with the xyloglucan/[a(1-4)GalAp] 5 substrate pair, while wild-type TmXET6.3 lacked this activity. Expression data obtained by real-time quantitative polymerase chain reaction of HvXET transcripts and a clustered heatmap of expression profiles of the gene family revealed that HvXET3 and HvXET6 co-expressed but did not share the monophyletic origin. Conversely, HvXET3 and HvXET4 shared this relationship, when we examined the evolutionary history of 419 glycoside hydrolase 16 family members, spanning monocots, eudicots and a basal Angiosperm. The discovered hetero-transglycosylation activity in HvXET3 and HvXET4 with the xyloglucan/[a(1-4)GalAp] 5 substrate pair is discussed against the background of roles of xyloglucan-pectin heteropolymers and how they may participate in spatial patterns of cell wall formation and remodelling , and affect the structural features of walls.

The construction of a sensitive electrochemical lectin-based immunosensor for detection of a prostate specific antigen (PSA) is shown here. Three lectins with different carbohydrate specificities were used in this study to glycoprofile PSA, which is the most common biomarker for prostate cancer (PCa) diagnosis. The biosensor showed presence of α-L-fucose and α-(2,6)-linked terminal sialic acid within PSA´s glycan with high abundance, while only traces of α-(2,3)-linked terminal sialic acid were found. MALDI TOF/TOF mass spectrometry was applied to validate results obtained by the biosensor with a focus on determination of a type of sialic acid linkage by two methods. The first direct comparison of electrochemical immunosensor assay employing lectins for PSA glycoprofiling with mass spectrometric techniques is provided here and both methods show significant agreement. Thus, electrochemical lectin-based immunosensor has potential to be applied for prostate cancer diagnosis.

BackgroundThe brain stem contains important nuclei that control cardiovascular function via the sympathetic nervous system (SNS), which is strongly influenced by nitric oxide. Its biological activity is also largely determined by oxygen free radicals. Despite many experimental studies, the role of AT1R-NAD(P)H oxidase-superoxide pathway in NO-deficiency is not yet sufficiently clarified. We determined changes in free radical signaling and antioxidant and detoxification response in the brain stem of young and adult Wistar rats during chronic administration of exogenous NO inhibitors.MethodsYoung (4 weeks) and adult (10 weeks) Wistar rats were treated with 7-nitroindazole (7-NI group, 10 mg/kg/day), a specific nNOS inhibitor, with NG-nitro-L-arginine-methyl ester (L-NAME group, 50 mg/kg/day), a nonspecific NOS inhibitor, and with drinking water (Control group) during 6 weeks. Systolic blood pressure was measured by non-invasive plethysmography. Expression of genes (AT1R, AT2R, p22phox, SOD and NOS isoforms, HO-1, MDR1a, housekeeper GAPDH) was identified by real-time PCR. NOS activity was detected by conversion of [3H]-L-arginine to [3H]-L-citrulline and SOD activity was measured using UV VIS spectroscopy.ResultsWe observed a blood pressure elevation and decrease in NOS activity only after L-NAME application in both age groups. Gene expression of nNOS (youngs) and eNOS (adults) in the brain stem decreased after both inhibitors. The radical signaling pathway triggered by AT1R and p22phox was elevated in L-NAME adults, but not in young rats. Moreover, L-NAME-induced NOS inhibition increased antioxidant response, as indicated by the observed elevation of mRNA SOD3, HO-1, AT2R and MDR1a in adult rats. 7-NI did not have a significant effect on AT1R-NADPH oxidase-superoxide pathway, yet it affected antioxidant response of mRNA expression of SOD1 and stimulated total activity of SOD in young rats and mRNA expression of AT2R in adult rats.ConclusionOur results show that chronic NOS inhibition by two different NOS inhibitors has age-dependent effect on radical signaling and antioxidant/detoxificant response in Wistar rats. While 7-NI had neuroprotective effect in the brain stem of young Wistar rats, L-NAME- induced NOS inhibition evoked activation of AT1R-NAD(P)H oxidase pathway in adult Wistar rats. Triggering of the radical pathway was followed by activation of protective compensation mechanism at the gene expression level.Electronic supplementary materialThe online version of this article (doi:10.1186/s12929-017-0366-4) contains supplementary material, which is available to authorized users.

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Mycobacterium tuberculosis, one of the deadliest pathogens in human history, is distinguished by a unique, multilayered cell wall, which offers the bacterium a high level of protection from the attacks of the host immune system. The primary structure of the cell wall core, composed of covalently linked peptidoglycan, branched heteropolysaccharide arabinogalactan, and mycolic acids, is well known, and numerous enzymes involved in the biosynthesis of its components are characterized. The cell wall biogenesis takes place at both cytoplasmic and periplasmic faces of the plasma membrane, and only recently some of the specific transport systems translocating the metabolic intermediates between these two compartments have been characterized [M. Jackson, C. M. Stevens, L. Zhang, H. I. Zgurskaya, M. Niederweis, Chem. Rev., 10.1021/acs.chemrev.0c00869 (2020)]. In this work, we use CRISPR interference methodology in Mycobacterium smegmatis to functionally characterize an ATP-binding cassette (ABC) transporter involved in the translocation of galactan precursors across the plasma membrane. We show that genetic knockdown of the transmembrane subunit of the transporter results in severe morphological changes and the accumulation of an aberrantly long galactan precursor. Based on similarities with structures and functions of specific O-antigen ABC transporters of gram-negative bacteria [C. Whitfield, D. M. Williams, S. D. Kelly, J. Biol. Chem. 295, 10593-10609 (2020)], we propose a model for coupled synthesis and export of the galactan polymer precursor in mycobacteria.

Congenital disorder of glycosylation type Ig (ALG12‐CDG) is a rare inherited metabolic disease caused by a defect in alpha‐mannosyltransferase 8, encoded by the ALG12 gene (22q13.33). To date, only 15 patients have been diagnosed with ALG12‐CDG globally. Due to a newborn Slovak patient's clinical and biochemical abnormalities, the isoelectric focusing of transferrin was performed with observed significant hypoglycosylation typical of CDG I. Furthermore, analysis of neutral serum N‐glycans by mass spectrometry revealed the accumulation of GlcNAc2Man5–7 and decreased levels of GlcNAc2Man8–9, which indicated impaired ALG12 enzymatic activity. Genetic analysis of the coding regions of the ALG12 gene of the patient revealed a novel homozygous substitution mutation c.1439T>C p.(Leu480Pro) within Exon 10. Furthermore, both of the patient's parents and his twin sister were asymptomatic heterozygous carriers of the variant. This comprehensive genomic and glycomic approach led to the confirmation of the ALG12 pathogenic variant responsible for the clinical manifestation of the disorder in the patient described.

AIM: Congenital disorders of glycosylation (CDG) belong to an expanding group of rare genetic metabolic disorders caused by defects in the complex chemical enzymatic process of glycosylation. The study is aimed at presenting a case report of a premature dysmorphic newborn, clinical presentation of the condition, the way it was diagnosed and treated, as well as its comparison with the known cases. RESULTS: The result of glycan analysis supports the assumption of a supposed glycosylation disorder and also specifi es a specifi c subtype: CDG-1, subtype ALG12-CDG (Ig). CONCLUSION: CDG have an extremely wide clinical spectrum and should be considered in any child with unexplained developmental delay, failure to thrive, seizures, and abnormalities in liver enzymes, coagulation and immunologic factors. The treatment of most forms of CDG depends upon numerous factors such as specifi c symptoms present, severity of the disorder, age and overall health of the patients and tolerance to certain medications or procedures. For these reasons, the treatment is specifi c for every individual. It is based on the symptoms and requires a coordination of efforts of a team of specialists (Tab. 4, Fig. 3, Ref. 19).

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