O-GlcNAcylation in Renal (Patho)Physiology.

Kidneys maintain internal milieu homeostasis through a well-regulated manipulation of body fluid composition. This task is performed by the correlation between structure and function in the nephron. Kidney diseases are chronic conditions impacting healthcare programs globally, and despite efforts, therapeutic options for its treatment are limited. The development of chronic degenerative diseases is associated with changes in protein O-GlcNAcylation, a post-translation modification involved in the regulation of diverse cell function. O-GlcNAcylation is regulated by the enzymatic balance between O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) which add and remove GlcNAc residues on target proteins, respectively. Furthermore, the hexosamine biosynthetic pathway provides the substrate for protein O-GlcNAcylation. Beyond its physiological role, several reports indicate the participation of protein O-GlcNAcylation in cardiovascular, neurodegenerative, and metabolic diseases. In this review, we discuss the impact of protein O-GlcNAcylation on physiological renal function, disease conditions, and possible future directions in the field.

[New variant in the ALG13 gene responsible for the congenital disorder of Is-type glycosylation in a male patient]. / Nueva variante en el gen ALG13 responsable del trastorno congénito de la glicosilación tipo Is en un paciente masculino.

INTRODUCTION: Congenital disorders of glycosylation (CDGs) are a group of inborn errors of glycan metabolism with multi-systemic manifestations. More than 100 different types of CDGs have been reported. The form involving the asparagine-linked glycosylation 13 (ALG13) gene is an uncommon X-linked form of these pathologies. OBJECTIVE: To describe the clinical features in one patient with ALG13-CDG and to compare them with previously reported cases. CLINICAL CASE: A 11-years-old boy, child of consangui neous parents, with hypotonia, severe developmental delay, intellectual disability, feeding difficulties, congenital heart disease (patent ductus arteriosus and mitral regurgitation), without epilepsy or coa gulation disorders. The metabolic screening showed unclear results, including N-glycosylation stu dies in plasma that were normal. Therefore, whole-exome sequencing (WES) was performed which identified a previously unreported variant in the ALG13 gene: c.428C > T (p.P143L) in hemizygous state; confirmed by Sanger sequencing. His mother was a carrier of the same variant. CONCLUSION: This is the first report of a Colombian patient with ALG13-CDG without epilepsy. The findings in this patient broaden the phenotypic spectrum of ALG13-CDG known to date and support that N- glycosylation disorders may be present in normal biochemical analysis. WES has become a cost- effective technique that allows the identification of disease-causing mutations in diseases with a broad phenotypic and genotypic spectrum.

Biosynthesis of oxygenated brasilane terpene glycosides involves a promiscuous N-acetylglucosamine transferase.

Investigation of the metabolome of the ascomycete Annulohypoxylon truncatum led to the identification of novel oxygenated brasilane glycosides and the revision of the stereochemistry of the brasilane A octahydro-1H-indene core scaffold to trans. The bra biosynthetic gene cluster containing five genes (braA-braE) was identified and verified by heterologous expression experiments in Aspergillus oryzae demonstrating that BraC is a multifunctional P450 monooxygenase. In vitro studies of BraB revealed it to be a very rare fungal UDP-GlcNAc dependent N-acetylglucosamine transferase. UDP-glucose is also accepted as a donor, and a broad acceptor substrate tolerance for various primary and secondary alcohols was observed.

O-GlcNAc impairs endothelial function in uterine arteries from virgin but not pregnant rats: The role of GSK3ß.

Increased O-Linked ß-N-acetylglucosamine (O-GlcNAc) is observed in several pathologies, and unbalanced O-GlcNAcylation levels favor endothelial dysfunction. Whether augmented O-GlcNAc impacts the uterine artery (UA) function and how it affects the UA during pregnancy remains to be elucidated. We hypothesized that glucosamine treatment increases O-GlcNAc, leading to uterine artery dysfunction and this effect is prevented by pregnancy. Pregnant (P) and non-pregnant (NP) Wistar rats were treated with glucosamine (300 mg/kg; i.p.) for 21 days. Concentration response-curves (CRC) to acetylcholine (in the presence or absence of L-NAME) and sodium nitroprusside were performed in UAs. In NP rats, glucosamine treatment increased O-GlcNAc expression in UAs accompanied by decreased endothelium-dependent relaxation, which was abolished by L-NAME. Endothelial nitric oxide synthase (eNOS) activity and total Akt expression were decreased by glucosamine-treatment in NP rats. Further, NP rats treated with glucosamine displayed increased glycogen synthase kinase 3 beta (GSK3ß) activation and O-GlcNAc-transferase (OGT) expression in the UA. P rats treated with glucosamine displayed decreased O-GlcNAc in UAs and it was accompanied by improved relaxation to acetylcholine, whereas eNOS and GSK3ß activity and total Akt and OGT expression were unchanged. Sodium nitroprusside-induced relaxation was not changed in all groups, indicating that glucosamine treatment led to endothelial dysfunction in NP rats. The underlying mechanism is, at least in part, dependent on Akt/GSK3ß/OGT modulation. We speculate that during pregnancy, hormonal alterations play a protective role in preventing O-GlcNAcylation-induced endothelial dysfunction in the UAs.

N-Acetylglucosaminyltransferase III (GnT-III) but not N-Acetylgalactosaminyltransferase-6 and 8 are Differentially Expressed in Invasive and In Situ Ductal Carcinoma of the Breast.

Mammary carcinoma is the most common malignant tumor in women, and it is the leading cause of mortality. In tumor context, glycosylation promotes post translational modifications necessary for cell progression, emerging as a relevant tumor hallmarker. This study aimed to analyze the association between polypeptide N-acetylgalactosaminyltransferase-6 (ppGalNAc-T6), -T8, N-acetylglucosaminyltransferase III (GnT-III) expression, Phaseolus vulgaris-leucoagglutinin (PHA-L), wheat germ agglutinin (WGA) and peanut agglutinin (PNA) staining with clinic-histopathological factors from patients with pure ductal carcinoma in situ (DCIS) and DCIS with invasive ductal carcinoma (DCIS-IDC) of breast. Formalin-fixed and paraffin-embedded samples (n = 109) were analyzed. In pure DCIS samples GnT-III was over-expressed in comedo lesions (p = 0.007). In DCIS-IDC, GnT-III expression was associated with high nuclear grade tumors (p = 0.039) while the presence of PHA-L and WGA were inversely related to HER-2 expression (p = 0.001; p = 0.036, respectively). These findings pointed to possible involvement of GnT-III, ppGalNAc-T8, L-PHA and WGA as probes in prognostic evaluation of DCIS.

O-GlcNAcylation reduces proximal tubule protein reabsorption and promotes proteinuria in spontaneously hypertensive rats.

Hypertensive individuals are at greater risk for developing chronic kidney disease (CKD). Reducing proteinuria has been suggested as a possible therapeutic approach to treat CKD. However, the mechanisms underlying the development of proteinuria in hypertensive conditions are incompletely understood. Cardiac and vascular dysfunction is associated with changes in the O-GlcNAcylation pathway in hypertensive models. We hypothesized that O-GlcNAcylation is also involved in renal damage, especially development of proteinuria, associated with hypertension. Using the spontaneously hypertensive rat (SHR) model, we observed higher renal cortex O-GlcNAcylation, glutamine-fructose aminotransferase (GFAT), and O-GlcNAc transferase (OGT) protein expression, which positively correlated with proteinuria. Interestingly, this was observed in hypertensive, but not pre-hypertensive, rats. Pharmacological inhibition of GFAT decreased renal cortex O-GlcNAcylation, proteinuria, and albuminuria in SHR. Using a proximal tubule cell line, we observed that increased O-GlcNAcylation reduced megalin surface expression and albumin endocytosis in vitro, and the effects were correlated in vivo Moreover, megalin is O-GlcNAcylated both in vitro and in vivo In conclusion, our results demonstrate a new mechanism involved in hypertension-associated proteinuria.

O-linked N-acetyl-glucosamine deposition in placental proteins varies according to maternal glycemic levels.

AIMS: Hyperglycemia increases glycosylation with O-linked N-acetyl-glucosamine (O-GlcNAc) contributing to placental dysfunction and fetal growth impairment. Our aim was to determine how O-GlcNAc levels are affected by hyperglycemia and the O-GlcNAc distribution in different placental regions. MAIN METHODS: Female Wistar rats were divided into the following groups: severe hyperglycemia (>300 mg/dL; n = 5); mild hyperglycemia (>140 mg/dL, at least than two time points during oral glucose tolerance test; n = 7) or normoglycemia (<120 mg/dL; n = 6). At 21 days of pregnancy, placental tissue was collected and processed for morphometry and immunohistochemistry analyses, or properly stored at -80 °C for protein quantification by western blot. KEY FINDINGS: Placental index was increased only in severe hyperglycemic rats. Morphometric analysis showed increased junctional zone and decreased labyrinth region in placentas exclusively from the severe hyperglycemic group. Proteins targeted by O-GlcNAc were detected in all regions, with increased O-GlcNAc levels in the hyperglycemic group compared to control and mild hyperglycemic rats. Proteins in endothelial and trophoblast cells were the main target for O-GlcNAc. Whereas no changes in O-GlcNAc transferase (OGT) expression were detected, O-GlcNAcase (OGA) expression was reduced in placentas from the severe hyperglycemic group and augmented in placentas from the mild hyperglycemic group, compared with their respective control groups. SIGNIFICANCE: Placental O-GlcNAc overexpression may contribute to placental dysfunction, as indicated by the placental index. Additionally, morphometric alterations, occurring simultaneously with increased O-GlcNAc accumulation in the placental tissue may contribute to placental dysfunction during hyperglycemia.

Early and Persistent O-GlcNAc Protein Modification in the Streptozotocin Model of Alzheimer's Disease.

 O-GlcNAc transferase (OGT), an enzyme highly expressed in brain tissue, catalyzes the addition of N-acetyl-glucosamine (GlcNAc) to hydroxyl residues of serine and threonine of proteins. Brain protein O-GlcNAcylation is diminished in Alzheimer's disease (AD), and OGT targets include proteins of the insulin-signaling pathway (e.g., insulin receptor susbtrate-1, IRS-1). We hypothesized that ICV streptozotocin (STZ) also affects O-GlcNAc protein modification. We investigated hippocampal metabolic changes in Wistar rats, particularly OGT levels and insulin resistance, as well as related astroglial activities, immediately after ICV STZ administration (first week) and later on (fourth week). We found an early (at one week) and persistent (at fourth week) decrease in OGT in the ICV STZ model of AD, characterized by a spatial cognitive deficit. Consistent with this observation, we observed a decrease in protein O-GlnNAc modification at both times. Increased phosphorylation at serine-307 of IRS-1, which is related to insulin resistance, was observed on the fourth week. The decrease in OGT and consequent protein O-GlnNAc modifications appear to precede the decrease in glucose uptake and increment of the glyoxalase system observed in the hippocampus. Changes in glial fibrillary acidic protein and S100B in the hippocampus, as well as the alterations in cerebrospinal fluid S100B, confirm the astrogliosis. Moreover, decreases in glutamine synthetase and glutathione content suggest astroglial dysfunction, which are likely implicated in the neurodegenerative cascade triggered in this model. Together, these data contribute to the understanding of neurochemical changes in the ICV STZ model of sporadic AD, and may explain the decreases in protein O-GlcNAc levels and insulin resistance observed in AD.

The Immunosuppressant Brasilicardin: Determination of the Biosynthetic Gene Cluster in the Heterologous Host Amycolatopsis japonicum.

Nocardia terpenica IFM 0406 is the producer of the immunosuppressants brasilicardins A-D. Brasilicardin is a promising compound because of its unique mode of action and its higher potency and reduced toxicity compared to today's standard drugs. However, production of brasilicardin is so far hampered as Nocardia terpenica IFM 0406 synthesizes brasilicardin in only low amounts and represents a human pathogen (biosafety level 2 BSL2). In order to achieve a safe and high yield production of brasilicardin A (BraA), the authors heterologously express the brasilicardin gene cluster in the nocardioform actinomycete Amycolatopsis japonicum (A. japonicum::bcaAB01), which is fast growing, genetically accessible and closely related to N. terpenica IFM 0406. In A. japonicum::bcaAB01, four brasilicardin congeners, intermediates of the BraA biosynthesis, are produced. Investigation of the genes flanking the previously defined brasilicardin biosynthetic gene cluster revealed two novel genes (bra0, bra12), which are involved in brasilicardin biosynthesis: bra12 encodes a transcriptional activator of the brasilicardin gene cluster. bra0 codes for a dioxygenase involved in methoxylation of brasilicardin. Based on this finding the authors are able to revise the proposed brasilicardin biosynthesis.

Computational insights into active site shaping for substrate specificity and reaction regioselectivity in the EXTL2 retaining glycosyltransferase.

Glycosyltransferases are enzymes that catalyze a monosaccharide transfer reaction from a donor to an acceptor substrate with the synthesis of a new glycosidic bond. They are highly substrate specific and regioselective, even though the acceptor substrate often presents multiple reactive groups. Currently, many efforts are dedicated to the development of biocatalysts for glycan synthesis and, therefore, a better understanding of how natural enzymes achieve this goal can be of valuable help. To gain a deeper insight into the catalytic strategies used by retaining glycosyltransferases, the wild type EXTL2 (CAZy family GT64) and four mutant forms (at positions 293 and 246) were studied using QM(DFT)/MM calculations and molecular dynamics simulations. Existing hypotheses on the roles of Arg293, an enigmatic residue in the CAZy family GT64 that seemed to contradict a mechanism through an oxocarbenium intermediate, and of Asp246 have been tested. We also provide a molecular interpretation for the results of site-directed mutagenesis experiments. Moreover, we have investigated why an Asp, and not a Glu like in the family GT6, is found on the ß-face of the transferred GlcNAc. It is predicted that an Asp246Glu mutant of EXTL2 would be unable to catalyze the α-1,4 transfer. The results herein presented clarify the roles that Arg293, Asp246 and Leu213 have at different stages of the catalytic process (for binding but also for efficient chemical reaction). Altogether, we provide a molecular view that connects the identity and conformation of these residues to the substrate specificity and regioselectivity of the enzyme, illustrating a delicate interplay between all these aspects.

Identification of novel biomarkers associated with poor patient outcomes in invasive breast carcinoma.

Breast carcinoma (BC) corresponds to 23 % of all cancers in women, with 1.38 million new cases and 460,000 deaths worldwide annually. Despite the significant advances in the identification of molecular markers and different modalities of treatment for primary BC, the ability to predict its metastatic behavior is still limited. The purpose of this study was to identify novel molecular markers associated with distinct clinical outcomes in a Brazilian cohort of BC patients. We generated global gene expression profiles using tumor samples from 24 patients with invasive ductal BC who were followed for at least 5 years, including a group of 15 patients with favorable outcomes and another with nine patients who developed metastasis. We identified a set of 58 differentially expressed genes (p ≤ 0.01) between the two groups. The prognostic value of this metastasis signature was corroborated by its ability to stratify independent BC patient datasets according to disease-free survival and overall survival. The upregulation of B3GNT7, PPM1D, TNKS2, PHB, and GTSE1 in patients with poor outcomes was confirmed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) in an independent sample of patients with BC (47 with good outcomes and eight that presented metastasis). The expression of BCL2-associated agonist of cell death (BAD) protein was determined in 1276 BC tissue samples by immunohistochemistry and was consistent with the reduced BAD mRNA expression levels in metastatic cases, as observed in the oligoarray data. These findings point to novel prognostic markers that can distinguish breast carcinomas with metastatic potential from those with favorable outcomes.

Changes in O-Linked N-Acetylglucosamine (O-GlcNAc) Homeostasis Activate the p53 Pathway in Ovarian Cancer Cells.

O-GlcNAcylation is a dynamic post-translational modification consisting of the addition of a single N-acetylglucosamine sugar to serine and threonine residues in proteins by the enzyme O-linked ß-N-acetylglucosamine transferase (OGT), whereas the enzyme O-GlcNAcase (OGA) removes the modification. In cancer, tumor samples present with altered O-GlcNAcylation; however, changes in O-GlcNAcylation are not consistent between tumor types. Interestingly, the tumor suppressor p53 is modified by O-GlcNAc, and most solid tumors contain mutations in p53 leading to the loss of p53 function. Because ovarian cancer has a high frequency of p53 mutation rates, we decided to investigate the relationship between O-GlcNAcylation and p53 function in ovarian cancer. We measured a significant decrease in O-GlcNAcylation of tumor tissue in an ovarian tumor microarray. Furthermore, O-GlcNAcylation was increased, and OGA protein and mRNA levels were decreased in ovarian tumor cell lines not expressing the protein p53. Treatment with the OGA inhibitor Thiamet-G (TMG), silencing of OGA, or overexpression of OGA and OGT led to p53 stabilization, increased nuclear localization, and increased protein and mRNA levels of p53 target genes. These data suggest that changes in O-GlcNAc homeostasis activate the p53 pathway. Combination treatment of the chemotherapeutic cisplatin with TMG decreased tumor cell growth and enhanced cell cycle arrest without impairing cytotoxicity. The effects of TMG on tumor cell growth were partially dependent on wild type p53 activation. In conclusion, changes in O-GlcNAc homeostasis activate the wild type p53 pathway in ovarian cancer cells, and OGA inhibition has the potential as an adjuvant treatment for ovarian carcinoma.

Ligand-mediated Galectin-1 endocytosis prevents intraneural H2O2 production promoting F-actin dynamics reactivation and axonal re-growth.

UNLABELLED: Axonal growth cone collapse following spinal cord injury (SCI) is promoted by semaphorin3A (Sema3A) signaling via PlexinA4 surface receptor. This interaction triggers intracellular signaling events leading to increased hydrogen peroxide levels which in turn promote filamentous actin (F-actin) destabilization and subsequent inhibition of axonal re-growth. In the current study, we demonstrated that treatment with galectin-1 (Gal-1), in its dimeric form, promotes a decrease in hydrogen peroxide (H2O2) levels and F-actin repolimerization in the growth cone and in the filopodium of neuron surfaces. This effect was dependent on the carbohydrate recognition activity of Gal-1, as it was prevented using a Gal-1 mutant lacking carbohydrate-binding activity. Furthermore, Gal-1 promoted its own active ligand-mediated endocytosis together with the PlexinA4 receptor, through mechanisms involving complex branched N-glycans. In summary, our results suggest that Gal-1, mainly in its dimeric form, promotes re-activation of actin cytoskeleton dynamics via internalization of the PlexinA4/Gal-1 complex. This mechanism could explain, at least in part, critical events in axonal regeneration including the full axonal re-growth process, de novo formation of synapse clustering, axonal re-myelination and functional recovery of coordinated locomotor activities in an in vivo acute and chronic SCI model. SIGNIFICANCE STATEMENT: Axonal regeneration is a response of injured nerve cells critical for nerve repair in human spinal cord injury. Understanding the molecular mechanisms controlling nerve repair by Galectin-1, may be critical for therapeutic intervention. Our results show that Galectin-1; in its dimeric form, interferes with hydrogen peroxide production triggered by Semaphorin3A. The high levels of this reactive oxygen species (ROS) seem to be the main factor preventing axonal regeneration due to promotion of actin depolymerization at the axonal growth cone. Thus, Galectin-1 administration emerges as a novel therapeutic modality for promoting nerve repair and preventing axonal loss.

High-fat diet increases O-GlcNAc levels in cerebral arteries: a link to vascular dysfunction associated with hyperlipidaemia/obesity?

Obesity and high fat intake induce alterations in vascular function and structure. Aberrant O-GlcNAcylation (O-GlcNAc) of vascular proteins has been implicated in vascular dysfunction associated with cardiovascular and metabolic diseases. In the present study, we tested the hypothesis that high-fat diet (HFD)-mediated increases in O-GlcNAc-modified proteins contribute to cerebrovascular dysfunction. O-GlcNAc-protein content was increased in arteries from male Wistar rats treated with a HFD (45% fat) for 12 weeks compared with arteries from rats on control diet (CD). HFD augmented body weight [(g) 550±10 compared with 502±10 CD], increased plasma triacylglycerols [(mg/dl) 160±20 compared with 95±15 CD] and increased contractile responses of basilar arteries to serotonin [5-hydroxytryptamine (5-HT)] [(pD2) 7.0±0.1 compared with 6.7±0.09 CD] and the thromboxane analogue 9,11-dideoxy-9α,11α-methanoepoxy prostaglandin F2α (U-46619) [(pD2) 7.2±0.1 compared with 6.8±0.09 CD]. Of importance, increased levels of O-GlcNAc [induced by 24 h-incubation of vessels with a potent inhibitor of O-GlcNAcase (OGA), O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PugNAc)] increased basilar artery contractions in response to U-46619 [(pD2) 7.4±0.07 compared with 6.8±0.08 CD] and 5-HT [(pD2) 7.5±0.06 compared with 7.1±0.1 CD]. Vessels from rats on the HFD for 12 weeks and vessels treated with PugNAc displayed increased phosphorylation of p38 (Thr(180/182)) and extracellular signal-regulated kinase 1/2 (Erk1/2) (Ser(180/221)). Increased 5HT-induced contractions in arteries from rats on the HFD or in arteries incubated with PugNAc were abrogated by mitogen-activated protein kinase (MAPK) inhibitors. Our data show that HFD augments cerebrovascular O-GlcNAc and this modification contributes to increased contractile responses and to the activation of the MAPK pathway in the rat basilar artery.

The role of N-glycans in colorectal cancer progression: potential biomarkers and therapeutic applications.

Changes in glycosylation, which is one of the most common protein post-translational modifications, are considered to be a hallmark of cancer. N-glycans can modulate cell migration, cell-cell adhesion, cell signaling, growth and metastasis. The colorectal cancer (CRC) is a leading cause of cancer-related mortality and the correlation between CRC progression and changes in the pattern of expression of N-glycans is being considered in the search for new biomarkers. Here, we review the role of N-glycans in CRC cell biology. The perspectives on emerging N-glycan-related anticancer therapies, along with new insights and challenges, are also discussed.

The mitochondrial O-linked N-acetylglucosamine transferase (mOGT) in the diabetic patient could be the initial trigger to develop Alzheimer disease.

Diabetes mellitus (DM) is considered a risk factor for the development of Alzheimer disease (AD); however, how DM favors evolution of AD is still insufficiently understood. Hyperglycemia in DM is associated to an increase in mitochondrial reactive oxygen species (ROS) generation, as well as damage of hippocampal cells, reflected by changes in morphological and mitochondrial functionality. Similar mitochondrial damage has been observed when amyloid beta (Aß) accumulates in the brain of AD patients. In DM, the excess of glucose in the brain induces higher activity of the hexosamine biosynthesis pathway (HBP), it synthesizes UDP-N-acetylglucosamine (UDP-GlcNAc), which is used by O-linked N-acetylglucosamine transferase (OGT) to catalyze O-GlcNAcylation of numerous proteins. Although O-GlcNAcylation plays an important role in maintaining structure and cellular functionality, chronic activity of this pathway has been associated with insulin resistance and hyperglycemia-induced glucose toxicity. Three different forms of OGT are known: nucleocytoplasmic (ncOGT), short (sOGT), and mitochondrial (mOGT). Previous reports showed that overexpression of ncOGT is not toxic to the cell; in contrast, overexpression of mOGT is associated with cellular apoptosis. In this work, we suggest that hyperglycemia in the diabetic patient could induce greater expression and activity of mOGT, modifying the structure and functionality of mitochondria in hippocampal cells, accelerating neuronal damage, and favoring the start of AD. In consequence, mOGT activity could be a key point for AD development in patients with DM.

Antisense glutaminase inhibition modifies the O-GlcNAc pattern and flux through the hexosamine pathway in breast cancer cells.

Glutamine behaves as a key nutrient for tumors and rapidly dividing cells. Glutaminase is the main glutamine-utilizing enzyme in these cells, and its activity correlates with glutamine consumption and growth rate. We have carried out the antisense L-type glutaminase inhibition in human MCF7 breast cancer cells, in order to study its effect on the hexosamine pathway and the pattern of protein O-glycosylation. The antisense mRNA glutaminase expressing cells, named ORF19, presented a 50% lower proliferation rate than parental cells, showing a more differentiated phenotype. ORF19 cells had an 80% reduction in glutamine:fructose-6-P amidotransferase activity, which is the rate-limiting step of the hexosamine pathway. Although the overall cellular protein O-glycosylation did not change, the O-glycosylation status of several key proteins was altered. O-glycosylation of O-GlcNAc transferase (OGT), the enzyme that links N-acetylglucosamine to proteins, was fivefold lower in ORF19 than in wild type cells. Inhibition of glutaminase also provoked a 10-fold increase in Sp1 expression, and a significant decrease in the ratio of O-glycosylated to total protein for both Sp1 and the Rpt2 proteasome component. These changes were accompanied by a higher Sp1 transcriptional activity. Proteome analysis of O-glycosylated proteins permitted the detection of two new OGT target proteins: the chaperonin TCP-1 theta and the oncogene Ets-related protein isoform 7. Taken together, our results support the hexosamine pathway and the O-glycosylation of proteins being a sensor mechanism of the nutritional and energetic states of the cell.

Isolation and characterization of the Golgi complex of the protozoan Trypanosoma cruzi.

In this study the Golgi complex of the epimastigote forms of Trypanosoma cruzi were isolated and characterized. Using well-controlled sonication to rupture the cells and centrifugation on a discontinuous sucrose density gradient, a highly enriched Golgi fraction was obtained. The Golgi fraction contained most of the beta-galactosyltransferase (beta-Gal transferase) and UDP-N-acetyl-glucosamine: polypeptide-alpha-N-acetyl-glucosaminyltransferase (O-alpha-GlcNAc transferase) activities with minimal contamination of other organelles, as observed by enzymatic assays and electron microscopy analysis. To characterize the Golgi from T. cruzi cells further, it was incubated with a monoclonal antibody against a 58 kDa protein involved in the association of the Golgi complex with microtubules in mammalian cells. Immunofluorescence microscopy showed that the 58 kDa protein is localized in the T. cruzi Golgi region, a result confirmed by high resolution scanning electron microscopy immunocytochemistry. Thus, our results show, for the first time, that the beta-Gal transferase, the O-alpha-GlcNAc transferase and the 58 kDa protein are present in the Golgi complex of T. cruzi and are novel biochemical markers which can be used in the characterization of this organelle in T. cruzi.

Involvement of gntS in the control of GntI, the main system for gluconate metabolism in Escherichia coli.

The initial steps of gluconate metabolism in E. coli, transport and phosphorylation, occur through duplicate activities. These activities have been included in two systems designated as GntI (main) and GntII (subsidiary), encoded by differently regulated operons located at the 76.4-77 and 95.3-96.9 regions on the map respectively. Despite recent molecular advances related to genetics and physiology of these systems, there is no information about the coordination of their expression when E. coli grows on gluconate. Under these conditions, the subsidiary gluconokinase (gntV gene, min 96.8) as well as the GntI activities are expressed in inducible form. Therefore it was of interest to find out if GntS, the positive regulator of gntV has a similar effect on GntI activities expression. Our results agree with this hypothesis. GntS, in addition to its regulatory action on the gntV gene, seems to assist, direct or indirectly, the expression of the GntI activities. A gntS E. coli mutant does not grow on gluconate but spontaneously pseudoreverts to a gluconate growing phenotype at high rate per cell generation when cultivated in rich media with or without gluconate or mineral medium containing any other suitable carbon source. In the pseudorevertants, the thermosensitive gluconokinase remains repressed while the GntI activities are inducibly expressed. At present, the location and nature of the gntS suppressor mutation are not known. Phage P1Kc mediated transductions have ruled out that it alters the gntT gene. This is the first report on GntI activities alteration due to a lesion located out of the bioH-asd region.