A new case of UDP-galactose transporter deficiency (SLC35A2-CDG): molecular basis, clinical phenotype, and therapeutic approach.

Congenital disorders of glycosylation (CDG) are a group of hereditary metabolic diseases characterized by abnormal glycosylation of proteins and lipids. Often, multisystem disorders with central nervous system involvement and a large variety of clinical symptoms occur. The main characteristics are developmental delay, seizures, and ataxia. In this paper we report the clinical and biochemical characteristics of a 5-year-old girl with a defective galactosylation of N-glycans, resulting in developmental delay, muscular hypotonia, epileptic seizures, inverted nipples, and visual impairment. Next generation sequencing revealed a de novo mutation (c.797G > T, p.G266V) in the X-chromosomal gene SLC35A2 (solute carrier family 35, UDP-galactose transporter, member A2; MIM 300896). While this mutation was found heterozygous, random X-inactivation of the normal allele will lead to loss of normal SLC35A2 activity in respective cells. The functional relevance of the mutation was demonstrated by complementation of UGT-deficient MDCK-RCA(r) and CHO-Lec8 cells by normal UGT-expression construct but not by the mutant version. The effect of dietary galactose supplementation on glycosylation was investigated, showing a nearly complete normalization of transferrin glycosylation.

Mutations in the SLC35C1 gene, contributing to significant differences in fucosylation patterns, may underlie the diverse phenotypic manifestations observed in leukocyte adhesion deficiency type II patients.

Congenital disorders of glycosylation (CDG) are a large family of genetic diseases resulting from defects in the synthesis of glycans and the attachment of glycans to macromolecules. The CDG known as leukocyte adhesion deficiency II (LAD II) is an autosomal, recessive disorder caused by mutations in the SLC35C1 gene, encoding a transmembrane protein of the Golgi apparatus, involved in GDP-fucose transport from the cytosol to the Golgi lumen. In this study, a cell-based model was used as a tool to characterize the molecular background of a therapy based on a fucose-supplemented diet. Such therapies have been successfully introduced in some (but not all) known cases of LAD II. In this study, the effect of external fucose was analyzed in SLC35C1 KO cell lines, expressing 11 mutated SLC35C1 proteins, previously discovered in patients with an LAD II diagnosis. For many of them, the cis-Golgi subcellular localization was affected; however, some proteins were localized properly. Additionally, although mutated SLC35C1 caused different α-1-6 core fucosylation of N-glycans, which explains previously described, more or less severe disorder symptoms, the differences practically disappeared after external fucose supplementation, with fucosylation restored to the level observed in healthy cells. This indicates that additional fucose in the diet should improve the condition of all patients. Thus, for patients diagnosed with LAD II we advocate careful analysis of particular mutations using the SLC35C1-KO cell line-based model, to predict changes in localization and fucosylation rate. We also recommend searching for additional mutations in the human genome of LAD II patients, when fucose supplementation does not influence patients' state.

Structural analysis of N-glycans from yellow lupin (Lupinus luteus) seed diphosphonucleotide phosphatase/phosphodiesterase.

N-linked oligosaccharide chains released by hydrazinolysis from yellow lupin seed diphosphonucleotide phosphatase/phosphodiesterase were fluorescence labeled and separated by high performance liquid chromatography (GlycoSep N and GlycoSep H columns). Exoglycosidase sequencing elucidated the structures of 24 separated N-glycans. Thirty percent of isolated glycans were found to be of high-mannose type (three to eight mannosyl residues), 42% were complex type and 26% belonged to paucimannosidic type. Among complex type glycans, structures with Lewis(a) epitope were identified. It is very unusual to find all types of plant N-glycans in one protein. Possible reasons for such a broad spectrum of N-glycan structures are discussed.

Characterization of diphosphonucleotide phosphatase/phosphodiesterase from yellow lupin (Lupinus luteus) seeds.

A phosphatase cleaving the pyrophosphate bond in diphosphonucleotides and phosphodiester bond in various phosphodiesters (pH optimum at 6.25) was purified from yellow lupin (Lupinus luteus L.) seeds. The enzyme is 75 kDa monomeric glycoprotein (pI=6.4) with 4.4% of carbohydrate (mannose, N-acetylglucosamine, fucose and xylose). Analysis of its partial amino acid sequence (8 peptides, 101 amino acid residues) together with no divalent cation requirements for catalysis points out that the purified enzyme is different from known plant pyrophosphate cleaving enzymes (apyrases and inorganic pyrophosphatases). Its physiological role could be related to a regulation of diphosphonucleotides level in plant metabolism.

Purification and characterization of acid phosphatase from yellow lupin (Lupinus luteus) seeds.

Acid phosphatase (EC 3.1.3.2) from yellow lupin (Lupinus luteus) seeds was purified to homogeneity by ammonium sulphate fractionation, affinity chromatography, cation-exchange chromatography, gel filtration or reverse-phase HPLC. The enzyme is a dimer with the 50 kD and 44 kD subunits and contains 7.3% of carbohydrate, forming at least four oligosaccharide chains. The optimum pH for the enzyme is 5.4. The apparent Km for p-nitrophenyl phosphate was estimated to be 0.28 mM and Vmax = 1780 IU/mg of protein. The purified phosphatase has the highest specific activities reported for any plant acid phosphatases measured for any native or synthetic substrate. The enzyme has broad specificity; however, cyclic nucleotides, pyrophosphate or phytate are not cleaved. It is inhibited by molybdate, fluoride and phosphate. There is no change in the enzyme activity in the presence of EDTA, phenanthroline and tartrate.

Oligosaccharides released by hydrazinolysis from Tamm-Horsfall protein of various human donors contain similar high-mannose glycans.

As pathophysiological functions claimed for Tamm-Horsfall protein (THP) are related to its sugar moiety, we examined influence of pregnancy and various diseases on high-mannose chains. Hydrazinolysis was used to liberate oligosaccharides from THP polypeptide backbone. After HPLC separation of fluorescently labelled glycans similar profiles of neutral oligosaccharides were observed in THP of healthy subjects, pregnant women, patients with Bartter's syndrome, patients with acute lymphoblastic leukemia and a patient with carbohydrate deficient glycoprotein syndrome. THP contains Man-5, Man-6 and Man-7 glycans, with the preponderant amount of Man-6 glycan (about 7% of total THP oligosaccharides). No statistically significant differences were found in THP high-mannose glycans profiles between control subjects and pregnant women or patients. It is likely that neither pregnancy nor the pathological conditions examined affect high-mannose chains. In our opinion hydrazinolysis as a method of oligosaccharides liberation, in contrast to enzymatic deglycosylation, is more appropriate for analysis of the sugar moiety of THP.

Lectin from Beauveria bassiana mycelium recognizes Thomsen-Friedenreich antigen and related structures.

A lectin was isolated from the mycelium of the stationary growing enthomopathogenic fungus Beauveria bassiana by extraction, chromatography on QAE-Sephadex A-25, salt precipitation, and hydrophobic chromatography on Phenyl-Sepharose 4B. The Beauveria bassiana lectin (BBL) is a 15 kDa glycoprotein rich in hydrophobic amino acids, without detectable amount of methionine. It contains 12.6% of carbohydrates including galactose and mannose. Isoelectric point was found at pH 7.1. The lectin is stable between pH 6 and 11, and at temperature under 50 degrees C. The activity of the lectin was not dependent on with Ca++, Mn++, Mg++ cations and was apparently not blood group ABO specific. The hemagglutination caused by the lectin was inhibited by alpha lactose (Gal beta 1-->4 Glc alpha), but not by beta lactose (Gal beta 1-->4 Glc beta). In direct ELISA the BBL preferentially reacted with some glycoproteins carrying O-linked sugar structure Gal beta 1-->3 GalNAc: strongly with human glycophorin A and weaker with mouse glycophorin, fetuin, IgA, ovine submaxillary mucin. On the other hand BBL did not react in direct ELISA with N-glycoproteins (alpha 1-acid glycoprotein, haptoglobin, fibronectin), however, N-glycoproteins could act as inhibitors of lectin-glycophorin A interaction. We observed also weak interaction with asialo-Tamm-Horsfall N-glycoprotein having unusual large, branched N-glycans with outer GalNAc beta 1-->4Gal sequence. Moreover, the interaction of BBL with highly sialylated preparations of glycoproteins was weaker than with asialo forms. Presented results indicate that BBL exhibits sugar binding specificity towards glycotope corresponding to Thomsen-Friedenreich antigen and its related sequences: Gal beta 1-->3 GalNAc > Neu Ac alpha 2-3 Gal beta 1-->3 (Neu Ac alpha 2-6) GalNAc > Gal beta 1-->4 Glc alpha.

Isolation and amino acid sequence of two trypsin isoinhibitors from duck pancreas.

DPTI II and DPTI IV, two trypsin inhibitors from duck pancreas, have been isolated by affinity chromatography on immobilized anhydrotrypsin, anion exchange and RP-HPLC. The complete amino acid sequence of both inhibitors was determined after reductive carboxymethylation and digestion with Staphylococcus aureus V8 protease or trypsin. The inhibitors were each found to be a single polypeptide chain comprised of 69 amino acid residues and their molecular masses were estimated at 7687 Da for DPTI II and 7668 Da for DPTI IV. The only difference in amino acid sequence between the two inhibitors is the replacement of Arg for His residue in the C-terminal position of DPTI IV.

Carbohydrate structures of haptoglobin in sera of healthy people and a patient with congenital disorder of glycosylation.

Haptoglobin is one of acute phase glycoproteins often used as markers in glycopathology studies. In this work the oligosaccharide structures of haptoglobin from 'healthy' subjects have been studied in detail, taking into consideration the possible dependence of glycosylation on the phenotype. About 75% of charged haptoglobin glycans were of biantennary complex structure, and some of them lacked one terminal sialic acid molecule. Triantennary structures made up almost 25% of the charged glycans pool, and highly branched tetrasialylated oligosaccharides did not exceed 1%. The main difference between haptoglobin derived from the sample of pooled 44 sera and from the 2-2 phenotype individual concerned the relative content of trisialylated oligosaccharide with one 2-3 linked sialic acid residue. The oligosaccharide profile of haptoglobin derived from serum of a patient suffering from congenital disorder of glycosylation was compared to 'healthy' controls. It was shown, that four main glycans are identical in patient and 'normal' haptoglobins. Some alterations were found in the relative content of mono-, bi-, and trisialylated glycans as well as in the appearance of some tracely abundant oligosaccharides in haptoglobin of the patient with congenital disorder of glycosylation.

[Clinical application of cyclooxygenase-2 inhibitors]. / Kliniczne zastosowania inhibitorów cyklooksygenazy-2.

Nonsteroidal antiinflammatory drugs (NSAIDs) are a group of compounds with similar therapeutic and side effects. Their therapeutic effects depend on blockade of prostaglandin synthesis through enzyme cyclooxygenase (COX) inhibition. Two isoforms of the enzyme cyclooxygenase have been identified: COX-1 and COX-2. Selective COX-2 inhibitors i.e. meloxicam, nimesulid, etodolac or highly selective COX-2 inhibitors i.e. celecoxib, rofecoxib have antiinflammatory and analgesic properties with less or no gastrointestinal or other NSAIDs-typical adverse effects. Highly selective COX-2 inhibitors may also be active in colonic polyposis, colorectal cancer and Alzheimer's disease.

[Antiarrhythmic properties of angiotensin converting enzyme inhibitors]. / Wlasciwosci przeciwarytmiczne inhibitorów konwertazy angiotensyny (IKA).

In animal experimental models angiotensin converting enzyme (ACE) inhibitors have been found to reduce arrhythmias. In patients with cardiovascular disorders treated with ACE inhibitors the reduction of incidence and the severity of arrhythmias as well as the decrease mortality have been observed. There is little evidence to prove that ACE inhibitors affect directly the electrophysiological function of a heart. It seems that antiarrhythmic effects of ACE inhibitors are indirect.

Fur homolog regulates Porphyromonas gingivalis virulence under low-iron/heme conditions through a complex regulatory network.

Porphyromonas gingivalis is a key pathogen responsible for initiation and progression of chronic periodontitis. Little is known about the regulatory mechanisms of iron and heme uptake that allow P. gingivalis to express virulence factors and survive in the hostile environment of the oral cavity, so we initiated characterization of a P. gingivalis Fur homolog (PgFur). Many Fur paralogs found in microbial genomes, including Bacteroidetes, confirm that Fur proteins have a tendency to be subjected to a sub- or even neofunctionalization process. PgFur revealed extremely high sequence divergence, which could be associated with its functional dissimilarity in comparison with other Fur homologs. A fur mutant strain constructed by insertional inactivation exhibited retarded growth during the early growth phase and a significantly lower tendency to form a homotypic biofilm on abiotic surfaces. The mutant also showed significantly weaker adherence and invasion to epithelial cells and macrophages. Transcripts of many differentially regulated genes identified in the fur mutant strain were annotated as hypothetical proteins, suggesting that PgFur can play a novel role in the regulation of gene expression. Inactivation of the fur gene resulted in decreased hmuY gene expression, increased expression of other hmu components and changes in the expression of genes encoding hemagglutinins and proteases (mainly gingipains), HtrA, some extracytoplasmic sigma factors and two-component systems. Our data suggest that PgFur can influence in vivo growth and virulence, at least in part by affecting iron/heme acquisition, allowing efficient infection through a complex regulatory network.

Oligosaccharide and polypeptide homology of lupin (Lupinus luteus L.) acid phosphatase subunits.

Peptide mapping of lupin acid phosphatase clearly demonstrated the homology between its two subunits. Sequenced tryptic peptides also showed 78% identity (92% similarity) to the red bean acid phosphatase. Peptides exclusive for the 50-kDa subunit are homologous to N-terminally located sequences in red bean acid phosphatase, leading to the assumption that the shorter subunit of lupin acid phosphatase is generated by the deletion of the N-terminal part of the longer subunit. Carbohydrate moiety was found to be identical in both subunits. Oligosaccharide chains released by hydrazinolysis from the both subunits were fluorescently labeled and separated by HPLC. The structure of oligosaccharides was elucidated by exoglycosidase sequencing. Seventeen percent of isolated glycans were found to be of the high-mannose type, while the rest belonged to plant complex-type structures. Most of the complex glycans were fucosylated and xylosylated; some were fucosylated or xylosylated only.

The sugar moiety of Tamm-Horsfall protein is affected by the carbohydrate-deficient glycoprotein type I syndrome. A case study.

As the sugar moiety of Tamm-Horsfall protein (THP) is affected by many pathological conditions, the aim of this study was to examine the influence of carbohydrate-deficient glycoprotein syndrome (CDG) on THP glycans. THP was isolated from urine of one patient with CDG type I and N-glycan profiling, analysis of monosaccharide content, determination of THP reactivity with specific lectins and with anti-THP antibodies were performed. THP of the CDG patient showed markedly lower amounts of all monosaccharides. Diminished amounts of lactosamine-type chains, galactose and alpha2,3 linked sialic acid were expressed in lower reactivity with PHA-L, DSA and MAA, respectively. These modifications were reflected in altered proportions of tetrasialylated and disialylated oligosaccharide chains. THP of the CDG patient reacted slightly more with anti-THP antibodies. Our results indicate that the CDG type I affects the THP sugar moiety and slightly enhances the THP immunoreactivity.

Evaluation of antiarrhythmic activity of captopril and enalaprilat in experimental cardiac arrhythmias in rabbits. Part II.

The influence of 21-day administration of captopril and enalaprilat on barium chloride and adrenaline-induced experimental arrhythmias was assessed. The experiments were performed on rabbits. Arrhythmias were evoked by two alternative arrhythmogen doses. The patterns of disturbances, their frequency and duration were evaluated on the basis of ECG examination. Antiarrhythmic properties of angiotensin converting enzyme inhibitors administered for 21 days were also compared with their effects after single administration. The results were subjected to statistic analysis. On the basis of the obtained results we were able to establish that repeated administration of enalaprilat decreases the frequency of barium chloride- and adrenaline-induced arrhythmias. Repeated administration of captopril and enalaprilat shortened the duration of adrenaline- and barium chloride-induced arrhythmias. Long-term enalaprilat administration was much more effective in preventing arrhythmias than its single dose, it also proved to be more efficient than either single or repeated administration of captopril.

Composition of the sugar moiety of Tamm-Horsfall protein in patients with urinary diseases.

The sugar moiety of Tamm-Horsfall protein (THP) is altered by pathological conditions. The aim of this study was to investigate the composition of THP glycans in urinary diseases. THP was isolated from the urine of patients with urinary tract infection (group A), glomerulonephritis or interstitial nephritis (group B), and Bartter's syndrome (BS) (group C). Monosaccharides, N-glycan profile, THP reactivity with specific lectins and some other proteins were analyzed. THP of patients from groups A, B, and C showed lower amounts of N-acetylgalactosamine (P<0.05, P<0.005, and P<0.05, respectively) than controls; this was reflected in lower reactivity with Phaseolus vulgaris lectin (P<0.005, P<0.05, and P<0.005). Reduced amounts of N-acetylglucosamine were noticed in groups A (P<0. 05) and B (P<0.05). In group A lower amounts of galactose and alpha2, 6-linked sialic acid, as determined by reactivity with Datura stramonium lectin (P<0.005) and Sambucus nigra lectin (P<0.005), were observed. In patients with BS there was a shift from tetrasialylated glycans towards less-sialylated chains. We found also that THP of all patients binds more strongly to IgG(1) (P<0.005, for all patient groups). Our results indicate that the urinary diseases examined affect the THP sugar moiety and the binding of THP to IgG(1).

Alterations of the sugar moiety of Tamm-Horsfall protein in children with malignancies of lymphoid cells.

The aim of this study was to examine whether the sugar moiety of Tamm-Horsfall protein (THP) is affected by pathological processes caused by acute lymphoblastic leukemia (ALL) or non-Hodgkin's lymphoma (NHL). The carbohydrate part of THP was studied by monosaccharide analysis, N-glycan profiling, and reactivity with specific lectins. Our results have shown that THP of ALL or NHL patients, in comparison with healthy subjects, have modified sugar chains. This is expressed in lower contents of N-acetylgalactosamine, alpha2,6-linked sialic acid and alpha1,6-linked fucose as well as in altered proportions of various N-glycans. We have shown that pathological processes also affect the carbohydrate unit of human immunoglobulin G (IgG) isolated from sera of ALL or NHL patients. As compared with healthy subjects, in IgG of the patient group, lower amounts of sialic acid and fucose were observed. These changes did not influence the biological properties of THP as judged by their unaltered ability to bind with interleukin-1alpha, alpha1-acid glycoprotein, serum albumin, transferrin, IgG1 and the light and heavy chains of IgG. Neither the in vivo alterations of IgG caused by ALL or NHL nor its in vitro modifications influence the interaction between IgG and THP.

Evaluation of antiarrhythmic activity of captopril and enalaprilat in experimental cardiac arrhythmias in rabbits. Part I.

The present study was undertaken to investigate the influence of captopril and enalaprilat--two different angiotensin converting enzyme (ACE) inhibitors--on experimental cardiac arrhythmias induced by barium chloride, ouabain or adrenaline. The research was carried out on rabbits. Captopril and enalaprilat were administered only once. Arrhythmias were evoked by three different doses of arrhythmogens: ED50 and two higher ones causing rhythm disturbances in 80-100% rabbits. The patterns of arrhythmias as well as their frequency and duration were evaluated on the basis of ECG examination. The results were subjected to statistic analysis. As a result of our research, we were able to establish that ACE inhibitors, when administered at a single dose, did not influence the patterns but changed the frequency and duration of barium chloride-, adrenaline- or ouabain-induced arrhythmias. Captopril and enalaprilat administered at a single dose decreased the frequency of barium chloride- or ouabain- but not adrenaline-induced arrhythmia. A single administration of captopril and enalaprilat limit the duration of arrhythmias caused by barium chloride, ouabain or adrenaline.

The effect of ethanol on arterial blood pressure, central venous pressure and ECG in rabbits treated with the single or multiple dose of amitriptyline or imipramine.

Amitriptyline and imipramine given in the single dose insignificantly depressed the arterial blood pressure but significantly elevated the central venous pressure, prolonged the PQ interval and widened the QRS complex. After a prolonged daily treatment, the subsequent 21st dose of either antidepressant significantly depressed the arterial blood pressure; amitriptyline also depressed the central venous pressure. When given chronically, amitriptyline induced rhythm disturbances and the flattening of T-wave, while imipramine caused the widening of the QRS complex, block of the left bundle branch, changes in the T-wave amplitude, elevation in the ST interval. An intravenous infusion of ethanol potentiated those changes. The impairment of atrioventricular conduction occurred more frequently after administration of ethanol jointly with amitriptyline than with imipramine. Physostigmine salicylate elevated the depressed arterial blood pressure, aggravated the impairment of conduction and potentiated rhythm disturbances caused by the interaction of ethanol with antidepressants. In the above interactions with ethanol imipramine was less toxic than amitriptyline.