Analysis of the microheterogeneity of the glycan chain of rat transferrin

To investigate the microheterogeneity of the glycan chain of rat transferrin, either the protein moiety was labeled with 125I or the sialyl residues with 3H. The molecule was then subjected to Con A chromatography. Three components were obtained. Each was enzymatically desialylated and sialyl/protein molar ratios were calculated. The native protein as well as the 3 components were also subjected to isoelectric focusing. The results indicated that rat transferrin may have 3 types of glycan chain: The major type (60%) corresponds to a molecular species with triantennary branching, while 30% consists of molecules with biantennary and 10% with tetraantennary branching. The last species has not been previously described.     

Carbohydrate structures of the third component of rat complement. Presence of both high-mannose and complex type oligosaccharide chains.

The kinetics of pyruvate kinase from Saccharomyces cerevisiae were studied in assays at pH 6.2 at 25 degrees C as a function of the concentrations of the substrates ADP, phosphoenolpyruvate and Mg2+ and the concentration of the effector fructose 1,6-bisphosphate. The enzyme was activated by 100 mM-K+ and 32 mM-NH4+ throughout. It was found that an increase in the fructose bisphosphate concentration from 24 microM to 1.2 mM brings about a transition from a sigmoidal to a non-inflected form in the relationships v = f([phosphoenolpyruvate]) and v = f([Mg2+]) together with a large increase in the affinity of these substrates for the enzyme. The binding behaviour of ADP is barely affected by the same change in effector concentration. By contrast, increase in fructose bisphosphate concentration below 24 microM increases the affinity of the enzyme for all its substrates and the sigmoidicity of the corresponding velocity-substrate-concentration relationships. As a result of this change in behaviour it has been found impossible to represent all the data by the exponential model for a regulatory enzyme, and it is suggested (supported by comparisons with previous work) that the failure may reflect a secondary action of the effector upon the enzyme.     

Determination of glycan structures and molecular masses of the glycovariants of serum transferrin from a patient with carbohydrate deficient syndrome type II

Serum transferrin from a child with carbohydrate deficient syndrome type II was isolated by immunoaffinity chromatography and separated into minor and major fractions by fast protein liquid chromatography. The structure of the glycans released from the major fraction by hydrazinolysis was established by application of methanolysis and 1H-NMR spectroscopy. The results led to the identification of an N-acetyllactosamininic type monosialylated, monoantennary Man(1-3) linked glycan. By electrospray-mass spectrometry analysis, the whole serum transferrin was separated into at least seven species (I to VII) with molecular masses ranging from 77 958 to 79 130 Da. On the basis of a polypeptide chain molecular mass of 75 143 Da, it was calculated that the major transferrin species III (78 247 Da) contains two monosialylated monoantennary glycans. The molecular mass of transferrin species V and VI (78 678 and 78 971 Da) suggests that one of their two glycans contains an additional N-acetyllactosamine and a sialylated N-acetyllactosamine units, respectively. Transferrin species I and V were found to correspond to the desialylated forms of species III and VI. The abnormal glycan structures can be explained by a defect in the N-acetylglucosaminyltransferase II activity [Charuk et al. (1995) Eur J Biochem 230: 797-805].     

GlycoSuiteDB: a new curated relational database of glycoprotein glycan structures and their biological sources

GlycoSuiteDB is a relational database that curates information from the scientific literature on glyco-protein derived glycan structures, their biological sources, the references in which the glycan was described and the methods used to determine the glycan structure. To date, the database includes most published O:-linked oligosaccharides from the last 50 years and most N:-linked oligosaccharides that were published in the 1990s. For each structure, information is available concerning the glycan type, linkage and anomeric configuration, mass and composition. Detailed information is also provided on native and recombinant sources, including tissue and/or cell type, cell line, strain and disease state. Where known, the proteins to which the glycan structures are attached are reported, and cross-references to the SWISS-PROT/TrEMBL protein sequence databases are given if applicable. The GlycoSuiteDB annotations include literature references which are linked to PubMed, and detailed information on the methods used to determine each glycan structure are noted to help the user assess the quality of the structural assignment. GlycoSuiteDB has a user-friendly web interface which allows the researcher to query the database using mono-isotopic or average mass, monosaccharide composition, glycosylation linkages (e.g. N:- or O:-linked), reducing terminal sugar, attached protein, taxonomy, tissue or cell type and GlycoSuiteDB accession number. Advanced queries using combinations of these parameters are also possible. GlycoSuiteDB can be accessed on the web at http://www.glycosuite.com.     

Molecular dynamics simulations of a monofucosylated biantennary glycan of the N-acetyllactosamine type: the human lactotransferrin glycan.

Molecular dynamics simulations were carried out to explore the conformational flexibility of the antennae of N-linked glycans. They were performed over 200 ps in vacuo on the complete disialylated monofucosylated biantennary glycan of the N-acetyllactosaminic type. Starting from a bird-conformation, the 3-D-structure evolved through 9 successive transitional states to a new, compact and energetically favorable conformation which had never been previously described. In this conformation, both antennae are organized in two coplanar loops rolled in a contrary direction and oriented perpendicularly to the plane of the di-N-acetyl chitobiose residue leading to a 'lobster conformation'. All the glycosidic linkages of the disialylated monofucosylated biantennary glycan, except the Fuc(alpha 1-6)GlcNAc(beta 1-), were modified by a phase transition. Particularly, the Man(beta 1-4) GlcNAc(beta 1-) linkage, which was previously described by NMR and X-ray diffraction as a rigid one, was involved in numerous conformational changes during 83 ps, even before the first transition phase. The freedom of mobility of the torsional angles of the Man(alpha 1-6)Man(beta 1-) linkage was limited, under these simulation conditions, to the angle psi which took three values: 30 degrees, 90 degrees and 180 degrees. Moreover, from 150 ps up to the end of the simulation, the value of the torsional angle omega of the NeuAc(alpha 2-6)Gal(beta 1-) linkage of the alpha-1,6-antenna continuously swung between 60 degrees and -60 degrees. Finally, we observed that the values of the torsional angles of the three linkages: NeuAc(alpha 2-6)Gal(beta 1-), Gal(beta 1-4)GlcNAc(beta 1-) and GlcNAc(beta 1-2)Man(beta 1-) of each of the two antennae were different, demonstrating their asymmetric conformation.     

Enzymological characterization of a putative canine analogue of primary hyperoxaluria type 1.

This paper concerns an enzymological investigation into a putative canine analogue of the human autosomal recessive disease primary hyperoxaluria type 1 (alanine:glyoxylate/serine:pyruvate aminotransferase deficiency). The liver and kidney activities of alanine:glyoxylate aminotransferase and serine:pyruvate aminotransferase in two Tibetan Spaniel pups with familial oxalate nephropathy were markedly reduced when compared with a variety of controls. There were no obvious deficiencies in a number of other enzymes including D-glycerate dehydrogenase/glyoxylate reductase which have been shown previously to be deficient in primary hyperoxaluria type 2. Immunoblotting of liver and kidney homogenates from oxalotic dogs also demonstrated a severe deficiency of immunoreactive alanine:glyoxylate aminotransferase. The developmental expression of alanine:glyoxylate/serine:pyruvate aminotransferase was studied in the livers and kidneys of control dogs. In the liver, enzyme activity and immunoreactive protein were virtually undetectable at 1 day old, but then increased to reach a plateau between 4 and 12 weeks. During this period the activity was similar to that found in normal human liver. The enzyme activities and the levels of immunoreactive protein in the kidneys were more erratic, but they appeared to increase up to 8 weeks and then decrease, so that by 36 weeks the levels were similar to those found at 1 day. The data presented in this paper suggest that these oxalotic dogs have a genetic condition that is analogous, at least enzymologically, to the human disease primary hyperoxaluria type 1.     

A new member of the troponin C superfamily: Comparison of the primary structures of rat oncomodulin and rat parvalbumin

When the amino-acid sequence of the 108-residue, rat tumour calcium-binding protein, oncomodulin, was aligned with that of rat muscle parvalbumin, 55 homologous positions were found, with an additional 33 single base-pair substitutions. This extensive homology, with virtual identity of the calcium-binding domains, signalled oncomodulin to be a member of the troponin C superfamily. The presence of Cys-18 and Phe-66 in oncomodulin, plus its isoelectric point of 3.9, suggest that this tumour protein is a beta-parvalbumin, rather than a muscle alpha-parvalbumin.     

GlycoSuiteDB: a curated relational database of glycoprotein glycan structures and their biological sources. 2003 update

GlycoSuiteDB is an annotated and curated relational database of glycan structures reported in the literature. It contains information on the glycan type, core type, linkages and anomeric configurations, mass, composition and the analytical methods used by the researchers to determine the glycan structure. Native and recombinant sources are detailed, including species, tissue and/or cell type, cell line, strain, life stage, disease, and if known the protein to which the glycan structures are attached. There are links to SWISS-PROT/TrEMBL and PubMed where applicable. Recent developments include the implementation of searching by 2D structure and substructure, disease and reference. The database is updated twice a year, and now contains over 7650 entries. Access to GlycoSuiteDB is available at http://www.glycosuite.com.     

Isolation and characterization of rat primary lung cells

Summary Lung cell culture may be useful as anin vitro alternative to study the susceptibility of the lung to various toxic agents. Lungs from female Wistar rats were enzymatically digested by recirculating perfusion through the pulmonary artery with a sequence of solutions containing deoxyribonuclease, chymopapain, pronase, collagenase, and elastase. Lung tissue was microdissected and resuspended and the cells obtained were washed by centrifugation. By this isolation method, 2×10⁸ cells per rat lung were obtained with an average viability of 97%. Lung cells cultured in medium containing antibiotics and serum maintained a viability of >70% for 5 d. Rat primary lung cells were exposed to various toxic agents and their viability was assessed by formazan production capacity after 18 h of incubation. Compared to rat and mouse hepatocyte cultures (EC⁵⁰=5.8 mM), rat primary lung cells were much more susceptible to hydrogen peroxide (EC₅₀=0.6 mM). All cell types were equally sensitive to the more potent toxicanttert-butylhydroperoxide (EC₅₀=0.1 mM). Paraquat was more toxic to lung cells (EC₅₀=0.03 mM) than to rat (EC₅₀=2.8 mM) and mouse (EC₅₀=0.2 mM) hepatocytes. In contrast, rat lung cells were less sensitive to sodium nitroprusside (EC₅₀=2.6 mM) compared to rat (EC₅₀=0.2 mM) and mouse (EC₅₀=0.03 mM) hepatocytes. Nitrofurantoin and menadione (at EC₅₀=0.04 mM and 0.006 mM, respectively) were more toxic to rat lung and liver cells than to murine hepatocytes (EC₅₀=0.2 mM and 0.04 mM, respectively). Our findings demonstrate the applicability of this rat primary lung cell culture for studying the effects of lung toxicants. Parts of the study had been presented orally at the meeting of the German Society of Toxicology and Pharmacology in Mainz (FRG), March 15–17, 1994.     

Molecular identification and characterization of a new type of bovine enterovirus

Bovine enteroviruses belong to the family Picornaviridae. Little is known about their pathogenic potential; however, they cause asymptomatic infections in cattle and are excreted in feces. In the present study, viruses isolated from environmental samples were sequenced. According to phylogenetic analyses and standard picornavirus nomenclature, these isolates constitute a new type of bovine enterovirus serogroup A.     

Purification of rat liver arylsulfatase A and its microheterogeneity assayed by crossed affinity-immunoelectrophoresis.

Arylsulfatase A (arylsulfatase sulfohydrolase) EC 3.1.6.1 was purified from rat liver by a procedure consisting of differential centrifugation, Con A-Sepharose and Blue Sepharose chromatography, PBE 94 chromatofocusing, DEAE-cellulose and gel filtration chromatography followed by preparative electrophoresis. A molecular mass of 132,000 was estimated by gradient PAGE. Particular proteins were detected by immunoelectrophoresis. Isoelectric focusing combined with immunoelectrophoresis gave two peaks of arylsulfatase A, with isoelectric points of pH 3.9 and 4.5. Microheterogeneity of rat liver arylsulfatase A was studied by affinity immunoelectrophoresis with 9 different lectins. The presence of concanavalin A-, Lens culinaris agglutinin-, Lotus tetragonolobus agglutinin- and wheat germ agglutinin-reactive forms permitted assessment of the types of carbohydrate moieties in arylsulfatase A.     

Molecular modeling of a disialylated monofucosylated biantennary glycan of theN-acetyllactosamine type

The conformations of a disialylated monofucosylated biantennary glycan of theN-acetyllactosamine type were analysed using the Tripos 5.3 force field from the Sybyl software currently used for molecular modelling. The conformation of each glycosidic linkage was calculated when included in oligosaccharide structures of up to 5 units and the influence of the glycosidic environment on the overall structure was measured. The study clearly shows that the conformation of a branched glycan cannot result from the simple addition of the different low energy conformers of each of the glycosidic linkages constituting the glycan structure. The asymmetrical conformation of the two antennae was demonstrated. The lowest energy conformations of the overall glycan structure were built and classified into 5 main models: the Y, T, bird and broken wing conformations already described and a new one called the back folded wing conformation.