Mutational analysis of basic residues in the N-terminus of the rRNA:m6A methyltransferase ErmC'.

Erm methyltransferases mediate the resistance to the macrolide-lincosamide-streptogramin B antibiotics via dimethylation of a specific adenine residue in 23S rRNA. The role of positively charged N-terminal residues of the ErmC' methyltransferase in RNA binding and/or catalysis was determined. Mutational analysis of amino acids K4 and K7 was performed and the mutants were characterized in in vivo and in vitro experiments. The K4 and K7 residues were suggested not to be essential for the enzyme activity but to provide a considerable support for the catalytic step of the reaction, probably by maintaining the optimum conformation of the transition state through interactions with the phosphate backbone of RNA.

Galectin-3 decreases in mice exposed to immobilization stress.

Using M3/38 monoclonal antibody we have analyzed effects of immobilization stress on the expression of galectin-3 in liver, spleen and peritoneal macrophages from adult RFM and C3H mice, as well as from aged C3H mice (total of 22 animals). In all analyzed tissues, immobilization stress caused a significant decrease in the expression of galectin-3, ranging from 14% to 47%. The decrease of galectin-3 was observed in both strains of mice, as well as in old animals. Moreover, the same range of decrease (approximately 50%) was observed when cells grown in vitro were exposed to subculturing, or heat-shock. Although the biological significance of this effect is not known, it is very interesting that a single episode of immobilization stress was sufficient to cause a significant decrease in galectin-3, implicating that this beta-galactoside-binding lectin might be involved in the physiological response to psychological stress.

Expression of galectin-3 in cells exposed to stress-roles of jun and NF-kappaB.

BACKGROUND/AIMS: Galectin-3 is an interesting intracellular lectin that appears to be involved in numerous physiological processes. We have analyzed expression of galectin-3 in glioblastoma cells exposed to heat-shock, alkylating agents, UV-C radiation and subculturing (trypsinization). METHODS: Protein levels of galectin-3 were measured by western-blot analysis using M3/38 monoclonal antibody. The involvement of transcription factors NF-kappaB and Jun in the induction of galectin-3 was addressed using specific inhibitor of NF-kappaB (zL(3)-vs) and antisense-jun oligonucleotides. RESULTS: Exposure of cells to heat-shock or subculturing (trypsinization) decreased levels of galectin-3 to approximately 50%. Alkylating damage and UV-C irradiation caused an increase in the expression of galectin-3. Both inhibition of Jun by antisense-jun oligonucleotides, and inhibition of NF-kappaB by specific proteasomal inhibitor attenuated the induction of galectin-3 by UV-light, but with somewhat different kinetics. CONCLUSIONS: We have found that different forms of cellular stress have different effects on the expression of galectin-3. Heat-shock and subculturing decrease, while alkylating agents and UV-light increase galectin 3. NF-kappaB and Jun were shown to be involved in the induction of galectin-3 by UV-light, which is a first demonstration that these transcriptional factors are involved in the regulation of galectin-3 expression.

Transfer to in vitro conditions influences expression and intracellular distribution of galectin-3 in murine peritoneal macrophages.

Galectin-3 is a beta-galactoside-binding lectin that has been implicated in numerous physiological processes, including mRNA splicing, cell differentiation, tumor metastasis and the stress response. We have studied effects of transfer of resident murine peritoneal macrophages to in vitro conditions on galectin-3 in different cell compartments. Galectin-3 was purified by immunoprecipitation with rat monoclonal antibody M3/38, and analyzed by immunoblotting using the same antibody. Transfer to in vitro conditions nearly doubled the total amount of galectin-3 in cells, and caused significant alterations in its intracellular distribution, indicating that galectin-3 is involved in the adaptation of peritoneal macrophages to in vitro conditions.

Purification and MALDI-MS characterization of stressin, a stress-associated glycoprotein.

Glycoconjugates have a whole spectrum of biological roles, from those that appear trivial to those that are crucial. Results accumulated in the past years indicate they might also play an important role in the response to stress, a complex physiological response of the human organism to various threats. We have recently identified stressin, a human serum glycoprotein, which was found to be increased under stress conditions. Here we report the purification of stressin from sera of professional soldiers and partial characterization of its protein and carbohydrate parts using lectin blotting and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Stressin was purified using a combination of ammonium sulfate precipitation, ion exchange chromatography, preparative gel electrophoresis and reverse-phase HPLC. It was found to be a highly glycosylated protein. Only 21.9 kDa (out of 36.7 kDa) was the protein part, whereas the remaining 40% of the mass originated from N-linked oligosaccharides. The carbohydrate part contained 12 sialic acids moieties, nearly 90% of which were lost due to post-source decay in the field-free tube. Tryptic fragments were produced from glycosylated and deglycosylated stressin, separated by reverse-phase HPLC and their exact molecular masses were determined using MALDI-MS. Comparison with tryptic maps of other proteins in computer databases indicated that stressin does not correspond to any already described protein.

Glycosylation of stress glycoprotein GP62 in cells exposed to heat-shock and subculturing.

GP62 is a member of the stress glycoprotein family that was proposed to have a chaperone-like function in the heat-shock response. Using lectin blotting we have studied glycosylation of GP62 and determined that in addition to heat-shock, even simple subculturing of cells is a sufficient stimulus to provoke induction of GP62. Interestingly, both kinetics of induction and glycosylation of GP62 induced by subculturing were different than when GP62 was induced by heat-shock. While GP62 induced by heat-shock was recognized by SNA, DSA and PHA-E lectins, and not by BSA I, Con A, RCA I, SJA, UEA I, VVA, and WGA lectins, GP62 induced by subculturing was also recognized by RCA I and WGA lectins.

Fucosylation of IgG heavy chains is increased in rheumatoid arthritis.

OBJECTIVES: Glycosylation of IgG was suggested to be important in the etiology of rheumatoid diseases. Most studies addressed the amount of galactose, but recently we showed that fucose is highly increased in the juvenile chronic arthritis. The objective of this study was to determine fucosylation of IgG heavy chains in patients with rheumatoid arthritis (RA). DESIGN AND METHODS: IgG was purified from sera of 29 RA patients and 17 matching controls using ammonium sulfate precipitation and ion exchange. Heavy chains were separated by denaturing polyacrylamide gel electrophoresis and their fucosylation analysed using fucose-specific UEA I lectin. RESULTS: Fucose was found to be approximately 40% increased in RA patients with very high statistical significance (p = 0.00095). CONCLUSIONS: Fucose on IgG heavy chains is significantly increased in patients with rheumatoid arthritis.

Stressin and natural killer cell activity in professional soldiers.

Chronic stress causes multiple biochemical and physiological changes in the human organism. Recently we have identified stressin, a human serum glycoprotein that was significantly increased in sera of prisoners released from Serbian concentration camps. To eliminate malnutrition and maltreatment as possible causes for the increased stressin concentration, we have analyzed stressin in sera of 40 professional soldiers after involvement in major military activity and compared it to stressin in sera of 20 control individuals. As expected, the sera of professional soldiers contained more than 2.2 times higher concentrations of stressin than control sera. It is interesting that, contrary to expectations, the natural killer cell activity of professional soldiers was normal or even increased. We hypothesize that this might be an effect of winning the war that could have, at least temporarily, erased the immunosuppressive effects of stress.

Fucosylation and galactosylation of IgG heavy chains differ between acute and remission phases of juvenile chronic arthritis.

Oligosaccharide structures are attached to nearly all membrane and serum proteins, and their composition changes significantly in many diseases. We have analysed glycosylation of IgG heavy chains in 34 patients with juvenile chronic arthritis and 13 control individuals. IgG was purified from 0.7 ml of serum, separated by electrophoresis and transferred on to polyvinylidene difluoride (PVDF) membrane. Ricinus communis agglutinin (RCA I) and Bandeirea simplicifolia (BSA II) and Ulex europaeus (UEA I) lectins were used to measure galactose, N-acetylglucosamine and fucose, respectively. While there was no significant difference in average levels of galactose and N-acetylglucosamine, patients with juvenile chronic arthritis had 2.4 times more fucose attached to IgG heavy chains than control individuals. A different picture emerged when patients were divided into those with acute disease and those in remission. Patients in whom juvenile chronic arthritis was currently active had significantly lower levels of galactose than those in remission, in whom galactose levels were comparable to the control group. Fucose levels in both groups of patients were significantly higher than in the control group. These results show that whereas de-galactosylation is a good test to detect and measure the activity of juvenile chronic arthritis, increased fucosylation is a much more reliable measure for diagnosis of the disease itself.

Changes of glycoprotein patterns in sera of humans under stress.

Stress exhibits adverse effects on many vital processes in which glycoproteins play a significant role(e.g. cell-cell/matrix interactions, immune response, neoplastic growth, implantation, prenatal development), yet only scarce attention has been directed towards studying stress induced changes in glycoprotein patterns. Using SDS-electrophoresis, blotting and digoxigenin-labelled lectins (Sambucus nigra agglutinin, Galanthus nivalis agglutinin, Datura stramonium agglutinin, Maackia amurensis agglutinin and peanut (Arachis hypogaea) agglutinin),sera were analysed from 30 individuals chosen randomly from a severely stressed population of 309 male volunteers with no specific medical symptoms. Significant changes were found in glycoprotein pattern and content, compared with healthy controls of matching age and sex. Occasionally minor non-specific deviations from the reference values for several analytes (haemoglobin, glucose, bilirubin and alanine aminotransferase) were detected in the tested group, but glycoprotein GP4S (Mr = 45 000), detected by Datura stramonium agglutinin and Sambucus nigra agglutinin, appeared in 96.7% of samples of the stressed population. The same population also revealed an approximately 500-fold increase of GP37 in comparison with the control sera. These results suggest that stress, as a non-specific syndrome, induces specific biochemical changes, which could be of diagnostic relevance as risk makers before any more serious symptoms of stress-related consequences have developed.

The photoreactive carbohydrate probe, a novel method for detection of lectins.

One of the main difficulties in the research of lectins is the absence of an adequate technique for their specific and routine detection. Here, we present a photoreactive carbohydrate-probe which could help to overcome this problem. The probe was comopsed by joining four segments: (i) a carbohydrate moiety, (ii) the digoxigenin tag, (iii) the photoreactive cross-linker and (iv) the lysyl-lysine backbone. After incubation with lectins in the dark, the probe can be activated and cross-linked to the lectins by illumination. The result is a lectin with covalently incorporated digoxigenin tag. Such a labelled lectin can be easily identified using anti-digoxigenin antibodies in a Western blot technique. This method is of high specificity and sensitivity and enables direct detection of lectins in complex mixtures, even whole cell homogenates.

Glycoprotein and ganglioside changes in human trophoblasts after exposure to pulsed Doppler ultrasound.

Changes in glycoprotein and ganglioside composition in human trophoblasts (eighth week of gestation) after in vitro exposure to pulsed Doppler ultrasound (pulse duration 1.22 microseconds; repetition frequency 11.1 kHz; center frequency 4 MHz; ISPPA = 175.5 W/cm2; ISPTA = 0.59 W/cm2) were investigated. Evacuated trophoblasts were divided in two halves and insonated for 10 min on top of a 6-cm layer of 5% gelatin in 50-mL tubes (Falcon) at 37 degrees C. One half of each trophoblast was sham insonated and served as an internal control. After insonation trophoblasts were maintained at 37 degrees C for 24 h. Glycoproteins were detected using alpha-D-mannose specific lectins from Galanthus nivalis and Narcissus pseudonarcissus. A decrease in the expression of mannose containing glycoprotein mgp47 and an increase in expression of mgp54 were observed. Ganglioside composition was also significantly altered. Concentrations of two gangliosides migrating similarly to GM2, and one similarly to GQ1, decreased by more than 75%. At the same time, concentrations of one ganglioside migrating similarly to GM3, and two other unidentified gangliosides increased two- to fourfold.

Identification and purification of a stress associated nuclear carbohydrate binding protein (M(r) 33,000) from rat liver by application of a new photoreactive carbohydrate probe.

A photoreactive alpha-D-glucose probe has been designed for the specific detection of carbohydrate binding proteins (CBPs). The probe consists of four parts: (i) an alpha-D-glucose moiety; (ii) the digoxigenin tag; (iii) the photoreactive cross-linker; and (iv) the lysyl-lysine backbone. After incubation with lectins in the dark, the probe is activated and cross-linked to the CBPs after being treated by several flashes. Using this method we have identified a new alpha-D-glucose CBP of M(r) = 33,000, termed CBP33, in the nuclei of rats exposed to transient immobilization stress. Monoclonal antibodies were raised against the partially purified protein and subsequently used to enrich CBP33. It was purified (> 2400-fold) to apparent homogeneity from a 0.6 M nuclear salt extract by two subsequent affinity chromatography steps (antibody-affinity as well as alpha-D-glucose affinity column).

Sperm motility and kinetics of dynein ATPase in astheno- and normozoospermic samples after stimulation with adenosine and its analogues.

We tested the effects of adenosine and 2-deoxyadenosine on the activation of human spermatozoa. In the asthenozoospermic group of patients adenosine produces an increase in sperm motility from 33.3 +/- 2.1% to 42.1 +/- 3.4%, progressive motility from 22.5 +/- 1.3% to 28.6 +/- 1.7% and forward progression rating from 2.1 +/- 0.2% to 2.8 +/- 0.1%. 2-Deoxyadenosine stimulated asthenozoospermic samples to a greater degree than adenosine. Sperm motility rose to 48.9 +/- 3.4%, progressive motility to 32.1 +/- 3.4% and forward progression rating to 3.0 +/- 0.1% following stimulation with 2-deoxy-adenosine. The kinetic parameters and basic characteristics of dynein ATPase were determined. The maximum activity of dynein ATPase, Vmax, was significantly different (P < 0.001) for asthenozoospermic and normozoospermic samples: 6.46 +/- 2.1 nmol Pi/mg/min and 16.99 +/- 3.7 nmol Pi/mg/min respectively. However, the enzyme affinity for ATP was not different. Stimulation of asthenozoospermic samples with adenosine and 2-deoxyadenosine caused an increase of Vmax (70-90% and 90-110% respectively) and no significant change in KM was observed. In order to block the nucleoside transporter and to eliminate the action of adenosine inside the cell, dipyridamole was used but the effects of adenosine were not neutralized. 5'-(N-ethylcarboxy-amido)-adenosine showed effects similar to those of adenosine, even when applied in 1 microM concentration. These results indicate that adenosine and its analogues stimulate sperm motility and activity of dynein ATPase, most probably via A2 receptors.

A non-radioactive, sensitive method for the detection of DNA fragmentation in apoptotic cells (rat pheochromocytoma PC12 and rat cortical cells).

A new method is described which is suitable for reliably analysing apoptotic fragmentation in small amounts of DNA. After isolation, DNA was labelled with biotin-4-dUTP using Klenow polymerase. Then DNA was size-separated by agarose gel electrophoresis, blot transferred and subsequently visualized by the streptavidin alkaline phosphatase-BCIP/NBT procedure. This non-radioactive method was used to detect apoptotic DNA in rat pheochromocytoma PC12 cells, treated with tributyltin (1 nM). While only 30 ng of DNA is required for analysis of apoptotic DNA using the new blot technique, 100-fold more material is needed to identify the fragmentation of DNA after separation by agarose gel electrophoresis and direct staining with ethidium bromide. In a further set of experiments, rat cortical cells were incubated with human immunodeficiency virus type 1 viral glycoprotein of M(r) of 120 kDa (gp120) to induce apoptosis. More than 0.3 ng of gp 120/ml are required to detect apoptotic DNA by the direct procedure; only 0.1 ng gp120/ml or less were sufficient to document clear DNA fragmentation using the non-radioactive blotting technique described here. These results demonstrate that the new procedure can be used to analyse very small amounts of apoptotic DNA and shows that gp120-induced apoptosis can be measured at low concentrations of the viral protein.

An assessment of some of the methods available for the determination of molecular weights of proteins as applied to aspartate aminotransferase from pig heart.

The isopotential specific volume of cytoplasmic aspartate aminotransferase from pig heart was found to be 0.763 ml g-1 whereas the value of the apparent specific volume obtained by summation of contributions from each type of amino acid in the protein is 0.735 ml g-1. Use of the experimentally determined isopotential specific volume largely abolishes the discrepancy between a previously reported value of the molecular weight of the native (dimeric) enzyme and that of the enzyme subunit obtained from its primary structure (46300). A new non-empirical method based on quantitative N-terminal analysis involving radioisotope dilution is described for the determination of subunit molecular weight of proteins. The method is capable of considerable accuracy and sensitivity. Some of the methods available for the determination of molecular weights ans subunit compositions of proteins are discussed.

Calorimetric and potentiometric characterization of the ionization behavior of ribonuclease A and its complex with 3'-cytosine monophosphate.

The proton association behavior of ribonuclease A and its complex with 3'-cytosine monophosphate has been thermodynamically characterized in the pH range 4--8 at 25 degrees, mu = 0.05. Calorimetric and potentiometric titration data have been used to estimate the apparent pK values and enthalpy values for protonation of the four histidine residues of the protein, deltaHp. In the free enzyme the pK values were deduced to be 5.0, 5.8, 6.6, and 6.7 and deltaHp deduced to be -6.5, -6.5, -6.5, and -24 kcal/mol for residues 119, 12, 105, and 48, respectively. For the nucleotide-enzyme complex it was concluded that the apparent pK values of residues 119, 12, and 48 increased to an average value of about 7.2, the deltaHp values remaining constant for all histidine groups except 48. It was also concluded that only the dianionic phosphate form of the nucleotide inhibitor is bound to the enzyme in this pH range. These results are consistent with a thermodynamic model for the binding reaction in which inhibitor-enzyme association is coupled to the ionization of three imidazole residues (12, 119, and 48) and the interaction between the negative phosphate moiety of the inhibitor and the positively charged residues 12 and 119 is purely electrostatic. However, the "interaction" with residue 48 probably involves a conformational rearrangement of the macromolecule.

The pH dependence of the thermodynamics of the interaction of 3'-cytidine monophosphate with ribonuclease A.

The apparent free energy (deltaGapp) and enthalpy changes (deltaHB) associated with the interaction of 3'-cytosine monophosphate (3'-CMP) and ribonuclease A (RNase) are reported for the pH range 4--9, T = 25 degrees, mu = 0.05. The pH dependence of deltaGapp and deltaHB has been interpreted in terms of coupled ionization of histidine residues 12, 48, and 119, assuming that only the dianionic form of the inhibitor is bound. The results of this analysis are consistent with the calorimetric and potentiometric titration results for the free enzyme and its 3'-CMP complex reported in the previous paper (M. Flogel and R. L. Biltonen ((1975), Biochemistry, preceding paper in this issue). This analysis allows the calculation of the thermodynamic quantities associated with hypothetical but clearly defined reactions (e.g., the reaction of the dianionic inhibitor with the completely protonated enzyme). It is concluded that the primary thermodynamic driving forces for the reaction are van der Waals interactions between the riboside moiety and the protein fabric and electrostatic interaction between the negatively charged phosphate group of the inhibitor and the positively charged histidine residues at the binding locus. It is also suggested that the binding reaction is weakly coupled (approximately to 0.5 kcal/mol) with a conformational change of the protein associated with protonation of residue 48. These results are consistent with the model originally proposed by G. G. Hammes ((1968), Adv. Protein Chem. 23, 1) and lend additional quantitative detail to the nature of the reaction.