K.V. Barinova

Sperm-specific glyceraldehyde-3-phosphate dehydrogenase is stabilized by additional proline residues and an interdomain salt bridge.

Sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDS) exhibits enhanced stability compared to the somatic isoenzyme (GAPD). A comparative analysis of the structures of these isoenzymes revealed characteristic features, which could be important for the stability of GAPDS: six specific proline residues and three buried salt bridges. To evaluate the impact of these structural elements into the stability of this isoenzyme, we obtained two series of mutant GAPDS: 1) six mutants each containing a substitution of one of the specific prolines by alanine, and 2) three mutants each containing a mutation breaking one of the salt bridges. Stability of the mutants was evaluated by differential scanning calorimetry and by their resistance towards guanidine hydrochloride (GdnHCl). The most effect on thermostability was observed for the mutants P326A and P164A: the Tm values of the heat-absorption curves decreased by 6.0 and 3.3°C compared to the wild type protein, respectively. The resistance towards GdnHCl was affected most by the mutation D311N breaking the salt bridge between the catalytic and NAD(+)-binding domains: the inactivation rate constant in the presence of GdnHCl increased six-fold, and the value of GdnHCl concentration corresponding to the protein half-denaturation decreased from 1.83 to 1.35M. Besides, the mutation D311N enhanced the enzymatic activity of the protein two-fold. The results suggest that the residues P164 (β-turn), P326 (first position of α-helix), and the interdomain salt bridge D311-H124 are significant for the enhanced stability of GAPDS. The salt bridge D311-H124 enhances stability of the active site of GAPDS at the expense of the catalytic activity.

Structural basis for the NAD binding cooperativity and catalytic characteristics of sperm-specific glyceraldehyde-3-phosphate dehydrogenase

Catalytic properties of enzymes used in biotechnology can be improved by eliminating those regulatory mechanisms that are not absolutely required for their functioning. We exploited mammalian glyceraldehyde-3-phosphate dehydrogenase as a model protein and examined the structural basis of the NAD(+) cooperative binding exhibited by its homologous isoenzymes: the somatic enzyme (GAPD) and the recombinant sperm-specific enzyme (dN-GAPDS). Moreover, we obtained a mutant dN-GAPDS, which misses the cooperativity, but exhibits a twofold increase in the specific activity instead (92 and 45 μmol NADH/min per mg protein for the mutant and the wild type proteins, respectively). Such an effect was caused by the disruption of the interdomain salt bridge D311-H124, which is located close to the active site of the enzyme. The thermal stability of the mutant protein also increased compared to the wild type form (heat absorption peak values were 70.4 and 68.6 °C, respectively). We expect our findings to be of importance for the purposes of biotechnological applications.

Glycation, glycolysis, and neurodegenerative diseases: Is there any connection?

This review considers the interrelation between different types of protein glycation, glycolysis, and the development of amyloid neurodegenerative diseases. The primary focus is on the role of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase in changing the concentration of carbonyl compounds – first and foremost, glyceraldehyde-3-phosphate and methylglyoxal. It has been suggested that various modifications of the enzyme – from the oxidation of the sulfhydryl groups of the active site to glycation with sugars – can lead to its inactivation, which causes a direct increase in glyceraldehyde-3-phosphate concentration and an indirect increase in the content of other aldehydes. This “primary inactivation” of glyceraldehyde-3-phosphate dehydrogenase promotes its glycation with aldehydes, including its own substrate, and a further irreversible decrease in its activity. Such a cycle can lead to numerous consequences – from the induction of apoptosis, which is activated by modified forms of the enzyme, to glycation of amyloidogenic proteins by glycolytic aldehydes. Of particular importance during the inhibition of glyceraldehyde-3-phosphate dehydrogenase is an increase in the content of the glycating compound methylglyoxal, which is much more active than reducing sugars (glucose, fructose, and others). In addition, methylglyoxal is formed by two pathways – in the cascade of reactions during glycation and from glycolytic aldehydes. The ability of methylglyoxal to glycate proteins makes it the main participant in this protein modification. We consider the effect of glycation on the pathological transformation of amyloidogenic proteins and peptides – β-amyloid peptide, α-synuclein, and prions. Our primary focus is on the glycation of monomeric forms of these proteins with methylglyoxal, although most works are dedicated to the analysis of the presence of “advanced glycation end products” in the already formed aggregates and fibrils of amyloid proteins. In our opinion, the modification of aggregates and fibrils is secondary in nature and does not play an important role in the development of neurodegenerative diseases. The glycation of amyloid proteins with carbonyl compounds can be one of the triggers of their transformation into toxic forms. The possible role of glycation of amyloidogenic proteins in the prevention of their modification by ubiquitin and the SUMO proteins due to a disruption of their degradation is separately considered.

S-glutathionylation of glyceraldehyde-3-phosphate dehydrogenase induces formation of C150-C154 intrasubunit disulfide bond in the active site of the enzyme

BACKGROUND Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a glycolytic protein involved in numerous non-glycolytic functions. S-glutathionylated GAPDH was revealed in plant and animal tissues. The role of GAPDH S-glutathionylation is not fully understood. METHODS Rabbit muscle GAPDH was S-glutathionylated in the presence of H2O2 and reduced glutathione (GSH). The modified protein was assayed by MALDI-MS analysis, differential scanning calorimetry, dynamic light scattering, and ultracentrifugation. RESULTS Incubation of GAPDH in the presence of H2O2 together with GSH resulted in the complete inactivation of the enzyme. In contrast to irreversible oxidation of GAPDH by H2O2, this modification could be reversed in the excess of GSH or dithiothreitol. By data of MALDI-MS analysis, the modified protein contained both mixed disulfide between Cys150 and GSH and the intrasubunit disulfide bond between Cys150 and Cys154 (different subunits of tetrameric GAPDH may contain different products). S-glutathionylation results in loosening of the tertiary structure of GAPDH, decreases its affinity to NAD+ and thermal stability. CONCLUSIONS The mixed disulfide between Cys150 and GSH is an intermediate product of S-glutathionylation: its subsequent reaction with Cys154 results in the intrasubunit disulfide bond in the active site of GAPDH. The mixed disulfide and the C150-C154 disulfide bond protect GAPDH from irreversible oxidation and can be reduced in the excess of thiols. Conformational changes that were observed in S-glutathionylated GAPDH may affect interactions between GAPDH and other proteins (ligands), suggesting the role of S-glutathionylation in the redox signaling. GENERAL SIGNIFICANCE The manuscript considers one of the possible mechanisms of redox regulation of cell functions.

Sperm-Specific Glyceraldehyde-3-Phosphate Dehydrogenase–An Evolutionary Acquisition of Mammals

This review is focused on the mammalian sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDS). GAPDS plays the major role in the production of energy required for sperm cell movement and does not perform non-glycolytic functions that are characteristic of the somatic isoenzyme of glyceraldehyde-3-phosphate dehydrogenase. The GAPDS sequence is composed of 408 amino acid residues and includes an additional N-terminal region of 72 a.a. that binds the protein to the sperm tail cytoskeleton. GAPDS is present only in the sperm cells of mammals and lizards, possibly providing them with certain evolutionary advantages in reproduction. In this review, studies concerning the problems of GAPDS isolation, its catalytic properties, and its structural features are described in detail. GAPDS is much more stable compared to the somatic isoenzyme, perhaps due to the necessity of maintaining the enzyme function in the absence of protein expression. The site-directed mutagenesis approach revealed the two GAPDS-specific proline residues, as well as three salt bridges, which seem to be the basis of the increased stability of this protein. As distinct from the somatic isoenzyme, GAPDS exhibits positive cooperativity in binding of the coenzyme NAD+. The key role in transduction of structural changes induced by NAD+ is played by the salt bridge D311–H124. Disruption of this salt bridge cancels GAPDS cooperativity and twofold increases its enzymatic activity instead. The expression of GAPDS was detected in some melanoma cells as well. Its role in the development of certain pathologies, such as cancer and neurodegenerative diseases, is discussed.

Low concentrations of hydrogen peroxide activate the antioxidant defense system in human sperm cells

The effect of low concentrations of hydrogen peroxide (10–100 µM) on sperm motility and on the activity of the sperm enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDS) was investigated. Incubation of semen samples with 10 and 100 µM hydrogen peroxide increased the content of spermatozoa with progressive motility by 20 and 18%, respectively, and enhanced the activity of GAPDS in the sperm cells by 27 and 20% compared to a semen sample incubated without additions. It was also found that incubation with 10 µM hydrogen peroxide increased the content of reduced glutathione (GSH) in sperm cells by 50% on average compared to that in the control samples. It is supposed that low concentrations of hydrogen peroxide activate the pentose phosphate pathway, resulting in NADPH synthesis and the reduction of the oxidized glutathione by glutathione reductase yielding GSH. The formed GSH reduces the oxidized cysteine residues of the GAPDS active site, increasing the activity of the enzyme, which in turn enhances the content of sperm cells with progressive motility. Thus, the increase in motile spermatozoa in the presence of low concentrations of hydrogen peroxide can serve as an indicator of normal functioning of the antioxidant defense system in sperm cells.

Binding of alpha-synuclein to partially oxidized glyceraldehyde-3-phosphate dehydrogenase induces subsequent inactivation of the enzyme

According to literature data, the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) co-localizes with alpha-synuclein in Lewy bodies in Parkinsons disease, which suggests the involvement of this protein in the development of synucleinopathies. The goal of the present work was to investigate the direct interaction between alpha-synuclein and GAPDH and to evaluate possible influence of this interaction on the catalytic properties of GAPDH. Molecular dynamic simulations predicted the binding of alpha-synuclein to the positively charged groove comprising NAD+-binding pocket of GAPDH. The formation of the complex between alpha-synuclein and GAPDH in vitro was confirmed by different experimental approaches. The binding of alpha-synuclein to GAPDH with partially oxidized active site cysteines resulted in the subsequent inactivation of the enzyme, decreased its thermostability and increased its propensity for aggregation. At the same time, the formation of the complex between GAPDH and monomeric alpha-synuclein prevented amyloid transformation of alpha-synuclein. This work presents the first evidence for the fact that the initial oxidation of GAPDH induces the binding of alpha-synuclein to the enzyme, leading to further inactivation of GAPDH and, as a consequence, inhibition of glycolysis. The described mechanism may contribute to the metabolic disorders that are characteristic for synucleinopathies.

Dimerization of Tyr136Cys alpha-synuclein prevents amyloid transformation of wild type alpha-synuclein.

Expression of human alpha-synuclein in E. coli cells is known to result in a mixture of the wild type alpha-synuclein and the protein containing Tyr136Cys substitution due to the translational error. The amount of Cys136 alpha-synuclein (Cys136-AS) may reach approximately 50% of the recombinant protein. The wild-type and Cys136-containing fractions of alpha-synuclein were separated using thiol-Sepharose, and their properties were investigated. In the absence of reducing agents, Cys136-AS forms dimers due to the disulfide bonding. Both wild-type and Cys136 alpha-synuclein preparations are prone to aggregate during prolonged incubation under shaking at pH 4 and 37°C, but only the wild-type alpha-synuclein produces amyloid aggregates. The aggregates produced by either monomeric or dimeric Cys136-AS do not exhibit amyloid properties according to the test with Thioflavin T. Moreover, an admixture of dimeric Cys136-AS prevents the amyloid transformation of the wild-type alpha-synuclein. CD spectroscopy analysis revealed an enhanced content of alpha-helical structures in the aggregates produced by dimeric Cys136-AS. The admixture of Cys136-AS in preparations of human recombinant alpha-synuclein can be a source of erroneous interpretation of experiments on amyloid transformation of this protein.

Denaturing action of adjuvant affects specificity of polyclonal antibodies

Influence of the immunization procedure on the specificity of the produced antibodies towards different conformations of the antigen was investigated. It was demonstrated that intravenous immunization of a rabbit with an adjuvant-free solution of recombinant sperm-specific glyceraldehyde-3-phosphate dehydrogenase (dN-GAPDS) resulted in production of antibodies recognizing only native conformation of dN-GAPDS and exhibiting no cross-reaction with somatic isoenzyme of glyceraldehyde-3-phosphate dehydrogenase. A subcutaneous immunization with human dN-GAPDS mixed with Freunds complete adjuvant yielded antibodies recognizing both native and denatured conformation of dN-GAPDS. The oil component of the adjuvant was shown to cause inactivation and partial denaturation of dN-GAPDS, leading to exposure of the epitopes that are masked in the native protein, which resulted in production of the antibodies to the denatured antigen. These results may be of importance for biochemical research that often require polyclonal antibodies recognizing different conformations of antigens.