pH-sensitive Proteins Research Articles

Histidine-Mediated pH-Sensitive Regulation of M-Ficolin:GlcNAc Binding Activity in Innate Immunity Examined by Molecular Dynamics Simulations

BackgroundM-ficolin, a pathogen recognition molecule in the innate immune system, binds sugar residues including N-acetyl-D-glucosamine (GlcNAc), which is displayed on invading microbes and on apoptotic cells. The cis and trans Asp282-Cys283 peptide bond in the M-ficolin, which was found to occur at neutral and acidic pH in crystal structures, has been suggested to represent binding and non-binding activity, respectively. A detailed understanding of the pH-dependent conformational changes in M-ficolin and pH-mediated discrimination mechanism of GlcNAc-binding activity are crucial to both immune-surveillance and clearance of apoptotic cells.Methodology/Principal FindingsBy immunodetection analysis, we found that the pH-sensitive binding of GlcNAc is regulated by a conformational equilibrium between the active and inactive states of M-ficolin. We performed constant pH molecular dynamics (MD) simulation at a series of pH values to explore the pH effect on the cis-trans isomerization of the Asp282-Cys283 peptide bond in the M-ficolin fibrinogen-like domain (FBG). Analysis of the hydrogen bond occupancy of wild type FBG compared with three His mutants (H251A, H284A and H297A) corroborates that His284 is indispensible for pH-dependent binding. H251A formed new but weaker hydrogen bonds with GlcNAc. His297, unlike the other two His mutants, is more dependent on the solution pH and also contributes to cis-trans isomerization of the Asp282-Cys283 peptide bond in weak basic solution.Conclusions/SignificanceConstant pH MD simulation indicated that the cis active isomer of Asp282-Cys283 peptide bond was predominant around neutral pH while the trans bond gradually prevailed towards acidic environment. The protonation of His284 was found to be associated with the trans-to-cis isomerization of Asp282-Cys283 peptide bond which dominantly regulates the GlcNAc binding. Our MD simulation approach provides an insight into the pH-sensitive proteins and hence, ligand binding activity.

HL-1 mouse cardiomyocyte injury and death after simulated ischemia and reperfusion: roles of pH, Ca2+-independent phospholipase A2, and Na+/H+ exchange

The Ca(2+)-independent phospholipase A(2) VI (iPLA(2)-VI) and the Na(+)/H(+) exchanger isoform 1 (NHE1) are highly pH-sensitive proteins that exert both protective and detrimental effects in cardiac ischemia-reperfusion. Here, we investigated the role of extracellular pH (pH(o)) in ischemia-reperfusion injury and death and in regulation and function of iPLA(2)-VI and NHE1 under these conditions. HL-1 cardiomyocytes were exposed to simulated ischemia (SI; 0.5% O(2), 8 mM K(+), and 20 mM lactate) at pH(o) 6.0 and 7.4, with or without 4 or 8 h of reperfusion (SI/R). Cytochrome c release and caspase-3 activation were reduced after acidic compared with neutral SI, whereas necrotic death, estimated as glucose-6-phosphate dehydrogenase release, was similar in the two conditions. Inhibition of iPLA(2)-VI activity by bromoenol lactone (BEL) elicited cardiomyocyte necrosis during normoxia and after acidic, yet not after neutral, SI. The isoform-selective enantiomers R- and S-BEL both mimicked the effect of racemic BEL after acidic SI. In contrast, inhibition of NHE activity by EIPA had no significant effect on necrosis after SI. Both neutral and acidic SI were associated with a reversible loss of F-actin and cortactin integrity. Inhibition of iPLA(2)-VI disrupted F-actin, cortactin, and mitochondrial integrity, whereas inhibition of NHE slightly reduced stress fiber content. iPLA(2)-VIA and NHE1 mRNA levels were reduced during SI and upregulated in a pH(o)-dependent manner during SI/R. This also affected the subcellular localization of iPLA(2)-VIA. Thus, the mode of cell death and the roles and regulation of iPLA(2)-VI and NHE1 are at least in part determined by the pH(o) during SI. In addition to having clinically relevant implications, these findings can in part explain the contradictory results obtained from previous studies of iPLA(2)-VIA and NHE1 during cardiac I/R.

Regulation and Role of the Presynaptic and Myocardial Na+/H+ Exchanger NHE1: Effects on the Sympathetic Nervous System in Heart Failure

In acute myocardial ischemia and in chronic heart failure, sympathetic activation with excessive norepinephrine (NE) release from and reduced NE reuptake into sympathetic nerve endings is a prominent cause of arrhythmias and cardiac dysfunction. The Na(+)/H(+) exchanger NHE1 is the predominant isoform in the heart. It contributes to cellular acid-base balance, and electrolyte, and volume homeostasis, and is activated in response to intracellular acidosis and/or activation of guanine nucleotide binding (G) protein-coupled receptors. NHE1 mediates its signaling via protein kinases A (PKA) or C (PKC). In cardiomyocytes, NHE1 is restricted to specialized membrane domains, where it regulates the activity of pH-sensitive proteins and modulates the driving force of the Na(+)/Ca(2+) exchanger. During acute ischemia/reperfusion and in heart failure the activity/amount of NHE1 is increased, leading to intracellular Ca(2+) overload and promoting structural (apoptosis, hypertrophy) and functional (arrhythmias, hypercontraction) myocardial damage. In sympathetic nerve endings, increased NHE1 activity results in the accumulation of axoplasmic Na(+) that diminishes the inward and/or favors the outward transport of NE via the neuronal norepinephrine transporter (NET). The increased NE levels within the nerve-muscle junction facilitate the sustained stimulation of myocardial alpha- and beta-adrenoceptors (ARs), which in turn aggravate the increases in myocardial NHE1 activity and the associated deleterious effects. Furthermore, the responsiveness of the beta-AR declines overtime, which results in further release of NE, initiating a vicious cycle. Accordingly, NHE1 is a potential candidate for targeted intervention to suppress this feedback loop.

CO2 central chemosensitivity: why are there so many sensing molecules?

CO2 central chemoreceptors (CCRs) play a critical role in respiratory and cardiovascular controls. Although the primary sensory cells and their neuronal networks remain elusive, recent studies have begun to shed insight into the molecular mechanisms of several pH sensitive proteins. These putative CO2/pH-sensing molecules are expressed in the brainstem, detect P(CO2) at physiological levels, and couple the P(CO2) to membrane excitability. Functional analysis suggests that multiple CO2/pH-sensing molecules are needed to achieve high sensitivity and broad bandwidth of the CCRs. In contrast to the diversity of pH sensitive molecules, molecular mechanisms for CO2 sensing are rather general. The sensing molecules detect pH changes rather than molecular CO2. One or a few titratable amino acid residues in these proteins are usually involved. Protonation of these residues may lead to a change in protein conformation that is coupled to a change in channel activity. Depending on the location of the protonation sites, a membrane protein can detect extra- and/or intracellular pH.

Frontiers in the measurement of cell and tissue pH.

Investigation into the causes and consequences of aberrant pH regulation is entirely dependent on the ability to measure this parameter with specificity and sensitivity. Since their beginnings in the 1950s, techniques for measuring cell and tissue pH have undergone a number of significant advances. Following each of these advances, new investigators have been brought into the field, and new discoveries have been made. From microelectrode and dye distribution studies, measurement of pH underwent a revolution with the advent of pH-sensitive dyes that could be loaded into the cytosol. A further significant advance came from the measurement of cell and tissue pH in whole organisms by magnetic resonance spectroscopy (MRS). Frontiers in pH measurement exist in the development of pH-sensitive proteins and pH-sensitive MR contrast agents. These are predicted to bring even more people into this fascinating field, and generate more important discoveries.

Activation of soluble polysaccharides with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) for use in protein-polysaccharide conjugate vaccines and immunological reagents. II. Selective crosslinking of proteins to CDAP-activated polysaccharides

Covalently linking protein to polysaccharides converts the anti-polysaccharide immune response from a T-cell independent response to one which is T-cell dependent. The organic cyanylating reagent 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) (Vaccine 14:190, 1996) has been used to activate polysaccharides, which can then be reacted with spacer reagents or directly with protein. We wished to explore ways in which proteins could be linked to CDAP-activated polysaccharides to conjugate in a more controlled and selective fashion. To this end, we examined the reaction of nucleophilic amino acids with CDAP-activated polysaccharides under basic and acidic conditions. We found that lysine, cysteine and histidine but not methionine, serine or tyrosine conjugated to CDAP-activated dextran. We also examined the reaction of various spacer reagents with CDAP-activated dextran as a function of pH. The addition of hexanediamine was highly pH dependent and maximal at pH 9.3. In contrast, the addition of adipic dihydrazide, which has a pKa of ca 2.5 was essentially independent of pH. By performing the conjugation reaction at pH 5, we were able to selectively couple hydrazides even in the presence of high concentrations of amines. Proteins derivatized with limited numbers of hydrazides could be conjugated to CDAP-activated polysaccharides at pH5, where the native protein was not reactive. Proteins could be derivatized with hydrazides on carboxyls using adipic dihydrazide and a water soluble carbodiimide or on amines using a mild two-step reaction. Tetanus toxoid-pneumococcal type 14 conjugates produced by coupling hydrazide-derivatized tetanus toxoid under acidic conditions induced anti-polysaccharide antibodies at titers comparable to that stimulated by conjugates produced using a basic coupling pH. Our data suggest that crosslinking was occurring only with the limited number of hydrazides on the protein and that we achieved limited and selective crosslinking between the protein and CDAP-activated polysaccharide. This work also demonstrates that CDAP-mediated conjugation to polysaccharides can be applied even to very pH sensitive proteins and polysaccharides.

Subcellular localization of the Na+/H+ exchanger NHE1 in rat myocardium.

The Na+/H+ exchanger NHE1 isoform is an integral component of cardiac intracellular pH homeostasis that is critically important for myocardial contractility. To gain further insight into its physiological significance, we determined its cellular distribution in adult rat heart by using immunohistochemistry and confocal microscopy. NHE1 was localized predominantly at the intercalated disk regions in close proximity to the gap junction protein connexin 43 of atrial and ventricular muscle cells. Significant labeling of NHE1 was also observed along the transverse tubular systems, but not the lateral sarcolemmal membranes, of both cell types. In contrast, the Na+-K+-ATPase alpha1-subunit was readily labeled by a specific mouse monoclonal antibody (McK1) along the entire ventricular sarcolemma and intercalated disks and, to a lesser extent, in the transverse tubules. These results indicate that NHE1 has a distinct distribution in heart and may fulfill specialized roles by selectively regulating the pH microenvironment of pH-sensitive proteins at the intercalated disks (e.g., connexin 43) and near the cytosolic surface of sarcoplasmic reticulum cisternae (e.g., ryanodine receptor), thereby influencing impulse conduction and excitation-contraction coupling.