Significance Of Amino Acid Residues Research Articles

An anti-picornaviral strategy based on the crystal structure of foot-and-mouth disease virus 2C protein.

The foot-and-mouth disease virus (FMDV) 2C protein shares conserved motifs with enterovirus 2Cs despite low sequence identity. Here, we determine the crystal structure of an FMDV 2C fragment to 1.83Å resolution, which comprises an ATPase domain, a region equivalent to the enterovirus 2C zinc-finger (ZFER), and a C-terminal domain harboring a loop (PBL) that occupies a hydrophobic cleft (Pocket) in an adjacent 2C molecule. Mutations at ZFER, PBL, and Pocket affect FMDV 2C ATPase activity and are lethal to FMDV infectious clones. Because the PBL-Pocket interaction between FMDV 2C molecules is essential for its functions, we design an anti-FMDV peptide derived from PBL (PBL-peptide). PBL-peptide inhibits FMDV 2C ATPase activity, binds FMDV 2C with nanomolar affinity, and disrupts FMDV 2C oligomerization. FMDV 2C targets lipid droplets (LDs) and induces LD clustering in cells, and PBL-peptide disrupts FMDV 2C-induced LD clustering. Finally, we demonstrate that PBL-peptide exhibits anti-FMDV activity in cells.

HIV-1 subtype C Tat exon-1 amino acid residue 24K is a signature for neurocognitive impairment.

Variation and differential selection pressures on Tat genes have been shown to alter the biological function of the protein, resulting in pathological consequences in a number of organs including the brain. We evaluated the impact of genetic variation and selection pressure on 147 HIV-1 subtype C Tat exon 1 sequences from monocyte-depleted peripheral lymphocytes on clinical diagnosis of neurocognitive impairment. Genetic analyses identified two signature amino acid residues, lysine at codon 24 (24K) with a frequency of 43.4% and arginine at codon 29 (29R) with a frequency of 34.0% in individuals with HIV-associated neurocognitive impairment. The analyses also revealed two signature residues, asparagine, 24N (31.9%), and histidine, 29H (21.3%), in individuals without neurocognitive impairment. Both codons, 24 and 29, were associated with high entropy but only codon 29 was under positive selection. The presence of signature K24 increased by 2.08 times the risk of neurocognitive impairment, 3.15 times higher proviral load, and 69% lower absolute CD4 T-cell count compared to those without the signature. The results support a linkage between HIV-1 C Tat N24K polymorphism, proviral load, immunosuppression, and neurocognitive impairment. The signature may induce more neurotoxic effects, which contributes to establishment and severity of HIV-associated neurocognitive impairment.

The Signature Amino Acid Residue Serine 31 of HIV-1C Tat Potentiates an Activated Phenotype in Endothelial Cells.

The natural cysteine to serine variation at position 31 of Tat in HIV-1C disrupts the dicysteine motif attenuating the chemokine function of Tat. We ask if there exists a trade-off in terms of a gain of function for HIV-1C Tat due to this natural variation. We constructed two Tat-expression vectors encoding Tat proteins discordant for the serine 31 residue (CS-Tat vs. CC-Tat), expressed the proteins in Jurkat cells under doxycycline control, and performed the whole transcriptome analysis to compare the early events of Tat-induced host gene expression. Our analysis delineated a significant enrichment of pathways and gene ontologies associated with the angiogenic signaling events in CS-Tat stable cells. Subsequently, we validated and compared angiogenic signaling events induced by CS- vs. CC-Tat using human umbilical vein endothelial cells (HUVEC) and the human cerebral microvascular endothelial cell line (hCMEC/D3). CS-Tat significantly enhanced the production of CCL2 from HUVEC and induced an activated phenotype in endothelial cells conferring on them enhanced migration, invasion, and in vitro morphogenesis potential. The ability of CS-Tat to induce the activated phenotype in endothelial cells could be of significance, especially in the context of HIV-associated cardiovascular and neuronal disorders. The findings from the present study are likely to help appreciate the functional significance of the SAR (signature amino acid residues) influencing the unique biological properties.

Structural significance of residues 158–160 in the H3N2 hemagglutnin globular head: A computational study with implications in viral evolution and infection

Influenza A H3N2 has been linked to annual outbreaks within the human population attributable to continuous structural changes. H3N2 HA contains well identified antigenic sites and receptor-binding sites (RBS) that are possibly correlated to viral evolution and infection. However, the structural significance of amino acid residues associated with both viral evolution and infection were not fully demonstrated. Throughout this study, we generated and analyzed H3N2 HA models that represented the clade 3C.2 population (comprised of clades 3C.2, 3C.2a, and 3C.21 from the transitioning 2014–2018 H3N2 strains) and 3C.3a (from the 2016 H3N2 strain). Model quality estimation, structural analyses and superimposition, and network analytics of H3N2 HA1 evolution were performed. We found that the structural properties of residues 158–160 could influence the overall HA backbone. More specifically, amino acid substitutions at residues 159–160 affected the amino acid orientation at residue 158, thereby, causing the overall HA backbone structure to vary. Our results were consistent with 1968–2018 HA1 evolution. Taken together, we propose that our results would highlight the structural significance of residues 158–160 in HA1 for both antigenic drift and RBS.

Fine epitope signature of antibody neutralization breadth at the HIV-1 envelope CD4-binding site.

Major advances in donor identification, antigen probe design, and experimental methods to clone pathogen-specific antibodies have led to an exponential growth in the number of newly characterized broadly neutralizing antibodies (bnAbs) that recognize the HIV-1 envelope glycoprotein. Characterization of these bnAbs has defined new epitopes and novel modes of recognition that can result in potent neutralization of HIV-1. However, the translation of envelope recognition profiles in biophysical assays into an understanding of in vivo activity has lagged behind, and identification of subjects and mAbs with potent antiviral activity has remained reliant on empirical evaluation of neutralization potency and breadth. To begin to address this discrepancy between recombinant protein recognition and virus neutralization, we studied the fine epitope specificity of a panel of CD4-binding site (CD4bs) antibodies to define the molecular recognition features of functionally potent humoral responses targeting the HIV-1 envelope site bound by CD4. Whereas previous studies have used neutralization data and machine-learning methods to provide epitope maps, here, this approach was reversed, demonstrating that simple binding assays of fine epitope specificity can prospectively identify broadly neutralizing CD4bs–specific mAbs. Building on this result, we show that epitope mapping and prediction of neutralization breadth can also be accomplished in the assessment of polyclonal serum responses. Thus, this study identifies a set of CD4bs bnAb signature amino acid residues and demonstrates that sensitivity to mutations at signature positions is sufficient to predict neutralization breadth of polyclonal sera with a high degree of accuracy across cohorts and across clades.

M Gene Reassortment in H9N2 Influenza Virus Promotes Early Infection and Replication: Contribution to Rising Virus Prevalence in Chickens in China.

Segment reassortment and base mutagenesis of influenza A viruses are the primary routes to the rapid evolution of high-fitness virus genotypes. We recently described a predominant G57 genotype of avian H9N2 viruses that caused countrywide outbreaks in chickens in China during 2010 to 2013, which led to the zoonotic emergence of H7N9 viruses. One of the key features of the G57 genotype is the replacement of the earlier A/chicken/Beijing/1/1994 (BJ/94)-like M gene with the A/quail/Hong Kong/G1/1997 (G1)-like M gene of quail origin. We report here the functional significance of the G1-like M gene in H9N2 viruses in conferring increased infection severity and infectivity in primary chicken embryonic fibroblasts and chickens. H9N2 virus housing the G1-like M gene, in place of the BJ/94-like M gene, showed an early surge in viral mRNA and viral RNA (vRNA) transcription that was associated with enhanced viral protein production and with an early elevated release of progeny virus comprising largely spherical rather than filamentous virions. Importantly, H9N2 virus with the G1-like M gene conferred extrapulmonary virus spread in chickens. Five highly represented signature amino acid residues (37A, 95K, 224N, and 242N in the M1 protein and 21G in the M2 protein) encoded by the prevalent G1-like M gene were demonstrated to be prime contributors to enhanced infectivity. Therefore, the genetic evolution of the M gene in H9N2 virus increases reproductive virus fitness, indicating its contribution to the rising virus prevalence in chickens in China.IMPORTANCE We recently described the circulation of a dominant genotype (genotype G57) of H9N2 viruses in countrywide outbreaks in chickens in China, which was responsible, through reassortment, for the emergence of H7N9 viruses that cause severe human infections. A key feature of the genotype G57 H9N2 virus is the presence of the quail-origin G1-like M gene, which had replaced the earlier BJ/94-like M gene. We found that H9N2 virus with the G1-like M gene, but not the BJ/94-like M gene, showed an early surge in progeny virus production and more severe pathology and extrapulmonary virus spread in chickens. Five highly represented amino acid residues in the M1 and M2 proteins derived from the G1-like M gene were shown to mediate enhanced virus infectivity. These observations enhance what we currently know about the roles of reassortment and mutations in virus fitness and have implications for assessing the potential of variant influenza viruses that can cause a rising prevalence in chickens.

The Evolving Profile of the Signature Amino Acid Residues in HIV-1 Subtype C Tat.

Using several HIV-1 tat exon 1 amino acid sequences available from public databases and additional sequences derived from a southern Indian clinical cohort, we compared the profile of the signature amino acid residues (SAR) between two different time periods, 1986-2004 and 2005-2014. The analysis identified eight positions as signature residues in subtype C Tat and demonstrated a changing pattern at four of these positions between the two periods. At three locations (histidine 29, serine 57, and proline 60), there appears to be a nonuniform negative selection against the SAR. The negative selection appears to be severe, especially against histidine 29 (p < .0001) and moderate against proline 60 (p < .0001). The negative selection against serine 57 is statistically insignificant and appears to have begun recently. At position 63, the frequency of signature residue glutamic acid increased over the past decade, although the difference was not significant. Importantly, at the three locations where the negative selection is in progress, the substitute amino acids are the generic residues present in most of the other HIV-1 subtypes. Our data demonstrate that viral evolution can subject specific amino acid residues to subtle and progressive selection pressures without affecting the prevalence of other amino acid residues.

Genomic signature and protein sequence analysis of a novel influenza A (H7N9) virus that causes an outbreak in humans in China

Very recently, a new avian flu outbreak in humans, which is caused by a novel H7N9 influenza A virus (AIV), was reported in China. As of April 13, 2013, 49 confirmed cases (mainly middle-aged to elderly males), including 11 deaths, were reported in China. Here we analyzed the genomic signatures and protein sequences of the human H7N9 AIVs. We found that the genomic signatures of A(H7N9) had high and low identity to avian and human IAVs, respectively, suggesting its avian origin. The signature amino acids of A(H7N9) had high identity to 1997 H5N1 and 2009 H1N1, but low identity to those influenza strains that caused pandemics before 1980. One of the key signature amino acids at 627 in PB2 mutated to lysine, which is associated with mammalian adaptation and increased virulence of the highly pathogenic avian influenza A(H5N1) virus. Besides, several other human-like signatures, including PB2-44S, PA-100A, PA-356R, and PA-409N are also found in this avian-origin A(H7N9) virus. The HA protein has the Q226L mutation, which is associated with increased binding to mammalian-like receptors bearing alpha 2,6 receptor in the human upper airway. The M2 protein contains the N31S mutation, suggesting its resistance to the M2 channel blockers amantadine and rimantadine. These findings suggest that this avian-origin AIV gains its bird-to-human, i.e., zoonotic, transmissibility and increased virulence, as well as drug-resistance, by mutating key signature amino acid residues and those in the functional domains of the viral proteins. Therefore, it is prudent to monitor the evolution of A(H7N9), as well as develop strategies to combat any potential epidemic or pandemic.

Functional diversification of ROK-family transcriptional regulators of sugar catabolism in the Thermotogae phylum

Large and functionally heterogeneous families of transcription factors have complex evolutionary histories. What shapes specificities toward effectors and DNA sites in paralogous regulators is a fundamental question in biology. Bacteria from the deep-branching lineage Thermotogae possess multiple paralogs of the repressor, open reading frame, kinase (ROK) family regulators that are characterized by carbohydrate-sensing domains shared with sugar kinases. We applied an integrated genomic approach to study functions and specificities of regulators from this family. A comparative analysis of 11 Thermotogae genomes revealed novel mechanisms of transcriptional regulation of the sugar utilization networks, DNA-binding motifs and specific functions. Reconstructed regulons for seven groups of ROK regulators were validated by DNA-binding assays using purified recombinant proteins from the model bacterium Thermotoga maritima. All tested regulators demonstrated specific binding to their predicted cognate DNA sites, and this binding was inhibited by specific effectors, mono- or disaccharides from their respective sugar catabolic pathways. By comparing ligand-binding domains of regulators with structurally characterized kinases from the ROK family, we elucidated signature amino acid residues determining sugar-ligand regulator specificity. Observed correlations between signature residues and the sugar-ligand specificities provide the framework for structure functional classification of the entire ROK family.

Unexpected Diversity of Chlorite Dismutases: a Catalytically Efficient Dimeric Enzyme from Nitrobacter winogradskyi

Chlorite dismutase (Cld) is a unique heme enzyme catalyzing the conversion of ClO(2)(-) to Cl(-) and O(2). Cld is usually found in perchlorate- or chlorate-reducing bacteria but was also recently identified in a nitrite-oxidizing bacterium of the genus Nitrospira. Here we characterized a novel Cld-like protein from the chemolithoautotrophic nitrite oxidizer Nitrobacter winogradskyi which is significantly smaller than all previously known chlorite dismutases. Its three-dimensional (3D) crystal structure revealed a dimer of two identical subunits, which sharply contrasts with the penta- or hexameric structures of other chlorite dismutases. Despite a truncated N-terminal domain in each subunit, this novel enzyme turned out to be a highly efficient chlorite dismutase (K(m) = 90 μM; k(cat) = 190 s(-1); k(cat)/K(m) = 2.1 × 10(6) M(-1) s(-1)), demonstrating a greater structural and phylogenetic diversity of these enzymes than was previously known. Based on comparative analyses of Cld sequences and 3D structures, signature amino acid residues that can be employed to assess whether uncharacterized Cld-like proteins may have a high chlorite-dismutating activity were identified. Interestingly, proteins that contain all these signatures and are phylogenetically closely related to the novel-type Cld of N. winogradskyi exist in a large number of other microbes, including other nitrite oxidizers.

Claudin Association with CD81 Defines Hepatitis C Virus Entry

Viruses initiate infection by attaching to molecules or receptors at the cell surface. Hepatitis C virus (HCV) enters cells via a multistep process involving tetraspanin CD81, scavenger receptor class B member I, and the tight junction proteins Claudin-1 and Occludin. CD81 and scavenger receptor class B member I interact with HCV-encoded glycoproteins, suggesting an initial role in mediating virus attachment. In contrast, there are minimal data supporting Claudin-1 association with HCV particles, raising questions as to its role in the virus internalization process. In the present study we demonstrate a relationship between receptor active Claudins and their association and organization with CD81 at the plasma membrane by fluorescence resonance energy transfer and stoichiometric imaging methodologies. Mutation of residues 32 and 48 in the Claudin-1 first extracellular loop ablates CD81 association and HCV receptor activity. Furthermore, mutation of the same residues in the receptor-inactive Claudin-7 molecule enabled CD81 complex formation and virus entry, demonstrating an essential role for Claudin-CD81 complexes in HCV infection. Importantly, Claudin-1 associated with CD81 at the basolateral membrane of polarized HepG2 cells, whereas tight junction-associated pools of Claudin-1 demonstrated a minimal association with CD81. In summary, we demonstrate an essential role for Claudin-CD81 complexes in HCV infection and their localization at the basolateral surface of polarized hepatoma cells, consistent with virus entry into the liver via the sinusoidal blood and association with basal expressed forms of the receptors.

Full Genome Analysis of an Emerging Cluster of Human Immunodeficiency Virus Type 1 Subtype B Infection in Hong Kong

With considerable capacity for genetic diversification, new HIV-1 genotypes have been reported over the years. Three HIV-1 isolates previously genotyped as B using gag and env sequences were completely sequenced and reanalyzed. Several amino acid mutations were found in vif, rev, and nef genes but not in gag or env sequences. These alterations have not previously been reported in Hong Kong. The investigation of phylogenetic relatedness revealed that a region of the vif of the studied Hong Kong isolates subtype B cluster contains several subtype D signature amino acid residues. Several unique mutations on vif in these three isolates were also identified.

Compartmentalization of HIV-1 within the Female Genital Tract Is Due to Monotypic and Low-Diversity Variants Not Distinct Viral Populations

BackgroundCompartmentalization of HIV-1 between the genital tract and blood was noted in half of 57 women included in 12 studies primarily using cell-free virus. To further understand differences between genital tract and blood viruses of women with chronic HIV-1 infection cell-free and cell-associated virus populations were sequenced from these tissues, reasoning that integrated viral DNA includes variants archived from earlier in infection, and provides a greater array of genotypes for comparisons.Methodology/Principal FindingsMultiple sequences from single-genome-amplification of HIV-1 RNA and DNA from the genital tract and blood of each woman were compared in a cross-sectional study. Maximum likelihood phylogenies were evaluated for evidence of compartmentalization using four statistical tests. Genital tract and blood HIV-1 appears compartmentalized in 7/13 women by ≥2 statistical analyses. These subjects' phylograms were characterized by low diversity genital-specific viral clades interspersed between clades containing both genital and blood sequences. Many of the genital-specific clades contained monotypic HIV-1 sequences. In 2/7 women, HIV-1 populations were significantly compartmentalized across all four statistical tests; both had low diversity genital tract-only clades. Collapsing monotypic variants into a single sequence diminished the prevalence and extent of compartmentalization. Viral sequences did not demonstrate tissue-specific signature amino acid residues, differential immune selection, or co-receptor usage.Conclusions/SignificanceIn women with chronic HIV-1 infection multiple identical sequences suggest proliferation of HIV-1-infected cells, and low diversity tissue-specific phylogenetic clades are consistent with bursts of viral replication. These monotypic and tissue-specific viruses provide statistical support for compartmentalization of HIV-1 between the female genital tract and blood. However, the intermingling of these clades with clades comprised of both genital and blood sequences and the absence of tissue-specific genetic features suggests compartmentalization between blood and genital tract may be due to viral replication and proliferation of infected cells, and questions whether HIV-1 in the female genital tract is distinct from blood.

Genetic characterization of type A foot-and-mouth disease virus 3A region in context of the reemergence of VP3 59-deletion lineage in India

The 3A region of foot-and-mouth disease virus has been implicated in host range and virulence. Here we analyzed the 3A region of serotype A virus in view of the emergence of a variant group in India with an amino acid deletion at an antigenically critical position of capsid protein, VP3. The 3A region exhibited extreme variability with 38% of the amino acid positions showing substitutions and the C-terminal third (127–151) region was most flexible. Genotype inclusive grouping of type A foot-and-mouth disease virus as observed in 1D region based phylogeny was much less apparent at 3A region possibly due to independent evolution of nonstructural and structural protein coding regions. Akin to the 1D region, the VP3 59-deletion group maintained its phylogenetic distinctness even at the 3A region and was found to be diverging with time. Twelve lineage specific signature amino acid residues, of which four were identified to be experiencing positive selection, indicates fixation of advantageous mutations in a lineage specific manner. Six positions, all located in the hypervariable C-terminal third, were identified to be under positive selection and were presumed to be imparting the virus certain advantage accounting for its adaptability to wide host spectrum and rapid dissemination. A significant change of Q 44H was noted only in the older lineage (VIIb) of the deletion group at a position where Q 44R mutation is associated with guinea pig adaptation. As this site has been detected to be under positive selection, such a lineage specific substitution is thought to have imparted certain temporary advantage to the virus during its possible adaptation in wild or some understudied domestic hosts and must not have seriously compromised fitness upon readaptation in bovines. A conserved hydrophobic transmembrane domain from position 59 to 76 could be predicted which possibly anchors 3A to intracellular membranes for successful interaction with RNA replication complex.

Characterization and signature pattern analysis of Korean clade HIV-1 using nef gene sequences

Phylogenetic studies of the HIV-1 gene sequences isolated from Korean patients have suggested that most of Korean isolates belong to the subtype B strain. This study aims to characterize the Korean clade by molecular phylogenetic analysis using all of the Korean nef gene sequences registered in the NCBI GenBank (N=422), in addition to 41 reference strains and 94 foreign isolates. Through phylogenetic analyses, we verified that most of the Korean isolates belonged to the subtype B, where 78.8% are clustered exclusively of foreign isolates. This cluster has been named the Korean clade subtype B (KCB) in order to distinguish it from other subtype B clusters. Genetic distance analysis suggested that the KCB cluster was more homogeneous and clearly distinctive from the non-Korean clade subtype B (NKCB). Comparison of consensus amino acid sequences from KCB and NKCB revealed that characteristic KCB signature amino acid patterns composed of 11 amino acid residues, whose frequencies in the KCB were significantly higher than in the NKCB. The KCB signature amino acid residues were critical in identifying KCB from NKCB, since substitution of the NKCB sequences with KCB signature amino acids relocated them to the Koran clade, and vice versa.

Structural and Functional Effects of Tryptophans Inserted into the Membrane-binding and Substrate-binding Sites of Human Group IIA Phospholipase A2

Phospholipase A(2) (PLA(2)) enzymes become activated by binding to biological membranes and hydrolyze phospholipids to free fatty acids and lyso-phospholipids, the precursors of inflammatory mediators. To understand the functional significance of amino acid residues at key positions, we have studied the effects of the substitution of Val(3) (membrane binding surface) and Phe(5) (substrate binding pocket) of human group IIA PLA(2) by tryptophan on the structure and function of the enzyme. Despite the close proximity of the sites of mutations, the V3W mutation results in substantial enhancement of the enzyme activity, whereas the F5W mutant demonstrates significantly suppressed activity. A structural analysis of all three proteins free in buffer and bound to membranes indicates that large differences in activities result from distinct conformational changes in PLA(2)s upon membrane binding. Although PLA(2) and the V3W mutant demonstrate a decrease in helical content and an increase in helix flexibility, the F5W mutant experiences partial distortion of the alpha-helical structure presumably resulting from the tendency of Trp(5) to insert into the membrane. Furthermore, whereas the PLA(2) and the V3W mutant bind to the membrane at similar and apparently productive-mode orientation, the F5W mutant binds to membranes with a distinctly different orientation. It is suggested that both the stimulatory effect of the V3W mutation and the inhibitory effect of the F5W mutation result from the high affinity of Trp for the membrane-water interface. Although Trp(3) at the membrane binding face of PLA(2) facilitates the proper membrane binding of the enzyme, Trp(5) in the internal substrate binding site causes partial unwinding of the N-terminal helix in order to interact with the membrane.

Solution structure of gamma-bungarotoxin: the functional significance of amino acid residues flanking the RGD motif in integrin binding.

Gamma-bungarotoxin, a snake venom protein isolated from Bungarus multicinctus, contains 68 amino acids, including 10 cysteine residues and a TAVRGDGP sequence at positions 30-37. The solution structure of gamma-bungarotoxin has been determined by nuclear magnetic resonance (NMR) spectroscopy. The structure is similar to that of the short-chain neurotoxins that contain three loops extending from a disulfide-bridged core. The tripeptide Arg-Gly-Asp (RGD) sequence is located at the apex of the flexible loop and is similar to that of other RGD-containing proteins. However, gamma-bungarotoxin only inhibits platelet aggregations with an IC50 of 34 microM. To understand its weak activity in inhibiting platelet aggregation, we mutated the RGD loop sequences of rhodostomin, a potent platelet aggregation inhibitor, from RIPRGDMP to TAVRGDGP, resulting in a 196-fold decrease in activity. In addition, the average Calpha-to-Calpha distance between R33 and G36 of gamma-bungarotoxin is 6.02 A, i.e., shorter than that of other RGD-containing proteins that range from 6.55 to 7.46 A. These results suggested that the amino acid residues flanking the RGD motif might control the width of the RGD loop. This structural difference may be responsible for its decrease in platelet aggregation inhibition compared with other RGD-containing proteins.

Cloning and expression analysis of 2-on-2 hemoglobin from soybean

Hemoglobins (Hbs) are heme proteins found in all five kingdoms of living organisms. In plants, three different classes of Hbs have been identified-nonsymbiotic Hbs from diverse species, symbiotic Hbs from nitrogen-fixing plants, and so-called 2-on-2 Hbs. Here, we report the cloning and expression analysis of the 2-on-2 Hb gene,GmGLB3, from soybean. TheGmGLB3 cDNA clone encodes a protein for 172 amino acid residues. Its deduced amino acid sequence shows the highest identity (74%) with 2-on-2 Hb fromMedicago truncatula. Multiple sequence alignment confirms the conserved and signature amino acid residues previously reported with plant 2-on-2 Hbs. Genomic Southern hybridization demonstrates thatGmGLB3 has two copies in the soybean genome. Based on our northern hybridization, theGmGLB3 gene is specifically expressed in root nodules, with levels increasing in the late stage during nodule development. Its transcript level is also increased under flooding and kinetin treatments in the roots, or under flooding and 2-iP treatments in the stems. However, no transcript is detected in the leaves regardless of treatment. Therefore, we propose that theGmGLB3 gene is specifically expressed in root nodules and that its expression in other plant organs is regulated by cytokinin and/or flooding.

Vertebrate agmatinases: what role do they play in agmatine catabolism?

Whereas agmatine in vertebrates may be derived from multiple sources such as the diet, endogenous synthesis via arginine decarboxylase, and possibly also from enteric bacteria, agmatinase is the only enzyme specific for agmatine catabolism. As it hydrolyzes a guanidino group within agmatine and also contains signature amino acid residues that act as ligand binding sites for the Mn(++) cofactor, agmatinase is classified as a member of the arginase superfamily. Very little information is available regarding how much agmatine in vertebrate species is catabolized by agmatinase versus other enzymes such as diamine and amine oxidases. Moreover, comparisons of primary sequences of several vertebrate agmatinases demonstrate that several residues essential for catalytic activity are not conserved in the mouse. This leads to the prediction that the agmatinase protein in mouse has little or no catalytic activity, not only raising questions about the physiologic routes of agmatine disposal in this organism, but also suggesting the existence of species-specific differences in mechanisms for regulating agmatine levels.

Structure-function study on gastric proton pump

H+, K(+)-ATPase is a proton pump responsible for gastric acid secretion. It actively transport proton and K+ coupled with the hydrolysis of ATP, resulting in the formulation of a 10(6) fold proton gradient across the plasma membrane of parietal cells. The pump belongs to a family of P-type ATPases which include the Na+ pump (Na+, K(+)-ATPase) and the Ca2+ pump (Ca(2+)-ATPase). This review focuses on the structure-function relationship of this proton pump by using functional antibodies, specific inhibitor(s), a fluorescent reagent and site-directed mutants. First we prepared monoclonal antibodies which modified the functions of the H+, K(+)-ATPase . One of the antibodies, HK2032 inhibited the H+, K(+)-ATPase activity and the chloride conductance in gastric vesicles opened by S-S cross-linking, suggesting that the chloride pathway is in the H+, K(+)-ATPase molecule, and that the H+, K(+)-ATPase is a multi-functional molecule. Other antibody, HK4001 inhibited the H+, K(+)-ATPase activity by inhibiting its phosphorylation step. By using this antibody we found an H+, K(+)-ATPase isoform in the rabbit distal colon. Second we found that scopadulcic acid B, a main ingredient of Paraguayan traditional herb, is an inhibitor specific for the H+, K(+)-ATPase. This compound inhibited the H+, K(+)-ATPase activity by stabilizing the K(+)-form of the enzyme. Third we studied the conformational changes of the H+, K(+)-ATPase by observing the fluorescence of FITC-labeled enzyme. H+, K(+)-ATPase did not utilize acetylphosphate instead the ATP as an energy source of active transport, suggesting that the energy transduction system is not common among P-type ATPases. Finally we constructed a functional expression system of the H+, K(+)-ATPase in human kidney cells. By using this functional expression system in combination with site-directed mutagenesis, we studied the significance of amino acid residues in the catalytic centers (a phosphorylation site and an ATP binding site) and the putative cation binding sites. We newly found the sites determining the affinity for cations.