Structural homology among the major 7s globulin subunits of soybean seed storage proteins

The soybean seed 7S globulin subunits, i.e. α, α′, β and γ-subunits of β-conglycinin, the γ-conglycinin subunit and the HI/HII and LII subunits of basic 7S globulin were purified and the NH₂-terminal amino acid sequences of all these subunits except the γ-subunit of β-conglycinin were determined. Only the NH₂-terminal regions of the α and α′-subunits showed high sequence homology. However, sequencing of tryptic peptides from the seven subunits revealed that internal region sequences were highly homologous among the four subunits of β-conglycinin. In contrast to the β-conglycinin subunits, no sequence homology was found among the other subunits. On the basis of these results, the major 7S globulin fraction is considered more heterogeneous in primary structure than another major globulin fraction, 11S globulin (glycinin), in soybean seeds.     

Thermodynamic analysis reveals that GTP binding affects the interaction between the alpha- and gamma-subunits of translation initiation factor 2

Eukaryotic and archaeal translation initiation factors 2, heterotrimers that consist of α-, β-, and γ-subunits, deliver methionylated initiator tRNA to a small ribosomal subunit in a manner that depends on GTP. To evaluate correlation of the function and association of the subunits, we used isothermal titration calorimetry to analyze the thermodynamics of the interactions between the α- and γ-subunits in the presence or absence of a nonhydrolyzable GTP analog or GDP. The α-subunits bound to the γ-subunit with large heat capacity change (ΔC_p) values. The ΔH and ΔC_p values for the interaction between the α- and γ-subunits varied in the presence of the GTP analog but not in the presence of GDP. These results suggest that the binding of both the α-subunit and GTP changes the conformation of the switch region of the γ-subunit and increases the affinity of the γ-subunit for tRNA.     

Potency of GABA at human recombinant GABAA receptors expressed in Xenopus oocytes: a mini review

GABA_A receptors are members of the ligand-gated ion channel superfamily that mediate inhibitory neurotransmission in the central nervous system. They are thought to be composed of 2 alpha (α), 2 beta (β) subunits and one other such as a gamma (γ) or delta (δ) subunit. The potency of GABA is influenced by the subunit composition. However, there are no reported systematic studies that evaluate GABA potency on a comprehensive number of subunit combinations expressed in Xenopus oocytes, despite the wide use of this heterologous expression system in structure–function studies and drug discovery. Thus, the aim of this study was to conduct a systematic characterization of the potency of GABA at 43 human recombinant GABA_A receptor combinations expressed in Xenopus oocytes using the two-electrode voltage clamp technique. The results show that the α-subunits and to a lesser extent, the β-subunits influence GABA potency. Of the binary and ternary combinations with and without the γ2L subunit, the α6/γ2L-containing receptors were the most sensitive to GABA, while the β2- or β3-subunit conferred higher sensitivity to GABA than receptors containing the β1-subunit with the exception of the α2β1γ2L and α6β1γ2L subtypes. Of the δ-subunit containing GABA_A receptors, α4/δ-containing GABA_A receptors displayed highest GABA sensitivity, with mid-nanomolar concentrations activating α4β1δ and α4β3δ receptors. At α4β2δ, GABA had low micromolar activity.     

Atomic force microscopy reveals the architecture of the epithelial sodium channel (ENaC)

The epithelial sodium channel (ENaC) is a member of the ENaC/degenerin superfamily. ENaC is a heteromultimer containing three homologous subunits (α, β, and γ); however, the subunit stoichiometry is still controversial. Here, we addressed this issue using atomic force microscopy imaging of complexes between isolated ENaC and antibodies/Fab fragments directed against specific epitope tags on the α-, β- and γ-subunits. We show that for α-, β- and γ-ENaC alone, pairs of antibodies decorate the channel at an angle of 120°, indicating that the individual subunits assemble as homotrimers. A similar approach demonstrates that αβγ-ENaC assembles as a heterotrimer containing one copy of each subunit. Intriguingly, all four subunit combinations also produce higher-order structures containing two or three individual trimers. The trimer-of-trimers organization would account for earlier reports that ENaC contains eight to nine subunits.     

The Pharmacology of the Benzodiazepine Site of the GABA-A Receptor is Dependent on the Type of γ-Subunit Present

Abstract

The pharmacology of native and recombinant GABA-A receptors containing either γ1, γ2 or γ3 subunits has been investigated. The pharmacology of native receptors has been investigated by immunoprecipitating receptors from solubilised preparations of rat brain with antisera specific for individual γ-subunits and analysing their radioligand binding characteristics. Receptors containing a γ1-subunit do not bind benzodiazepine radioligands with high affinity. Those containing either a γ2 or γ3 subunit bind [³H]flumazenil with high affinity. Some compounds compete for these binding sites with multiple affinities, reflecting the presence of populations of receptors containing several different types of α-subunit. Photoaffinity-labelling of GABA-A receptors from a cell line stably expressing GABA-A receptors of composition α₁β3γ2 followed by immunoprecipitation of individual subunits revealed that the α and γ but not the β-subunit could be irreversibly labelled by [³H]flunitrazepam.

The properties of recombinant receptors have been investigated in oocytes expressing γ1, γ2, or γ3 subunits in combination with an α and a β-subunit. Some compounds such as zolpidem, DMCM and flunitrazepam show selectivity for receptors containing different γ-subunits. Others such as CL 218,872 show no selectivity between receptors containing different γ-subunits but exhibit selectivity for receptors containing different α-subunits. These data taken together suggest that the benzodiazepine site of the GABA-A receptor is formed with contributions from both the α and γ-subunits.     

Early embryonic development of the freshwater shrimp Caridina multidentata (Crustacea,Decapoda, Atyidae)

Decapod crustaceans show a great diversity of developmental modes at all levels. In particular, early cleavage varies from total via mixed to superficial modes and from determinate cleavage with a stereotyped pattern to indeterminate cleavage. However, the ground pattern of early decapod development is not clear. To address this problem, we studied the early embryonic development of the caridean shrimp Caridina multidentata with a combination of confocal laser scanning microscopy, scanning electron microscopy, 4D microscopy and 3-D reconstruction software. Despite a yolk-rich egg, the cleavage is holoblastic and shows a distinct pattern of blastomere arrangement, characterized by two interlocking cell bands. This resembles the conditions in dendrobranchiate shrimps, which most likely are the sister group to Pleocyemata to which C. multidentata belongs. Hence, our results offer the possibility to assume total cleavage with blastomeres arranged in two interlocking cell bands as ancestral cleavage mode for Decapoda.     

Unique assignment of inter-subunit association in GABAA α1β3γ2 receptors determined by molecular modeling

Recent publications defined requirements for inter-subunit contacts in a benzodiazepine-sensitive GABA_A receptor (GABA_ARα1β3γ2). There is strong evidence that the heteropentameric receptor contains two α1, two β3, and one γ2 subunit. However, the available data do not distinguish two possibilities: When viewed clockwise from an extracellular viewpoint the subunits could be arranged in either γ2β3α1β3α1 or γ2α1β3α1β3 configurations. Here we use molecular modeling to thread the relevant GABA_AR subunit sequences onto a template of homopentameric subunits in the crystal structure of the acetylcholine binding protein (AChBP). The GABA_A sequences are known to have 15-18% identity with the acetylcholine binding protein and nearly all residues that are conserved within the nAChR family are present in AChBP. The correctly aligned GABA_A sequences were threaded onto the AChBP template in the γ2β3α1β3α1 or γ2α1β3α1β3  arrangements. Only the γ2α1β3α1β3 arrangement satisfied three known criteria: (1) α1 His¹⁰² binds at the γ2 subunit interface in proximity to γ2 residues Thr¹⁴², Phe⁷⁷, and Met¹³⁰; (2) α1 residues 80-100 bind near γ2 residues 91-104; and (3) α1 residues 58-67 bind near the β3 subunit interface. In addition to predicting the most likely inter-subunit arrangement, the model predicts which residues form the GABA and benzodiazepine binding sites.     

Subunit compositions of crustacean haemocyanins are species-specific: evidence from non-decapod species

Electrophoretic examination of dissociated haemocyanin subunits from a number of amphipod, decapod and isopod crustaceans supports the hypothesis that subunit composition is species-specific, despite marked within-species variation in many species. General patterns of heterogeneity on native PAGE gels were also evident between groupings within the Amphipoda. Gammarid amphipods could be split into two groups; one characterised by a high degree of heterogeneity and the other by a low degree of heterogeneity. The talitrid amphipods generally displayed a low degree of heterogeneity similar to, although still distinct from, the second gammarid category. Haemocyanin from the Hyalidae, a family allied to the talitrids was highly heterogeneous, similar to the first gammarid group and unlike the talitrids. Isopod haemocyanin banding patterns were more similar to one another than to any of the amphipod or decapod species examined. In general, the molecular weights of the amphipod Hcs tended to be greater than those of the isopods, with the decapods being lowest of all. It is suggested that Hc subunit heterogeneity may be a useful tool for investigating speciation and speciation events, and for reliably separating very closely-related species (e.g. Gammarus spp.), purely on the basis of their Hc subunit compositions.     

The complete mitochondrial genome of the Hawaiian anchialine shrimp Halocaridina rubra Holthuis, 1963 (Crustacea: Decapoda: Atyidae)

Shrimp of the family Atyidae are important members of nearly all tropical (and most temperate) fresh and brackish water ecosystems in the world. To date, a complete mitochondrial genome from this important crustacean group has not been reported. Here, we present the complete mitochondrial DNA sequence of the Hawaiian atyid Halocaridina rubra [Holthuis, L.B., 1963. On red coloured shrimps (Decapoda, Caridea) from tropical land-locked saltwater pools. Zool. Meded.16, 261-279.] (Crustacea: Decapoda: Atyidae). The genome is a circular molecule of 16,065 bp and encodes the 37 mitochondrial genes (13 protein-coding, 22 tRNAs, and two rRNAs) typically found in the metazoa. Gene order and orientation in the H. rubra mitochondrial genome is syntenic with most malacostracans that have been examined to date. Of special note is the absence of the dihydrouridine (DHU) arm stem from tRNA(Tyr) and the use of CCG as an initiation codon for cytochrome oxidase subunit I (COI); these represent the first reported examples of such phenomena in the Malacostraca. Phylogenetic analyses utilizing complete mitochondrial sequences from other malacostracans place H. rubra as sister to Macrobrachium rosenbergii, which also belongs to the Infraorder Caridea. However, the placement of this infraorder, as well as the Infraorder Dendrobrachiata, in the phylogeny of the Decapoda varied depending on outgroup selection. Data from additional mitochondrial genomes, such as basal decapods like the Stenopodidea, should contribute to a better overall understanding of decapod phylogenetics.     

The complete mitochondrial genomes of two common shrimps (Litopenaeus vannamei and Fenneropenaeus chinensis) and their phylogenomic considerations

Complete mitochondrial genomes have proven extremely valuable in helping to understand the evolutionary relationships among metazoans. However, uneven taxon sampling may lead to unclear or even erroneous phylogenetic topologies. The decapod crustaceans are relatively well-sampled, but sampling is still uneven within this group. We have sequenced the mitochondrial genomes of two shrimps Litopenaeus vannamei and Fenneropenaeus chinensis. As seen in other metazoans, the genomes contain a standard set of 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes and an AT-rich non-coding region. The gene arrangements are consistent with the pancrustacean ground pattern. Both the pattern of gene rearrangements and phylogenomic analyses using concatenated nucleic acid and amino acid sequences of the 13 mitochondrial protein-coding genes strengthened the support that Caridea and Palinura are primitive members of Pleocyemata. These sequences, in combination with two previously published penaeid mitochondrial genomes, suggest that genera within the family Penaeidae have the following relationship: (((Penaeus+Fenneropenaeus)+Litopenaeus)+Marsupenaeus). The analyses of nucleic acid and amino acid sequences of the mitochondrial genomes also strongly support the monophyly of Penaeidae, Brachyura and Pleocyemata. In addition, the analyses of the average Ka/Ks in the 13 mitochondrial protein-coding genes of penaeid shrimps indicated a strong purifying selection within this group.     

cAMP,not Ca2+/calmodulin,regulates the phosphorylation of acetylcholine receptor in Torpedo californica electroplax

The regulation of the phosphorylation of the acetylcholine receptor in electroplax membranes from Torpedo californica and of purified acetylcholine receptor was investigated. The phosphorylation of the membrane-bound acetylcholine receptor was not stimulated by Ca²⁺/calmodulin, nor was it inhibited by EGTA, but it was stimulated by the catalytic subunit of cAMP-dependent protein kinase, and was blocked by the protein inhibitor of cAMP-dependent protein kinase. Purified acetylcholine receptor was not phosphorylated by Ca²⁺/calmodulin-dependent protein kinase activity in electroplax membranes, nor by partially purified Ca²⁺/calmodulin-dependent protein kinases from soluble or particulate fractions from the electroplax. Of the four acetylcholine receptor subunits, termed α, β, γ and δ, only the γ- and δ-subunits were phosphorylated by the cAMP-dependent protein kinase (+cAMP), or by its purified catalytic subunits.     

Co-expression of γ2 Subunits Hinders Processing of N-Linked Glycans Attached to the N104 Glycosylation Sites of GABAA Receptor β2 Subunits

GABA_A receptors, the major mediators of fast inhibitory neuronal transmission, are heteropentameric glycoproteins assembled from a panel of subunits, usually including α and β subunits with or without a γ2 subunit. The α1β2γ2 receptor is the most abundant GABA_A receptor in brain. Co-expression of γ2 with α1 and β2 subunits causes conformational changes, increases GABA_A receptor channel conductance, and prolongs channel open times. We reported previously that glycosylation of the three β2 subunit glycosylation sites, N32, N104 and N173, was important for α1β2 receptor channel gating. Here, we examined the hypothesis that steric effects or conformational changes caused by γ2 subunit co-expression alter the glycosylation of partnering β2 subunits. We found that co-expression of γ2 subunits hindered processing of β2 subunit N104 N-glycans in HEK293T cells. This γ2 subunit-dependent effect was strong enough that a decrease of γ2 subunit expression in heterozygous GABRG2 knockout (γ2^+/?) mice led to appreciable changes in the endoglycosidase H digestion pattern of neuronal β2 subunits. Interestingly, as measured by flow cytometry, γ2 subunit surface levels were decreased by mutating each of the β2 subunit glycosylation sites. The β2 subunit mutation N104Q also decreased GABA potency to evoke macroscopic currents and reduced conductance, mean open time and open probability of single channel currents. Collectively, our data suggested that γ2 subunits interacted with β2 subunit N-glycans and/or subdomains containing the glycosylation sites, and that γ2 subunit co-expression-dependent alterations in the processing of the β2 subunit N104 N-glycans were involved in altering the function of surface GABA_A receptors.     

Biochemical and molecular characterisation of hemocyanin from the amphipod <Emphasis Type="Italic">Gammarus roeseli</Emphasis>: complex pattern of hemocyanin subunit evolution in Crustacea

Hemocyanin is a copper-containing respiratory protein that is widespread within the arthropod phylum. Among the Crustacea, hemocyanins are apparently restricted to the Malacostraca. While well-studied in Decapoda, no hemocyanin sequence has been known from the ’lower’ Malacostraca. The hemocyanin of the amphipod Gammarus roeseli is a hexamer that consists of at least five distinct subunits. The complete cDNA sequence of one subunit and a tentative partial sequence of another subunit have been determined. The complete G. roeseli hemocyanin subunit comprises 2,150 bp, which translates in a protein of 672 amino acids with a molecular mass of 76.3 kDa. Phylogenetic analyses show that, in contrast to previous assumptions, the amphipod hemocyanins do not belong to the α-type of crustacean hemocyanin subunits. Rather, amphipod hemocyanins split from the clade leading to α and γ-subunits most likely at the time of separation of peracarid and eucarid Crustacea about 300 million years ago. Molecular clock analyses further suggest that the divergence of β-type subunits and other crustacean hemocyanins occurred around 315 million years ago (MYA) in the malacostracan stemline, while α- and γ-type subunits separated 258 MYA, and pseudohemocyanins and γ-subunits 210 million years ago.     

Crystal structure of the intact archaeal translation initiation factor 2 demonstrates very high conformational flexibility in the alpha- and beta-subunits

In Eukarya and Archaea, translation initiation factor 2 (eIF2/aIF2), which contains three subunits (α, β, and γ), is pivotal for binding of charged initiator tRNA to the small ribosomal subunit. The crystal structure of the full-sized heterotrimeric aIF2 from Sulfolobus solfataricus in the nucleotide-free form has been determined at 2.8-Å resolution. Superposition of four molecules in the asymmetric unit of the crystal and the comparison of the obtained structures with the known structures of the aIF2αγ and aIF2βγ heterodimers revealed high conformational flexibility in the α- and β-subunits. In fact, the full-sized aIF2 consists of a rigid central part, formed by the γ-subunit, domain 3 of the α-subunit, and the N-terminal α-helix of the β-subunit, and two mobile “wings,” formed by domains 1 and 2 of the α-subunit, the central part, and the zinc-binding domain of the β-subunit. High structural flexibility of the wings is probably required for interaction of aIF2 with the small ribosomal subunit. Comparative analysis of all known structures of the γ-subunit alone and within the heterodimers and heterotrimers in nucleotide-bound and nucleotide-free states shows that the conformations of switch 1 and switch 2 do not correlate with the assembly or nucleotide states of the protein.     

Purification and reversible subunit dissociation of overproduced Escherichia coli phenylalanyl-tRNA synthetase

Phenylalanyl-tRNA synthetase (EC 6.1.1.20) has been purified to homogeneity from a 100-fold overproducing Escherichia coli strain carrying a hybrid pBR322 plasmid containing the pheS-pheT locus. The purified enzyme is identical to the phenylalanyl-tRNA synthetase isolated from an haploid strain. The enzyme was found to dissociate in the presence of 0.5 M NaSCN and the α- and β-subunits composing the native α₂β₂ enzyme were separated by gel filtration. Neither isolated subunit showed significant catalytic activity. A complex indistinguishable from the native enzyme with full catalytic activity is recovered upon mixing the subunits. The N- and C-terminal sequences and the amino acid composition of each subunit were determined. They are compared to the available data concerning the primary structure of the subunits, as deduced from nucleotide sequencing of the pheS-pheT operon.     

Assembly of GABAA receptors (Review)

GABA_A receptors are the major inhibitory transmitter receptors in the central nervous system. They are chloride ion channels that can be opened by γ-aminobutyric acid (GABA) and are the targets of action of a variety of pharmacologically and clinically important drugs. GABA_A receptors are composed of five subunits that can belong to different subunit classes. The existence of 19 different subunits gives rise to the formation of a large variety of distinct GABA_A receptor subtypes in the brain. The majority of GABA_A receptors seems to be composed of two α, two β and one γ subunit and the occurrence of a defined subunit stoichiometry and arrangement in αβγ receptors strongly indicates that assembly of GABA_A receptors proceeds via defined pathways. Based on the differential ability of subunits to interact with each other, a variety of studies have been performed to identify amino acid sequences or residues important for assembly. Such residues might be involved in direct protein-protein interactions, or in stabilizing direct contact sites in other regions of the subunit. Several homo-oligomeric or hetero-oligomeric assembly intermediates could be the starting point of GABA_A receptor assembly but so far no unequivocal assembly mechanism has been identified. Possible mechanisms of assembly of GABA_A receptors are discussed in the light of recent publications.     

Replacement of a terminal cytochrome c oxidase by ubiquinol oxidase during the evolution of acetic acid bacteria

The bacterial aerobic respiratory chain has a terminal oxidase of the heme-copper oxidase superfamily, comprised of cytochrome c oxidase (COX) and ubiquinol oxidase (UOX); UOX evolved from COX. Acetobacter pasteurianus, an α-Proteobacterial acetic acid bacterium (AAB), produces UOX but not COX, although it has a partial COX gene cluster, ctaBD and ctaA, in addition to the UOX operon cyaBACD. We expressed ctaB and ctaA genes of A. pasteurianus in Escherichia coli and demonstrated their function as heme O and heme A synthases. We also found that the absence of ctaD function is likely due to accumulated mutations. These COX genes are closely related to other α-Proteobacterial COX proteins. However, the UOX operons of AAB are closely related to those of the β/γ-Proteobacteria (γ-type UOX), distinct from the α/β-Proteobacterial proteins (α-type UOX), but different from the other γ-type UOX proteins by the absence of the cyoE heme O synthase. Thus, we suggest that A. pasteurianus has a functional γ-type UOX but has lost the COX genes, with the exception of ctaB and ctaA, which supply the heme O and A moieties for UOX. Our results suggest that, in AAB, COX was replaced by β/γ-Proteobacterial UOX via horizontal gene transfer, while the COX genes, except for the heme O/A synthase genes, were lost.     

Immunochemical Localization of GABA<Subscript>A</Subscript> Receptor Subunits in the Freshwater Polyp <Emphasis Type="Italic">Hydra vulgaris</Emphasis> (Cnidaria,Hydrozoa)

γ-aminobutyric acid (GABA) receptors, responding to GABA positive allosteric modulators, are present in the freshwater polyp Hydra vulgaris (Cnidaria, Hydrozoa), one of the most primitive metazoans to develop a nervous system. We examined the occurrence and distribution of GABA_A receptor subunits in Hydra tissues by western blot and immunohistochemistry. Antibodies against different GABA_A receptor subunits were used in Hydra membrane preparations. Unique protein bands, inhibited by the specific peptide, appeared at 35, 60, ～50 and ～52 kDa in membranes incubated with α3, β1, γ3 or δ antibodies, respectively. Immunohistochemical screening of whole mount Hydra preparations revealed diffuse immunoreactivity to α3, β1 or γ3 antibodies in tentacles, hypostome, and upper part of the gastric region; immunoreactive fibers were also present in the lower peduncle. By contrast, δ antibodies revealed a strong labeling in the lower gastric region and peduncle, as well as in tentacles. Double labeling showed colocalization of α3/β1, α3/γ3 and α3/δ immunoreactivity in granules or cells in tentacles and gastric region. In the peduncle, colocalization of both α3/β1 and α3/γ3 immunoreactivity was found in fibers running horizontally above the foot. These data indicate that specific GABA_A receptor subunits are present and differentially distributed in Hydra body regions. Subunit colocalization suggests that Hydra GABA receptors are heterologous multimers, possibly sub-serving different physiological activities.     

Large Conductance Calcium-Activated Potassium Channels: Their Expression and Modulation of Glutamate Release from Nerve Terminals Isolated from Rat Trigeminal Caudal Nucleus and Cerebral Cortex

Large conductance, calcium-activated potassium channels [big potassium (BK) channel] consist of a tetramer of pore-forming α-subunit and distinct accessory β-subunits (β1–4) that modify the channel’s properties. In this study, we analyzed the effects of BK channel activators and blockers on glutamate and γ-aminobutyric acid (GABA) release from synaptosomes isolated from the cerebral cortices or trigeminal caudal nuclei (TCN) of rats. Real-time polymerase chain reaction was used to characterize BK channel α and β(1–4) subunit expression in the cortex and in the trigeminal ganglia (TG), whose neurons project primary terminal afferents into the TCN. Immunocytochemistry was used to localize these subunits on cortical and TCN synaptosomes. The BK channels regulating [³H]D-aspartate release from primary afferent nerve terminals projecting into the TCN displayed limited sensitivity to iberiotoxin, whereas those expressed on cortical synaptosomes were highly sensitive to this toxin. BK channels did not appear to be present on GABAergic nerve terminals from the TCN since [³H]-γ-aminobutyric acid release in this model was unaffected by BK channel activators or blockers. Gene expression studies revealed expression levels of the α subunit in the TG that were only 31.2 ± 2.1 % of those found in cortical tissues. The β4 subunit was the accessory subunit expressed most abundantly in both the cortex and TG. Levels of β1 and β2 were low in both these areas although β2 expression in the TG was higher than that found in the cortex. Immunocytochemistry experiments showed that co-localization of α and β4 subunits (the accessory subunit most abundantly expressed in both brain areas) was more common in TCN synaptosomes than in cortical synaptosomes. On the basis of these findings, it is reasonable to hypothesize that BK channels expressed on glutamatergic terminals in the TCN and cortex have distinct pharmacological profiles, which probably reflect different α and β subunit combinations. Channels in the cortex seem to be composed mainly of α subunits and to a lesser degree by α and β4 subunits, whereas in the TG the α + β4 combination seems to prevail (although α and/or α + β2 channels cannot be excluded). In light of the BK channels’ selective control of excitatory transmission and their pharmacological diversity, their effects on primary glutamatergic afferents projecting to TCN represent a potential target for drug therapy of migraines and other types of orofacial pain.