Hexameric Protein Research Articles

RNA helicases and remodeling proteins

It is becoming increasingly clear that RNA molecules play a major role in all aspects of metabolism. The conformational state and stability of RNA are controlled by RNA remodeling proteins, which are ubiquitous motor proteins in the cell. Here, we review advances in our understanding of the structure and function of three major structural families of RNA remodeling proteins, the hexameric ring proteins, the processive monomeric RNA translocase/helicases, and the functionally diverse DEAD-box remodeling proteins. New studies have revealed molecular mechanisms for coupling between ATP hydrolysis and unwinding, the physical basis for regulatory control by cofactors, and novel functions for RNA remodeling proteins.

PDGFR Expression Distinguishes GFAP-Expressing Neural Stem Cells from PDGF-Responsive Neural Precursors in the Adult Periventricular Area

Jackson et al. (2006) have reported that adult glial fibrillary acid protein (GFAP)-expressing neural stem cells (NSCs) also express platelet-derived growth factor (PDGF) receptor-α (PDGFRα), and that their stimulation by PDGF induced the formation of a glioma-like mass. Here, we reexamined the relationship between PDGFRα and GFAP expression within the three-dimensional organization of the adult periventricular area. Using four independent PDGFRα antibodies, we found that adult mouse GFAP-expressing NSCs and PDGFRα-expressing cells represent two distinct populations of neural precursors. Examination of the adult periventricular area in a mouse line that expresses nuclear-localized enhanced green fluorescent protein under the control of the PDGFRα promoter confirmed that GFAP-expressing NSCs do not express PDGFRα. Furthermore, PDGF-responsive neural precursors were found at least one cell layer subjacent to the ependymal layer, and were evenly distributed across the lateral ventricular wall, which contrasts with the reported patchy and often ependymal localization of adult GFAP-expressing NSCs. Adult human PDGFRα-expressing neural precursors were also found not to express GFAP. PDGF-responsive neural precursors, but not GFAP-expressing NSCs, responded to infusions of PDGF by generating glioma-like masses. Our results do not support the view that GFAP-expressing NSCs are the origin of glioma-like masses that form after intraventricular PDGF infusion.

A257T Linker Region Mutant of T7 Helicase-Primase Protein Is Defective in DNA Loading and Rescued by T7 DNA Polymerase

The helicase and primase activities of the hexameric ring-shaped T7 gp4 protein reside in two separate domains connected by a linker region. This linker region is part of the subunit interface between monomers, and point mutations in this region have deleterious effects on the helicase functions. One such linker region mutant, A257T, is analogous to the A359T mutant of the homologous human mitochondrial DNA helicase Twinkle, which is linked to diseases such as progressive external opthalmoplegia. Electron microscopy studies show that A257T gp4 is normal in forming rings with dTTP, but the rings do not assemble efficiently on the DNA. Therefore, A257T, unlike the WT gp4, does not preassemble on the unwinding DNA substrate with dTTP without Mg(II), and its DNA unwinding activity in ensemble assays is slow and limited by the DNA loading rate. Single molecule assays measured a 45 times slower rate of A257T loading on DNA compared with WT gp4. Interestingly, once loaded, A257T has almost WT-like translocation and DNA unwinding activities. Strikingly, A257T preassembles stably on the DNA in the presence of T7 DNA polymerase, which restores the ensemble unwinding activity of A257T to ∼75% of WT, and the rescue does not require DNA synthesis. The DNA loading rate of A257T, however, remains slow even in the presence of the polymerase, which explains why A257T does not support T7 phage growth. Similar types of defects in the related human mitochondrial DNA helicase may be responsible for inefficient DNA replication leading to the disease states.

Synaptophysin Regulates the Kinetics of Synaptic Vesicle Endocytosis in Central Neurons

Despite being the most abundant synaptic vesicle membrane protein, the function of synaptophysin remains enigmatic. For example, synaptic transmission was reported to be completely normal in synaptophysin knockout mice; however, direct experiments to monitor the synaptic vesicle cycle have not been carried out. Here, using optical imaging and electrophysiological experiments, we demonstrate that synaptophysin is required for kinetically efficient endocytosis of synaptic vesicles in cultured hippocampal neurons. Truncation analysis revealed that distinct structural elements of synaptophysin differentially regulate vesicle retrieval during and after stimulation. Thus, synaptophysin regulates at least two phases of endocytosis to ensure vesicle availability during and after sustained neuronal activity.

Funnel-like Hexameric Assembly of the Periplasmic Adapter Protein in the Tripartite Multidrug Efflux Pump in Gram-negative Bacteria

Gram-negative bacteria expel diverse toxic chemicals through the tripartite efflux pumps spanning both the inner and outer membranes. The Escherichia coli AcrAB-TolC pump is the principal multidrug exporter that confers intrinsic drug tolerance to the bacteria. The inner membrane transporter AcrB requires the outer membrane factor TolC and the periplasmic adapter protein AcrA. However, it remains ambiguous how the three proteins are assembled. In this study, a hexameric model of the adapter protein was generated based on the propensity for trimerization of a dimeric unit, and this model was further validated by presenting its channel-forming property that determines the substrate specificity. Genetic, in vitro complementation, and electron microscopic studies provided evidence for the binding of the hexameric adapter protein to the outer membrane factor in an intermeshing cogwheel manner. Structural analyses suggested that the adapter covers the periplasmic region of the inner membrane transporter. Taken together, we propose an adapter bridging model for the assembly of the tripartite pump, where the adapter protein provides a bridging channel and induces the channel opening of the outer membrane factor in the intermeshing tip-to-tip manner.

Abstract 421: Epidermal growth factor-like repeats of tenascin C alter melanoma cell migration both directly and through alteration of the ECM

Abstract In melanoma, the most aggressive type of skin cancer, invasion through the extracellular matrix (ECM) is a harbinger of dissemination and mortality. Highly upregulated in both primary invasive and metastatic melanoma is tenascin C (TNC), a hexameric ECM protein with a multidomain structure composed of the globular N-terminal domain, 14.5 epidermal growth factor (EGF)-like repeats, fibronectin-III like repeats and the C-terminal globular fibrinogen-like domain. Our group previously showed that the EGF-like repeats of TNC are able to bind the EGF receptor (EGFR) with μM affinity and preferentially activate the motogenic signaling cascades. Both normal melanocytes and melanoma cells migrate faster on surfaces coated with TNC. Therefore, we hypothesize that the increased levels of TNC in melanomas promote tumor cell migration and invasion heralding dissemination through enhanced EGFR signaling. We expressed EGF-like repeats of TNC (TNC-EGFL) in melanoma cell lines already expressing endogenous TNC and observed change in the cell morphology, and more importantly a prominent anti-adhesive phenotype. TNC-EGFL expressing cells attached with a four-hour lag compared to parental cell lines and detached to a greater extent in inverted centrifugation adhesion assays. This was not due to decreased viability of TNC-EGFL expressing cells as determined by dye exclusion assay. As this phenotype could not be reverted by adding anti-EGFR antibody, we speculate that EGFR signaling might be intracellular. In two-dimensional migration assays, TNC-EGFL expressing cells closed a gap more slowly, possibly due to inability to properly attach during migration, emphasizing the need of concerted action of different TNC domains in regulating migration. On the other hand, quantitative PCR array analysis of a panel of ECM and adhesion molecules revealed changes in matrix metalloproteinase (MMP) expression, specifically MMP-2, MMP-9 and MMP-14, and increase in TGF β1 expression, proteins associated with melanoma invasiveness and vasculogenic mimicry. These results imply that TNC-EGFL repeats are involved in melanoma invasion both by directly influencing cell migration and indirectly by altering the tumor microenvironment and may have implications for development of anti tumor therapies directed at the extracellular matrix. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 421. doi:10.1158/1538-7445.AM2011-421

Single Chain Forms of the Enhancer Binding Protein PspF Provide Insights into Geometric Requirements for Gene Activation

Genetic information in the DNA is accessed by the molecular machine RNA polymerase following a highly conserved process, invariably involving the transition between double-stranded and single-stranded DNA states. In the case of the bacterial enhancer-dependent RNA polymerase (which is essential for adaptive responses and bacterial pathogenesis), the DNA melting event depends on specialized hexameric AAA+ ATPase activators. Involvement of such factors in transcription was demonstrated 25 years ago, but why these activators need to be hexameric, whether all the subunits operate identically, what is the contribution of each of the six subunits within the hexamer (structural, functional, or both), and how many active subunits are required for transcription activation remain open questions. Using engineered single-chain polypeptides covalently linking two or three subunits of the activator (allowing the subunit distribution within a hexamer to be fixed), we now show that (i) individual subunits have differential contributions to the activities of the oligomer and (ii) only a fraction of the subunits within the hexameric ATPase is directly required for gene activation. We establish that nucleotide-dependent coordination across three subunits of the hexameric bacterial enhancer binding proteins (bEBPs) is necessary for engagement and remodeling of the closed complex (RPc). Outcomes revealed features of bEBP, distinguishing their mode of action from fully processive AAA+ proteins or from simple bimodal switches. We now propose that the hexamer functions with asymmetric organization, potentially involving a split planar (open ring) or spiral character.

Functional Implications of an Intermeshing Cogwheel-like Interaction between TolC and MacA in the Action of Macrolide-specific Efflux Pump MacAB-TolC

Macrolide-specific efflux pump MacAB-TolC has been identified in diverse gram-negative bacteria including Escherichia coli. The inner membrane transporter MacB requires the outer membrane factor TolC and the periplasmic adaptor protein MacA to form a functional tripartite complex. In this study, we used a chimeric protein containing the tip region of the TolC α-barrel to investigate the role of the TolC α-barrel tip region with regard to its interaction with MacA. The chimeric protein formed a stable complex with MacA, and the complex formation was abolished by substitution at the functionally essential residues located at the MacA α-helical tip region. Electron microscopic study delineated that this complex was made by tip-to-tip interaction between the tip regions of the α-barrels of TolC and MacA, which correlated well with the TolC and MacA complex calculated by molecular dynamics. Taken together, our results demonstrate that the MacA hexamer interacts with TolC in a tip-to-tip manner, and implies the manner by which MacA induces opening of the TolC channel.

Exciton Delocalization and Energy Transport Mechanisms in R-Phycoerythrin

Energy transport mechanisms in R-Phycoerythrin (RPE), a light harvesting protein located at the top of the phycobilisome antenna in red algae, are investigated using nonlinear optical spectroscopies and theoretical models. The RPE hexamer possesses a total of 30 bilin pigments, which can be subdivided into three classes based on their molecular structures and electronic resonance frequencies. Of particular interest to this study is the influence of exciton delocalization on the real-space paths traversed by photoexcitations as they concentrate on the lowest energy pigment sites. Transient grating measurements show that significant nuclear relaxation occurs at delay times less than 100 fs, whereas energy transport spans a wide range of time scales depending on the proximity of the initial and final states involved in the process. The fastest energy transport dynamics within the RPE complex are close to 1 ps; however, evidence for sub-100 fs exciton self-trapping is also obtained. In addition, photon echo experiments reveal vibronic interactions with overdamped and underdamped nuclear modes. To establish signatures of exciton delocalization, energy transport is simulated using both modified Redfield and Förster theories, which respectively employ delocalized and localized basis states. We conclude that exciton delocalization occurs between six pairs of phycoerythrobilin pigments (i.e., dimers) within the protein hexamer. It is interesting that these dimers are bound in locations analogous to the well-studied phycocyanobilin dimers of cyanobacterial allophycocyanin and c-phycocyanin in which wave function delocalization is also known to take hold. Strong conclusions regarding the electronic structures of the remaining pigments cannot be drawn based on the present experiments and simulations due to overlapping resonances and broad spectroscopic line widths, which prevent the resolution of dynamics at particular pigment sites.

A Quantitative Analysis of the Effect of Nucleotides and the M Domain on the Association Equilibrium of ClpB

ClpB is a hexameric molecular chaperone that, together with the DnaK system, has the ability to disaggregate stress-denatured proteins. The hexamer is a highly dynamic complex, able to reshuffle subunits. To further characterize the biological implications of the ClpB oligomerization state, the association equilibrium of the wild-type (wt) protein and of two deletion mutants, which lack part or the whole M domain, was quantitatively analyzed under different experimental conditions, using several biophysical [analytical ultracentrifugation, composition-gradient (CG) static light scattering, and circular dichroism] and biochemical (ATPase and chaperone activity) methods. We have found that (i) ClpB self-associates from monomers to form hexamers and higher-order oligomers that have been tentatively assigned to dodecamers, (ii) oligomer dissociation is not accompanied by modifications of the protein secondary structure, (iii) the M domain is engaged in intersubunit interactions that stabilize the protein hexamer, and (iv) the nucleotide-induced rearrangement of ClpB affects the protein oligomeric core, in addition to the proposed radial extension of the M domain. The difference in the stability of the ATP- and ADP-bound states [ΔΔG(ATP-ADP) = -10 kJ/mol] might explain how nucleotide exchange promotes the conformational change of the protein particle that drives its functional cycle.

Comparative Proteomic Analysis of Hemocyanins inDinocras cephalotesandPerla marginata(Plecoptera)

Hemocyanins are large oligomeric respiratory proteins found in many arthropods and mollusks. The overall expression of hemocyanin mRNA, revealed by studies on Plecoptera hemocyanin sequencing, has raised the question of whether the protein is expressed or not. In fact, the presence of expressed hemocyanin has only been reported in the literature for one species, Perla marginata (Panzer, 1799). In this paper, we report the presence of hemocyanin and hexamerin proteins in Dinocras cephalotes (Curtis, 1827), a species closely related to P. marginata. To assess the presence of hemocyanin, we used a reproducible and highly sensitive method based on liquid chromatography tandem mass spectrometry. We conclude that regardless of its putative function (respiratory, immune defense, storage protein), the hemocyanin is actually expressed in species in which its mRNA is present.

Molecular basis of LMAN1 in coordinating LMAN1-MCFD2 cargo receptor formation and ER-to-Golgi transport of FV/FVIII

AbstractThe LMAN1-MCFD2 (lectin, mannose binding 1/multiple coagulation factor deficiency protein 2) cargo receptor complex transports coagulation factors V (FV) and VIII (FVIII) from the endoplasmic reticulum (ER) to the ER-Golgi intermediate compartment (ERGIC). LMAN1 (ERGIC-53) is a hexameric transmembrane protein with a carbohydrate recognition domain (CRD) on the ER luminal side. Here, we show that mutations in the first beta sheet of the CRD abolish MCFD2 binding without affecting the mannose binding, suggesting that LMAN1 interacts with MCFD2 through its N-terminal beta sheet, consistent with recently reported crystal structures of the CRD-MCFD2 complex. Mutations in the Ca2+- and sugar-binding sites of the CRD disrupt FV and FVIII interactions, without affecting MCFD2 binding. This interaction is independent of MCFD2, as LMAN1 mutants defective in MCFD2 binding can still interact with FVIII. Thus, the CRD of LMAN1 contains distinct, separable binding sites for both its partner protein (MCFD2) and the cargo proteins (FV/FVIII). Monomeric LMAN1 mutants are defective in ER exit and unable to interact with MCFD2, suggesting that the oligomerization of LMAN1 is necessary for its cargo receptor function. These results point to a central role of LMAN1 in regulating the binding in the ER and the subsequent release in the ERGIC of FV and FVIII.

Purified Hexameric Epstein-Barr Virus-Encoded BARF1 Protein for Measuring Anti-BARF1 Antibody Responses in Nasopharyngeal Carcinoma Patients

WHO type III nasopharyngeal carcinoma (NPC) is highly prevalent in Indonesia and 100% associated with Epstein-Barr virus (EBV). NPC tumor cells express viral proteins, including BARF1, which is secreted and is considered to have oncogenic and immune-modulating properties. Recently, we found conserved mutations in the BARF1 gene in NPC isolates. This study describes the expression and purification of NPC-derived BARF1 and analyzes humoral immune responses against prototype BARF1 (B95-8) and purified native hexameric BARF1 in sera of Indonesian NPC patients (n = 155) compared to healthy EBV-positive (n = 56) and EBV-negative (n = 16) individuals. BARF1 (B95-8) expressed in Escherichia coli and baculovirus, as well as BARF1-derived peptides, did not react with IgG or IgA antibodies in NPC. Purified native hexameric BARF1 protein isolated from culture medium was used in enzyme-linked immunosorbent assay (ELISA) and revealed relatively weak IgG and IgA responses in human sera, although it had strong antibody responses to other EBV proteins. Higher IgG reactivity was found in NPC patients (P = 0.015) than in regional Indonesian controls or EBV-negative individuals (P < 0.001). IgA responses to native BARF1 were marginal. NPC sera with the highest IgG responses to hexameric BARF1 in ELISA showed detectable reactivity with denatured BARF1 by immunoblotting. In conclusion, BARF1 has low immunogenicity for humoral responses and requires native conformation for antibody binding. The presence of antibodies against native BARF1 in the blood of NPC patients provides evidence that the protein is expressed and secreted as a hexameric protein in NPC patients.

The first characterized asparaginase from a basidiomycete, Flammulina velutipes

Flammulina velutipes enjoys high popularity as an edible mushroom in Asian cuisines. Investigating the secretion of peptidases in nutrient media enriched with gluten, an enzyme was noticed that catalyzed the deamidation of L-asparagine and L-glutamine. The enzyme was purified to electrophoretic homogeneity by foaming and SEC. PAGE analysis revealed a protein of about 85 kDa with 13 kDa subunits indicating a hexameric protein. Degenerated primers were deduced from peptide fragments and the complete coding sequence of 372 bp was determined. The gene of Flammulina velutipes asparaginase (FvNase) over expressed in E. coli achieved an L-asparagine-hydrolyzing activity of 16 U/mL in crude extract, which was five times higher than its L-glutamine-hydrolyzing ability. The enzyme showed a pH-optimum at pH 7, remarkable tolerance towards elevated temperature and sodium chloride concentration in both the native and recombinant form, and no significant homology to any conserved domains of published asparaginases or glutaminases.

Conserved mutation of Epstein-Barr virus-encoded BamHI-A Rightward Frame-1 (BARF1) gene in Indonesian nasopharyngeal carcinoma

BackgroundBamHI-A rightward frame-1 (BARF1) is a carcinoma-specific Epstein-Barr virus (EBV) encoded oncogene. Here we describe the BARF1 sequence diversity in nasopharyngeal carcinoma (NPC), other EBV-related diseases and Indonesian healthy EBV carriers in relation to EBV genotype, viral load and serology markers. Nasopharyngeal brushings from 56 NPC cases, blood or tissue from 15 other EBV-related disorders, spontaneous B cell lines (LCL) from 5 Indonesian healthy individuals and several prototype EBV isolates were analysed by PCR-direct sequencing.ResultsMost NPC isolates revealed specific BARF1 nucleotide changes compared to prototype B95-8 virus. At the protein level these mutations resulted in 3 main substitutions (V29A, W72G, H130R), which are not considered to cause gross tertiary structure alterations in the hexameric BARF1 protein. At least one amino acid conversion was detected in 80.3% of NPC samples compared to 33.3% of non-NPC samples (p < 0.001) and 40.0% of healthy LCLs (p = 0.074). NPC isolates also showed more frequent codon mutation than non-NPC samples. EBV strain typing revealed most isolates as EBV type 1. The viral load of either NPC or non-NPC samples was high, but only in non- NPC group it related to a particular BARF1 variant. Serology on NPC sera using IgA/EBNA-1 ELISA, IgA/VCA-p18 ELISA and immunoblot score showed no relation with BARF1 sequence diversity (p = 0.802, 0.382 and 0.058, respectively). NPC patients had variable antibody reactivity against purified hexameric NPC-derived BARF1 irrespective of the endogenous BARF1 sequence.ConclusionThe sequence variation of BARF1 observed in Indonesian NPC patients and controls may reflect a natural selection of EBV strains unlikely to be predisposing to carcinogenesis. The conserved nature of BARF1 may reflect an important role in EBV (epithelial) persistence.

Myosin IIb Regulates Actin Dynamics during Synaptic Plasticity and Memory Formation

Reorganization of the actin cytoskeleton is essential for synaptic plasticity and memory formation. Presently, the mechanisms that trigger actin dynamics during these brain processes are poorly understood. In this study, we show that myosin II motor activity is downstream of LTP induction and is necessary for the emergence of specialized actin structures that stabilize an early phase of LTP. We also demonstrate that myosin II activity contributes importantly to an actin-dependent process that underlies memory consolidation. Pharmacological treatments that promote actin polymerization reversed the effects of a myosin II inhibitor on LTP and memory. We conclude that myosin II motors regulate plasticity by imparting mechanical forces onto the spine actin cytoskeleton in response to synaptic stimulation. These cytoskeletal forces trigger the emergence of actin structures that stabilize synaptic plasticity. Our studies provide a mechanical framework for understanding cytoskeletal dynamics associated with synaptic plasticity and memory formation.

The structures of mutant forms of Hfq fromPseudomonas aeruginosareveal the importance of the conserved His57 for the protein hexamer organization

The bacterial Sm-like protein Hfq forms homohexamers both in solution and in crystals. The monomers are organized as a continuous beta-sheet passing through the whole hexamer ring with a common hydrophobic core. Analysis of the Pseudomonas aeruginosa Hfq (PaeHfq) hexamer structure suggested that solvent-inaccessible intermonomer hydrogen bonds created by conserved amino-acid residues should also stabilize the quaternary structure of the protein. In this work, one such conserved residue, His57, in PaeHfq was replaced by alanine, threonine or asparagine. The crystal structures of His57Thr and His57Ala Hfq were determined and the stabilities of all of the mutant forms and of the wild-type protein were measured. The results obtained demonstrate the great importance of solvent-inaccessible conserved hydrogen bonds between the Hfq monomers in stabilization of the hexamer structure.

Characterization of recombinant prolyl aminopeptidase from Aspergillus oryzae

Prolyl aminopeptidase (PAP) degrades only amino-terminal proline from peptides. The food-grade fungus Aspergillus oryzae produces this enzyme only in small amounts. In this paper, we present efficient production of recombinant PAP with an overexpression system of A. oryzae and characterization of its biochemical properties. The gene encoding PAP was overexpressed as a His-tag fusion protein under a taka-amylase gene (amyB) promoter with a limited expressing condition in A. oryzae. The PAP activity in the mycelia grown in rich medium containing glucose (repressing condition) was twice that in starch (inducing condition). The enzyme prepared as cell-free extract was partially purified through two-step column chromatography. The PAP was estimated to be a hexameric protein and exhibited salt tolerance against NaCl of up to 4 mol l(-1). Aspergillus oryzae PAP was produced under the repressing condition of amyB promoter in a PAP-overexpressing strain and purified 1800-folds. Overproduction of PAP under promoter-inducing conditions led to an increase in inactive PAP, possibly because of irregular folding. PAP with a high specific activity and salt tolerance may be used effectively in the manufacturing processes of fermented foods.

Differential Gene Expression and Protein Abundance Evince Ontogenetic Bias toward Castes in a Primitively Eusocial Wasp

Polistes paper wasps are models for understanding conditions that may have characterized the origin of worker and queen castes and, therefore, the origin of paper wasp sociality. Polistes is “primitively eusocial” by virtue of having context-dependent caste determination and no morphological differences between castes. Even so, Polistes colonies have a temporal pattern in which most female larvae reared by the foundress become workers, and most reared by workers become future-reproductive gynes. This pattern is hypothesized to reflect development onto two pathways, which may utilize mechanisms that regulate diapause in other insects. Using expressed sequence tags (ESTs) for Polistes metricus we selected candidate genes differentially expressed in other insects in three categories: 1) diapause vs. non-diapause phenotypes and/or worker vs. queen differentiation, 2) behavioral subcastes of worker honey bees, and 3) no a priori expectation of a role in worker/gyne development. We also used a non-targeted proteomics screen to test for peptide/protein abundance differences that could reflect larval developmental divergence. We found that foundress-reared larvae (putative worker-destined) and worker-reared larvae (putative gyne-destined) differed in quantitative expression of sixteen genes, twelve of which were associated with caste and/or diapause in other insects, and they also differed in abundance of nine peptides/proteins. Some differentially-expressed genes are involved in diapause regulation in other insects, and other differentially-expressed genes and proteins are involved in the insulin signaling pathway, nutrient metabolism, and caste determination in highly social bees. Differential expression of a gene and a peptide encoding hexameric storage proteins is especially noteworthy. Although not conclusive, our results support hypotheses of 1) larval developmental pathway divergence that can lead to caste bias in adults and 2) nutritional differences as the foundation of the pathway divergence. Finally, the differential expression in Polistes larvae of genes and proteins also differentially expressed during queen vs. worker caste development in honey bees may indicate that regulatory mechanisms of caste outcomes share similarities between primitively eusocial and advanced eusocial Hymenoptera.

Identification of 86kDa protein as methionine rich hexamerin in the rice moth, Corcyra cephalonica

We cloned the complete cDNA of methionine rich hexamerin from rice moth, Corcyra cephalonica using RACE strategy. The amplicon size was 2.5 kb with an ORF of 2.31 kb. The cDNA clone showed high percentage of methionine (4.8%), which is consistent with the previously reported sequences in other insects. The Southern and Northern analysis carried out showed that methionine rich hexamerin in rice moth is a single copy gene. Multiple alignment analysis of amino acid sequence revealed that the cDNA clone is most similar to Plodia interpunctella hexamerin storage protein (74% identity). The calculated isoelectric point is 9.2. The deduced amino acid sequence corresponded to 86 kDa subunit of hexamerin protein in rice moth. The 86 kDa protein (methionine rich subunit) was purified and polyclonal antibodies were raised against the subunit to check the specificity of the purified subunit. The developmental profile of 86 kDa subunit during the larval stages both in the fat body and haemolymph show that it is present at a higher concentration during the LLI (late-last instar) larval stage compared to the previous stages. The present work carried out shows that the methionine rich hexamerin cloned is the 86 kDa subunit of hexamerin which was identified previously by our group in rice moth, C. cephalonica.