A convenient method for preparation of biologically active recombinant CHH of the kuruma prawn, Marsupenaeus japonicus, using the bacterial expression system

Crustacean hyperglycemic hormone (CHH) not only plays an important role in the modulation of hemolymph glucose level but also functions in other biological events including molting, reproduction and stress response. Of the six CHHs characterized in Marsupenaeus japonicus, an expression system for recombinant Pej-SGP-VII (rPej-SGP-VII-amide) has not yet been established. Here, we established a procedure using a Nus-tag for solubilization, thereby soluble and biologically active rPej-SGP-VII-amide could successfully be obtained by a simpler procedure than previous ones used for producing other recombinant Pej-SGPs (Pej-SGP-I, III and IV). It was found that rPej-SGP-VII-amide thus obtained had the correct arrangement of intramolecular disulfide bonds and helix-rich secondary structure. The established expression system for rPej-SGP-VII-amide may be applicable for the preparation of other recombinant CHHs.     

Effect of a glycine residue insertion into crustacean hyperglycemic hormone on hormonal activity

Crustacean hyperglycemic hormone (CHH) and molt-inhibiting hormone (MIH) have similar amino acid sequences and therefore comprise a peptide family referred to as the CHH family. All MIHs unexceptionally have an additional glycine residue at position 12, which is lacking in all CHHs. In order to understand the relevance of the absence of the glycine residue for hyperglycemic activity, a mutant CHH having a glycine residue insertion was prepared, and its hyperglycemic activity was assessed. This mutant CHH had the same disulfide bond arrangement as the recombinant CHH produced in Escherichia coli cells, and exhibited a similar circular dichroism spectrum to the recombinant CHH, indicating that the two CHHs possessed similar conformations. The mutant CHH showed a hyperglycemic effect weaker than the recombinant CHH by about one order of magnitude. These results suggest that the insertion of a glycine residue is one of the indices for structural and functional divergence of the CHH family peptides.     

Functional Assessment of Residues in the Amino- and Carboxyl-Termini of Crustacean Hyperglycemic Hormone (CHH) in the Mud Crab Scylla olivacea Using Point-Mutated Peptides

To assess functional importance of the residues in the amino- and carboxyl-termini of crustacean hyperglycemic hormone in the mud crab Scylla olivacea (Sco-CHH), both wild-type and point-mutated CHH peptides were produced with an amidated C-terminal end. Spectral analyses of circular dichroism, chromatographic retention time, and mass spectrometric analysis of the recombinant peptides indicate that they were close in conformation to native CHH and were produced with the intended substitutions. The recombinant peptides were subsequently used for an in vivo hyperglycemic assay. Two mutants (R13A and I69A rSco-CHH) completely lacked hyperglycemic activity, with temporal profiles similar to that of vehicle control. Temporal profiles of hyperglycemic responses elicited by 4 mutants (I2A, F3A, D12A, and D60A Sco-CHH) were different from that elicited by wild-type Sco-CHH; I2A was unique in that it exhibited significantly higher hyperglycemic activity, whereas the remaining 3 mutants showed lower activity. Four mutants (D4A, Q51A, E54A, and V72A rSco-CHH) elicited hyperglycemic responses with temporal profiles similar to those evoked by wild-type Sco-CHH. In contrast, the glycine-extended version of V72A rSco-CHH (V72A rSco-CHH-Gly) completely lost hyperglycemic activity. By comparing our study with previous ones of ion-transport peptide (ITP) and molt-inhibiting hormone (MIH) using deleted or point-mutated mutants, detail discussion is made regarding functionally important residues that are shared by both CHH and ITP (members of Group I of the CHH family), and those that discriminate CHH from ITP, and Group-I from Group-II peptides. Conclusions summarized in the present study provide insights into understanding of how functional diversification occurred within a peptide family of multifunctional members.     

Functional analysis of crustacean Hyperglycemic Hormone by in vivo assay with wild-type and mutant recombinant proteins

The neuro-endocrine X-organ sinus-gland complex regulates important crustacean physiological processes, such as growth, reproduction and molting. Its major products are the neuropeptides of the cHH/MIH/GIH family. Until now the structure-function relationships of these neuropeptides were established by sequence comparison. To study the functional relevance of conserved amino acid residues or peptide motifs, we generated point and deletion mutants of the Norway lobster Nephrops norvegicus cHH. The wild type mature neuropeptide cHH and its mutant forms were expressed in bacteria as fusion proteins and assayed in vivo to assess their hyperglycemic activity. The wild type cHH had a hyperglycemic activity similar to that of cHH present in an eyestalk extract, and it was blocked by an anti-recombinant cHH antibody. Bioassays of cHHs, obtained by a progressive deletion of five highly conserved motifs, showed that the only deleted cHH, which conserves a hyperglycemic activity, is the one lacking the C-terminal motif, but still retaining all the motifs reported to be important for functional specificity and three-dimensional structure. All the cHH point mutants lacked a hyperglycemic activity. These results identify amino acid residues that are required for the hyperglycemic activity of cHH.     

Disulfide and secondary structures of recombinant human granulocyte colony stimulating factor

Molecular characteristics and secondary structures of recombinant methionyl human granulocyte colony stimulating factor produced by genetically engineered Escherichia coli are described. Limited radiolabeling of the protein with tritiated iodoacetate and determination of the labeled residue revealed that this recombinant protein contains only one free cysteine at position 17 which is not essential for activity. The free cysteine is inaccessible to modification unless the molecule is unfolded under denaturing conditions. The molecule forms two disulfide bridges which were assigned as Cys(36)-Cys(42) and Cys(64)-Cys(74) based on the results of isolation and characterization of disulfide-containing peptides obtained from a subtilisin digest of the intact protein. CD analyses and secondary structure prediction suggest that the molecule is abundant in alpha-helical structures.     

19F NMR of 5-fluorouracil-substituted transfer RNA transcribed in vitro: resonance assignment of fluorouracil-guanine base pairs.

5-Fluorouracil is readily incorporated into active tRNA(Val) transcribed in vitro from a recombinant phagemid containing a synthetic E. coli tRNA(Val) gene. This tRNA has the expected sequence and a secondary and tertiary structure resembling that of native 5-fluorouracil-substituted tRNA(Val), as judged by 19F NMR spectroscopy. To assign resonances in the 19F spectrum, mutant phagemids were constructed having base changes in the tRNA gene. Replacement of fluorouracil in the T-stem with cytosine, converting a FU-G to a C-G base pair, results in the loss of one downfield peak in the 19F NMR spectrum of the mutant tRNA(Val). The spectra of other mutant tRNAs having guanine for adenine substitutions that convert FU-A to FU-G base pairs all have one resonance shifted 4.5 to 5 ppm downfield. These results allow assignment of several 19F resonances and demonstrate that the chemical shift of the 19F signal from base-paired 5-fluorouracil differs considerably between Watson-Crick and wobble geometry.     

Foldability, enzymatic activity, and interacting ability of barnase mutants obtained by permutation of secondary structure units

Barnase, a well-characterized ribonuclease, has been decomposed into six modules (M1-M6) or secondary structure units (S1-S6). We have studied the foldability and activity of the barnase mutants obtained by permutation of the four internal modules (M2-M5) or secondary structure units (S2-S5) to investigate whether permutation of these building blocks is a useful way to create foldable and/or functional proteins. In this study, we found that one of the secondary structure unit mutants was expressed in Escherichia coli only when His102 was substituted by alanine, which is a catalytic residue of wild-type barnase. This mutant (S2354H102A) had ordered conformations, which unfolded cooperatively during urea-induced unfolding experiments. S2354H102A interacted with other barnase mutants to show a distinct RNase activity, although its own activity was quite weak. This interaction was specific, because S2354H102A interacted with only barnase mutants having His 102 and certain orders of the secondary structure units giving a distinct RNase activity. These results suggest that secondary structure units permuted in barnase mutants maintain their intrinsic "interacting ability" that is used for the folding of wild-type barnase, and the units can form certain conformations that complement those of the appropriate counterparts. Seven of 23 secondary structure unit mutants and only 2 of 23 module mutants had RNase activity. On the basis of the results of analyses of foldability and RNase activity of the mutants performed in this and previous studies, we conclude that secondary structure units are more suitable than modules as building blocks to create novel foldable and/or functional proteins in the case of barnase.     

Methods of preparation of recombinant proteins-cytokines. IV. Renaturation of recombinant human interleukin-3

Renaturation of recombinant human interleukin-3 produced as inclusion bodies in the transformed cells of Escherichia coli was studied and optimized. Importance was shown of removing from the protein solution the hydrophobic cellular components causing irreversible aggregation of the protein under renaturation conditions. An effect of pH on the secondary structure of the denatured protein was revealed by CD spectroscopy. It was thereby found that at pH 8.5, which is the optimal value for denaturation, the protein has the secondary structure most close to the native one. The isolation according to the scheme proposed allows preparation of interleukin-3 in 50% yield with 99% purity and biological activity 2 x 10(7) U/mg.     

Immunological comparison of native and recombinant egg allergen, ovalbumin, expressed in Escherichia coli

Chicken ovalbumin is one of the major egg white allergens which causes IgE-mediated food hypersensitivity. A gene encoding for chicken ovalbumin (Gad dI) was isolated from chicken oviduct by PCR amplification and was cloned under the control of T5 promoter fused with a six-histidine tag at the N-terminal end. Escherichia coli harbouring this construct expressed high quantities of the recombinant protein in the form of soluble fraction. The protein was purified using affinity chromatography on a Ni(2+)-nitrilotriacetic acid agarose column and was further purified to homogeneity by ion exchange chromatography. Homogeneity was confirmed through SDS-PAGE, Western blot and secondary conformation analysis. The reactivity of the recombinant and native protein was tested against six egg allergic human patient's sera and the IgE and IgG binding activity was tested using both Western blot and ELISA. When compared to native ovalbumin, the recombinant protein had similar binding activity in immunoblotting, but slightly increased activity by ELISA. Circular dichroism revealed that the recombinant protein had a slightly less compact structure than the native form. Both antigens exhibited a similar immunogenicity in mice.     

Uncovering the beginning of diabetes: the cellular redox status and oxidative stress as starting players in hyperglycemic damage

Early hyperglycemic insult can lead to permanent, cumulative damage that might be one of the earliest causes for a pre-diabetic situation. Despite this, the early phases of hyperglycemic exposure have been poorly studied. We have previously demonstrated that mitochondrial injury takes place early on upon hyperglycemic exposure. In this work, we demonstrate that just 1 h of hyperglycemic exposure is sufficient to induce increased mitochondrial membrane potential and generation. This is accompanied (and probably caused) by a decrease in the cells’ NAD⁺/NADH ratio. Furthermore, we show that the modulation of the activity of parallel pathways to glycolysis can alter the effects of hyperglycemic exposure. Activation of the pentose phosphate pathway leads to diminished effects of glucose on the above parameters, either by removing glucose from glycolysis or by NADPH generation. We also demonstrate that the hexosamine pathway inhibition also leads to a decreased effect of excess glucose. So, this work demonstrates the need for increased focus of study on the reductive status of the cell as one of the most important hallmarks of initial hyperglycemic damage.     

4alpha-Phorbol negates the inhibitory effects of phorbol-12-myristate-13-acetate on human cilia and alters the phosphorylation of PKC

RIO1 and Rio-related proteins display little similarity of primary sequence with conventional protein kinases. Based on secondary structure alignments, we show that it contains the domain structure (subdomains I-XI) and conserved secondary structure elements found in conventional protein kinases. We show that recombinant wild-type Rio1p isolated from Escherichia coli displays kinase activity which depends on autophosphorylation and magnesium or manganese as ATP-activating ions. An initial biochemical characterization of Rio1p is presented.     

UDP-galactose 4-epimerase from Kluyveromyces fragilis: equilibrium unfolding studies

UDP-galactose 4-epimerase from yeast (Kluyveromyces fragilis) is a homodimer of total molecular mass 150 kDa having possibly one mole of NAD/dimer acting as a cofactor. The molecule could be dissociated and denatured by 8 M urea at pH 7.0 and could be functionally reconstituted after dilution with buffer having extraneous NAD. The unfolded and refolded equilibrium intermediates of the enzyme between 0-8 M urea have been characterized in terms of catalytic activity, NADH like characteristic coenzyme fluorescence, interaction with extrinsic fluorescence probe 1-anilino 8-naphthelene sulphonic acid (ANS), far UV circular dichroism spectra, fluorescence emission spectra of aromatic residues and subunit dissociation. While denaturation monitored by parameters associated with active site region e.g. inactivation and coenzyme fluorescence, were found to be cooperative having delta G between -8.8 to -4.4 kcals/mole, the overall denaturation process in terms of secondary and tertiary structure was however continuous without having a transition point. At 3 M urea a stable dimeric apoenzyme was formed having 65% of native secondary structure which was dissociated to monomer at 6 M urea with 12% of the said structure. The unfolding and refolding pathways involved identical structures except near the final stage of refolding where catalytic activity reappeared.     

Expression and applications of recombinant crustacean hyperglycemic hormone from eyestalks of white shrimp (Litopenaeus vannamei) against bacterial infection

Crustacean hyperglycemic hormone (CHH) has many functions to regulate carbohydrate metabolism, ecdysis and reproduction including ion transport in crustaceans. The cDNA encoding CHH peptides containing 369 bp open reading frame encoding 122 amino acids was cloned from eyestalk of white shrimp (Litopenaeus vannamei) and was produced by a bacterial expression system. The biological activity of recombinant L. vannamei crustacean hyperglycemic hormone (rLV-CHH) was tested. The hemolymph glucose level of shrimp increased two-fold at 1h after the rLV-CHH injection and then returned to normal after 3h. In addition to the effect of rLV-CHH administration (25 μg/shrimp) on immunological responses of white shrimp against pathogenic bacteria, Vibrio harveyi was studied. Results showed that the blood parameters of shrimp injected with rLV-CHH; the THC, PO activity, serum protein level and clearance ability to V. harveyi, were also higher than those of Neg-protein and PBS-injected shrimp. The survival of shrimp injected with rLV-CHH was significantly higher (66.0%) than shrimp that injected with Neg-protein (33.3%) and PBS (28.9%) after 14 days. It is possible that the administration of rLV-CHH in L. vannamei exhibited a higher immune response related to resistance against V. harveyi infection.     

Cloning and characterization of a molt-inhibiting hormone-like peptide from the prawn Marsupenaeus japonicus

Recently, it was demonstrated by PCR amplification that an additional molt-inhibiting hormone (MIH)-like peptide was present in the kuruma prawn Marsupenaeus japonicus. In this study, a cDNA encoding this peptide designated Pej-MIH-B was cloned. The Pej-MIH-B gene was expressed strongly in the nerve cord, and weakly in the eyestalk. It was possible to isolate Pej-MIH-B from the sinus glands in the eyestalks. The recombinant Pej-MIH-B expressed in Escherichia coli showed low molt-inhibiting activity, but did not exhibit hyperglycemic activity. These results suggest that Pej-MIH-B does not function as MIH or CHH intrinsically, but may have some unknown functions.     

The lysozyme from insect (Manduca sexta) is a cold-adapted enzyme

Enzymatic activity is dependent on temperature, although some proteins have evolved to retain activity at low temperatures at the expense of stability. Cold adapted enzymes are present in a variety of organisms and there is ample interest in their structure-function relationships. Lysozyme (E.C. 3.2.1.17) is one of the most studied enzymes due to its antibacterial activity against Gram positive bacteria and is also a cold adapted protein. In this work the characterization of lysozyme from the insect Manduca sexta and its activity at low temperatures is presented. Both M. sexta lysozymes natural and recombinant showed a higher content of alpha-helix secondary structure compared to that of hen egg white lysozyme and a higher specific enzymatic activity in the range of 5-30 degrees C. These results together with measured thermodynamic activation parameters support the designation of M. sexta lysozyme as a cold adapted enzyme. Therefore, the insect recombinant lysozyme is feasible as a model for structure-function studies for cold-adapted proteins.     

Nuclease S1-sensitive sites on superhelical DNA molecules carrying the LTR region of Moloney murine leukemia virus

The long terminal repeat (LTR) from proviral DNA of Moloney murine leukemia virus (Mo-MLV) was cloned on a derivative of pBR322, and after introducing superhelical torsions into the resulting recombinant, the sites of conformational transition were investigated by the nuclease S1-digestion method. With an increase in the negative linking differences, fourteen dominant cutting sites were identified, of which two were mapped inside the LTR and one at the 3' end of the LTR. By searching the sequence data, all these sites were localized in the regions having either palindromic sequences or AT-rich sequences. Free energy calculation for the local secondary structure on one strand indicated that nuclease S1 attacked the palindromic sequence regions which could form relatively stable hairpin structures. Under the conditions used, no correlation was found between the S1-sensitive sites and the potential Z-DNA-forming regions, including those within the enhancer sequence.     

Secondary structure of a crustacean neuropeptide hormone family by means of CD

Three hormonal neuropeptides have been purified from the sinus gland of the Mexican crayfish Procambarus bouvieri by means of a single-step HPLC method: The molt-inhibiting hormone (MIH) and two isoforms of the crustacean hyperglycemic hormone (CHH-B and CHH-C). Compositional analysis and partial characterization of three neuropeptides revealed such a high degree of homology that we consider them to be members of a family. Circular dichroic spectra of the three neuropeptides showed that the secondary structure of both isoforms of the CHH are very similar, but that there are important differences in secondary structure between MIH and the CHHs, especially in helix content and in disordered regions.     

Recombinant sterol esterase from Ophiostoma piceae: an improved biocatalyst expressed in Pichia pastoris

ABSTRACT: BACKGROUND: The ascomycete Ophiostoma piceae produces a sterol esterase (OPE) with high affinity towards p-nitrophenol, glycerol and sterol esters. Its hydrolytic activity on natural mixtures of triglycerides and sterol esters has been proposed for pitch biocontrol in paper industry since these compounds produce important economic losses during paper pulp manufacture. RESULTS: Recently, this enzyme has been heterologously expressed in the methylotrophic yeast Pichia pastoris, and the hydrolytic activity of the recombinant protein (OPE*) studied. After the initial screening of different clones expressing the enzyme, only one was selected for showing the highest production rate. Different culture conditions were tested to improve the expression of the recombinant enzyme. Complex media were better than minimal media for production, but in any case the levels of enzymatic activity were higher (7-fold in the best case) than those obtained from O. piceae. The purified enzyme had a molecular mass of 76 kDa, higher than that reported for the native enzyme under SDS-PAGE (60 kDa). Steadystate kinetic characterization of the recombinant protein showed improved catalytic efficiency for this enzyme as compared to the native one, for all the assayed substrates (p-nitrophenol, glycerol, and cholesterol esters). Different causes for this were studied, as the increased glycosylation degree of the recombinant enzyme, their secondary structures or the oxidation of methionine residues. However, none of these could explain the improvements found in the recombinant protein. N-terminal sequencing of OPE* showed that two populations of this enzyme were expressed, having either 6 or 8 amino acid residues more than the native one. This fact affected the aggregation behaviour of the recombinant protein, as was corroborated by analytical ultracentrifugation, thus improving the catalytic efficiency of this enzyme. CONCLUSION: P. pastoris resulted to be an optimum biofactory for the heterologous production of recombinant sterol esterase from O. piceae, yielding higher activity levels than those obtained with the saprophytic fungus. The enzyme showed improved kinetic parameters because of its modified N-terminus, which allowed changes in its aggregation behaviour, suggesting that its hydrophobicity has been modified.     

The Effect of Conditions of the Expression of the Recombinant Outer Membrane Phospholipase А<Subscript>1</Subscript> from <Emphasis Type="Italic">Yersinia pseudotuberculosis</Emphasis> on the Structure and Properties of Inclusion Bodies

The effect of the concentration of an inducer (IPTG) and the time of induction at 37°С on the heterologous synthesis of the mature membrane protein phospholipase А₁ (PldA) from Yersinia pseudotuberculosis in the form of inclusion bodies (IBs) and on the physicochemical and structural characteristics of IBs has been studied. The sizes, shape, stability (solubility in urea and detergents, resistance against proteolysis), the secondary structure of the protein of IBs, and the presence of amyloid structures have been determined by electron microscopy, dynamic light scattering, and optical spectroscopy. It was found that IBs have a shape close to spherical and a rough surface and are cleaved by proteinase K. The protein contained in IBs has an ordered secondary structure with a high content of β-structure. As the inducer concentration and the time of expression increase, the conformation of the recombinant protein in IBs undergoes changes, as indicated by an increase in the stability of IBs and a decrease in the enzymatic activity of the protein. When IBs are dissolved in 0.06% SDS and 5 M urea, the recombinant protein retains the secondary structure in a partially modified form, and the addition of a zwitterionic detergent at a micellar concentration does not transform the protein conformation into the native one.     

Anti-CHH antibody causes impaired hyperglycemia in Penaeus monodon

Crustacean hyperglycemic hormone (CHH) plays a major role in controlling glucose level in the haemolymph and also triggers important events during molting and reproductive cycles. In Penaeus monodon, three types of CHH, namely Pem-CHH1, Pem-CHH2 and Pem-CHH3, have been previously characterized. In this study, mouse polyclonal antibody was raised against recombinant Pem-CHH1 that was expressed in Escherichia coli. The anti-Pem-CHH1 antibody recognized all three types of Pem-CHHs but did not cross-react with either related hormone, molt-inhibiting hormone of P. monodon, or unrelated human growth hormone. The hyperglycemic activity in the extract from the eyestalk neural tissues was significantly depleted after incubating with anti-Pem-CHH antibody. Direct injection of the antibody into shrimp caused about 30-50% reduction in the haemolymph glucose level. The result demonstrates the ability of anti-Pem-CHH1 antibody to deplete the activity of CHH in vivo, and thus provides a possibility of using anti-Pem-CHH1 antibody to inhibit the hormone activity as a strategy to modulate growth and reproduction in this species.