Bacterial expression of two human aryl sulfotransferases

The effect of replacing a single codon in the N-terminal of human aryl sulfotransferase (HAST) 1 and 3 with one that is more commonly found in E. coli genes was assessed. The pKK233-2 E. coli expression vector was employed and the polymerase chain reaction (PCR) was used to introduce the 5' nucleotide substitution, at the same time maintaining the fidelity of the amino acid sequence. The data indicates that this change had a minimal effect on protein production, subcellular localization or, in the case of HAST3, catalytic activity. In general, the pKK233-2 E. coli vector has been less than optimal for expressing human sulfotransferase cDNAs. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.

Spectroscopic identification of a dinuclear metal centre in manganese(II)-activated aminopeptidase P from Escherichia coli: implications for human prolidase

Electron paramagnetic resonance (EPR) spectra and X-ray absorption (EXAFS and XANES) data have been recorded for the manganese enzyme aminopeptidase P (AMPP, PepP protein) from Escherichia coli. The biological function of the protein, a tetramer of 50-kDa subunits, is the hydrolysis of N-terminal Xaa-Pro peptide bonds. Activity assays confirm that the enzyme is activated by treatment with Mn2+. The EPR spectrum of Mn2+-activated AMPP at liquid-He temperature is characteristic of an exchange-coupled dinuclear Mn(II) site, the Mn-Mn separation calculated from the zero-field splitting D of the quintet state being 3.5 (+/- 0.1) Angstrom. In the X-ray absorption spectrum of Mn2+-activated AMPP at the Mn K edge, the near-edge features are consistent with octahedrally coordinated Mn atoms in oxidation state +2. EXAFS data, limited to k less than or equal to 12 Angstrom(-1) by traces of Fe in the protein, are consistent with a single coordination shell occupied predominantly by O donor atoms at an average Mn-ligand distance of 2.15 Angstrom, but the possibility of a mixture of O and N donor atoms is not excluded. The Mn-Mn interaction at 3.5 Angstrom, is not detected in the EXAFS, probably due to destructive interference from light outer-shell atoms. The biological function, amino acid sequence and metal-ion dependence of E. coli AMPP are closely related to those of human prolidase, an enzyme that specifically cleaves Xaa-Pro dipeptides. Mutations that lead to human prolidase deficiency and clinical symptoms have been identified. Several known inhibitors of prolidase also inhibit AMPP. When these inhibitors are added to Mn2+-activated AMPP, the EPR spectrum and EXAFS remain unchanged. It can be inferred that the inhibitors either do not bind directly to the Mn centres, or substitute for existing Mn ligands without a significant change in donor atoms or coordination geometry. The conclusions from the spectroscopic measurements on AMPP have been verified by, and complement, a recent crystal structure analysis.

Leu(10) of alpha-conotoxin PnIB confers potency for neuronal nicotinic responses in bovine chromaffin cells

Two alpha-conotoxins PnIA and PnIB (previously reported as being mollusc specific) which differ in only two amino acid residues (AN versus LS at residues 10 and 11, respectively), show markedly different inhibition of the neuronal nicotinic acetylcholine receptor response in bovine chromaffin cells, a mammalian preparation. Whereas alpha-conotoxin PnIB completely inhibits the nicotine-evoked catecholamine release at 10 mu M, with IC50 = 0.7 mu M, alpha-conotoxin PnIA is some 30-40 times less potent. Two peptide analogues, [A10L]PnIA and [N11S]PnIA were synthesized to investigate the extent to which each residue contributes to activity. [A10L]PnIA (IC50 = 2.0 mu M) completely inhibits catecholamine release at 10 mu M whereas [N11S]PnIA shows Little inhibition. In contrast, none of the peptides inhibit muscle-type nicotinic responses in the rat hemi-diaphragm preparation. We conclude that the enhanced potency of alpha-conotoxin PnIB over alpha-conotoxin PnIA in the neuronal-type nicotinic response is principally determined by the larger, more hydrophobic leucine residue at position 10 in alpha-conotoxin PnIB. (C) 2000 Elsevier Science B.V. All rights reserved.

Synthesis and structure elucidation of kappa-casein (45-87) - A two dimensional nuclear magnetic resonance study

We have shown that 44 amino acid residues N-terminal segment of kappa-casein exhibits considerable a-helical structure. This prompted us to investigate the structures of the remaining segments of kappa-casein. Thus, in this study the chemical synthesis and structure elucidation of the peptide 45-87 amino acid residues of kappa-casein is reported. The peptide was assembled using solid phase peptide synthesis methodology on pam resin, cleaved via HF, freeze dried and, after purification, characterised by mass spectrometry (observed m/z 4929; calculated mit 4929.83). The amino acid sequence of the peptide is: CKPVALINNQFLPYPYYAKPAAVRSPAQILQWQVLSNTVPAKA Its structure elucidation has been carried out using circular dichroism (CD) and nuclear magnetic resonance (NMR) techniques. CD spectrum of the peptide shows it to be a random structure in water but in 30% trifluoroethanol the peptide exhibits considerable structure. The 1D and 2D NMR spectra corroborated the results of CD. The structure elucidation of the peptide using TOCSY and NOESY NMR techniques will be discussed.

Conotoxin TVIIA, a novel peptide from the venom of Conus tulipa - 1. Isolation, characterization and chemical synthesis

A novel conotoxin belonging to the 'four-loop' structural class has been isolated from the venom of the piscivorous cone snail Conus tulipa. It was identified using a chemical-directed strategy based largely on mass spectrometric techniques. The new toxin, conotoxin TVIIA, consists of 30 amino-acid residues and contains three disulfide bonds. The amino-acid sequence was determined by Edman analysis as SCSGRDSRCOOVCCMGLMCSRGKCVSIYGE where O = 4-transl-hydroxyproline. Two under-hydroxylated analogues, [Pro10]TVIIA and [Pro10,11]TVIIA, were also identified in the venom of C. tulipa. The sequences of TVIIA and [Pro10]TVIIA were further verified by chemical synthesis and coelution studies with native material. Conotoxin TVIIA has a six cysteine/four-loop structural framework common to many peptides from Conus venoms including the omega-, delta- and kappa-conotoxins. However, TVIIA displays little sequence homology with these well-characterized pharmacological classes of peptides, but displays striking sequence homology with conotoxin GS, a peptide from Conus geographus that blocks skeletal muscle sodium channels. These new toxins and GS share several biochemical features and represent a distinct subgroup of the four-loop conotoxins.

Comparative surface accessibility of a pore-lining threonine residue (T6') in the glycine and GABA(A) receptors

The substituted cysteine accessibility method was used to probe the surface exposure of a pore-lining threonine residue (T6') common to both the glycine receptor (GlyR) and gamma-aminobutyric acid, type A receptor (GABAAR) chloride channels. This residue lies close to the channel activation gate, the ionic selectivity filter, and the main pore blocker binding site. Despite their high amino acid sequence homologies and common role in conducting chloride ions, recent studies have suggested that the GlyRs and GABA(A)Rs have divergent open state pore structures at the 6' position. When both the human alpha1(T6'C) homomeric GlyR and the rat alpha1(T6'C)beta1(T6'C) heteromeric GABA(A)R were expressed in human embryonic kidney 293 cells, their 6' residue surface accessibilities differed significantly in the closed state. However, when a soluble cysteine-modifying compound was applied in the presence of saturating agonist concentrations, both receptors were locked into the open state. This action was not induced by oxidizing agents in either receptor. These results provide evidence for a conserved pore opening mechanism in anion-selective members of the ligand-gated ion channel family. The results also indicate that the GABA(A)R pore structure at the 6' level may vary between different expression systems.

Physico-chemical characterization and synthesis of neuronally active alpha-conotoxins

The high speciFIcity of alpha-conotoxins for different neuronal nicotinic acetylcholine receptors makes them important probes for dissecting receptor subtype selectivity. New sequences continue to expand the diversity and utility of the pool of available alpha-conotoxins. Their identification and characterization depend on a suite of techniques with increasing emphasis on mass spectrometry and microscale chromatography, which have benefited from recent advances in resolution and capability. Rigorous physicochemical analysis together with synthetic peptide chemistry is a prerequisite for detailed conformational analysis and to provide sufficient quantities of alpha-conotoxins for activity assessment and structure-activity relationship studies.

Stabilization of native protein fold by intein-mediated covalent cyclization

A mutant version of the N-terminal domain of Escherichia coli DnaB helicase was used as a model system to assess the stabilization against unfolding gained by covalent cyclization. Cyclization was achieved in vivo by formation of an amide bond between the N and C termini with the help of a split mini-intein. Linear and circular proteins were constructed to be identical in amino acid sequence. Mutagenesis of Phe102 to Glu rendered the protein monomeric even at high concentration. A difference in free energy of unfolding, DeltaDeltaG, between circular and linear protein of 2.3(+/-0.5) kcal mol(-1) was measured at 10degreesC by circular dichroism. A theoretical estimate of the difference in conformational entropy of linear and circular random chains in a three-dimensional cubic lattice model predicted DeltaDeltaG = 2.3 kcal mol(-1), suggesting that stabilization by protein cyclization is driven by the reduced conformational entropy of the unfolded state. Amide-proton exchange rates measured by NMR spectroscopy and mass spectrometry showed a uniform, approximately tenfold decrease of the exchange rates of the most slowly exchanging amide protons, demonstrating that cyclization globally decreases the unfolding rate of the protein. The amide proton exchange was found to follow EX1 kinetics at near-neutral pH, in agreement with an unusually slow refolding I measured by stopped-flow circular dichroism. rate of less than 4 min(-1) The linear and circular proteins differed more in their unfolding than in their folding rates. Global unfolding of the N-terminal domain of E. coli DnaB is thus promoted strongly by spatial separation of the N and C termini, whereas their proximity is much less important for folding. (C) 2005 Elsevier Ltd. All rights reserved.

Characterisation of the recently cloned rat noradrenaline transporter - Comparison with bovine and human transporters

The aims of this study were to characterize the recently cloned rat norepinephrine transporter (NET) in more detail and in particular to study possible species differences in its pharmacological properties compared with the human and bovine NETs. The study was carried out by measuring the uptake of [3H]norepinephrine in COS-7 cells expressing the NET after transient transfection with rat, human, or bovine NET cDNA. There were small but significant differences between the rat NET and the human or bovine NETs with respect to the affinities of sodium ions (greater for rat than for bovine) of the substrates norepinephrine, epinephrine, and 1-methyl-4-phenylpyridinium (greater for human than for rat), and of the inhibitor cocaine (greater for human and bovine than for rat), whereas the affinities of dopamine and of most inhibitors, including tricyclic antidepressants, showed no species differences. The fact that the affinities for some substrates, cocaine and sodium ions exhibited small but significant interspecies differences among the rat, human, and bovine NETs suggests that ligand recognition, the translocation process, and sodium ion dependence are influenced differentially by just a few amino acid exchanges in the primary sequences of the transporters. On the other hand, the lack of any major differences in the pharmacological properties of the rat, human, and bovine NETs in this study suggests that data obtained in previous studies on rat tissues and bovine cells can be extrapolated, in all except the most quantitative analyses, to the properties of the human NET.

The structure of versutoxin (delta-atracotoxin-Hv1) provides insights into the binding of site 3 neurotoxins to the voltage-gated sodium channel

Background: Versutoxin (delta-ACTX-Hv1) is the major component of the venom of the Australian Blue Mountains funnel web spider, Hadronyche versuta. delta-ACTX-Hv1 produces potentially fatal neurotoxic symptoms in primates by slowing the inactivation of voltage-gated sodium channels; delta-ACTX-Hv1 is therefore a useful tool for studying sodium channel function. We have determined the three-dimensional structure of delta ACTX-Hv1 as the first step towards understanding the molecular basis of its interaction with these channels. Results: The solution structure of delta-ACTX-Hv1, determined using NMR spectroscopy, comprises a core beta region containing a triple-stranded antiparallel beta sheet, a thumb-like extension protruding from the beta region and a C-terminal 3(10) helix that is appended to the beta domain by virtue of a disulphide bond. The beta region contains a cystine knot motif similar to that seen in other neurotoxic polypeptides. The structure shows homology with mu-agatoxin-l, a spider toxin that also modifies the inactivation kinetics of vertebrate voltage-gated sodium channels. More surprisingly, delta-ACTX-Hv1 shows both sequence and structural homology with gurmarin, a plant polypeptide. This similarity leads us to suggest that the sweet-taste suppression elicited by gurmarin may result from an interaction with one of the downstream ion channels involved in sweet-taste transduction. Conclusions: delta-ACTX-Hv1 shows no structural homology with either sea anemone or alpha-scorpion toxins, both of which also modify the inactivation kinetics of voltage-gated sodium channels by interacting with channel recognition site 3. However, we have shown that delta-ACTX-Hv1 contains charged residues that are topologically related to those implicated in the binding of sea anemone and alpha-scorpion toxins to mammalian voltage-gated sodium channels, suggesting similarities in their mode of interaction with these channels.

Insulin-like growth factor 2 cDNA cloning and ontogeny of gene expression in the liver of the marsupial brushtail possum (Trichosurus vulpecula)

The cDNA sequence for insulin-like growth factor 2 (IGF-2) was determined from the liver of the marsupial brushtail possum (Trichosurus vulpecula) using reverse transcription followed by polymerase chain reaction (RT-PCR) with gene-specific primers. The 359 bp of possum sequence encompassed the mature peptide, 27 bp of the signal peptide, and 125 bp of the E-peptide. Alignment of the deduced amino acid sequence with those from other species indicated that the mature peptide was 71 amino acids in length, 4 amino acids longer than most other mammals. At both the nucleotide and amino acid levels there was a high degree of sequence identity with IGF-2 from other mammalian and nonmammalian species. Amino acid identity ranged from 94.4% with a variant form of human IGF-2 to 80.3% with zebrafinch IGF-2. Northern analysis revealed that radiolabeled possum IGF-2, cDNA hybridized to multiple transcripts in the liver of both adult possums and 150-day-old pouch young and that the overall level of expression was greater in pouch young. Semiquantitative RT-PCR with total RNA from liver samples of pouch young aged 12 to 150 days postpartum and adults confirmed that IGF-2 gene expression was two to three times more abundant in pouch young than in adults but there was no significant change in the level of expression during pouch life. Unlike other mammalian species, in which there is a decline in levels of liver IGF-2 gene expression around the time of birth, levels in the marsupial brushtail possum remain elevated for at least 150 days after birth. This suggests that the decline in liver IGF-2 expression in marsupials and eutherians occurs at a similar stage of development and may reflect a role for this growth factor during the postnatal growth and development of the marsupial, (C) 2001 Academic Press.

Projections from the substantia nigra pars reticulata to the motor thalamus of the rat: Single axon reconstructions and immunohistochemical study

This is a study in the rat of the distribution of specific neurotransmitters in neurones projecting from the substantia nigra reticulata (SNR) to the ventrolateral (VL) and ventromedial (VM) thalamic nuclei. Individual axons projecting from the SNR to these thalamic nuclei have also been reconstructed following small injection of the anterograde tracer dextran biotin into the the SNR. Analysis of reconstructions revealed two populations of SNR neurones projecting onto the VL and VM thalamic nuclei. One group projects directly onto the VM and VL, and the other projects to the VM/VL and to the parafascicular nucleus. In another set of experiments Fluoro-Gold was injected into the VL/VM to label SNR projection neurones retrogradely, and immunohistochemistry was performed to determine the distribution of choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT), gamma -aminobutyric acid (GABA), and glutamate in Fluoro-Gold-labelled SNR projection neurones. Most SNR-VL/VM thalamic projection neurones were immunoreactive to acetylcholine or glutamate, whereas only 25% of the projection neurones were found to be immunoreactive to GABA. (C) 2001 Wiley-Liss, Inc.

A novel variant genotype C of hepatitis B virus identified in isolates from Australian Aborigines: complete genome sequence and phylogenetic relatedness

There have been no reports of DNA sequences of hepatitis B virus (HBV) strains from Australian Aborigines, although the hepatitis B surface antigen (HBsAg) was discovered among them. To investigate the characteristics of DNA sequences of HBV strains from Australian Aborigines, the complete nucleotide sequences of HBV strains were determined and subjected to molecular evolutionary analysis. Serum samples positive for HBsAg were collected from five Australian Aborigines. Phylogenetic analysis of the five complete nucleotide sequences compared with DNA sequences of 54 global HBV isolates from international databases revealed that three of the five were classified into genotype D and were most closely related in terms of evolutionary distance to a strain isolated from a healthy blood donor in Papua New Guinea. Two of the five were classified into a novel variant genotype C, which has not been reported previously, and were closely related to a strain isolated from Polynesians, particularly in the X and Core genes. These two strains of variant genotype C differed from known genotype C strains by 5.9-7.4% over the complete nucleotide sequence and 4.0-5.6 % in the small-S gene, and had residues Arg(122), Thr(127) and Lys(160) characteristic of serotype ayw3, which have not been reported previously in genotype C. In conclusion, this is the first report of the characteristics of complete nucleotide sequences of HBV from Australian Aborigines. These results contribute to the investigation of the worldwide spread of HBV, the relationship between serotype and genotype and the ancient common origin of Australian Aborigines.

Genomic organization of the CC chemokine MIP-3 alpha/CCL20/LARC/EXODUS/SCYA20, showing gene structure, splice variants, and chromosome localization

We describe the genomic organization of a recently identified CC chemokine, MIP3 alpha /CCL20 (HGMW-approved symbol SCYA20). The MIP-3 alpha /CCL20 gene was cloned and sequenced, revealing a four exon, three intron structure, and was localized by FISK analysis to 2q35-q36. Two distinct cDNAs were identified, encoding two forms of MIP-3 alpha /CCL20, Ala MLP-3 alpha /CCL20 and Ser MIP-3 alpha /CCL20, that differ by one amino acid at the predicted signal peptide cleavage site. Examination of the sequence around the boundary of intron 1 and exon 2 showed that use of alternative splice acceptor sites could give rise to Ata MIP-3 alpha /CCL20 or Ser MIP-3 alpha /CCL20. Both forms of MIP-3cr/CCL20 were chemically synthesized and tested for biological activity. Both flu antigen plus IL-a-activated CD4(+) and CD8(+) T lymphoblasts and cord blood-derived dendritic cells responded to Ser and Ala MIP-3 alpha /CCL20. T lymphocytes exposed only to IL-2 responded inconsistently, while no response was detected in naive T lymphocytes, monocytes, or neutrophils. The biological activity of Ser MIP-3 alpha /CCL20 and Ala MIP-3 alpha /CCL20 and the tissue-specific preference of different splice acceptor sites are not yet known. (C) 2001 Academic Press.

Atm knock-in mice harboring an in-frame deletion corresponding to the human ATM 7636del9 common mutation exhibit a variant phenotype

ATM, the gene mutated in the human immunodeficiency disorder ataxia-telangiectasia (A-T), plays a central role in recognizing ionizing radiation damage in DNA and in controlling several cell cycle checkpoints. We describe here a murine model in which a nine-nucleotide in-frame deletion has been introduced into the Atm gene by homologous recombination followed by removal of the selectable marker cassette by Cre-loxP site-specific, recombination-mediated excision. This mouse, Abm-Delta SRI, was designed as a model of one of the most common deletion mutations (7636de19) found in A-T patients. The murine Atm deletion results in the loss of three amino acid residues (SRI; 2556-2558) but produces near full-length detectable Atm protein that lacks protein kinase activity. Radiosensitivity was observed in Atm-Delta SRI mice, whereas the immunological profile of these mice showed greater heterogeneity of T-cell subsets than observed in Atm(-/-) mice. The life span of Atm-Delta SRI mice was significantly longer than that of Atm(-/-) mice when maintained under nonspecific pathogen-free conditions. This can be accounted for by a lower incidence of thymic lymphomas in Atm-Delta SRI mice up to 40 weeks, after which time the animals died of other causes. The thymic lymphomas in Atm-Delta SRI mice were characterized by extensive apoptosis, which appears to be attributable to an increased number of cells expressing Fas ligand. A variety of other tumors including B-cell lymphomas, sarcomas, and carcinomas not seen in Atm(-/-) mice were observed in older Atm-Delta SRI animals. Thus, expression of mutant protein in Atm-Delta SRI knock-in mice gives rise to a discernibly different phenotype to Atm(-/-) mice, which may account for the heterogeneity seen in A-T patients with different mutations.