Identification of a second mutation in the protein-coding sequence of the Z type alpha 1-antitrypsin gene

This study reports the entire nucleotide sequence of the protein coding region sequence of the alpha 1-antitrypsin (alpha 1AT) Z gene, a common form of the alpha 1AT gene associated with serum alpha 1AT deficiency. In addition to Glu342 to Lys342 mutation in exon V which has been previously identified by peptide analysis, another point mutation (GTG to GCG in exon III) in the gene sequence predicts a second amino acid substitution (Val213 to Ala213) in the Z protein. This Val213 to Ala213 mutation was confirmed to be a general finding in Z type alpha 1AT gene by evaluating genomic DNA from 40 Z haplotypes using synthetic oligonucleotide gene probes directed toward the mutated exon III sequences in the Z gene. Furthermore, the exon III Val213 to Ala213 mutation eliminates a BstEII restriction endonuclease site in the alpha 1AT Z gene, allowing rapid identification of this Val213 to Ala213 substitution at the genomic DNA level. Surprisingly, when genomic DNA samples from individuals thought to be homozygous for the M1 gene (the most common alpha 1AT normal haplotype) were evaluated with BstEII, 23% of the M1 haplotypes were BstEII site negative, thus identifying a new form of M1 (i.e. M1(Ala213], likely identical to M1 but with an isoelectric focusing "silent" amino acid substitution (Val213 to Ala213). Although the relative importance of the newly identified exon III Val213 to Ala213 mutation to the pathogenesis of the abnormalities associated with the Z gene is not known, it is likely that M1(Ala213) gene represents a common "normal" polymorphism of the alpha 1AT gene that served as an evolutionary intermediate between the M1(Val213) and Z genes.     

Platelet Glutamate and Aspartate Uptake in Huntington's Disease

Abstract A possible involvement of amino acid uptake mechanisms in the etiology of the human neurodegenerative disease, Huntington's disease (HD), was investigated. Measurement of glutamate (Glu) and aspartate (Asp) uptake was performed in blood platelets, which have previously been shown to constitute a peripheral model system for central amino acid uptake processes. Analyses of Glu and Asp accumulation at 10⁻⁷ M and kinetic examination of the high affinity site for Glu indicate no significant differences between control and HD platelets. A genetically determined defect in amino acid uptake therefore does not seem to underlie the nerve cell loss observed in HD patients.     

Primary structures of M6 and M7 of mugiline beta (Mugil japonicus)

Mugiline beta isolated from mature sperm nuclei of the Formosan grey mullet, belonging to Perciformes, was fractionated into seven components (M1-M7), by chromatography on CM-Sephadex C-25. The amino acid sequences of the two major components (M6 and M7) were then determined. M6 contained 33 amino acid residues per molecule: Arg, 21; Thr, 1; Ser, 1; Glu, 1; Pro, 3; Ala, 2; Val, 2; Met, 0.3 and Ile, 1.7. The amino acid sequence of M6 is: Pro-Arg-Arg-Arg-Arg-Glu-Thr-Ser-Arg-Pro-Ile-Arg-Arg-Arg-Arg-Arg-Ala-Pro- Ile (Met)-Arg-Arg-Arg-Arg-Arg-Val-Val-Arg-Arg-Arg-Arg. Isoleucine at position 22 is partially replaced by methionine. M7 had an amino acid sequence similar to that of M6 except that glutamic acid at position 6 of M6 was replaced by glutamine. A high degree of homology in the sequences was found between mugiline beta from mullet and thynnine from tuna fish, which also belongs to Perciformes.     

Peptide phosphorylation by calcium-dependent protein kinase from maize seedlings.

Ca2+-dependent protein kinase (CDPK-1) was purified from maize seedlings, and its substrate specificity studied using a set of synthetic peptides derived from the phosphorylatable sequence RVLSRLHS15VRER of maize sucrose synthase 2. The decapeptide LARLHSVRER was found to be efficiently phosphorylated as a minimal substrate. The same set of peptides were found to be phosphorylated by mammalian protein kinase Cbeta (PKC), but showed low reactivity with protein kinase A (PKA). Proceeding from the sequence LARLHSVRER, a series of cellulose-membrane-attached peptides of systematically modified structure was synthesised. These peptides had hydrophobic (Ala, Leu) and ionic (Arg, Glu) amino acids substituted in each position. The phosphorylation of these substrates by CDPK-1 was measured and the substrate specificity of the maize protein kinase characterised by the consensus sequence motif A/L-5X-4R-3X-2X-1SX+1R+2Z+3R+4, where X denotes a position with no strict amino acid requirements and Z a position strictly not tolerating arginine compared with the other three varied amino acids. This motif had a characteristic sequence element RZR at positions +2 to +4 and closely resembled the primary structure of the sucrose synthase phosphorylation site. The sequence surrounding the phosphorylatable serine in this consensus motif was similar to the analogous sequence K/RXXS/TXK/R proposed for mammalian PKC, but different from the consensus motif RRXS/TX for PKA.     

Molecular genetic characterization of H5N1 influenza virus strains isolated from poultry in the Kurgan region in 2005

In the second half of 2005, a large-scale outbreak of influenza in poultry and wild birds was caused by a highly pathogenic H5N1 influenza virus in Russia. The level of pathogenicity is a polygenic trait, and most individual genes contribute to the influenza A virus pathogenicity in birds, animals, and humans. The full-length nucleotide sequences were determined for H5N1 strains isolated in the Kurgan region (Western Siberia). The structure of viral proteins was analyzed using the deduced amino acid sequences. The receptor-binding site of hemagglutinin (HA) in strains A/chicken/Kurgan/05/2005 and A/duck/Kurgan/08/2005 was typical for avian influenza viruses and contained Glu and Gly at positions 226 and 228, respectively. The structure of the basic amino acid cluster located within the HA cleavage site was identical in all isolates: QGERRRKKR. According to the neuraminidase structure, all H5N1 isolates from the Kurgan region were assigned to the Z genotype. Amino acid residues typical for the avian influenza virus were revealed in 30 out of 32 positions of M1, M2, NP, PA, and PB2, determining the host range specificity. One of the strains contained Lys at position 627 of PB2. Isolates from the Kurgan region were shown to have a remantadine-sensitive genotype. Both strains contained Glu at position 92 of NS1, indicating that the virus is interferon-resistant. Phylogenetic analysis related the Kurgan isolates to subclade 2 of clade 2 of highly pathogenic H5N1 influenza viruses.     

Somatostatin Potently Stimulates In Vivo Striatal Dopamine and γ-Aminobutyric Acid Release by a Glutamate-Dependent Action

Abstract: We have used in vivo microdialysis in anaesthetised rats to investigate whether somatostatin (SRIF) can play a neuromodulatory role in the striatum. When 100 n M SRIF was retrodialysed for 15 min, it increased concentrations of dopamine (DA) by 28-fold, γ-aminobutyric acid (GABA) by eightfold, and glutamate (Glu) by sixfold as well as those of aspartate (Asp) and taurine (Tau). These effects were both calcium- and tetrodotoxin-sensitive. Lower (10 or 50 n M ) and higher (1 µ M ) SRIF concentrations were less effective. Rapid sampling showed that whereas Asp and Glu concentrations were raised for 3 min at the start of 15-min SRIF infusions, those of DA were increased for 12 min. A second 15-min application of 100 n M SRIF given 135 min after the first application failed to increase transmitter release. An NMDA receptor antagonist, 2-amino-5-phosphonopentanoic acid (200 µ M ), blocked SRIF (100 n M )-evoked Asp, Glu, Tau, and GABA release and reduced that of DA. An α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate antagonist, 6,7-dinitroquinoxaline-2,3-dione (100 µ M ), blocked SRIF-induced DA and Tau release and reduced that of Asp, Glu, and GABA. These results show that SRIF increases DA, Glu, Asp, GABA, and Tau release in the rat striatum and suggest that its actions on DA and GABA release are mainly mediated through increased excitatory amino acid release.     

Genetically engineered human interleukin-6 variant with enhanced stability

A recombinant human interleukin-6 mutant with enhanced conformational stability toward denaturant was obtained by site-specific mutagenesis. The clone was identified as having a single amino acid substitution of Lys70Glu. When urea-induced denaturation was monitored by the change in fluorescence intensity at 360 nm, Lys70Glu mutation shifted the midpoint of unfolding transition from 5.8 M (wild type) to 6.6 M urea. This mutation did not impair the biological activity.     

Beta-hydroxyaspartic acid in vitamin K-dependent proteins

A method for the quantitation of beta-hydroxyaspartic acid in proteins is described. After hydrolysis in 6 M HCl, the beta-hydroxyaspartic acid released is quantitated on an automatic amino acid analyzer employing a pH 2.0 eluting buffer and postcolumn reaction with o-phthalaldehyde for detection. The sensitivity is about 0.01 nmol. Among vitamin K-dependent proteins, factor IX, factor X, protein C, and protein Z each contain about one residue of beta-hydroxyaspartic acid whereas protein S contains two or three residues. Prothrombin lacks beta-hydroxyaspartic acid as do a number of non-vitamin K-dependent proteins also analyzed.     

Cloning and characterization of Thermotoga maritima beta-glucuronidase

The putative beta-glucuronidase from Thermotoga maritima, comprising 563 amino acid residues conjugated with a Hisx6 tag, was cloned and expressed in Escherichia coli. The enzyme has a moderately broad specificity, hydrolysing a range of p-nitrophenyl glycoside substrates, but has greatest activity on p-nitrophenyl beta-D-glucosiduronic acid (kcat=68 s(-1), kcat/K(M)= 4.5x10(5) M(-1) s(-1)). The enzyme also shows a relatively broad pH-dependence with activity from pH4.5 to 7.5 and a maximum at pH6.5. As expected the enzyme is stable towards heat denaturation, with a half life of 3h at 85 degrees C, in contrast to the mesophilic E. coli enzyme, which has a half life of 2.6h at 50 degrees C. The identity of the catalytic nucleophile was confirmed as Glu476 within the sequence VTEFGAD by trapping the glycosyl-enzyme intermediate using the mechanism-based inactivator, 2-deoxy-2-fluoro-beta-D-glucosyluronic acid fluoride and identifying the labeled peptide in peptic digests by HPLC-MS/MS methodologies. Consistent with this, the Glu476Ala mutant was shown to be hydrolytically inactive. The acid/base catalyst was confirmed as Glu383 by generation and kinetic analysis of enzyme mutants modified at that position, Glu383Ala and Glu383Gln. The demonstration of activity rescue by azide is consistent with the proposed role for this residue. This enzyme therefore appears suitable for use in enzymatic oligosaccharide synthesis in either the transglycosylation mode or by use of glycosynthase and thioglycoligase approaches.     

A single gene codes for two forms of rat nucleolar protein B23 mRNA

Protein B23 (38 kDa, pI = 5.1) is an abundant RNA-associated nucleolar phosphoprotein and putative ribosome assembly factor. A full length cDNA clone (lambda JH1) encoding a major expressed form of rat protein B23, now designated B23.1, was reported recently (Chang, J. H., Dumbar, T. S., and Olson, M. O. J. (1988) J. Biol. Chem. 263, 12824-12837). In this paper the isolation from a rat brain library and sequence of a cDNA clone (lambda JH2) coding for a second form (B23.2) of protein B23 is reported. Isoforms B23.1 and B23.2 are polypeptides of 292 and 257 amino acids, respectively. The 5'-untranslated regions of the two cDNAs and the amino-terminal 255 amino acids of the proteins are identical in the two isoforms. However, the 3'-untranslated regions of the mRNAs are completely different, and the dipeptide Gly-Gly in B23.1 (residues 256 and 257) is replaced by Ala-His in B23.2 indicating that the former is not a precursor of the latter. The finding of AGGT sequences in the 3' regions of lambda JH1 suggest the presence of intron-exon boundaries at the point where the two cDNAs begin to differ. To investigate the origin of the two isoforms, two rat genomic libraries were screened with oligonucleotide probes based on sequences from the unique regions of the two cDNAs. One of the genomic clones isolated (lambda JH125) contained a 6.5-kilobase fragment encoding the 3' end of both cDNAs. lambda JH125 contains four exons designated W, X, Y, and Z in the order indicated. Exons W and X encode 36 amino acids at the carboxyl terminus of B23.2, whereas exons W, Y, and Z encode the carboxyl-terminal 71 amino acid residues of B23.1. Exons X and Z each contain distinct 3'-untranslated sequences in which are found polyadenylation signals. These data suggest that two different mRNAs are formed by alternative splicing of separate 3' segments onto a common 5' region.     

Extraction, purification and characterization of the sperm protamines of the dog-fish Scylliorhinus caniculus

Dog-fish sperm nuclei contain four low molecular weight basic proteins called scylliorhinines. Protein Z3 is a typical arginine-rich protamine, whilst the three other components, Z1, Z2 and S4, are characterized by high arginine and cysteine contents. In contrast to protamine Z3, which can be directly solubilized by 0.25 M HCl, the three other protamines must be reduced and alkylated before acid extraction. They were further purified by ion-exchange chromatography on carboxymethyl-cellulose. The amino acid compositions and the N-terminal sequences reveal significant differences between scylliorhinines, particularly in their molecular size and amino acid diversity. Moreover, they show no common feature with other sperm-specific protamines previously described.     

汉坦病毒中国疫苗株Z37M片段的克隆及序列分析

汉坦病毒Z37株是从褐家鼠体内分离到的,用于生产双价肾综合征出血热疫苗的病毒毒株之一,血清分型为SEO型。利用RT-PCR方法扩增Z37株M基因片段cDNA,克隆入质粒载体,进行核苷酸序列测定及分析。Z37株M基因片段由3651个核苷酸组成,只有一个开放读码框架,共编码1133个氨基酸。与HTN型病毒(76-118、A9、HV-114)的核苷酸和氨基酸同源性分别为71.8%～72.1%、76.2%～76.7%,与SEO型(R22、L99、80-39)的核苷酸和氨基酸同源性分别为95.3%～96.1%、95.3%～98.9%。这一结果的获得进一步从分子水平确定了Z37株的型别,并为研制M基因片段重组疫苗打下基础。     

Muscarinic toxin 7 selectivity is dictated by extracellular receptor loops

Muscarinic toxin 7 (MT7) is a mamba venom protein antagonist with extremely high selectivity for the M1 muscarinic acetylcholine receptor. To map the sites for the interaction of MT7 with muscarinic receptors we have used chimeric M1:M3 receptors and site-directed mutagenesis of the M3 and M4 receptor subtypes. Two Glu residues in M1, one in extracellular loop 2 and one in extracellular loop 3, were found to be important for the high affinity binding of MT7. Substitution of the corresponding Lys residues in the M3 receptor with Glu converted the M3 mutant to an MT7 binding receptor, albeit with lower affinity compared with M1. A Phe --> Tyr substitution in extracellular loop 2 of M3 together with the 2 Glu mutations generated a receptor with an increased MT7 affinity (apparent Ki = 0.26 nM in a functional assay) compared with the M1 receptor (apparent Ki = 1.31 nM). The importance of the identified amino acid residues was confirmed with a mutated M4 receptor constructs. The results indicate that the high selectivity of MT7 for the M1 receptor depends on very few residues, thus providing good prospects for future design and synthesis of muscarinic receptor-selective ligands.     

Complete primary structure of a calcium-dependent serine proteinase capable of degrading extracellular matrix proteins

A novel calcium-dependent serine proteinase (CASP) secreted from malignant hamster embryo fibroblast Ni 12C2 degrades extracellular matrix proteins. A complementary DNA encoding CASP has been isolated with the use of oligonucleotide probes synthesized based on partial amino acid sequences of CASP. The complete amino acid sequence of CASP revealed that it has a serine active site at the C-terminal side. Glu rich and proEGF homologous sites are found at the N-terminal site suggesting that it is structurally similar to blood coagulation factors such as IX, X and an anti-coagulation factor, protein C.     

Glutamate 346 of human Na+-H+ exchanger NHE1 is crucial for modulating both the affinity for Na+ and the interaction with amiloride derivatives

A NHE1 variant that exhibits very high resistance to (3-methyl sulfonyl-4-piperidinobenzoyl) guanidine methane sulfonate (HOE694), a potent inhibitor of Na(+)-H(+) exchangers, was selected and characterized. Sequencing of the coding region corresponding to the N-terminal domain of this variant revealed the presence of only one mutation located within membrane-spanning segment 9 (M9). This base pair change replaces a glutamate (Glu) with an aspartate (Asp). We reproduced this amino acid change in wild-type NHE1 and found that this mutation alone is responsible for the huge decrease in sensitivity to the HOE694 compound and to ethylisopropylamiloride (EIPA). We found that the NHE1-Glu(346)Asp mutant was more than 2000-fold more resistant to HOE694 and up to 300-fold more resistant to EIPA than wild-type NHE1, with the size, rather than the charge, of the amino acid in position 346 having the greatest effect. Interestingly, its affinity for Na(+) was at least 4-fold lower than that of wild-type NHE1. Mutation of amino acids in the vicinity of Glu(346) did not change the sensitivity of mutated NHE1 proteins to inhibitors. We suggest there is a direct interaction of Glu(346) or involvement of Glu(346) in a coordination site with NHE inhibitors and with Na(+).     

Peripheral region for core cross-beta plays important role in amyloidogenicity

The role of the peripheral sequence neighboring the core cross-beta region was investigated using a peptide library constructed with all possible combinations of Lys, Glu, Ser, and Leu at three residue positions (X1-X3) forming the N-terminal region linked to the amyloid core sequence of the barnase-derived segment (A4-K22). By means of CD spectra and thioflavin T binding assay for 64 peptides, not only the composition but also the sequence in the peripheral region were found to be responsible for amyloid formation. The preferences of amino acid residues in the peripheral region of the amyloid-forming peptides were in the order of Leu approximately SerGlu>Lys. A balance of positive and negative charges was found to be essential for amyloid formation, suggesting that the electrostatic interaction at the surface of the amyloid fibrils is relevant to their stability. On the basis of the maximum fluorescence wavelength of fibril-bound thioflavin T, the highly amyloidogenic peptides were classified into two classes, which exhibited the sequence preferences of (Leu, Ser/Glu, and Leu) and (Glu, Leu, and Ser) for the peripheral sequence (X1, X2, and X3). The former class can be rationally assigned to the structural model with deep grooves along the fibril axis. Thus, the peripheral sequence regulates the manner of molecular packing in the fibrils as well as the amyloidogenicity. In addition, the chains of the peripheral sequence are most likely to form thioflavin T binding sites.     

Simultaneous determination of vigabatrin and amino acid neurotransmitters in brain microdialysates by capillary electrophoresis with laser-induced fluorescence detection

Capillary electrophoresis with laser-induced fluorescence detection (CE-LIFD) coupled to in vivo microdialysis sampling was used in order to monitor simultaneously a drug and several neurotransmitters in the brain extracellular fluid. Determination of the antiepileptic drug vigabatrin and the amino acid neurotransmitters glutamate (Glu), l-aspartate (l-Asp) and gamma-aminobutyric acid (GABA) was performed on low-concentration samples which were derivatized with naphthalene-2,3-dicarboxaldehyde (NDA) and separated using a pH 9.2 75 mM sodium borate running buffer containing 60 mM sodium dodecyl sulfate (SDS) and 5mM hydroxypropyl-beta-cyclodextrin (HP-beta-CD). Glu, l-Asp and vigabatrin derivatized at a concentration of 1.0 x 10(-9) M, and GABA derivatized at a concentration of 5.0 x 10(-9) M, produced peaks with signal-to-noise ratios of 8:1, 8:1, 4:1 and 5:1, respectively. The nature of the neurotransmitter peaks found in rat brain microdialysates was confirmed by both electrophoretic and pharmacological validations. This method was used for monitoring vigabatrin and amino acid neurotransmitters in microdialysates from the rat striatum during intracerebral infusion of the drug and revealed rapid vigabatrin-induced changes in GABA and Glu levels. This original application of CE-LIFD coupled to microdialysis represents a powerful tool for pharmacokinetic/pharmacodynamic investigations.     

Review of Glycosylation Engineering of Biopharmaceuticals: Methods and Protocols

The glycoform profile of a glycoprotein is non-templated, i.e., is not encoded within the genome or otherwise predetermined; however, it is estimated that ~50% of human genes having an open reading frame encode a –N-X-S/T- amino acid sequence, where X represents any amino acid other than proline, that comprises a potential site (sequon) for N-linked glycosylation of the translated protein. N-linked glycosylation is both a co- and post-translational modification. The complex oligosaccharide GlcNAc2Man9Glu3 may be added at a –N-X-S/T- sequon as the polypeptide chain emerges from the ribosome tunnel. Local secondary structure determines whether oligosaccharide is added and the extent of addition. Higher occupancy is observed for –N-X-T- sequons than at –N-X-S- sequons, and the efficiency of addition can be further influenced by adjacent amino acid residues.     

Structure-function studies of Na,K-ATPase. Site-directed mutagenesis of the border residues from the H1-H2 extracellular domain of the alpha subunit

It has recently been shown that replacement of the border residues (Gln-111 and Asn-122) of the H1-H2 extracellular domain of the sheep Na,K-ATPase alpha subunit with the charged amino acids Arg and Asp generates a ouabain-resistant enzyme (Price, E. M. and Lingrel, J. B. (1988) Biochemistry 27, 8400-8408). In order to further study structure-function relationships in Na,K-ATPase, six additional mutations have been made at these border positions. Two of these mutants were single amino acid substitutions (Gln-111 to Arg or Asn-122 to Asp). These mutations change one or the other H1-H2 border residue to a charged amino acid. The remaining substitutions were double mutants in which both of the H1-H2 border residues were simultaneously changed to charged amino acids. Changes were made which introduced either positively charged amino acids (Lys at positions 111 and 122), negatively charged amino acids (Glu at positions 111 and 122) or oppositely charged amino acids (Lys at position 111 and Glu at 122; Asp at position 111 and Arg at 122) at the borders of the H1-H2 extracellular domain. HeLa cells transfected with any of these sheep Na,K-ATPase alpha subunit mutants were able to grow in concentrations of ouabain that were toxic to untransfected cells or cells transfected with the wild type sheep alpha subunit. Crude membranes isolated from the transfectants were analyzed for ouabain inhibitable Na,K-ATPase activity. All of the transfectants contained a relatively ouabain-resistant component of enzyme activity, with the ouabain I50 values ranging from 4 x 10(-3) M to 1 x 10(-6) M. The most resistant enzyme was the double mutant that contained Asp at position 111 and Arg at 122, whereas the least resistant were the enzymes containing the single amino acid substitutions. There was no correlation between the type of charged amino acid present at the border position and the degree of ouabain resistance. These data demonstrate the functional importance, in terms of ouabain binding, of the border positions of the H1-H2 extracellular domain of the Na,K-ATPase alpha subunit.