Isolation and characterization of cDNA clones encoding a low molecular weight nonmuscle tropomyosin isoform

cDNA clones encoding rat fibroblast tropomyosin 4 (TM-4) were isolated and characterized. DNA sequence analysis was carried out to determine the sequence of the protein. The derived amino acid sequence revealed that rat fibroblast TM-4 was found to contain 248 amino acids. The amino acid sequence of rat fibroblast TM-4 was compared with two other low molecular weight TM isoforms, equine platelet beta-TM and a human fibroblast TM. Rat TM-4 exhibited 98% sequence identity with the equine platelet TM but only 75% identity with the human fibroblast TM isoform. The high degree of conservation between the rat and equine proteins indicates that they belong to the same isotype of TM. Comparison of the amino acid sequences of the three low molecular TM isoforms along the length of the proteins reveals regions that are strongly conserved and regions that have considerably diverged. In the regions from amino acid residues 1 to 148 and 176 to 221, amino acid substitutes are moderate. The most variant regions in the sequence are in the middle part of the proteins from amino acids 149 to 175 and at the carboxyl-terminal region of the proteins from amino acids 222 to 248. The differences in the sequence of the rat and platelet TMs compared to the human TM may define distinct functional domains among the low molecular weight TMs. In addition, expression of tropomyosin was studied in a variety of tissues and transformed cells. We also demonstrate that at least three separate genes encode tropomyosins expressed in rat fibroblasts.     

A Transmembrane Polar Interaction Is Involved in the Functional Regulation of Integrin αLβ2

Integrins are heterodimeric transmembrane (TM) receptors formed by noncovalent associations of α and β subunits. Each subunit contains a single α-helical TM domain. Inside-out activation of an integrin involves the separation of its cytoplasmic tails, leading to disruption of αβ TM packing. The leukocyte integrin αLβ2 is required for leukocyte adhesion, migration, proliferation, cytotoxic function, and antigen presentation. In this study, we show by mutagenesis experiments that the packing of αLβ2 TMs is consistent with that of the integrin αIIbβ3 TMs. However, molecular dynamics simulations of αLβ2 TMs in lipids predicted a polar interaction involving the side chains of αL Ser1071 and β2 Thr686 in the outer-membrane association clasp (OMC). This is supported by carbonyl vibrational shifts observed in isotope-labeled αLβ2 TM peptides that were incorporated into lipid bilayers. Molecular dynamics studies simulating the separation of αLβ2 tails showed the presence of polar interaction during the initial perturbation of the inner-membrane association clasp. When the TMs underwent further separation, the polar interaction was disrupted. OMC polar interaction is important in regulating the functions of β2 integrins because mutations that disrupt the OMC polar interaction generated constitutively activated αLβ2, αMβ2, and αXβ2 in 293T transfectants. We also show that the expression of mutant β2 Thr686Gly in β2-deficient T cells rescued cell adhesion to intercellular adhesion molecule 1, but the cells showed overt elongated morphologies in response to chemokine stromal-cell-derived factor 1α treatment as compared to wild-type β2-expressing cells. These two TM polar residues are totally conserved in other members of the β2 integrins in humans and across different species. Our results provide an example of the stabilizing effect of polar interactions within the low dielectric environment of the membrane interior and demonstrate its importance in the regulation of αLβ2 function.     

Purification,properties and cDNA cloning of neoverrucotoxin (neoVTX), a hemolytic lethal factor from the stonefish Synanceia verrucosa venom

A proteinaceous toxin with hemolytic and lethal activities, named neoverrucotoxin (neoVTX), was purified from the venom fluid of stonefish Synanceia verrucosa and its primary structure was elucidated by a cDNA cloning technique. NeoVTX is a dimeric 166 kDa protein composed of α-subunit (702 amino acid residues) and β-subunit (699 amino acid residues) and lacks carbohydrate moieties. Its hemolytic activity is inhibited by anionic lipids, especially potently by cardiolipin. These properties are comparable to those of stonustoxin (SNTX) previously purified from S. horrida. Alignment of the amino acid sequences also reveals that the neoVTX α- and β-subunits share as high as 87 and 95% sequence identity with the SNTX α- and β-subunits, respectively. The distinct differences between neoVTX and SNTX are recognized only in the numbers of Cys residues (18 for neoVTX and 15 for SNTX) and free thiol groups (10 for neoVTX and 5 for SNTX). In contrast, neoVTX considerably differs from verrucotoxin (VTX), a tetrameric 322 kDa glycoprotein, previously purified from S. verrucosa. In addition, the sequence identity of the neoVTX β-subunit with the reported VTX β-subunit is 90%, being lower than that with the SNTX β-subunit.     

A beta-defensin 1-like antimicrobial peptide from the tree shrew, Tupaia belangeri

A novel beta-defensin 1-like antimicrobial peptide (β-defensin 1TB) containing 36 amino acid residues was purified and characterized from the serum of the tree shrew, Tupaia belangeri. Its amino acid sequence was determined as DHYLCVKNEGICLYSSCPSYTKIEGTCYGGKAKCCK, by Edman degradation, mass spectrometry analysis, and cDNA cloning. Evolution analysis indicated that β-defensin 1TB showed maximal similarity to the β-defensin 1 identified from cotton-top tamarin, Saguinus oedipus. β-defensin 1TB exerted potential antimicrobial activities against wide spectrum of microorganisms including Gram-negative and -positive bacteria and fungi. It showed little hemolitic activity to human or rabbit red cells. To the best of our knowledge, this is the first report of antimicrobial peptide from Tupaiidae.     

The cDNA sequence of three hemocyanin subunits from the garden snail Helix lucorum

Hemocyanins are blue copper containing respiratory proteins residing in the hemolymph of many molluscs and arthropods. They can have different molecular masses and quaternary structures. Moreover, several molluscan hemocyanins are isolated with one, two or three isoforms occurring as decameric, didecameric, multidecameric or tubule aggregates. We could recently isolate three different hemocyanin isopolypeptides from the hemolymph of the garden snail Helix lucorum (HlH). These three structural subunits were named α_D-HlH, α_N-HlH and β-HlH. We have cloned and sequenced their cDNA which is the first result ever reported for three isoforms of a molluscan hemocyanin. Whereas the complete gene sequence of α_D-HlH and β-HlH was obtained, including the 5′ and 3′ UTR, 180 bp of the 5′ end and around 900 bp at the 3′ end are missing for the third subunit. The subunits α_D-HlH and β-HlH comprise a signal sequence of 19 amino acids plus a polypeptide of 3409 and 3414 amino acids, respectively. We could determine 3031 residues of the α_N-HLH subunit. Sequence comparison with other molluscan hemocyanins shows that α_D-HlH is more related to Aplysia californicum hemocyanin than to each of its own isopolypeptides. The structural subunits comprise 8 different functional units (FUs: a, b, c, d, e, f, g, h) and each functional unit possesses a highly conserved copper-A and copper-B site for reversible oxygen binding. Potential N-glycosylation sites are present in all three structural subunits. We confirmed that all three different isoforms are effectively produced and secreted in the hemolymph of H. lucorum by analyzing a tryptic digest of the purified native hemocyanin by MALDI-TOF and LC-FTICR mass spectrometry.     

Structural and functional properties of the non-muscle tropomyosins

Summary The non-muscle tropomyosins (TMs), isolated from such tissues as platelets, brain and thyroid, are structurally very similar to the muscle TMs, being composed of two highly -helical subunits wound around each other to form a rod-like molecule. The non-muscle TMs are shorter than the muscle TMs; sequence analysis demonstrates that each subunit of equine platelet TM consists of 247 amino acids, 37 fewer than for skeletal muscle TM. The major differences in sequence between platelet and skeletal muscle TM are found near the amino and carboxyl terminal ends of the proteins. Probably as the result of such alterations, the non-muscle TMs aggregate in a linear end-to-end manner much more weakly than do the muscle TMs. Since end-to-end interactions are responsible for the highly cooperative manner in which TM binds to actin, the non-muscle TMs have a lower affinity for actin filaments than do the muscle TMs. However, the attachment of other proteins to actin (e.g. the Tn-I subunit of skeletal muscle troponin or the S-1 subfragment of skeletal muscle myosin) can increase the affinity of actin filaments for non-muscle TM. The non-muscle TMs interact functionally with the Tn-I component of skeletal muscle troponin to inhibit the ATPase activity of muscle actomyosin and with whole troponin to regulate the muscle actomyosin ATPase in a Ca⁺⁺-dependent manner, even though one of the binding sites for troponin on skeletal TM is missing in non-muscle TM. A novel actomyosin regulatory system can be produced using Tn-I, calmodulin and non-muscle TM; in this case inhibition is released when the non-muscle TM detaches from the actin filament in the presence of Ca⁺⁺. Although it has not yet been demonstrated that the non-muscle TMs participate in a Ca⁺⁺-dependent contractile regulatory system in vivo it does appear that they are associated with actin filaments in vivo.     

Antimicrobial function of the GAPDH-related antimicrobial peptide in the skin of skipjack tuna,Katsuwonus pelamis

A 3.4 kDa of antimicrobial peptide was purified from an acidified skin extract of skipjack tuna, Katsuwonus pelamis, by preparative acid-urea–polyacrylamide gel electrophoresis and C₁₈ reversed-phase HPLC. A comparison of the N-terminal amino acid sequence of the purified peptide with that of other known polypeptides revealed high sequence homology with the YFGAP (Yellowfin tuna Glyceraldehyde-3-phosphate dehydrogenase-related Antimicrobial Peptide); thus, this peptide was identified as the skipjack tuna GAPDH-related antimicrobial peptide (SJGAP). SJGAP showed potent antimicrobial activity against Gram-positive bacteria, such as Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus, and Streptococcus iniae (minimal effective concentrations [MECs], 1.2–17.0 μg/mL), Gram-negative bacteria, such as Aeromonas hydrophila, Escherichia coli D31, and Vibrio parahaemolyticus (MECs, 3.1–12.0 μg/mL), and against Candida albicans (MEC, 16.0 μg/mL) without significant hemolytic activity. Antimicrobial activity of this peptide is heat-stable but salt-sensitive. According to the secondary structural prediction and the homology modeling, this peptide consists of three secondary structural motifs, including one α-helix and two parallel β-strands, and forms an amphipathic structure. This peptide showed neither membrane permeabilization ability nor killing ability, but did display a small degree of leakage ability. These results suggest that SJGAP acts through a bacteriostatic process rather than bactericidal one. SJGAP is another GAPDH-related antimicrobial peptide isolated from skipjack tuna and likely plays an important role for GAPDH in the innate immune defense of tuna fish.     

Tubulin Genes in the Algal Protist Euglena gracilis

ABSTRACT Alpha- and beta-tubulin cDNA were selected from a Euglenaλgt11 expression library, recloned and either sequenced (α-tubulin cDNA) or hybridized to Euglena RNA and DNA (α- and β-tubulin cDNA). RNA for hybridization was extracted at 30 minute intervals after flagellar amputation and quantitated for cDNA binding. Unlike previous reports on most other flagellates, no net increase in either α- or β-tubulin RNA could be detected during regeneration—suggesting steady state or constitutive tubulin RNA synthesis. Incubation of the cDNA with genomic DNA after restriction digestion produced patterns of hybridization consistent with the presence of one to two kinds each of the α- and β-tubulin genes. The deduced amino acid sequence of the α-tubulin cDNA was more than 90% identical to the α-tubulins of Trypanosoma, Chlamydomonas, Naegleria, Tetrahymena and higher plants. The carboxy terminus of the α-tubulin cDNA and the previously sequenced β-tubulin of Euglena showed greatest identity to the carboxy terminus of the tubulins from Trypanosoma brucei. The sequence data for α and β-tubulins of Euglena provides direct evidence for the similarity of two gene products from euglenas and trypanosomes and adds support to earlier suggestions that these organisms are phylogenetically related.     

Cloning, sequence analysis and crystal structure determination of a miraculin-like protein from Murraya koenigii

Earlier, the purification of a 21.4 kDa protein with trypsin inhibitory activity from seeds of Murraya koenigii has been reported. The present study, based on the amino acid sequence deduced from both cDNA and genomic DNA, establishes it to be a miraculin-like protein and provides crystal structure at 2.9 Å resolution. The mature protein consists of 190 amino acid residues with seven cysteines arranged in three disulfide bridges. The amino acid sequence showed maximum homology and formed a distinct cluster with miraculin-like proteins, a soybean Kunitz super family member, in phylogenetic analyses. The major differences in sequence were observed at primary and secondary specificity sites in the reactive loop when compared to classical Kunitz family members. The crystal structure analysis showed that the protein is made of twelve antiparallel β-strands, loops connecting β-strands and two short helices. Despite similar overall fold, it showed significant differences from classical Kunitz trypsin inhibitors.     

Salt Induces Expression of RH3.2A, Encoding an H3.2-type Histone H3 Protein in Rice (Oryza sativa L.)

Histone H3 is one of the four histones, along with H2A, H2B, and H4, which form the eukaryotic nucleosome octamer core. In this study, a new gene RH3.2A encoding an H3.2-type histone H3 protein from rice (Oryza sativa L.) was reported. RH3.2A was cloned through RT-PCR from salt-treated rice seedlings. This gene encoded a protein of 136 amino acid residues that were similar to some plant histone H3 proteins reported previously. However, the cDNA sequence of RH3.2A and other rice H3 genes were different. Alignment of RH3.2A encoding protein with other plant histone H3 proteins revealed that three amino acid residues (32, 88, and 91) were markedly different between H3.1-type and H3.2-type proteins. The mRNA expression analysis of RH3.2A revealed that RH3.2A gene was upregulated by salt stress in rice roots and ABA treatment in seedlings. The potential role of RH3.2A during salt stress was discussed.     

Molecular cloning and nucleotide sequence of tuna growth hormone cDNA

cDNA for mRNA of tuna growth hormone (GH) was cloned by screening a cDNA library constructed from tuna pituitary gland poly(A)⁺ RNA. The nucleotide sequence of cDNA (911 bases) revealed an open reading frame of 615 nucleotides, including a sequence (51 bases) for a possible secretory protein leader peptide. Noncoding regions were found in the nucleotide sequences up- (5′-terminal: 65 bases) and down- (3′-terminal: 231 bases) stream of the open reading frame. An amino-acid sequence deduced from the nucleotide sequence of the cDNA was identical with that determined in the purified tuna GH. Tuna GH was composed of 187 amino acids, and had a calculated molecular weight of 21275. Amino-acid sequencing showed that there was one possible N-glycosylation site at Asn (Asn-Cys-Thr). Tuna GH showed amino-acid sequence homologies with chum salmon (67%), yellow tail (90%) and with human (32%) growth hormones.     

Cloning and analysis of the cDNA encoding the rod G-protein transducin α, β1 and γ1 subunits from the canine retina

The canine (Canis familiaris) retinal rod transducin (G_T) α, β1 and γ1 subunits were sequenced. Cloning of the cDNAs was accomplished by polymerase chain reaction (PCR) using degenerate and wild type retinal cDNA libraries as templates. The deduced amino acid sequences were highly similar to rod transducins from other species: G_Tα differed by 5 amino acids from the corresponding human sequence, whereas β1 and γ1 were identical to human sequences. The coding sequence of rod transducin was evaluated as a possible cause for the recessively inherited retinal rod-cone degeneration: there were no nucleotide differences between the wild type and retinal degenerate strains.     

Biochemical,pharmacological and genomic characterisation of Ts IV,an α-toxin from the venom of the South American scorpion Tityus serrulatus

The venom of the scorpion, Tityus serrulatus, was fractionated to investigate the chemical and pharmacological properties of its α-toxin content. Three α-toxins (Ts III, Ts IV and Ts V) were purified by conventional chromatography (gel filtration and ion-exchange chromatography), followed by immunoaffinity chromatography. Competition experiments using reference α- and β-toxins suggested that these α-toxins were contaminated with around 0.01% of β-toxin. The sequence of the first 30 amino acids of Ts IV was established. Using an oligonucleotide probe, a cDNA encoding its precursor was cloned from a venom gland cDNA library. The primary structure deduced from the cDNA nucleotide sequence provides possible explanations for the polymorphism of these three molecules.     

Multiple β-defensin isoforms identified in early developmental stages of the teleost Paralichthys olivaceus

The β-defensin-like gene and its cloned isoforms (fBDI-1 to -5) were identified in an expressed sequence tag (EST) library from the early developmental stages of the olive flounder, Paralichthys olivaceus. The fBDI cDNA clones show identical amino acid sequences in 24 residues of the signal peptide and 38 residues of the mature peptide; however, the propiece region varies in sequence and length, from 5 to 15 amino acid residues. The predicted molecular weight of the mature peptide is 3.83 kDa, and its predicted isoelectric point is 4.1, showing anionic properties. The genomic organisation of the isoforms was analysed using bacterial artificial chromosome (BAC) DNA containing the fBDI gene. Southern blotting and sequence analyses of fBDI BAC DNA confirmed that the fBDI isoforms cluster at the same locus and exhibit the conserved gene organisation reported for other fish defensin genes. The fBDI mRNA was expressed constitutively in early developmental stages after hatching, and pathogen challenge induced fBDI expression in the head kidney of juvenile fish. We also produced a recombinant fBDI peptide (smfBD) using the expression plasmid pET32 and examined its bioactivity toward Escherichia coli.     

A Mesorhizobium lipopolysaccharide (LPS) specific lectin (CRL) from the roots of nodulating host plant, Cicer arietinum

A 30 kDa rabbit erythrocyte agglutinating glycoprotein isolated and characterized from the roots of Cicer arietinum and designated as cicer root lectin (CRL). Hemagglutination activity of CRL is strongly inhibited by cell surface LPS of nodulating cicer specific Rhizobium. CRL agglutinates mesorhizobial cells and not Escherichia coli or yeast cells. It binds to immobilized LPS of cicer specific Rhizobium only. The primary structure of CRL as predicted by peptide mass fingerprinting by MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) indicated ∼54% amino acid sequence homology with C. arietinum seedling lectin (Accession no. gi/3204123) and ∼26% with C. arietinum (Accession no. gi/110611256), and Pisum sativum (Accession nos. gi/230612, gi/6729956, gi/126148) lectins. These suggested CRL to be a member of vegetative tissue lectin. Circular dichroism analysis indicated that the secondary structure of CRL consists of 48% β-sheets, 26% random coils, and 11% α-helix. CRL has six isoforms of closely associated molecular mass with differential acidic pI of 5.30, 5.20, 5.15, 5.05, 5.00, 4.80. Identity of these isoforms was confirmed from their binding with cicer specific Rhizobium LPS. All the isoforms of CRL are differentially glycosylated as identified by deglycosylation and monosaccharide analysis using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). All these results suggest that unlike other plant lectins CRL is a LPS-binding lectin.     

Identification and expression analysis of the Steinernema carpocapsae elastase-like serine protease gene during the parasitic stage

A cDNA encoding elastase was isolated from Steinernema carpocapsae by suppression subtractive hybridization and rapid amplification of 5′ cDNA ends. The predicted protein contained a 19-aa signal peptide, a 44-aa N-terminal propeptide, and a 264-aa mature protein with a predicted molecular mass of 28,949 Da and a theoretical pI of 8.88. BLAST analysis showed 27-35% amino acid sequence identity to serine proteases from insects, mammals, fish and other organisms. The Sc-ela gene contains three exons and two introns with at least two copies in the S. carpocapsae genome. Expression analysis indicated that the Sc-ela gene was upregulated during the initial parasitic stage. Sequence comparison and evolutionary marker analysis revealed that Sc-ELA was a member of the elastase serine protease family with potential degradative, developmental and fibrinolytic activities. Homology modeling showed that Sc-ELA adopts a two β-barrel fold typical of trypsin-like serine proteases, and phylogenetic analysis indicates that Sc-ELA branched off early during elastase evolution.     

Integrin αIIbβ3 inside-out activation: an in situ conformational analysis reveals a new mechanism

Integrins are a family of heterodimeric adhesion receptors that transmit signals bi-directionally across the plasma membranes. The transmembrane domain (TM) of integrin plays a critical role in mediating transition of the receptor from the default inactive to the active state on the cell surfaces. In this study, we successfully applied the substituted cysteine scanning accessibility method to determine the intracellular border of the integrin α(IIb)β(3) TM in the inactive and active states in living cells. We examined the aqueous accessibility of 75 substituted cysteines comprising the C terminus of both α(IIb) and β(3) TMs, the intracellular membrane-proximal regions, and the whole cytoplasmic tails, to the labeling of a membrane-permeable, cysteine-specific chemical biotin maleimide (BM). The active state of integrin α(IIb)β(3) heterodimer was generated by co-expression of activating partners with the cysteine-substituted constructs. Our data revealed that, in the inactive state, the intracellular lipid/aqueous border of α(IIb) TM was at Lys(994) and β(3) TM was at Phe(727) respectively; in the active state, the border of α(IIb) TM shifted to Pro(998), whereas the border of β(3) TM remained unchanged, suggesting that complex conformational changes occurred in the TMs upon α(IIb)β(3) inside-out activation. On the basis of the results, we propose a new inside-out activation mechanism for integrin α(IIb)β(3) and by inference, all of the integrins in their native cellular environment.