Primary Structures of Actinoporins from Sea Anemone <Emphasis Type="Italic">Oulactis orientalis</Emphasis>

Partial amino acid sequences of the actinoporins Or-A (136 aa) and Or-G (144 aa) isolated from the Sea of Japan sea anemone Oulactis orientalis were determined by sequencing the clones obtained by the amplification of genomic DNA and cDNA with primers specific to the N-terminal regions of the O. orientalis actinoporin sequences and to the C-terminal region, which is highly conservative in all the known actinoporin sequences. The complete structures of Or-A (165 aa) and Or-G (173 aa) were elucidated by sequencing the cDNA clones obtained by the rapid amplification of 3′-ends of cDNA. A comparative analysis of the amino acid sequences of the Oulactis actinoporins with those of actinoporins from tropical species revealed considerable differences in the structures of their N-terminal fragments and their membrane-binding sites. We believe that these differences could explain the lower hemolytic activities of Or-A and Or-G compared to those of actinoporins from the tropical species.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 4, 2005, pp. 357–362.Original Russian Text Copyright © 2005 by Il’ina, Monastyrnaya, Isaeva, Guzev, Rasskazov, Kozlovskaya     

Actinoporins from the Sea of Japan anemone Oulactis orientalis: isolation and partial characterization

Two cytolytic toxins (cytolysins Or-A and Or-G) were isolated from the Sea of Japan anemone Oulactis orientalis and characterized. Their purification scheme involved a hydrophobic chromatography on Polychrom 1, a gel filtration on Akrilex P-4, a cation-exchange chromatography on CM-32 cellulose, and a reversed-phase HPLC on a Nucleosil C18 column. The molecular masses of Or-A and Or-G were determined by SDS-PAGE in 14% PAG to be ca. 18 kDa. The absence of Cys residues and a high content of basic amino acid residues are characteristic of their amino acid compositions. The hemolytic activities of Or-A and Or-G were found to be 295.86 and 322.58 HU/mg, respectively; these are by three orders of magnitude lower than those of sphingomyelin-inhibitable cytolysins from the tropic sea anemones. The amino acid sequences of the N-terminal fragments of Or-A and Or-G were determined to be ATFRVLAK and GAIIAGAA, respectively. Action of the cytolysins on the erythrocyte membrane is inhibited by exogenous sphingomyelin. They form ion channels in bilayer lipid membranes with the conductivity of 16, 32, and 40 pSm in 0.1 M NaCl and 168, 240, and 320 pSm in 1 M NaCl at pH 7.2. Therefore, they were attributed to the group of actinoporins.     

Actinoporins from the Sea of Japan anemone <Emphasis Type="Italic">Oulactis orientalis</Emphasis>: Isolation and partial characterization

Two cytolytic toxins (cytolysins Or-A and Or-G) were isolated from the Sea of Japan anemone Oulactis orientalis and characterized. Their purification scheme involved a hydrophobic chromatography on Polychrom-1, a gel filtration on Akrilex P-4, a cation-exchange chromatography on CM-32 cellulose, and a reverse-phase HPLC on a Nucleosil C₁₈ column. The molecular masses of Or-A and Or-G were determined by SDS-PAGE in 14% PAG to be ca. 18 kDa. The absence of Cys residues and a high content of basic amino acid residues are characteristic of their amino acid compositions. The hemolytic activities of Or-A and Or-G were found to be 295.86 and 322.58 HU/mg, respectively; these are by three orders of magnitude lower than those of sphingomyelin-inhibitable cytolysins from the tropic sea anemones. The amino acid sequences of the N-terminal fragments of Or-A and Or-G were determined to be ATFRVLAK and GAIIAGAA, respectively. Action of the cytolysins on the erythrocyte membrane is inhibited by exogenous sphingomyelin. They form ion channels in bilayer lipid membranes with the conductivity of 16, 32, and 40 pSm in 0.1 M NaCl and 168, 240, and 320 pSm in 1 M NaCl at pH 7.2. Therefore, they were attributed to the group of actinoporins.Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 1, 2005, pp. 39–48.Original Russian Text Copyright © 2005 by Ilina, Monastyrnaya, Sokotun, Egorov, Nazarenko, Likhatskaya, Kozlovskaya.     

New actinoporins from sea anemone <Emphasis Type="Italic">Heteractis crispa</Emphasis>: Cloning and functional expression

A new actinoporin Hct-S4 (molecular mass 19,414 ± 10 Da) belonging to the sphingomyelin-inhibited α-pore forming toxin (α-PFT) family was isolated from the tropical sea anemone Heteractis crispa (also called Radianthus macrodactylus) and purified by methods of protein chemistry. The N-terminal nucleotide sequence (encoding 20 amino acid residues) of actinoporin Hct-S4 was determined. Genes encoding 18 new isoforms of H. crispa actinoporins were cloned and sequenced. These genes form a multigene Hct-S family characterized by presence of N-terminal serine in the mature proteins. Highly conserved residues comprising the aromatic phosphorylcholine-binding site and significant structure-function changes in the N-terminal segment (10–27 amino acid residues) of actinoporins were established. Two expressed recombinant actinoporins (rHct-S5 and rHct-S6) were one order less hemolytically active than native actinoporins.     

Characterization of the cytochrome P-450IID subfamily in bovine liver. Nucleotide sequences and microheterogeneity.

To elucidate the molecular mechanisms underlying drug detoxification, the structures of the members of the microsomal cytochrome P-450IID subfamily were analyzed by isolating, mapping and sequencing cytochrome P-450IID (CYP2D) cDNA clones from bovine liver. The screening was performed under nonstringent conditions so that most of the P-450IID subfamily members could be obtained. 114 of the 147 positive clones were classified into four groups on the basis of their restriction-enzyme maps. The maps of the four groups were highly similar, however, the clones of one group contained an insertion of approximately 500 bp in the coding region. Analysis of partial nucleotide sequences of several representative clones from each group showed that the bovine P-450IID subfamily in liver consisted of several, not many, highly similar members, differing by less than 7% in their nucleotide sequences. The location of the insertion found in the minor group corresponded to intron 7 and the GT/AG rule was found at the exon/intron boundary, suggesting that intron 7 was retained in this group. The complete nucleotide sequences of two clones from the major group were examined to determine the structures of the P-450IID subfamily in bovine liver. A full-length cDNA clone (1615 bp) and a partial cDNA clone (1538 bp) contained open reading frames encoding 500 and 487 amino acid residues, respectively. The partial clone lacked the nucleotide sequence corresponding to the first 13 N-terminal amino acid residues. The deduced amino acid sequences of the two clones were 98% similar, and 80% and 68% similar to those from human CYP2D6 and rat CYP2D1, respectively. Comparisons of the amino acid sequences of the P-450IID subfamily members showed the highly conserved C-terminal region of their molecules and the high similarity between the members in one species, especially in cattle and man.     

The primary structure of human gamma-glutamyl transpeptidase

A cDNA hybridizable to that of rat gamma-glutamyl transpeptidase (GGT) was cloned from a cDNA library of human fetal liver. The insert of the cDNA clone contained 1866 bp consisting of an open reading frame (ORF) of 1709 bp (569 amino acids (aa), N-terminal portion truncated) and a 135-bp 3'-untranslated region followed by a polyadenylated tail. In parallel, amino acid sequences of N-terminal portions of heavy and light chains of a purified human GGT were determined. Two stretches of amino acid sequences identical to the N-terminal sequences of heavy and light chains were found in the ORF. We therefore concluded that the clone is a cDNA for human GGT. From the amino acid sequence deduced from cDNA, the heavy and the light chains of the purified enzyme are estimated to be composed of 351 aa (Mr 38,336) and of 189 aa (Mr 20,000), respectively. The heavy chain is preceded by a signal peptide of at least 29 aa presumed to be cleaved by bromelain treatment. Six putative N-glycosylation sites are present in the heavy subunit region and one in the light subunit region. Primary structure and hydrophobicity profile are closely similar to those of rat GGT.     

Nuclear genes for cytochrome c oxidase subunits of Neurospora crassa. Isolation and characterization of cDNA clones for subunits IV, V, VI, and possibly VII

We obtained cDNA clones for cytochrome oxidase subunits IV, V, VI, and possibly VII by constructing a lambda gt11 library of Neurospora crassa cDNA and probing it with antiserum directed against Neurospora cytochrome oxidase holoenzyme. Positive clones were further characterized with antisera directed against individual cytochrome oxidase subunits and subsequently by DNA sequencing. The clones for subunits IV and V encode proteins with regions matching the known N-terminal amino acid sequences of purified Neurospora cytochrome oxidase subunits IV and V, respectively. The sequences of these clones provide the first evidence that cytochrome oxidase subunits IV and V are made as precursors with N-terminal extensions in Neurospora. The N-terminal extensions encoded by these clones share homology, and are rich in arginine, as are signal sequences of other mitochondrially destined proteins. The subunit VI clone codes for the carboxyl terminus of a protein homologous to the carboxy termini of yeast cytochrome oxidase subunit VI and bovine cytochrome oxidase subunit Va. The subunit VII clone contains an open reading frame for a 47-residue protein, the expected size for subunit VII. However, the protein coded by this clone has an unusual amino acid composition. Whether this clone represents an authentic cytochrome oxidase subunit is not established.     

Cloning of Complementary and Genomic DNAs Encoding Echotoxins,Proteinaceous Toxins from the Salivary Gland of Marine Gastropod <Emphasis Type="Italic">Monoplex echo</Emphasis>

Echotoxins are hemolytic and lethal proteinaceous toxins of about 25 kDa that are contained in the salivary gland of marine gastropod Monoplex echo. In this study, complementary DNAs (cDNAs) encoding four echotoxins (2, A, B1 and B2) were isolated from the cDNA library constructed from the M. echo salivary gland and completely sequenced. Although the amino acid sequence identity between the four echotoxins and actinoporins (20 kDa hemolysins from sea anemones) was very low (12–16%), some amino acid residues important for the biological activity of actinoporins were well conserved in the echotoxins. In the case of echotoxin 2, the genomic DNA corresponding to the coding region was amplified. Sequencing data revealed that the echotoxin 2 gene is devoid of introns within the coding region as reported for the actinoporin genes. These results suggest that echotoxins have evolved from actinoporins or both toxins have evolved from the same ancestor.     

Metallothionein cDNA, promoter, and genomic sequences of the tropical green mussel, Perna viridis.

The primary structure of the cDNA and metallothionein (MT) genomic sequences of the tropical green mussel (Perna viridis) was determined. The complete cDNA sequences were obtained using degenerate primers designed from known metallothionein consensus amino acid sequences from the temperate species Mytilus edulis. The amino acid sequences of P. viridis metallothionein deduced from the coding region consisted of 72 amino acids with 21 cysteine residues and 9 Cys-X-Cys motifs corresponding to Type I MT class of other species. Two different genomic sequences coding for the same mRNA were obtained. Each putative gene contained a unique 5'UTR and two unique introns located at the same splice sites. The promoters for both genes were different in length and both contained metal responsive elements and active protein-binding sites. The structures of the genomic clones were compared with those of other species. J. Exp. Zool. 284:445-453, 1999.     

Molecular cloning of a cDNA for human delta-aminolevulinate dehydratase

A cDNA encoding human delta-aminolevulinic acid dehydratase (ALA-D; EC 4.2.1.24), the second enzyme in the heme biosynthetic pathway, was isolated from a human liver cDNA expression library. Of the original 17 clones selected with anti-ALA-D antibody, only four expressed anti-ALA-D epitopes as assessed by rescreening with antibody preabsorbed with purified antigen. Subsequent screening of the antibody-positive clones with mixed oligodeoxynucleotide (oligo) probes, synthesized to correspond to human N-terminal and bovine active-site peptide sequences, identified three clones which hybridized only with the oligo probes for the bovine amino acid (aa) sequences. Restriction endonucleases analysis revealed that these three clones contained the same 800-bp cDNA insert. This insert was recloned into bacteriophage M13mp18 and mp19 and sequenced by primer extension. The aa sequence predicted from the partial nucleotide sequence was found to be essentially colinear with the sequences of four bovine ALA-D peptides, totaling 35 non-overlapping aa residues.     

Characterization of long cDNA clones from human adult spleen. II. The complete sequences of 81 cDNA clones.

To accumulate information on the coding sequences (CDSs) of unidentified genes, we have conducted a sequencing project of human long cDNA clones. Both the end sequences of approximately 10,000 cDNA clones from two size-fractionated human spleen cDNA libraries (average sizes of 4.5 kb and 5.6 kb) were determined by single-pass sequencing to select cDNAs with unidentified sequences. We herein present the entire sequences of 81 cDNA clones, most of which were selected by two approaches based on their protein-coding potentialities in silico: Fifty-eight cDNA clones were selected as those having protein-coding potentialities at the 5'-end of single-pass sequences by applying the GeneMark analysis; and 20 cDNA clones were selected as those expected to encode proteins larger than 100 amino acid residues by analysis of the human genome sequences flanked by both the end sequences of cDNAs using the GENSCAN gene prediction program. In addition to these newly identified cDNAs, three cDNA clones were isolated by colony hybridization experiments using probes corresponding to known gene sequences since these cDNAs are likely to contain considerable amounts of new information regarding the genes already annotated. The sequence data indicated that the average sizes of the inserts and corresponding CDSs of cDNA clones analyzed here were 5.0 kb and 2.0 kb (670 amino acid residues), respectively. From the results of homology and motif searches against the public databases, functional categories of the 29 predicted gene products could be assigned; 86% of these predicted gene products (25 gene products) were classified into proteins relating to cell signaling/communication, nucleic acid management, and cell structure/motility.     

Mass isolation of cuticle protein cDNAs from wing discs of Bombyx mori and their characterizations

Multiple cloning of cuticle protein genes was performed by sequencing of cDNAs randomly selected from a cDNA library of wing discs just before pupation, and nine different cuticular protein genes were identified. Thirty-one clones of a cuticle protein gene were identified from the 1050 randomly sequenced clones; about 3% were cuticle protein genes in the W3-stage wing disc cDNA library. The sequence diversity of the deduced amino acid sequences of isolated Bombyx cuticle genes was examined along with the expression profiles. The deduced amino acid sequences of the nine cuticle protein genes contained a putative signal peptide at the N-terminal region and a very conserved hydrophilic region known as the R and R motif. The developmental expression of cuticle genes was classified into two types: pupation (five clones were expressed only around pupation) and pupation and mid-pupal (four clones were expressed around this stage). All the isolated genes were expressed in the head, thoracic, and abdominal regions of the epidermis at different levels around pupation, but no expression was observed in the epidermis at the fourth molting stage.     

Tauropine dehydrogenase from the marine sponge Halichondria japonica is a homolog of ornithine cyclodeaminase/mu-crystallin

The partial amino acid sequence including the N- and C-terminal portions of tauropine dehydrogenase (EC 1.5.1.23) from the marine sponge Halichondria japonica was determined by enzymatic cleavages followed by peptide sequencing. This information was used to design degenerate primers for amplification of cDNA encoding the tauropine dehydrogenase. The cDNA included 1231 nucleotides with an open reading frame of 1002 nucleotides that encodes a protein of 334 amino acid residues. From the peptide and nucleotide sequencing, the mature tauropine dehydrogenase was estimated to consist of 333 amino acid residues with an acetylated N-terminal serine residue and no intrachain disulfide bonds. The primary structure of the H. japonica enzyme showed apparent similarity with a homolog of ornithine cyclodeaminase from Rhizobium meliloti and other proteins of the ornithine cyclodeaminase/mu-crystallin family, but it showed no significant similarity with the known sequences of octopine dehydrogenases and tauropine dehydrogenases from marine invertebrates. These findings indicate that opine dehydrogenases in marine invertebrates are not all homologous.     

Analysis of nucleotide sequences of two ligninase cDNAs from a white-rot filamentous fungus, Phanerochaete chrysosporium

An analysis of nucleotide sequences of two types of ligninase cDNAs isolated from the basidiomycete Phanerochaete chrysosporium, designated CLG4 and CLG5, are presented here. The amino acid sequences of the corresponding ligninase proteins, designated LG4 and LG5, respectively, have been deduced from the cDNA sequences. Mature ligninases LG4 and LG5 are preceded by leader sequences containing 28 and 27 amino acids (aa), respectively, and each contains 344 aa residues. The estimated Mrs of mature LG4 and LG5 are 36,540 and 36,607, respectively. Potential N-glycosylation site(s) with the general sequence Asn-X-Thr/Ser are found in both LG4 and LG5. Nucleotide sequence homology between the coding region of CLG4 and CLG5 is 71.5%, whereas the amino acid sequence homology between the two ligninases is 68.5%. The codon usage of ligninases is extremely biased in favor of codons rich in cytosine and guanine. Amino acid sequences of two tryptic peptides of ligninase H8 have exactly matching sequences in ligninase LG5. Also, the sequences of the oligodeoxynucleotide probes, which correspond to the sequences in the tryptic peptides of ligninase H8 and which were used in isolating the ligninase clones from the cDNA library, have exactly matching sequences in CLG5. The experimentally determined N-terminal sequence of purified ligninase H8 is found in the deduced N-terminal amino acid sequence of LG5. These results suggest that CLG5 encodes ligninase H8 and that CLG4 represents a related but different ligninase gene.     

Cloning of the cDNAs coding for cat growth hormone and prolactin

Characterization of the prolactin (PRL) amino acid (aa) or cDNA sequences has not been reported for any member of the Felidae family. We cloned cat growth hormone (cGH) and cat PRL (cPRL) cDNA sequences from a feline pituitary cDNA library. High homology between species allowed bovine PRL (bPRL) and bGH cDNA clones to be used to identify clones encoding the 229-aa cPRL and 216-aa cGH sequences. The cGH protein is most homologous to pig and dog GH. Similarly, cPRL shares the most aa identity to pig PRL (pPRL). Northern blot analysis revealed the mRNA size for cGH and cPRL to be approx. 1 and 1.1 kb, respectively. These results reveal that GH and PRL from the Felidae family are highly conserved to other families of GH and PRL.     

The α-glucuronidase-encoding gene of Trichoderma reesei

The Trichoderma reesei cDNA coding for α-glucuronidase (GLRI), which releases glucuronic acid attached to xylose units of xylan, was cloned and sequenced. The deduced N-terminal amino acid (aa) sequence of the protein was verified by sequencing of the purified GLRI. The aa sequence of the GLRI displayed no similarity with any aa sequence available in the data bases.     

Isolation of cDNA clones for human erythrocyte membrane sialoglycoproteins alpha and delta.

We have isolated almost full-length cDNA clones corresponding to human erythrocyte membrane sialoglycoproteins alpha (glycophorin A) and delta (glycophorin B). The predicted amino acid sequence of delta differs at two amino acid residues from the sequence determined by peptide sequencing. The sialoglycoprotein delta clone we have isolated contains an interrupting sequence within the region that gives rise to the cleaved N-terminal leader sequence for the protein and represents a product that is unlikely to be inserted into the erythrocyte membrane. Comparison of the cDNA sequences of alpha and delta shows very strong homology at the DNA level within the coding regions. The two mRNA sequences are closely related and differ by a number of clearly defined insertions and deletions.     

Expressed Sequence Tags from Developing Castor Seeds

To expand the availability of genes encoding enzymes and structural proteins associated with storage lipid synthesis and deposition, partial nucleotide sequences, or expressed sequence tags (ESTs), were obtained for 743 cDNA clones derived from developing seeds of castor (Ricinus communis L.). Enrichment for seed-specific cDNA clones was obtained by selecting clones that did not detectably hybridize to first-strand cDNA from leaf mRNA. Similarly, clones that hybridized to storage proteins or other highly abundant mRNA species from developing seeds were selected against. To enrich for endomembrane-associated proteins, some clones were selected for sequencing by immunological screening with antibodies prepared against partially purified endoplasmic reticulum membranes. Comparison of the deduced amino acid sequences of the ESTs with the public data bases resulted in the assignment of putative identities of 49% of the clones selected by differential hybridization and 71% of the clones selected by immunological screening. Open reading frames in 100 of the ESTs exhibited higher homology to 78 different nonplant gene products than to any previously known plant gene product.     

Cloning, characterization and nucleotide sequences of two cDNAs encoding human pancreatic trypsinogens

Two cDNA clones encoding two major human trypsinogen isozymes were isolated from a human pancreatic cDNA library. The deduced amino acid (aa) sequences of the two trypsinogen precursors are found to have 89% sequence homology, and have the same number of aa (247), including 15 aa for a signal peptide and 8 aa for an activation peptide. Southern blot analysis of human genomic DNA using the cloned cDNA as a probe, revealed that the human trypsinogen genes constitute a multigene family of more than ten genes.