Cloning and nucleotide sequence of a novel 28-kDa protein from the mantle muscle of the squid Todarodes pacificus with homology to tropomyosin

In recent studies, we found autodegradation of collagen from the mantle muscle of the squid Todarodes pacificus and also that the 28- and 25-kDa proteins are closely related to this phenomenon [Connect. Tissue Res. 45 (2004) 109-121]. We obtained partial sequences of three internal portions of this protein, which suggested that 25-kDa protein is a partially degraded form of the 28-kDa protein. We determined the full cDNA sequence of this protein by the degenerate polymerase chain reaction (PCR) using the information of amino acid sequences. The deduced amino acid sequence corresponding to the 212-bp cDNA contained all of the amino acid identified from the 28-kDa protein. Rapid amplification of cDNA ends (RACE) and squid mantle muscle RNA allowed cloning of the full 522-bp sequence, corresponding to a protein of 174 amino acids. A database search indicated that this is a new protein that shares 27-34% identity with tropomyosins from various animals. Structural prediction suggested that it possesses heptad repeats that form coiled-coil structures. We expressed a recombinant protein encoded by the 212-bp cDNA in Escherichia coli and used it to generate a polyclonal antibody. Western blotting with this antibody showed that the 28-kDa protein is expressed in fin, tentacle, and mantle muscle, but not in liver.     

Suppressed expression of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) in an irradiation-attenuated Plasmodium berghei XAT strain

Plasmodium berghei XAT (XAT) is a non-reversible, non-lethal type malaria parasite strain derived from the highly virulent lethal P. berghei NK65 (NK65) by X-irradiation. The difference in polypeptide expression between NK65 and XAT was examined in this study. Western blot patterns of the parasite polypeptides showed that a 30-kDa polypeptide was not detected in XAT. In the present paper, we focused the study on the difference in the expression of the 30-kDa polypeptide between XAT and NK65. Although several other significant differences were noted in the spots shown by two-dimensional gel electrophoresis, the 30-kDa polypeptide was isolated by means of preparative 2D-gel electrophoresis followed by HPLC, and N-terminal amino acid sequence of the polypeptide was eventually determined. Complementary DNA clones encoding the 30-kDa polypeptide were isolated and characterized. Full-length cDNA clones from XAT encoded a protein of 231 amino acid residues with a 693-bp open reading frame. The deduced amino acid sequence exhibited 67% identity with that for P. falciparum hypoxanthine-guanine phosphoribosyltransferase (HGPRT; EC 2.4.2.8), suggesting that this protein is P. berghei HGPRT. Northern blot analysis revealed that expression of HGPRT in XAT was only one-eighth of that in NK65. This finding indicates that HGPRT gene expression is markedly suppressed in XAT. The amino acid sequence of HGPRT from NK65 was identical to that from XAT. This finding showed that the amino acid sequence of XAT-HGPRT was not mutated and had not undergone deletion.     

Cloning,characterization, and expression of cDNA encoding a lipase from Kurtzmanomyces sp. I-11

A cDNA clone of the lipase secreted by Kurtzmanomyces sp. I-11 was isolated from a cDNA library of this yeast by PCR screening using oligonucleotide primers designed on the basis of the partial amino acid sequence of the lipase. The cloned cDNA (lip1) encoded a hydrophobic protein of 484 amino acids, where the first 20 amino acids and the following 6 amino acid sequences were predicted to be the signal sequence for secretion and a pro-sequence, respectively. The deduced amino acid sequence of the Kurtzmanomyces lipase was most similar to Candida antarctica DSM 3855 lipase A (74% identity) and weakly to other lipases. The consensus pentapeptide (-Gly-X-Ser-X-Gly-) that forms a part of the interfacial lipid recognition site in lipases was conserved. A high level of lipase was produced by Pichia pastoris transformed with the lip1 cDNA, indicating that the cloned cDNA indeed encodes a lipase.     

A family of small inducible proteins secreted by leukocytes are members of a new superfamily that includes leukocyte and fibroblast-derived inflammatory agents, growth factors, and indicators of various activation processes

We have isolated and characterized four cDNA clones that encode mRNA expressed more abundantly in Con A-activated mouse helper T cells than by resting T cells. One mRNA encoded a approximately 14-kDa protein with a hydrophobic N-terminal sequence and was abundantly expressed by the Th 2 subset of Th cells, but was not expressed by Th 1 cells. The remaining three mRNA encoded related approximately 8-kDa secreted proteins that are part of a family of small, secreted, and inducible mouse and human proteins. This family of proteins is itself distantly related to another family of growth and inflammatory factors that are associated with various lymphoid and fibroblast activation phenomena. One of the small, inducible, secreted proteins has a predicted mature N terminus identical to that of the previously described macrophage inflammatory protein.     

Protein synthesis at the blood-brain barrier. The major protein secreted by amphibian choroid plexus is a lipocalin.

Among the proteins secreted by choroid plexus of vertebrates, one protein is much more abundant than all others. In mammals, birds, and reptiles this protein is transthyretin, a tetramer of identical 15-kDa subunits. In this study choroid plexus from frogs, tadpoles, and toads incubated in vitro were found to synthesize and secrete one predominant protein. However, this consisted of one single 20-kDa polypeptide chain. It was expressed throughout amphibian metamorphosis. Part of its amino acid sequence was determined and used for construction of oligonucleotides for polymerase chain reaction. The amplified DNA was used to screen a toad choroid plexus cDNA library. Full-length cDNA clones were isolated and sequenced. The derived amino acid sequence for the encoded protein was 183 amino acids long, including a 20-amino acid presegment. The calculated molecular weight of the mature protein was 18,500. Sequence comparison with other proteins showed that the protein belonged to the lipocalin superfamily. Its expression was highest in choroid plexus, much lower in other brain areas, and absent from liver. Since no transthyretin was detected in proteins secreted from amphibian choroid plexus, abundant synthesis and secretion of transthyretin in choroid plexus must have evolved only after the stage of the amphibians.     

Primary mesenchyme cells of the sea urchin embryo require an autonomously produced, nonfibrillar collagen for spiculogenesis

A collagen molecule in the sea urchin embryo was characterized by analysis of a 2.7-kb cDNA clone. This clone, Spcoll, was obtained by screening a gastrula stage Strongylocentrotus purpuratus cDNA library with a 237-bp genomic clone encoding a collagen-like sequence previously isolated by Venkatesan et al. (1986). DNA sequence analysis of the cDNA clone demonstrated the nonfibrillar nature of the encoded molecule--13 interruptions of the Gly-X-Y repeat motif were found in the 85-kDa open reading frame. The mRNA of approximately 9 kb accumulated specifically in mesenchyme cells of the embryo through development to the pluteus larva. Polyclonal antibodies generated against a Spcoll-beta-galactosidase fusion protein were utilized to identify and localize the native Spcoll. This collagen molecule of approximately 210 kDa was deposited into the blastocoel by the primary mesenchyme cells. When primary mesenchyme cells were cultured in vitro, Spcoll was secreted into the media and accumulated at sites of cell-substrate interaction. Addition of anti-Spcoll antibodies to primary mesenchyme cell cultures selectively inhibited spiculogenesis, whereas other antibodies had no inhibitory effect. Since collagen is not a component of the organic matrix of spicules (Benson et al., 1986), these results suggest that the autonomous production of Spcoll by differentiating mesenchyme cells in turn influences the point in differentiation at which these cell initiate biomineralization.     

Cloning and characterization of a cDNA from Aspergillus parasiticus encoding an O-methyltransferase involved in aflatoxin biosynthesis.

Aflatoxins are polyketide-derived secondary metabolites produced by the fungi Aspergillus flavus and Aspergillus parasiticus. Among the catalytic steps in the aflatoxin biosynthetic pathway, the conversion of sterigmatocystin to O-methylsterigmatocystin and the conversion of dihydrosterigmatocystin to dihydro-O-methylsterigmatocystin are catalyzed by an S-adenosylmethionine-dependent O-methyltransferase. A cDNA library was constructed by using RNA isolated from a 24-h-old culture of wild-type A. parasiticus SRRC 143 and was screened by using polyclonal antiserum raised against a purified 40-kDa O-methyltransferase protein. A clone that harbored a full-length cDNA insert (1,460 bp) containing the 1,254-bp coding region of the gene omt-1 was identified by the antiserum and isolated. The complete cDNA sequence was determined, and the corresponding 418-amino-acid sequence of the native enzyme with a molecular weight of 46,000 was deduced. This 46-kDa native enzyme has a leader sequence of 41 amino acids, and the mature form of the enzyme apparently consists of 377 amino acids and has a molecular weight of 42,000. Direct sequencing of the purified mature enzyme from A. parasiticus SRRC 163 showed that 19 of 22 amino acid residues were identical to the amino acid residues in an internal region of the deduced amino acid sequence of the mature protein. The 1,460-bp omt-1 cDNA was cloned into an Escherichia coli expression system; a Western blot (immunoblot) analysis of crude extracts from this expression system revealed a 51-kDa fusion protein (fused with a 5-kDa beta-galactosidase N-terminal fragment).(ABSTRACT TRUNCATED AT 250 WORDS)     

Mitochondrial DNA polymerases from yeast to man: a new family of polymerases

We report the sequence of a 4.5-kb cDNA clone isolated from a human melanoma library which bears high amino acid sequence identity to the yeast mitochondrial (mt) DNA polymerase (Mip1p). This cDNA contains a 3720-bp open reading frame encoding a predicted 140-kDa polypeptide that is 43% identical to Mip1p. The N-terminal part of the sequence contains a 13 glutamine stretch encoded by a CAG trinucleotide repeat which is not found in the other DNA polymerases γ (Pol γ). Multiple amino acid sequence alignments with Pol γ from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Drosophila melanogaster, Xenopus laevis and Mus musculus show that these DNA polymerases form a family strongly conserved from yeast to man and are only loosely related to the Family A DNA polymerases.     

Nucleotide sequence of the insecticidal protein gene of Bacillus thuringiensis strain aizawai IPL7 and its high-level expression in Escherichia coli

A DNA fragment carrying the insecticidal protein gene of Bacillus thuringiensis subsp. aizawai IPL7 was cloned from a 78-kb plasmid. The nucleotide sequence revealed that the cloned DNA fragment contained a 3465-bp protein-coding region with 156-bp 5'-flanking, and 168-bp 3'-flanking regions. The open reading frame encoded a 130,690 Da protein consisting of 1155 amino acid residues. Nucleotide sequence comparison of the aizawai gene with the published berliner 1715 gene showed only 8 nt changes in the coding regions. It was found that 72 bp of the 5'-flanking sequence of the cloned aizawai gene was responsible for constitutive expression of the 130-kDa protein gene in Escherichia coli. The expression was greatly enhanced by introducing the tac promoter upstream from the 72-bp 5'-flanking region of the aizawai gene. Under optimal conditions, the 130-kDa insecticidal protein amounted to 38% of the total cellular protein.     

Identification of the cDNA clone which encodes the 58-kDa protein containing the amino acid sequence of rat liver non-specific lipid-transfer protein (sterol-carrier protein 2). Homology with rat peroxisomal and mitochondrial 3-oxoacyl-CoA thiolases

The relationship between the rat liver non-specific lipid-transfer protein (nsLTP) and the 58-kDa protein cross-reactive with anti-nsLTP antibodies, was investigated by cDNA analysis. A 1945-bp cDNA clone was isolated which encodes a 58.7-kDa protein. This protein is identical to the 58-kDa immunoreactive protein determined by N-terminal sequence analysis of the purified 58-kDa protein. It consists of 546 amino acid residues, of which the 123 C-terminal residues are identical to the sequence of nsLTP. The N-terminal 400 amino acid residues of the 58.7-kDa protein were found to have 23.5% identity with the sequence of both mitochondrial and peroxisomal rat 3-oxoacyl-CoA thiolases, including a hypothetical substrate-binding site. The cDNA insert hybridizes with 1.1-kb, 1.7-kb, 2.4-kb and 3.0-kb mRNA species in RNA isolated from various rat tissues and from Chinese hamster ovary (CHO) cells. Southern blot analysis suggests that these mRNA species are generated from a single gene. Mutant CHO cells, deficient in peroxisomes, lack nsLTP. We have found that the mRNA encoding nsLTP is still present in these cells, which suggests that the absence of this protein is related to the lack of peroxisomes.     

Cloning of cDNA encoding a soybean allergen, Gly m Bd 28K

A cDNA clone encoding a soybean allergen, Gly m Bd 28K, has been isolated. The clone has a 1567-bp cDNA insert with a 1419-bp open reading frame and a 148-bp 3'-untranslated region, followed by a polyadenylation tail. The open reading frame was shown to encode a polypeptide composed of 473 amino acids. The chemically determined amino acid sequences of the peptides obtained from the allergen, including its N-terminal peptide, were shown to be contained in the N-terminal region of the amino acid sequence deduced from the cDNA, showing that the first half of the cDNA encodes the allergen with a preceding segment of 21 amino acids. The peptide fragment including the allergen was expressed as a fusion protein with glutathione S-transferase in Escherichia coli and immunoblotted with the sera of soybean-sensitive patients and the monoclonal antibody against the allergen. Furthermore, homology analyses demonstrate that the polypeptide for the cDNA exhibits high homology with the MP27/MP32 proteins in pumpkin seeds and the carrot globulin-like protein. This finding suggests that the polypeptide may consist of a 21-amino acid segment as a part of the signal peptide and the proprotein, which may be converted to two mature proteins, Gly m Bd 28K and a 23-kDa protein, during the development of soybean cotyledons.     

cDNA cloning of ovine pulmonary SP-A, SP-B, and SP-C: isolation of two different sequences for SP-B

Pulmonary surfactant promotes alveolar stability by lowering the surface tension at the air-liquid interface in the peripheral air spaces. The three surfactant proteins SP-A, SP-B, and SP-C contribute to dynamic surface properties involved during respiration. We have cloned and sequenced the complete cDNAs for ovine SP-A and SP-C and two distinct forms of ovine SP-B cDNAs. The nucleotide sequence of ovine SP-A cDNA consists of 1,901 bp and encodes a protein of 248 amino acids. Ovine SP-C cDNA contains 809 bp, predicting a protein of 190 amino acids. Ovine SP-B is encoded by two mRNA species, which differ by a 69-bp in-frame deletion in the region coding for the active airway protein. The larger SP-B cDNA comprises 1,660 bp, encoding a putative protein of 374 amino acids. With the sequences reported, a more complete analysis of surfactant regulation and the determination of their physiological function in vivo will be enabled.     

Myristic acid is the NH2-terminal blocking group of the 43-kDa protein of Torpedo nicotinic post-synaptic membranes

The NH2-terminal blocking group of the 43-kDa peripheral membrane protein (43-kDa protein) of Torpedo post-synaptic membranes has been identified as myristic acid. To identify that blocking group pure 43-kDa protein was digested with trypsin and the blocked tryptic peptide was isolated by reverse phase HPLC. That peptide coeluted with and had the same amino acid composition as a synthetic peptide, myristoyl-Gly-Gln-Asp-Gln-Thr-Lys, the structure of the amino terminus predicted from the protein sequence deduced from a cDNA clone. The presence of myristate was confirmed by the precise molecular mass of the peptide, 886.5266, determined by fast atom bombardment mass spectroscopy.     

Molecular cloning of a member of the Fasciola hepatica saposin-like protein family

A 436-bp complementary DNA (cDNA) was isolated from an adult Fasciola hepatica cDNA expression library by screening with the serum from a rabbit infected with F. hepatica for 4 wk. The deduced amino acid sequence encoded by this cDNA is an 11.5-kDa polypeptide that has significant homology to F. hepatica NK-lysin protein, to several members of saposin-like or NK-lysin protein families, as well as 3 amoebapore precursors of Entamoeba histolytica. The most striking feature observed within this protein, denoted FhSAP-2, is the presence of 6 conserved cysteine residues arranged within 5 amphipathic alpha-helical domains and the presence of 7 hydrophobic residues in strictly conserved positions. Using enzyme-linked immunosorbent assay it was found that rFhSAP-2 is highly reactive with sera from rabbits infected with F. hepatica for 2-14 wk as well as with sera from humans with chronic fascioliasis. An anti-rFhSAP-2 rabbit antiserum reacted with F. hepatica excretory-secretory antigens by Western blot, revealing a major 11.5-kDa and 2 minor 46- and 67-kDa antigenic polypeptides. This suggests that FhSAP-2 may be an antigen released from cytoplasmic storage granules present within F. hepatica parasites. rFhSAP-2 also exhibits a strong lytic activity on human erythrocytes and peripheral blood mononuclear cells. This suggests that cell lysis could be 1 of the biological functions of this protein.     

Effects of pre- and pro-sequence of thaumatin on the secretion by Pichia pastoris

Thaumatin is a 22-kDa sweet-tasting protein containing eight disulfide bonds. When thaumatin is expressed in Pichia pastoris using the thaumatin cDNA fused with both the alpha-factor signal sequence and the Kex2 protease cleavage site from Saccharomyces cerevisiae, the N-terminal sequence of the secreted thaumatin molecule is not processed correctly. To examine the role of the thaumatin cDNA-encoded N-terminal pre-sequence and C-terminal pro-sequence on the processing of thaumatin and efficiency of thaumatin production in P. pastoris, four expression plasmids with different pre-sequence and pro-sequence were constructed and transformed into P. pastoris. The transformants containing pre-thaumatin gene that has the native plant signal, secreted thaumatin molecules in the medium. The N-terminal amino acid sequence of the secreted thaumatin molecule was processed correctly. The production yield of thaumatin was not affected by the C-terminal pro-sequence, and the pro-sequence was not processed in P. pastoris, indicating that pro-sequence is not necessary for thaumatin synthesis.     

Co- and post-translational processing of the hevein preproprotein of latex of the rubber tree (Hevea brasiliensis)

Hevein is a chitin-binding protein of 43 amino acids found in the lutoid body-enriched fraction of rubber tree latex. A hevein cDNA clone (HEV1) (Broekaert, W., Lee, H.-i., Kush, A., Nam, C.-H., and Raikhel, N. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 7633-7637) encodes a putative signal sequence of 17 amino acids followed by a polypeptide of 187 amino acids. Interestingly, this polypeptide has two distinct domains: an amino-terminal domain of 43 amino acids, corresponding to mature hevein, and a carboxyl-terminal domain of 144 amino acids. To investigate the mechanisms involved in processing of the protein encoded by HEV1, three domain-specific antisera were raised against fusion proteins harboring the amino-terminal domain (N domain), carboxyl-terminal domain (C domain), and both domains (NC domain). Translocation experiments using an in vitro translation system show that the first 17-amino acid sequence encoded by the cDNA functions as a signal peptide. Immunoblot analysis of proteins extracted from lutoid bodies demonstrates that a 5-kDa protein comigrated with purified mature hevein and cross-reacted with N domain- and NC domain-specific antibodies. A 14-kDa protein was recognized by C domain- and NC domain-specific antibodies. A 20-kDa protein was cross-reactive with all three antibodies. Microsequencing data further suggest that the 5-kDa (amino-terminal domain) and 14-kDa (carboxyl-terminal domain) proteins are post-translational cleavage products of the 20-kDa polypeptide (both domains) which corresponds to the proprotein encoded by HEV1. In addition, it was found that the amino-terminal domain could provide chitin-binding properties to a fusion protein bearing it either amino terminally or carboxyl terminally.