Cloning and sequence analysis of a cDNA encoding a Brazil nut protein exceptionally rich in methionine

The primary amino acid sequence of an abundant methionine-rich seed protein found in Brazil nut (Bertholletia excelsa H.B.K.) has been elucidated by protein sequencing and from the nucleotide sequence of cDNA clones. The 9 kDa subunit of this protein was found to contain 77 amino acids of which 14 were methionine (18%) and 6 were cysteine (8%). Over half of the methionine residues in this subunit are clustered in two regions of the polypeptide where they are interspersed with arginine residues. In one of these regions, methionine residues account for 5 out of 6 amino acids and four of these methionine residues are contiguous. The sequence data verifies that the Brazil nut sulfur-rich protein is synthesized as a precursor polypeptide that is considerably larger than either of the two subunits of the mature protein. Three proteolytic processing steps by which the encoded polypeptide is sequentially trimmed to the 9 kDa and 3 kDa subunit polypeptides have been correlated with the sequence information. In addition, we have found that the sulfur-rich protein from Brazil nut is homologous in its amino acid sequence to small water-soluble proteins found in two other oilseeds, castor bean (Ricinus communis) and rapeseed (Brassica napus). When the amino acid sequences of these three proteins are aligned to maximize homology, the arrangement of cysteine residues is conserved. However, the two subunits of the Brazil nut protein contain over 19% methionine whereas the homologous proteins from castor bean and rapeseed contain only 2.1% and 2.6% methionine, respectively.     

Partial characterization of vitellin and localization of vitellogenin production in the terrestrial isopod, Armadillidium vulgare

Vitellins were purified separately from ovaries and eggs of the isopod, Armadillidium vulgare. Ovarian vitellin consisted of at least six proteins with relative molecular masses of 205, 200, 185, 180, 122 and 112 kDa. The larger four proteins disappeared in eggs within a week after oviposition and a 59-kDa protein appeared thereafter. The amino-terminal amino acid sequences of these vitellin proteins were identical except for the ovarian 112 kDa, egg 112 kDa and 59 kDa proteins, and showed considerable similarity to those of known vitellogenins from other animals. Comparison of tryptic peptide maps of the 122 and 112 kDa proteins from eggs on reversed-phase HPLC and sequence identification of two randomly selected peaks having the same retention times indicated that two peptides were mostly similar in sequence. PCR-assisted cloning of the 5' region of a cDNA (591 bp) encoding vitellogenin revealed the presence of a signal peptide consisting of 16 amino acid residues and clarified the structural relationship among the protein components except for the ovarian 112 kDa and the egg 59 kDa proteins. Northern blot analysis revealed that the fat body is the main vitellogenin producing organ.     

Protein sequence analysis, cloning, and expression of flammutoxin, a pore-forming cytolysin from Flammulina velutipes. Maturation of dimeric precursor to monomeric active form by carboxyl-terminal truncation

Flammutoxin (FTX), a 31-kDa pore-forming cytolysin from Flammulina velutipes, is specifically expressed during the fruiting body formation. We cloned and expressed the cDNA encoding a 272-residue protein with an identical N-terminal sequence with that of FTX but failed to obtain hemolytically active protein. This, together with the presence of multiple FTX family proteins in the mushroom, prompted us to determine the complete primary structure of FTX by protein sequence analysis. The N-terminal 72 and C-terminal 107 residues were sequenced by Edman degradation of the fragments generated from the alkylated FTX by enzymatic digestions with Achromobacter protease I or Staphylococcus aureus V8 protease and by chemical cleavages with CNBr, hydroxylamine, or 1% formic acid. The central part of FTX was sequenced with a surface-adhesive 7-kDa fragment, which was generated by a tryptic digestion of FTX and recovered by rinsing the wall of a test tube with 6 M guanidine HCl. The 7-kDa peptide was cleaved with 12 M HCl, thermolysin, or S. aureus V8 protease to produce smaller peptides for sequence analysis. As a result, FTX consisted of 251 residues, and protein and nucleotide sequences were in accord except for the lack of the initial Met and the C-terminal 20 residues in protein. Recombinant FTX (rFTX) with or without the C-terminal 20 residues (rFTX271 or rFTX251, respectively) was prepared to study the maturation process of FTX. Like natural FTX, rFTX251 existed as a monomer in solution and assembled into an SDS-stable, ring-shaped pore complex on human erythrocytes, causing hemolysis. In contrast, rFTX271, existing as a dimer in solution, bound to the cells but failed to form pore complex. The dimeric rFTX271 was converted to hemolytically active monomers upon the cleavage between Lys(251) and Met(252) by trypsin.     

Identification,cloning, and sequencing of the genes involved in the conversion of D,L-2-amino-delta2-thiazoline-4-carboxylic acid to L-cysteine in Pseudomonas sp. strain ON-4a

The newly isolated strain Pseudomonas sp. ON-4a converts D,L-2-amino-delta2-thiazoline-4-carboxylic acid to L-cysteine via N-carbamoyl-L-cysteine. A genomic DNA fragment from this strain containing the gene(s) encoding enzymes that convert D,L-2-amino-delta2-thiazoline-4-carboxylic acid into L-cysteine was cloned in Escherichia coli. Transformants expressing cysteine-forming activity were selected by growth of an E. coli mutant defective in the cysB gene. A positive clone, denoted CM1, carrying the plasmid pCM1 with an insert DNA of approximately 3.4 kb was obtained, and the nucleotide sequence of a complementing region was analyzed. Analysis of the sequence found two open reading frames, ORF1 and ORF2, which encoded proteins of 183 and 435 amino acid residues, respectively. E. coli DH5alpha harboring pTrCM1, which was constructed by inserting the subcloned sequence into an expression vector, expressed two proteins of 25 kDa and 45 kDa. From the analyses of crude extracts of E. coli DH5alpha carrying deletion derivatives of pTrCM1 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by enzymatic activity, it was found that the 25-kDa protein encoded by ORF1 was the enzyme L-2-amino-delta2-thiazoline-4-carboxylic acid hydrolase, which catalyzes the conversion of L-2-amino-delta2-thiazoline-4-carboxylic acid to N-carbamoyl-L-cysteine, and that the 45-kDa protein encoded by ORF2 was the enzyme N-carbamoyl-L-cysteine amidohydrolase, which catalyzes the conversion of N-carbamoyl-L-cysteine to L-cysteine.     

Cloning and Characterization of Replication Protein A p32 Complementary DNA in Zebrafish (Danio rerio)

Replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein (70, 32, and 14 kDa) that is an essential component of the DNA replication fork. A complementary DNA encoding zebrafish RPA 32-kDa subunit was isolated by screening a zebrafish embryo lambda APII cDNA library with a human RPA p32 cDNA probe. The zebrafish RPA p32 cDNA consisted of 1097 bp encoding 272 amino acid residues. The deduced amino acid sequence shows high similarity to mouse and human RPA p32. In vitro phosphorylation of zebrafish RPA protein by Cdc2 kinase was shown. A recombinant protein of zebrafish RPA p32 containing a short histidine tag at the NH(2)-terminus was overexpressed in Escherichia coli BL21(DE3) pLys using an inducible T7 expression system, and was purified by Ni-NTA affinity chromatography. In this article, cloning of the zebrafish RPA p32 cDNA is reported in relation to the study of DNA replication in fish.     

The glycine cleavage system. Molecular cloning of the chicken and human glycine decarboxylase cDNAs and some characteristics involved in the deduced protein structures

A cDNA encoding chicken glycine decarboxylase (pCP15b) was isolated using an antibody specific to this protein. Additional cDNAs were cloned with the aid of the genomic fragments obtained by using the pCP15b cDNA probe. No initiator methionine codon is found in the currently elucidated cDNA sequence, and an ATG codon in an exon is assigned to this role. The precursor glycine decarboxylase deduced from the 3514-base pair nucleotide sequence is comprised of 1,004 amino acids (Mr = 111,848). The 1,020 amino acid residues are encoded for the precursor form of human glycine decarboxylase (Mr = 112,869) in the 3,783-base long cDNA sequence of two 1.9-kilobase pair cDNAs with a pentanucleotide overlap. The pyridoxal phosphate binding site lysine and a glycine-rich region, which is suggested to be responsible for the attachment of the phosphate moiety of pyridoxal phosphate, are found in close proximity in both the chicken and human enzymes. This region essential for the enzyme action is suggested to be embedded in a segment rich in beta-turns and random coils and is surrounded by conserved and repetitive amino acid sequences. It is suggested that these structures are involved in the organization of the active site of glycine decarboxylase.     

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.     

Insect allantoinase: cDNA cloning, purification, and characterization of the native protein from the cat flea, Ctenocephalides felis

Allantoinase catalyses the hydrolysis of allantoin to allantoic acid. This reaction is a step in the purine degradation pathway, which produces nitrogenous waste for excretion. A cDNA encoding full-length allantoinase was cloned from a Ctenocephalides felis hindgut and Malpighian tubule (HMT) cDNA library. The cDNA encoded a 483 amino acid protein that had 43% identity with the bullfrog Rana catesbeiana allantoinase and contained the conserved histidine and aspartic acid residues required for zinc-binding and catalytic activity. Unlike the bullfrog allantoinase, the C. felis allantoinase sequence was predicted to contain a 22 amino acid signal sequence, which targets the protein to the secretory pathway. Expression of the mRNA was detected by Northern blot in the first, third, and wandering larval stages as well as in fed and unfed adults, but was not seen in eggs or pupae. In adults, mRNA encoding allantoinase was detected only in the HMT tissues. Immunohistochemistry performed using affinity-purified rabbit immune serum generated against purified recombinant flea allantoinase showed that the native protein localized to the HMT tissues in adult fleas. The anti-allantoinase serum recognized two proteins in an adult flea soluble protein extract, one migrating at 56 kDa and the other at 53 kDa. The two proteins were separated by gel filtration chromatography and were both associated with allantoinase activity. The difference in size appeared to be due to a difference in glycosylation of the proteins. The 53 kDa protein was further purified to near homogeneity by affinity chromatography and retained allantoinase activity. A comparison of the sizes of the native and recombinant C. felis proteins indicated that the 53 kDa native protein may be the product of a post-translational cleavage event, possibly at the putative 22 amino acid signal sequence at the N-terminus of the protein.     

Primary structure, genomic organization and expression of the major secretory protein of murine epididymis, ME1

The mouse cDNA and its genomic clones encoding the epididymal secretory glycoprotein ME1 were identified. The Me1 gene spans 15kb with four exons and three introns. The deduced amino-acid sequence of the ME1 cDNA revealed that it consists of 149 amino acid residues, which contain a signal peptide characteristic of secretory proteins, six cysteine residues and a proline-rich region conserved in the orthologous proteins. Northern blot analysis revealed that 1.3kb ME1 mRNA is highly expressed in the mouse epididymis. The polyclonal antibodies generated against human HE1 (ME1 orthologous protein) expressed in bacteria reacted with approximately 17 to 25kDa components in mouse epididymis crude extract. The reduction of the molecular mass of the recombinant ME1 protein with the digestion of glycopeptidase A indicated that it is modified by Asn-linked glycosylation.     

Isolation, characterization and molecular cloning of the bark lectins from Maackia amurensis

A detailed study was made of the bark lectins of the legume tree Maackia amurensis using a combination of protein purification and cDNA cloning. The lectins, which are the most abundant bark proteins, are a complex mixture of isoforms composed of two types of subunits of 32 and 37 kDa, respectively. Isolation and characterization of the homotetrameric isoforms indicated that the 32 kDa subunit exhibits a 100-fold stronger haemagglutinating activity than the 37 kDa subunit. Molecular cloning confirmed that the two lectin subunits are encoded by different genes. The 32 kDa subunit is apparently encoded by a single gene, whereas two highly homologous genes encode the 37 kDa subunit. A comparison of the deduced amino acid sequences of the bark lectin cDNAs and the previously described cDNA encoding the seed haemagglutinin demonstrated that they are encoded by different genes. Abbreviations: LECMAHb, cDNA clone encoding Maackia amurensis bark haemagglutinin; LECMALb, cDNA clone encoding Maackia amurensis bark leucoagglutinin; MALb, Maackia amurensis bark leucoagglutinin; MAHb, Maackia amurensis bark haemagglutinin This revised version was published online in November 2006 with corrections to the Cover Date.