Two I84 family protease inhibitors from Chinese razor clams Sinonovacula constricta expressed in response to environmental challenges
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Abstract In order to determine the primary structure of banana shrimp, Penaeus merguiensis , vitellogenin (Vg), we previously purified vitellin (Vt) from the ovaries of vitellogenic females, and chemically analyzed the N‐terminal amino acid sequence of its 78 kDa subunit. In this study, a cDNA from this species encoding Vg was cloned based on the N‐terminal amino acid sequence of the major 78 kDa subunit of Vt and conserved sequences of Vg/Vt from other crustacean species. The complete nucleotide sequence of Vg cDNA was achieved by RT‐PCR and 5′ and 3′ rapid amplification of cDNA ends (RACE) approaches. The full‐length Vg cDNA consisted of 7,961 nucleotides. The open reading frame of this cDNA encoding a precursor peptide was comprised of 2,586 amino acid residues, with a putative processing site, R‐X‐K/R‐R, recognized by subtilisin‐like endoproteases. The deduced amino acid sequence was obtained from the Vg cDNA and its amino acid composition showed a high similarity to that of purified Vt. The deduced primary structure, of P . merguiensis Vg was 91.4% identical to the Vg of Penaeus semisulcatus and was also related to the Vg sequences of six other crustacean species with identities that ranged from 86.9% to 36.6%. In addition, the amino acid sequences corresponding to the signal peptide, N‐terminal region and C‐terminal region of P . merguiensis Vg were almost identical to the same sequences of the seven other reported crustacean species. Results from RT‐PCR analysis showed that Vg mRNA expression was present in both the ovary and hepatopancreas of vitellogenic females but was not detected in other tissues including muscle, heart, and intestine of females or in the hepatopancreas of mature males. These results indicate that the Vg gene may be expressed only by mature P . merguiensis females and that both the ovary and hepatopancreas are possible sites for Vg synthesis in this species of shrimp. Mol. Reprod. Dev. © 2006 Wiley‐Liss, Inc.
Vitellogenin
Protein primary structure
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The nucleotide sequence of the PHO5 gene of the yeast, Saccharomyces cerevisiae, which encodes repressible acid phosphatase (APase) was determined. Comparison of N-terminal amino acid sequence deduced from the nucleotide sequence with that of the purified repressible APase revealed the existence of a putative signal peptide in the precursor protein. The signal peptide was shown to contain 17 amino acid residues and its structural features were quite similar to those of higher eukaryotic and prokaryotic signal peptides. The nucleotide sequence of 5' and 3' noncoding flanking regions of the PHO5 gene are also discussed.
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We have isolated several recombinant clones carrying the complementary DNA (cDNA) sequence of the rainbow trout (rt) Salmo gairdneri growth hormone (GH) mRNA by immunoblot screening using an antiserum to chum salmon (Oncorhyncus keta) GH. The nucleotide sequence of one of the rtGH cDNA clones (pAF51) was determined. The rt cDNA sequence in pAF51 encodes a hybrid polypeptide of 199 amino acid residues containing 9 amino acid residues of the bacterial beta-galactosidase, one residue from the codon at the junction of the beta-galactosidase gene, and the rtGH cDNA sequence, an additional residue from the presumptive signal peptide of the pre-rtGH and the entire sequence of the mature rtGH (188 amino acid residues). Pairwise matrix comparisons of the hydropathy profiles of bovine, human, rat, and rainbow trout GH polypeptides indicate that regions of similarity exist between the rtGH and mammalian GH. In particular, there are two major regions of similarity found near the amino-terminal region and at the carboxy-terminal region. These regions correspond to hydrophilic domains of the GH molecules. The possible significance of these domains is discussed.
Coding region
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Signal peptidases, the endoproteases that remove the amino-terminal signal sequence from many secretory proteins, have been isolated from various sources. Seven signal peptidases have been purified, two from E. coli, two from mammalian sources, and three from mitochondrial matrix. The mitochondrial enzymes are soluble and function as a heterogeneous dimer. The mammalian enzymes are isolated as a complex and share a common glycosylated subunit. The bacterial enzymes are isolated as monomers and show no sequence homology with each other or the mammalian enzymes. The membrane-bound enzymes seem to require a substrate containing a consensus sequence following the - 3, - 1 rule of yon Heijne at the cleavage site; however, processing of the substrate is strongly influenced by the hydrophobic region of the signal peptide. The enzymes appear to recognize an unknown three-dimensional motif rather than a specific amino acid sequence around the cleavage site. The matrix mitochondrial enzymes are metallo-endopeptidases; however, the other signal peptidases may belong to a unique class ofproteases as they are resistant to chelators and most protease inhibitors. There are no data concerning the substrate binding site of these enzymes. In vivo, the signal peptide is rapidly degraded. Three different enzymes in Escherichia coli that can degrade a signal peptide in vitro have been identified. The intact signal peptide is not accumulated in mutants lacking these enzymes, which suggests that these peptidases individually are not responsible for the degredation of an intact signal peptide in vivo. It is speculated that signal peptidases and signal peptide hydrolases are integral components of the secretory pathway and that inhibition of the terminal steps can block translocation.
Signal peptidase
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A mixture of tetradecamer oligodeoxyribonucleotides complementary to the codons specifying the carboxyl-terminal sequence, Ile-His-Pro-Phe-His, of angiotensin was chemically synthesized as two pools and used for the isolation of a cDNA clone specific for angiotensinogen from a cDNA bank of rat liver mRNA sequences. The two pools (oligo 1 and oligo 2), each containing 24 oligodeoxyribonucleotides, were first used as primers to initiate reverse transcription of rat liver mRNA. One of the pools (oligo 1) was found to prime a specific 32P-labeled cDNA of approximately 160 nucleotides that contained the anticoding sequence corresponding exactly to the amino acid sequence of rat angiotensin. This cDNA, in turn, was used to rescreen cDNA clones that were isolated by initially selecting the rat liver cDNA bank by hybridization with the oligo 1 mixture. One clone thus obtained, designated pRag16, was subjected to nucleotide sequence analysis and verified to contain a nearly full-length cDNA sequence coding for rat angiotensinogen precursor. The deduced amino acid sequence indicates that the precursor molecular consists of angiotensinogen of 453 amino acid residues and a putative signal peptide of 24 amino acid residues. The predicted molecular weight and amino acid composition of angiotensinogen agree well with those determined by using the purified protein. An angiotensin moiety is located at the amino-terminal part of angiotensinogen, preceded directly by the signal peptide and followed by a large carboxyl-terminal sequence that contains two internally homologous sequences and three potential glycosylation sites.
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We have determined the complete nucleotide sequence for cDNA of rat homologues of human eosinophil major basic protein (MBP) and eosinophil cationic protein (ECP) using the rapid amplification of cDNA ends (RACE) procedure. Nucleotide sequence of cDNA of rat MBP revealed that mRNA of rat MBP encodes a protein containing 227 amino acids which has three functional domains; namely, the signal peptide, the acidic peptide that contains numerous acidic amino acids and the mature MBP, as in human, guinea pig and mouse MBP. In addition, cDNA of a rat homologue of human ECP was also cloned. The deduced amino acid sequence revealed that this gene encodes a putative protein with a molecular weight of 15.5 kD which has ribonuclease activity. The homology of amino acid sequence between the rat homologue and the murine eosinophil-associated ribonucleases (EARs) was high (65%). Therefore, we named this rat homologue ‘rat EAR-1’.
Identification
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Abstract Based on the amino acid sequence of a bradykinin‐potentiating peptide (Bpp) (peptide K‐12) from scorpion Buthus occitanus, a full‐length cDNA sequence encoding the precursor of a novel venom peptide (named BmKbpp) related to this Bpp, has been isolated and analyzed. The cDNA encodes a precursor of 72 amino acid residues, including a signal peptide of 22 residues and an extra Arg‐ArgArg tail at the C‐terminal end of the precursor, which have to be removed in the processing step. The C‐terminal region (21 residues) of the precursor is homologous (57% identical) with the sequence of peptide K‐12. Thus, according to the primary structure of the BmKbpp precursor, there may be a propeptide between the signal peptide and the putative mature BmKbpp at the C‐terminal region of the precursor.
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A human Colony Stimulating Factor-1 (CSF-1 or Macrophage-CSF) encoding cDNA was previously isolated from the pancreatic tumor cell line MIA-PACA-2 (I). This cDNA encodes a protein of 26 Kd molecular weight. To isolate a murine cDNA homologous to human M-CSF, a probe from the coding region of the human cDNA was prepared by nick-translation. A recotnbinant lambda phage library made from the mRNA of the murine fibroblast cell line. L929, was screened with the human M-CSF cDNA probe The cDNA isolated from the murine library has an open reading frame of 1656 nucleotides which encodes a protein of 552 amino acids. The first 32 amino acids encode a putative signal peptide which when cleaved yields a mature protein beginning with the amino acid sequence: Lys-Glu-Val-Ser. This deduced sequence is identical to the amino terminal residues reported for M-CSF as purified from L-cell supernatant? (2). The mature protein deduced from the cDNA sequence has a molecular weight of 57.261 d. All ten of the cysteine residues are found in approximately the amino-terminal half of the molecule. There are three potential relinked glycosylation sites (Asn-X-Thr/Ser) located at residues 154, 172, and 378. From the first amino acid encoded by the cDNA to residue 180 and from residue 476 to the cartxwy-terminal residue, approximately 80% homology is found between the murine and human cDNA's at both the nucleotide and amino acid levels. For comparison the deduced human M-CSF protein sequence is aligned below that of the murine sequence. The open reading frame in the murine cDNA encodes an additional 295 amino acids which are not found in the deduced amino-acid sequence of human M-CSF (I). However, higher molecular weight forms of human M-CSF mRNA have also been identified by Northern analysis of MIA-PACA-2 mRNA using the 26 Kd encoding cDNA as a probe (3). Furthermore, the murine M-CSF encoding cDNA we isolated was incorporated into an animal cell expression vector under SV40 early promoter transcrlptional control. When the vector was transfected Into the simian cell line, COS-7, murine M-CSF biological activity, as measured on mouse bone marrow cells, was observed in the transfected-culture supernalants
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