The pro- and mature forms of the E. coli K-12 outer membrane phospholipase A are identical.

The nucleotide sequence of the pldA gene, coding for the outer membrane (OM) phospholipase A of Escherichia coli K-12, and flanking sequences, was determined. Data were obtained from sequences of overlapping deletions which had been generated in vitro from both ends of the gene, using DNase I in the presence of Mn2+ and Bal31 nuclease. The deduced amino acid sequence of the pldA gene product is the first primary sequence of a membrane-bound phospholipase. The complete PldA protein contains 260 amino acids, which include a putative signal sequence, and has a calculated mol. wt. of 29 946 similar to that of the purified protein. Furthermore we found the N terminus of the purified protein to be blocked and the overall amino acid composition to be consistent with the one deduced from the complete pldA gene. Analysis of proteins synthesized in minicells with a pldA coding plasmid in the presence of 8% ethanol did not reveal any new bands on polyacrylamide gels, whereas the control beta-lactamase clearly showed its unprocessed form under the same conditions. These data are consistent with the empirical prediction from the primary sequence, that the PldA protein lacks any signal peptidase 'target' site. We therefore conclude that the PldA protein is exported to the OM without proteolytic removal of the signal peptide.     

Amino acid sequence of a specific antigenic peptide of protein B23

A specific antigenic peptide was obtained from protein B23 (Mr/pI = 37,000/5.1) after 30 min of digestion with staphylococcal V8 protease (10 micrograms/ml/mg protein B23). The antigenic peptide was purified by DEAE-cellulose chromatography and high pressure liquid chromatography on a reverse-phase C18 column. The antigenic peptide contains 14.7 and 18.7 mol% of glutamic acid and lysine, respectively. Amino acid sequence analysis showed that the peptide has 68 amino acids and is located on the carboxyl-terminal sequence of protein B23. The sequence is Ser-Phe-Lys-Lys-Gln-Glu-Lys-Thr-Pro-Lys-Thr-Pro- Lys-Gly-Pro-Ser-Ser-Val-Glu-Asp-Ile-Lys-Ala-Lys-Met-Gln-Ala-Ser-Ile-Glu- Lys-Gly- Gly-Ser-Leu-Pro-Lys-Val-Glu-Ala-Lys-Phe-Ile-Asn-Tyr-Val-Lys-Asn-Cys-Phe- Arg-Met- Thr-Asp-Gln-Glu-Ala-Ile-Gln-Asp-Leu-Trp-Gln-Trp-Arg-Lys-Ser-Leu-Cooh. Extensive digestion of the antigenic peptide with V8 protease, trypsin, or chymotrypsin results in loss of the antigenic activity. Three cloned cDNAs (hpB1, hpB2, and hpB7) which code for the 82 amino acids at the COOH terminus of protein B23 and the 3' non-translating sequence were identified and characterized. All three clones have identical nucleotide sequences coding for the antigenic portion of the protein (68 amino acids at the COOH terminus), the stop codon, and the 3' non-translated region. However, mutation of 6 nucleotide bases of one clone (hpB2) caused changes in 4 amino acids in the sequence just preceding the immunoreactive region. The result suggests the presence of at least 2 immunologically similar but distinct proteins which are both recognized by the anti-B23 antibody.     

N-terminal amino acid sequence of human urinary prokallikrein

The N-terminal amino acid sequences of human urinary prokallikrein and kallikrein have been determined. Their amino acid sequences are as follows. (Formula; see text) The results showed that prokallikrein comprises an additional seven amino acids at the amino terminus of the kallikrein, of which the sequence is (H2N)Ala-Pro-Pro-Ile-Gln-Ser-Arg(COOH). Comparison of the structure of this peptide with those of other proteins revealed extensive sequence identity with the propeptide portions of rat and mouse tissue kallikreins, that were predicted from the preproenzyme-encoded nucleotide sequences. The amino acid sequence of the peptide was also highly homologous to that of the propeptide portion of EGF-binding protein, that was predicted from the nucleotide sequence, and that of the alpha-subunit of NGF. The N-terminal amino acid sequence of kallikrein was completely identical to the reported one (Lottspeich, F., et al. (1979) Hoppe-Seyler's Z. Physiol. Chem. 360, 1947-1950) and shows considerable amino acid sequence homology with the porcine and rat pancreatic kallikreins. As far as the present results are concerned, it is strongly indicated that the inactive kallikrein in human urine is a tissue type prokallikrein which is activated on the release of the N-terminal peptide consisting of seven amino acids.     

Phospholipid transfer protein: full-length cDNA and amino acid sequence in maize. Amino acid sequence homologies between plant phospholipid transfer proteins

We have determined the primary structure of a phospholipid transfer protein (PLTP) isolated from maize seeds. This protein consists of 93 amino acids and shows internal homology originating in the repetition of (do)decapeptides. By using antibodies against maize PLTP, we have isolated from a cDNA library one positive clone (6B6) which corresponds to the incomplete nucleotide sequence. Another cDNA clone (9C2) was obtained by screening a size-selected library with 6B6. Clone 9C2 (822 base pairs) corresponds to the full-length cDNA of the phospholipid-transfer protein whose mRNA contains 0.8 kilobase. Southern blot analysis shows that the maize genome may contain several PLTP genes. In addition, the deduced amino acid sequence of clone 9C2 reveals the presence of a signal peptide. The significance of this signal peptide (27 amino acids) might be related to the function of the phospholipid-transfer protein. The amino acid sequence of maize PLTP was compared to those isolated from spinach leaves or castor bean seeds which exhibit physicochemical properties close to those of the maize protein. A high homology was observed between the three sequences. Three domains can be distinguished: a highly charged central core (around 40-60), a very hydrophobic N-terminal sequence characteristic of polypeptide-membrane interaction, and a hydrophilic C terminus. A model for plant phospholipid-transfer proteins is proposed in which the phospholipid molecule is embedded within the protein with its polar moiety interacting with the central hydrophilic core of the protein, whereas the N-terminal region plunges within the membrane in the transfer process.     

Inhibition of HIV-1 infection by an intramolecular antisense peptide to T20 in gp160

Antisense amino acids are amino acids which can be translated from the corresponding anti-codons of a sense amino acid. Antisense peptides encoded by the noncoding DNA strand have a tendency to interact with each other. We have demonstrated that antisense peptide sequences are present intramolecularly, and these may contribute to the folding and maintenance of the tertiary structure of a protein. T20 is a synthetic peptide with an amino acid sequence in the gp41 of HIV-1 and has been demonstrated to be a potent inhibitor of HIV-1 infection. We searched for intramolecular peptide sequences which are antisense to portions of T20. A synthetic peptide (TA-1L) consisting of amino acids 84 to 97 of gp160, which contains an antisense peptide sequence (TA-1) to T20, was shown to inhibit HIV-1(IIIB) infection of MT-4 cells. Interaction of these antisense peptides could be involved in sustaining HIV-1 infectivity. The TA-1L site, which exists in the C1 domain of gp160, is highly homologous among strains of HIV-1, especially at TA-1 and in the amino acids flanking the C terminus. Although the TA-1 sites of 18 out of 30 HIV-1 strains were antisense to the T20 region, those of the remaining 12 strains, including HIV-1(MN), were not. However, TA-1L inhibited infection by HIV-1(MN), which has no antisense peptide in T20 corresponding to TA-1, although the inhibitory effect was weaker. TA-1L may thus also interfere with the gp160 interaction with CD4, which has an antisense sequence to TA-1.     

Characterization and expression of the cDNA encoding a new kind of phospholipid transfer protein, the phosphatidylglycerol/phosphatidylinositol transfer protein from Aspergillus oryzae: evidence of a putative membrane targeted phospholipid transfer protein in fungi

The full-length cDNA of a phospholipid transfer protein (PLTP) was isolated from Aspergillus oryzae by a RACE-PCR procedure using degenerated primer pool selected from the N-terminal sequence of the purified phosphatidylinositol/phosphatidylglycerol transfer protein (PG/PI-TP). The cDNA encodes a 173 amino acid protein of 18823 Da. The deduced amino acid sequence from position 38 to 67 is 100% identical to the N-terminal sequence (first 30 amino acids) of the purified PG/PI-TP. This amino acid sequence is preceded by a leader peptide of 37 amino acids which is predicted to be composed of a signal peptide of 21 amino acids followed by an extra-sequence of 16 amino acids, or a membrane anchor protein signal (amino acid 5-29). This strongly suggests that the PG/PI-TP is a targeted protein. The deduced mature protein is 138 amino acids long with a predicted molecular mass of 14933 Da. Comparison of the deduced PG/PI-TP sequence with other polypeptide sequences available in databases revealed a homology with a protein deduced from an open reading frame coding for an unknown protein in Saccharomyces cerevisiae (36% identity and 57% similarity). Apart from this homology, the PG/PI-TP is unique and specific to the filamentous fungi on the basis of comparison of PLTP protein sequences. Northern blot analysis of RNA isolated from A. oryzae cultures grown on glucose or glucose supplemented with phospholipids suggests that the PG/PI-TP is transcribed by only one RNA species and allows us to show that expression of the protein is regulated at the messenger RNA level.     

Complete nucleotide sequence of the haemagglutinin gene from a human influenza virus of the Hong Kong subtype.

The complete nucleotide sequence has been determined for a cloned double-stranded DNA copy of the haemagglutinin gene from the human influenza strain A/NT/60/68/29C, a laboratory-isolated variant of A/NT/60/68, an early strain of the Hong Kong subtype. The gene is 1765 nucleotides long and contains information sufficient to code for a protein of 566 amino acids, which includes a hydrophobic leader peptide (16 residues), HA1 (328), HA2 (221) and an arginine residue which joins the HA subunits. Comparison of the predicted amino acid sequence for 29C haemagglutinin with protein sequence data available for HA from other influenza strains shows that no potential coding information is lost by processing of the mRNA. A comparison of the amino acid sequences predicted from the gene sequences for 29C and fowl plague virus haemagglutinins, (1) indicates the extent to which changes can occur in the primary sequence of different regions of the protein, while maintaining essential structure and function.     

cDNA cloning, functional expression and cellular localization of rat liver mitochondrial electron-transfer flavoprotein-ubiquinone oxidoreductase protein

A 64 kD protein was enriched from rat liver mito-chondria during the purification of choline dehydro-genase (CHDH)[1]. Homologous comparison and func-tional experiments demonstrated that the protein was electron-transfer flavoprotein-ubiquinone oxidoreduc-tase protein (ETF-QO). The N-terminal sequence determination of rat liver ETF-QO protein purified by various methods did not provide unequivocal result. However, when the protein was digested with V8 protease, peptide fragments could b…     

A statistical view of FMRFamide neuropeptide diversity

FMRFamide-like peptide (FLP) amino acid sequences have been collected and statistically analyzed. FLP amino acid composition as a function of position in the peptide is graphically presented for several major phyla. Results of total amino acid composition and frequencies of pairs of FLP amino acids have been computed and compared with corresponding values from the entire GenBank protein sequence database. The data for pairwise distributions of amino acids should help in future structure-function studies of FLPs. To aid in future peptide discovery, a computer program and search protocol was developed to identify FLPs from the GenBank protein database without the use of keywords.     

Isolation, characterization and cDNA cloning of a one-lobed transferrin from the ascidian Halocynthia roretzi

Transferrin was isolated from plasma of the ascidian Halocynthia roretzi by ion-exchange chromatography. The molecular weight of the plasma transferrin was determined to be 52K by SDS-polyacrylamide gel electrophoresis and gel filtration. Ascidian plasma transferrin was found to bind one mole of iron ion per mole of protein. The reductive S-pyridylethylated transferrin was subjected to Edman degradation analysis for determination of the N-terminal amino acid sequence, and it was also subjected to proteolytic fragmentation to yield peptide fragments, whose amino acid sequences were determined by Edman degradation analysis. Using the above amino acid sequences, a cDNA clone (1880 base pairs) encoding a protein of 372 amino acids containing a signal peptide of 21 amino acids was isolated from an H. roretzi hepatopancreas cDNA library. The reduced amino acid sequence contains the same sequences of the peptide fragments. A comparison of the amino acid sequence of ascidian transferrin with those of other members of the transferrin family revealed that the ascidian transferrin is composed of only the N-terminal lobe of two-lobed vertebrate transferrins. Thus, a one-lobed transferrin is present in the ascidian H. roretzi.     

Primary amino acid sequence of bovine dopamine beta-hydroxylase

Fifty-eight tryptic and Staphylococcus aureus V8 protease generated peptides from bovine dopamine beta-hydroxylase were isolated by reverse-phase high pressure liquid chromatography and sequenced. These peptide sequences were compared with the deduced amino acid sequences of bovine and human dopamine beta-hydroxylase obtained from the cloned cDNAs. Bovine peptide sequences had five differences with the sequence derived from the bovine cDNA, and four of the changes could be accounted for by a single base change in the DNA. N-terminal sequence analysis of the bovine enzyme indicated that it contained two N termini, one of which is 3 amino acids longer than the other and begins with the sequence Ser-Ala-Pro. The amino acid sequences deduced from the bovine and human cDNAs are 19 and 25 amino acids longer, respectively, and these additional amino acids represent leader peptide sequences. Two bovine peptide sequences contained glycosylation sites and gave positive tests for carbohydrate residues, and two others contained the consensus sequence for a glycosylation site but were negative in the carbohydrate test. The bovine enzyme contains 6 Trp, as compared with 7 in the bovine cDNA and 8 in the human cDNA. The protein and bovine cDNA contain 24 Tyr each, as compared with 26 in the human cDNA. These numbers indicate that the true epsilon 1% 280 = 8.95, and, therefore, that it is 28% lower than the previously determined value. The data also identify 5 His-containing regions that may be involved in Cu2+ coordination at the active site.     

乙型肝炎病毒X蛋白的优势氨基酸序列与热点突变位点的生物信息学分析

乙型肝炎病毒X蛋白(hepatitis B virus X protein,HBx)全长154个氨基酸,与肝癌发生密切相关.为确定HBx的优势氨基酸序列和热点突变位点,在GenBank中下载所有HBx的氨基酸序列13950条,剔除插入突变、缺失突变和起始密码子非甲硫氨酸的序列,最后保留7126条.通过分析这7126条序列,计算出HBx每个位点的氨基酸分布情况,出现频率最高的氨基酸为该位点的优势氨基酸,其他氨基酸为突变氨基酸.154个位点的优势氨基酸组成HBx优势氨基酸序列.突变率>10.0%的热点突变位点有32个.其中第36、42、44、87、88和127位氨基酸有4种(突变率>1.0%)以上突变形式,具有较高的多态性.与肝癌密切相关的K130M/V131I双突变率为34.7%.通过7126条HBx序列与优势序列的同源性比较,随机选出其中50条序列(2条与优势序列同源性<75%,48条同源性为76%~99%),与23条参考序列及优势序列共同构建系统发生树.结果显示,HBx优势氨基酸序列属于基因型C,这与基因型C为全球主要流行型一致.本研究首次系统性分析了GenBank中HBx的优势序列,确定了32个HBx热点突变位点和6个多态性较高的位点,为基于HBx突变的基础和应用研究奠定了基础.     

cDNA cloning,functional expression and cellular localization of rat liver mitochondrial electron-transfer flavoprotein-ubiquinone oxidoreductase protein

A membrane-bound protein was purified from rat liver mitochondria. After being digested with V8 protease, two peptides containing identical 14 amino acid residue sequences were obtained. Using the 14 amino acid peptide derived DNA sequence as gene specific primer, the cDNA of correspondent gene 5′-terminal and 3′-terminal were obtained by RACE technique. The full-length cDNAthat encoded a protein of 616 amino acids was thus cloned, which included the above mentioned peptide sequence. The full length cDNA was highly homologous to that of human ETF-QO, indicating that it may be the cDNA of rat ETF-QO. ETF-QO is an iron sulfur protein located in mitochondria inner membrane containing two kinds of redox center: FAD and [4Fe-4S] center. After comparing the sequence from the cDNA of the 616 amino acids protein with that of the mature protein of rat liver mitochondria, it was found that the N terminal 32 amino acid residues did not exist in the mature protein, indicating that the cDNA was that of ETF-QOp. When the cDNA was expressed in Saccharomyces cerevisiae with inducible vectors, the protein product was enriched in mitochondrial fraction and exhibited electron transfer activity (NBT reductase activity) of ETF-QO. Results demonstrated that the 32 amino acid peptide was a mitochondrial targeting peptide, and both FAD and iron-sulfur cluster were inserted properly into the expressed ETF-QO. ETF-QO had a high level expression in rat heart, liver and kidney. The fusion protein of GFP-ETF-QO co-localized with mitochondria in COS-7 cells.     

Isolation and expression in Escherichia coli of a cDNA clone encoding porcine pancreatic elastase

We have cloned a DNA that is complementary to the messenger RNA that encodes porcine pancreatic elastase 1 from pancreas using rat pancreatic elastase 1 cDNA as a probe. This complementary DNA contains the entire protein coding region of 798 nucleotides which encodes an elastase of 266 amino acids, and 22 and 136 nucleotides of the 5' and 3'-untranslated sequences. When this deduced amino acid sequence was compared with known amino acid sequences, a carboxy-terminal 240 amino acids long peptide was found to be identical with a mature form of porcine pancreatic elastase 1, except for two amino acids. The porcine enzyme contains the same number of amino acid residues as the rat enzyme, and their amino acid sequences are 85% homologous. Taking the above findings together, we conclude that the cloned cDNA encodes a mature enzyme of 240 amino acids including a leader and activation peptide of 26 amino acids. We expressed the cloned porcine pancreatic elastase 1 cDNA in E. coli as a lac-fused protein. The resulting fused protein showed enzymatic activity and immunoreactivity toward anti-elastase serum.     

cDNA sequence and predicted primary structure of the gamma subunit from the ATP synthase from Chlamydomonas reinhardtii

The 1701-base nucleotide sequence (not including the poly(A) tail) of a cDNA for the gamma subunit of the ATP synthase from Chlamydomonas reinhardtii was determined. A start translation sequence, 23 bases in from the 5' end, initiates an 1074-base-long open reading frame. The sequence of the first 21 amino acids at the amino-terminal end of the mature gamma subunit from C. reinhardtii was determined and compared to the deduced amino acid sequence of the open reading frame. From this it was determined that the mature protein contains 323 amino acids, with the first 35 amino acids probably being part of the transit peptide. The length of the mature protein is the same as that for the mature gamma subunit from spinach, for which only a few of the amino acids of the transit peptide are known. The similarity of the two mature proteins at the nucleotide level is 56% while at the amino acid level it is 77%. In addition, the 3 cysteines, which in spinach are involved in the energy-linked catalytic functions of the ATP synthase, are conserved in the predicted amino acid sequence for the gamma subunit from C. reinhardtii. In contrast, the mature C. reinhardtii gamma subunit contains 3 additional cysteine residues not found in the spinach gamma subunit.     

Primary structure of acetylcholinesterase: implications for regulation and function

A cDNA encoding acetylcholinesterase (AChE) (EC 3.1.1.7) from Torpedo californica was isolated and from its nucleotide sequence the entire amino acid sequence of the processed protein and a portion of the leader peptide has been deduced. Approximately 70% of the tryptic peptides from the catalytic subunit of the 11 S form have been sequenced, and a comparison of the peptide sequences with the sequence inferred from the cDNA suggests that the cDNA sequence derives from mRNA for the 11 S form of the enzyme. The amino acid sequence is preceded by a hydrophobic leader peptide and contains an open reading frame encoding for 575 amino acids characteristic of a secreted globular protein. Eight cysteines, most of which are disulfide linked, are found along with four potential sites of N-linked glycosylation. The active-site serine is located at residue 200. Local homology is found with other serine hydrolases in the vicinity of the active site, but the enzyme shows striking global homology with the COOH-terminal portion of thyroglobulin. Further comparison of the amino acid sequences of the individual enzyme forms with other cDNA clones that have been isolated should resolve the molecular basis for polymorphism of the AChE species.     

Cloning of a major heparan sulfate proteoglycan from brain and identification as the rat form of glypican.

We have obtained the complete coding sequence of a highly conserved heparan sulfate proteoglycan which we previously characterized biochemically after isolation from rat brain. An open reading frame of 558 amino acids encodes a protein with a molecular mass of 62 kDa containing three peptide sequences present in the isolated proteoglycan. The total sequence obtained is 3.5 kb long, including 1.6 kb of 3'-untranslated sequence and 0.2 kb of 5'-untranslated sequence. The deduced amino acid sequence and the 3'- and 5'-untranslated sequences have 89% and 66-80% identity, respectively, with those of a phosphatidylinositol-anchored human lung fibroblast heparan sulfate proteoglycan (glypican) for which mRNA is detectable in a large number of human cell lines. Our data therefore demonstrate that this major heparan sulfate proteoglycan of brain is the rat form of glypican.     

Nucleotide sequence of complementary DNA and derived amino acid sequence of murine complement protein C3

The nucleotide sequences coding for murine complement component C3 have been determined from a cloned genomic DNA fragment and several overlapping cloned complementary DNA fragments. The amino acid sequence of the protein was deduced. The mature beta and alpha subunits contain 642 and 993 amino acids respectively. Including a 24 amino acid signal peptide and four arginines in the beta-alpha transition region, which are probably not contained in the mature protein, the unglycosylated single chain precursor protein preproC3 would have a molecular mass of 186 484 Da and consist of 1663 amino acid residues. The C3 messenger RNA would be composed of a 56 +/- 2 nucleotide long 5' non-translated region, 4992 nucleotides of coding sequence, and a 3' non-translated region of 39 nucleotides, excluding the poly A tail. The beta chain contains only three cysteine residues, the alpha chain 24, ten of which are clustered in the carboxy terminal stretch of 175 amino acids. Two potential carbohydrate attachment sites are predicted for the alpha chain, none for the beta chain. From a comparison with human C3 cDNA sequence (of which over 80% has been determined) an extensive overall sequence homology was observed. Human and murine preproC3 would be of very similar length and share several noteworthy properties: the same order of the subunits in the precursor, the same basic residue multiplet in the beta-alpha transition region, and a glutamine residue in the thioester region. The equivalent position of the known factor I cleavage sites in human C3 alpha could be located in the murine C3 alpha chain and the size and sequence of the resulting peptide were deduced. A comparison of the amino acid sequences of murine C3 and human alpha 2-macroglobulin is given. Several areas of strong sequence homology are observed, and we conclude that the two genes must have evolved from a common ancestor.     

Structural gene and complete amino acid sequence of Pseudomonas aeruginosa IFO 3455 elastase.

The DNA encoding the elastase of Pseudomonas aeruginosa IFO 3455 was cloned, and its complete nucleotide sequence was determined. When the cloned gene was ligated to pUC18, the Escherichia coli expression vector, bacteria carrying the gene exhibited high levels of both elastase activity and elastase antigens. The amino acid sequence, deduced from the nucleotide sequence, revealed that the mature elastase consisted of 301 amino acids with a relative molecular mass of 32,926 daltons. The amino acid composition predicted from the DNA sequence was quite similar to the chemically determined composition of purified elastase reported previously. We also observed nucleotide sequence encoding a signal peptide and "pro" sequence consisting of 197 amino acids upstream from the mature elastase protein gene. The amino acid sequence analysis revealed that both the N-terminal sequence of the purified elastase and the N-terminal side sequences of the C-terminal tryptic peptide as well as the internal lysyl peptide fragment were completely identical to the deduced amino acid sequences. The pattern of identity of amino acid sequences was quite evident in the regions that include structurally and functionally important residues of Bacillus subtilis thermolysin.