Amino acid sequence around the active-site selenocysteine of rat liver glutathione peroxidase

Glutathione peroxidase (glutathione:hydrogen-peroxide oxidoreductase, EC 1.11.1.9) from rat liver was purified to at least 95% purity. A peptide of 16 amino acids, including the active-site selenocysteine (SeCys) residue, was isolated from a tryptic digest by reverse-phase HPLC and gel filtration. The amino acid sequence of the first 46 residues of glutathione peroxidase was obtained from the overlapping sequences of the tryptic selenopeptide and the intact subunit. The selenocysteine residue was located at residue 41, and the sequence of the tryptic selenopeptide was Val-Leu-Leu-Ile-Glu-Asn-Val-Ala-Ser-Leu-SeCys-Gly-Thr-Thr-Thr-Arg. The sequence was analyzed by computer for homology with other proteins, but no closely related sequences were found. Glutathione peroxidase is the first selenoprotein for which sequence data have been obtained, and the methods described should be applicable to mapping and to sequence analysis of Se-containing peptides from other selenoproteins.     

Selection of functional signal peptide cleavage sites from a library of random sequences.

The export of proteins to the periplasmic compartment of bacterial cells is mediated by an amino-terminal signal peptide. After transport, the signal peptide is cleaved by a processing enzyme, signal peptidase I. A comparison of the cleavage sites of many exported proteins has identified a conserved feature of small, uncharged amino acids at positions -1 and -3 relative to the cleavage site. To determine experimentally the sequences required for efficient signal peptide cleavage, we simultaneously randomized the amino acid residues from positions -4 to +2 of the TEM-1 beta-lactamase enzyme to form a library of random sequences. Mutants that provide wild-type levels of ampicillin resistance were then selected from the random-sequence library. The sequences of 15 mutants indicated a bias towards small amino acids. The N-terminal amino acid sequence of the mature enzyme was determined for nine of the mutants to assign the new -1 and -3 residues. Alanine was present in the -1 position for all nine of these mutants, strongly supporting the importance of alanine at the -1 position. The amino acids at the -3 position were much less conserved but were consistent with the -3 rules derived from sequence comparisons. Compared with the wild type, two of the nine mutants have an altered cleavage position, suggesting that sequence is more important than position for processing of the signal peptide.     

The amino-acid sequence of bovine glutathione peroxidase

The amino-acid sequence of the seleno-enzyme glutathione peroxidase from bovine erythrocytes was completely determined. Fragmentation of the carboxymethylated protein comprised cleavages with trypsin, with endoproteinase Lys-C, and with cyanogen bromide in 70% formic acid. The resulting peptides were separated by reversed-phase high-performance chromatography or by gel filtration. For sequence determination automated solid or liquid phase techniques of Edman degradation were used. The proper alignment of fragments was experimentally proven in all but one instance. In this case, consistent indirect evidence was provided. The monomer of glutathione peroxidase was shown to consist of 198 amino acids representing a molecular mass ob about 21 900 Da. The active site selenocysteine was localized at position 45. In addition, four cysteine residues were found at positions 74, 91, 111, and 152. The N-terminal part of the sequence obtained revealed a pronounced homology with a partial sequence of the rat liver enzyme. Moreover, tentative sequence data predicted from X-ray crystallographic analysis of bovine glutathione peroxidase were found to agree in about 80% of the residues with the sequence presented. Differences between the predicted and the experimentally determined sequence are discussed.     

Structure of the horseradish peroxidase isozyme C genes

We have isolated, cloned and characterized three cDNAs and two genomic DNAs corresponding to the mRNAs and genes for the horseradish (Armoracia rusticana) peroxidase isoenzyme C (HPR C). The amino acid sequence of HRP C1, deduced from the nucleotide sequence of one of the cDNA clone, pSK1, contained the same primary sequence as that of the purified enzyme established by Welinder [FEBS Lett. 72, 19-23 (1976)] with additional sequences at the N and C terminal. All three inserts in the cDNA clones, pSK1, pSK2 and pSK3, coded the same size of peptide (308 amino acid residues) if these are processed in the same way, and the amino acid sequence were homologous to each other by 91-94%. Functional amino acids, including His40, His170, Tyr185 and Arg183 and S-S-bond-forming Cys, were conserved in the three isozymes, but a few N-glycosylation sites were not the same. Two HRP C isoenzyme genomic genes, prxC1 and prxC2, were tandem on the chromosomal DNA and each gene consisted of four exons and three introns. The positions in the exons interrupted by introns were the same in two genes. We observed a putative promoter sequence 5' upstream and a poly(A) signal 3' downstream in both genes. The gene product of prxC1 might be processed with a signal sequence of 30 amino acid residues at the N terminus and a peptide consisting of 15 amino acid residues at the C terminus.     

Isolation of a Drosophila gene coding for a protein containing a novel phosphatidylserine-binding motif

To elucidate the molecular basis of the binding of proteins to the membrane phospholipid phosphatidylserine (PS), we characterized PS-binding peptides isolated from a phage display library. Amino acid sequences deduced from the nucleotide sequences of over 60 phage clones isolated revealed that there was no common primary structure among these peptides, but all peptides were rich in basic amino acid residues. In particular, 15 clones encoded peptides that contained contiguous arginine residues. Characterization of two such peptides in more detail showed that they bound to PS, and to a much lower extent to other phospholipids, including phosphatidylinositol, phosphatidylethanolamine, and phosphatidylcholine. Unlike other Ca2+-dependent PS-binding proteins, these peptides did not require Ca2+ for binding to PS, and the addition of Ca2+ did not alter the phospholipid specificity. Substitution of one of the two RR sequences in one peptide by alanine had no effect, but that of both sequences completely abolished the activity. Furthermore, we identified a Drosophila gene coding for a presumed nuclear protein that shares an amino acid sequence, including a RR residue, with one of the two PS-binding peptides. This protein bound to PS partly depending on the presence of the RR residue. These results allowed us to conclude that an amino acid sequence including contiguous arginine residues is a novel motif that defines Ca2+-independent PS-binding activity.     

Plant aldolase: cDNA and deduced amino-acid sequences of the chloroplast and cytosol enzyme from spinach

We report the sequences of full-length cDNAs for the nuclear genes encoding the chloroplastic and cytosolic fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) from spinach. A comparison of the deduced amino-acid sequences with one another and with published cytosolic aldolase sequences of other plants revealed that the two enzymes from spinach share only 54% homology on their amino acid level whereas the homology of the cytosolic enzyme of spinach with the known sequences of cytosolic aldolases of maize, rice and Arabidopsis range from 67 to 92%. The sequence of the chloroplastic enzyme includes a stroma-targeting N-terminal transit peptide of 46 amino acid residues for import into the chloroplast. The transit peptide exhibits essential features similar to other chloroplast transit peptides. Southern blot analysis implies that both spinach enzymes are encoded by single genes.     

Amino acid sequence specificities of an adhesive recognition signal

Synthetic peptides derived from the cell-binding domain of fibronectin have previously been found to inhibit fibronectin-mediated adhesion in vitro competitively and reversibly, as well as inhibiting cell migratory events in vivo. The amino acid sequence specificity required for this inhibitory activity has been examined further using variations of the originally identified active peptide sequences. The most active small peptide was found to be the pentapeptide Gly-Arg-Gly-Asp-Ser. Although the tetrapeptide Arg-Gly-Asp-Ser was found to retain substantial activity, it was approximately threefold less active. An "inverted" peptide sequence with these same four amino acids arranged in the mirror symmetrical sequence Ser-Asp-Gly-Arg was found to be nearly as active as the forward sequence. However, the same inverted tetrapeptide sequence embedded in a synthetic decapeptide derived from a sequence of histocompatibility antigens has minimal activity, suggesting the importance of adjacent sequences in modifying the activity of such peptides. Neither substitution of amino acids of the same charge nor reversal of the positions of the two charged amino acids retains biological activity. Decreasing the spacing between the charged residues also causes a loss of activity. Our results suggest the hypothesis that this adhesive recognition signal consists of a specific arrangement of one acidic and one basic charged group and additional information provided by adjacent amino acids.     

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.     

Pattern of repeating aromatic residues in synexin. Similarity to the cytoplasmic domain of synaptophysin

Synexin was isolated from bovine liver by high resolution cation exchange chromatography and fragmented with cyanogen bromide or trypsin. Peptides were isolated and their amino acid sequences partially determined. Twenty percent of the synexin sequence was determined in one contiguous sequence of 61 residues and a nonoverlapping sequence of 20 residues. The sequence is characterized by a hexapeptide repeat of the form YPXXXX occurring eight times in series, with phenylalanine substituting for tyrosine in two positions. The intervening amino acids (X) are predominantly proline, glycine and alanine. This pattern of periodic aromatic residues suggests the presence of a novel secondary structure and is similar to repeats present in synaptophysin, gliadin and type II keratin.     

Partial amino acid sequence of porcine elastase II. Active site and the activation peptide regions

The partial amino acid sequence of porcine elastase II, isolated from crude trypsin Type II, was determined. The enzyme consists of two polypeptide chains, a light chain composed of 11 residues, and a heavy chain (Mr = 23 500) with four intrachain disulfide bridges; the two chains are held together by one interchain disulfide bond. Elastase II was fragmented into several peptides by chemical cleavages with CNBr at the two methionine residues, 99 and 180 (chymotrypsinogen numbering), and with hydroxylamine at the peptide bond following DIP-Ser195. About 50% of the sequence was determined and the positions of 120 amino acids were located, including the light chain residues and most of the active site residues. The partial sequence shows 64% difference between porcine elastase II and elastase I and only 26% difference between porcine elastase II and bovine chymotrypsin B.     

Isolation and characterization of a novel PHGPx gene in Raphanus sativus

A full-length cDNA encoding putative phospholipid hydroperoxide glutathione peroxidase (PHGPx) was cloned from Raphanus sativus. The cDNA, designated RsPHGPx, includes an open reading frame which encodes 197 amino acid residues. The alignment of amino acid sequences showed that RsPHGPx had the highest sequence homology to plant PHGPx and contained an N-terminal extension characteristic of a mitochondrial targeting peptide. Northern blot analysis indicated that RsPHGPx was constitutively and ubiquitously expressed during radish development, and its expression was differently regulated by various stress conditions. The expression of RsPHGPx in a yeast PHGPx-deletion mutant significantly rescued the mutant sensitivity to oxidation-sensitive linolenic acid, just as the yeast PHGPx3 gene did. This suggested that RsPHGPx encodes a functional PHGPx protein.     

Cloning and sequence analysis of the gene for glucodextranase from Arthrobacter globiformis T-3044 and expression in Escherichia coli cells

The gld gene for glucodextranase from Arthrobacter globiformis T-3044 was cloned by using a combination of gene walking and probe methods and expressed on the recombinant plasmid pGD8, which was constructed with pUC118, in Escherichia coli cells. The enzyme gene consisted of a unique open reading frame of 3,153 bp. The comparison of the DNA sequence data with the N-terminal and 6 internal amino acid sequences of the purified enzyme secreted from A. globiformis T-3044 suggested the enzyme was translated from mRNA as a secretory precursor with a signal peptide of 28 amino acids residues. The deduced amino acids sequence of the mature enzyme contained 1,023 residues, resulting in a polypeptide with a molecular mass of 107,475 daltons. The deduced sequence showed about 38% identity to that of the glucoamylase from Clostridium sp. G0005. The glucodextranase activity of transformant harboring pGD8 was about 40 mU/ml at 30 degrees C for a 16-h culture. Although the GDase that was produced from the transformant was shorter than authentic GDase by 2 amino acid residues at the N-terminal end side, its enzymatic properties were almost same as the authentic one. Two kinds of genes, dex1 and dex2, for endo-dextranases from A. globiformis T-3044 were also cloned into Escherichia coli cells. The N-terminal of the purified endo-dextranase from A. globiformis T-3044 agreed with the deduced amino acid sequence, after the 33rd alanine residue, of only the dex1 gene for edo-dextranase. This result suggests that the endo-dextranase is translated from mRNA as a secretory precursor with a signal peptide of 32 amino acids residues. The deduced sequence of endo-dextranase 1 and endo-dextranase 2 showed about 93% and 65% identity with that of known endo-dextranase from Arthrobacter sp. CB-8, respectively.     

Trypanothione reductase of Trypanosoma congolense: gene isolation, primary sequence determination, and comparison to glutathione reductase

The gene encoding trypanothione reductase, the redox disulfide-containing flavoenzyme that is unique to the parasitic trypanosomatids (Shames et al., 1986), has been isolated from the cattle pathogen Trypanosoma congolense. Library screening was carried out with inosine-containing oligonucleotide probes encoding sequences determined from two active site peptides isolated from the purified Crithidia fasciculata enzyme. The nucleotide sequence of the gene was determined according to the dideoxy chain termination method of Sanger. The structural gene is 1476 nucleotides long and encodes 492 amino acids. We have identified the active site peptide containing the redox-active disulfide, a peptide corresponding to the histidine-467 region of human erythrocyte glutathione reductase, as well as the flavin binding domain that is highly conserved in all disulfide-containing flavoprotein reductase enzymes. Alignment of five tryptic peptides (80 residues) isolated from the C. fasciculata trypanothione reductase with the primary sequence of the T. congolense enzyme showed 88% homology with 76% identity. Additionally, a sequence comparison of the glutathione reductase from Escherichia coli or human erythrocytes to T. congolense trypanothione reductase reveals greater than 50% homology. A search for the amino acid residues in the primary sequence of trypanothione reductase functionally active in binding/catalysis in human erythrocyte glutathione reductase shows that only the two arginine residues (Arg-37 and Arg-347), shown by X-ray crystallographic data to hydrogen bond to the GS1 glutathione glycyl carboxylate, are absent.     

Cloning and nucleotide sequence of a bovine pancreatic preprocarboxypeptidase A cDNA

A full-length cDNA clone encoding bovine pancreatic preprocarboxypeptidase A was isolated and sequenced. The 1405-base pair insert contains a 26-nucleotide 5'-noncoding region, a 1260-nucleotide open reading frame and a 76-nucleotide 3'-noncoding fragment plus a poly(A) tail of at least 43 nucleotides. The open reading frame encodes a protein of 419 amino acids, including the 16 amino acid signal peptide. The mature enzyme (309 residues) has two additional C-terminal amino acids, as compared with the amino acid sequence of the protein which was reported more than 20 years ago. In addition, four residues deduced from the nucleotide sequence differed from those identified in the reported amino acid sequence from their net charge: Asp-89, Asp-114, Gln-122, and Asp-185 instead of Asn-89, Asn-114, Glu-122, and Asn-185, respectively. A high degree of identity exists between the nucleotide sequences (81.3%), on the one hand, and the amino acid sequences (78.3%), on the other hand, of bovine preprocarboxypeptidase A and rat preprocarboxypeptidase A1.     

Conservation of functional residues between yeast and E. coli inorganic pyrophosphatases

The alignments of the amino acid sequences of inorganic pyrophosphatase (PPase) from Saccharomyces cerevisiae (Y1-PPase, 286 amino acids) and Escherichia coli (E-PPase, 175 amino acids) are examined in the light of crystallographic and chemical modification results placing specific amino acid residues at the active site of the yeast enzyme. The major results are: (1) the full E-PPase sequence aligns within residues 28-225 of Y1-PPase, raising the possibility that the N-terminal and C-terminal portions of Y1-PPase may not be essential for activity, and (2) that whereas the overall identity between the two sequences is only modest (22-27% depending on the choice of alignment parameters), of some 17 putative active site residues, 14-16 are identical between Y-PPase and E-PPase. PPase thus appears to be an example of enzymes from widely divergent species that conserve common functional elements within the context of substantial overall sequence variation.     

Effect of deletions within the leader peptide of pre-ornithine transcarbamylase on mitochondrial import

The uptake of the cytoplasmically synthesized mammalian enzyme, ornithine transcarbamylase, into mitochondria is directed by an N-terminal peptide of 32 amino acids. We have investigated some of the structural requirements for the import of the enzyme from rat liver into isolated mitochondria and into mitochondria of COS cells transfected with cDNA encoding the precursor form of ornithine transcarbamylase. Deletion of 21 amino acids from the N terminus of the leader peptide blocked the import of the precursor; deletion of 5 amino acids at positions 15-19 from the N terminus of the leader peptide had no deleterious effect on the import of the enzyme, nor on the processing and assembly of subunits in mitochondria. The region deleted contained three of eight basic residues in the leader peptide suggesting that specific structural elements containing basic residues, rather than the general basic nature of the leader, may be involved in mitochondrial import.     

Avian adipose lipoprotein lipase: cDNA sequence and reciprocal regulation of mRNA levels in adipose and heart

cDNA clones for chicken adipose lipoprotein lipase were isolated from an expression library in lambda gt11 by antibody screening and characterized by hybridization selection and nucleotide sequencing. Based on the cDNA sequence and on N-terminal sequence analysis of the purified enzyme, chicken adipose lipoprotein lipase is a mature protein of 465 amino acids with a signal peptide of 19 or 25 amino acids, depending on which of two methionine residues is used for translation initiation. The predicted amino-acid sequence was found to be 73-77% identical to the four known mammalian adipose lipoprotein lipase sequences, with conservation of position of cysteine residues and putative functional domains, and number of potential N-glycosylation sites. Chicken lipoprotein lipase differs from mammalian lipoprotein lipases with respect to the position of one N-glycosylation site and the presence of an additional 15-17 C-terminal amino acids. 32P-labeled cDNA clones hybridized to mRNA species of 3.7 and 4.0 kb in Northern blots of heart and adipose, but not of liver RNA. In chickens that were fasted for 48 h and then refed, lipoprotein lipase mRNA levels in adipose increased to a maximal level of 350% that of controls at 10 h, whereas heart lipoprotein lipase mRNA levels fell to 40% of controls at 14 h. Concomitantly, no changes in total RNA were observed. Thus, avian lipoprotein lipase is subject to reciprocal pretranslational regulation in adipose and heart.     

Vacuolar protein sorting in fission yeast: cloning, biosynthesis, transport, and processing of carboxypeptidase Y from Schizosaccharomyces pombe.

PCR was used to isolate a carboxypeptidase Y (CPY) homolog gene from the fission yeast Schizosaccharomyces pombe. The cloned S. pombe cpy1+ gene has a single open reading frame, which encodes 950 amino acids with one potential N-glycosylation site. It appears to be synthesized as an inactive pre-pro protein that likely undergoes processing following translocation into appropriate intracellular organelles. The C-terminal mature region is highly conserved in other serine carboxypeptidases. In contrast, the N-terminal pro region containing the vacuolar sorting signal in CPY from Saccharomyces cerevisiae shows fewer identical residues. The pro region contains two unusual repeating sequences; repeating sequence I consists of seven contiguous repeating segments of 13 amino acids each, and repeating sequence II consists of seven contiguous repeating segments of 9 amino acids each. Pulse-chase radiolabeling analysis revealed that Cpy1p was initially synthesized in a 110-kDa pro-precursor form and via the 51-kDa single-polypeptide-chain intermediate form which has had its pro segment removed is finally converted to a heterodimer, the mature form, which is detected as a 32-kDa protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Like S. cerevisiae CPY, S. pombe Cpy1p does not require the N-linked oligosaccharide moiety for vacuolar delivery. To investigate the vacuolar sorting signal of S. pombe Cpy1p, we have constructed cpy1+-SUC2 gene fusions that direct the synthesis of hybrid proteins consisting of N-terminal segments of various lengths of S. pombe Cpy1p fused to the secreted enzyme S. cerevisiae invertase. The N-terminal 478 amino acids of Cpy1 are sufficient to direct delivery of a Cpy1-Inv hybrid protein to the vacuole. These results showed that the pro peptide of Cpy1 contains the putative vacuolar sorting signal.     

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.