Isolation and sequence determination of rat cardiac natriuretic peptide

We have isolated a cardiac natriuretic peptide of 5K daltons from the rat atrium and determined its amino acid sequence. The 5K cardiac natriuretic peptide was elucidated to be a 45-amino acid peptide with the sequence of S-Q-D-S-A-F-R-I-Q-E-R-L-R-N-S-K-M-A-H-S-S-S-C-F-G-Q-K-I-D-R-I-G-A-V-S-R- L-G-C-D - G-L-R-L-F by sequencing the native peptide and its lysyl endopeptidase digests. The sequence of this peptide was identical to the amino acid sequence [51-95] of the rat brain natriuretic peptide (BNP) precursor deduced from the cDNA sequence. The 5K cardiac natriuretic peptide, or BNP[51-95], was identified as the major storage and secretory form derived from the BNP precursor in the rat heart.     

Peptide YY. Structure of the precursor and expression in exocrine pancreas

Peptide YY is a 36-residue gastrointestinal hormone which inhibits both pancreatic and gastric secretion. We have isolated a cDNA encoding the peptide YY precursor by screening a rat intestinal lambda gt11 cDNA library with an antiserum directed against the porcine hormone. The nucleotide sequence of the cDNA encodes a 98-residue protein (molecular weight, 11, 121) which has an amino acid sequence identical to that of porcine peptide YY. Rat peptide YY is preceded immediately by a signal sequence and followed by a cleavage-amidation sequence Gly-Lys-Arg plus 31 additional amino acids. Thus the peptide YY precursor is similar in structure to that of two related peptides, pancreatic polypeptide and neuropeptide Y. RNA blot hybridizations reveal that the peptide YY gene is much more actively expressed in pancreas than previously realized. In situ hybridizations localized peptide YY cells exclusively to the exocrine pancreas. The abundance of peptide YY in one of its target organs, the pancreas, suggests a paracrine mechanism for peptide YY in regulating pancreatic enzyme secretion.     

Aplysia californica neurons R3-R14: primary structure of the myoactive histidine-rich basic peptide and peptide I

The R3-R14 neurons of the marine mollusc Aplysia are neuroendocrine cells that express a gene encoding peptides I, II and histidine-rich basic peptide (HRBP), a myoactive peptide that excites Aplysia heart and enhances gut motility in vitro. Peptide II has been chemically characterized (35), but the complete primary structures of peptide I and HRBP have not been established by amino acid sequence analysis. HRBP, peptide I, and the prohormone (proHRBP) were therefore purified from acid extracts of Aplysia californica neural tissue using sequential gel filtration and reverse-phase high-performance liquid chromatography and chemically characterized. Amino acid sequence analysis demonstrated that HRBP was a 43-residue peptide whose sequence was: less than Glu-Val-Ala-Gln-Met-His-Val-Trp-Arg-Ala-Val-Asn-His-Asp-Arg-Asn-His-Gly- Thr-Gly - Ser-Gly-Arg-His-Gly-Arg-Phe-Leu-Ile-Arg-Asn-Arg-Tyr-Arg-Tyr-Gly-Gly-Gly- His-Leu - Ser-Asp-Ala-COOH. Compositional and sequence analyses of peptide I and proHRBP demonstrated that peptide I was a 26-residue peptide with the following sequence: NH2-Glu-Glu-Val-Phe-Asp-Asp-Thr-Asp-Val-Gly-Asp-Glu-Leu-Thr-Asn-Ala- Leu-Glu-Ser-Val-Leu-Thr-Asp-Phe-Lys-Asp-COOH. These results demonstrated that the pro-HRBP sequence predicted by nucleotide sequence analysis of a cDNA clone (24) was in fact synthesized in R3-R14 neurons. Hydrophilicity and hydrophobicity profiles of preproHRBP, combined with charge distribution profiles and predictive secondary structural analysis, showed that cleavage at dibasic sequences was strongly associated with peaks of hydrophilicity in alpha-helical regions of the preprohormone.     

Isolation and structures of glucagon and glucagon-like peptide from catfish pancreas

Both glucagon and the structurally similar glucagon-like peptide proteolytically derived from preproglucagon were purified from the endocrine pancreas of the channel catfish (Ictalurus punctata). This study represents the first report of the isolation of glucagon-like peptide from any source. Peptide sequences of glucagon-like peptide from other species have only been deduced from the cDNA sequences for preproglucagon. The sequence of the 34-residue glucagon-like peptide was found to be HADGTYTSDVSSYLQDQAAKDFITWLKSGQPKPE. Catfish glucagon-like peptide shares sequence identity at 26 of 31 residues with the putative glucagon-like peptide from anglerfish preproglucagon II. The mass of catfish glucagon-like peptide was found by fast atom bombardment-mass spectrometry to be 3785, identical with the value predicted by sequence analysis. This suggests that no post-translational modification occurs beyond proteolytic processing. The sequence of catfish glucagon was determined to be HSEGTFSNDYSKYLETRRAQDFVQWLM(N,S). Catfish glucagon exhibits a high degree of immunologic similarity with porcine glucagon by radioimmunoassay, whereas catfish glucagon-like peptide does not.     

The primary structure of the cytotoxin alpha-sarcin

The primary structure of the cytotoxin alpha-sarcin was determined. Eighteen of the 19 tryptic peptides were purified; the other peptide has arginine only. The complete sequence of 17 of the peptides was determined; the sequence of the remaining peptide was determined in part. The sequence of the 39 NH2-terminal residues was obtained by automated Edman degradation. The carboxyl-terminal amino acids were identified after carboxypeptidase treatment. The assignment of the amino acids in the tryptic peptides was confirmed and their alignment established from the sequence of the secondary tryptic peptides obtained after cleavage of citraconylated alpha-sarcin, from the sequence of a 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine peptide, from the sequence of a chymotryptic peptide, and from the sequence of a peptide obtained with Staphylococcus aureus V8 protease. alpha-Sarcin contains 150 amino acid residues; the molecular weight is 16,987. There are disulfide bridges between cysteine residues at positions 6 and 148 and between residues 76 and 132.     

多肽内切酶鉴定牙龈卟啉单胞菌血凝素2结合位点

目的利用多肽内切酶分析牙龈卟啉单胞菌凝血素2(Porphyromonas gingivalishemagglutinin-2,PgHA-2)与氯化血红素结合位点的氨基酸序列。方法Endoproteinase Lys-C多肽内切酶水解获得与氯化血红素结合的功能性的多肽片段,质谱技术鉴定多肽片段的氨基酸序列。结果质谱鉴定与氯化血红素结合的多肽片段的氨基酸序列为YAVNDGFPGDHYAVMISK。结论进一步明确了HA-2与氯化血红素结合位点的氨基酸序列,为牙周病的预防和治疗方法的改进奠定基础。     

Suppression and reversal of allergic encephalomyelitis in guinea pigs with a non-encephalitogenic analogue of the tryptophan region of the myelin basic protein.

The administration of synthetic peptide S42 leads to suppression and reversal of experimental allergic encephalomyelitis (EAE) induced in guinea pigs by myelin basic protein. Peptide S42 contains a linear sequence of 21 amino acid residues, H-Phe-Ser-Trp-Gln-Lys-Phe-Ser-Trp-Gln-Lys-Phe-Ser-Trp-Gln-Lys-Phe-Ser-Trp-Gln-Lys-Gly-OH, made up of four repeating unit sequences of H-Phe-Ser-Trp-Gln-Lys-OH in addition to a C-terminal glycine. Injected at relatively high doses, peptide S42 is non-encephalitogenic. It induces delayed-type hypersensitivity which is not followed by EAE, and elicits delayed-type hypersensitivity responses in peptide S42, encephalitogenic trytophan peptide, or BP-challenged animals for either of the three antigens. The repeating unit sequence of peptide S42 is analogous to the encephalitogenic tryptophan region of the BP molecules . The sequence homology is responsible for cellular recognition of this antigen by the skin test assay and suggests in vivo interaction between peptide S42 and EAE-inducing cells leading to suppression and reversal of disease.     

The complete amino acid sequence of the pediocin-like antimicrobial peptide leucocin C

The pediocin-like antimicrobial peptide leucocin C produced by a strain of Leuconostoc mesenteroides has been purified using a recently developed rapid two-step procedure. The complete and corrected amino acid sequence of the peptide has been determined by Edman degradation of the intact peptide and a C-terminal fragment generated by cleavage with Asp-N endoprotease. Leucocin C contained 43 residues with the following sequence: KNYGNGVHCTKKGCSVDWGYAWTNIANNSVMNGLTGGNAGWHN. The molecular weight of leucocin C as determined by mass spectrometry was 4595, which is consistent with the theoretical molecular weight of 4596 calculated from the sequence. Moreover, the molecular weights of the two fragments generated by cleavage with Asp-N were also consistent with the determined sequence.     

Structural design and characterization of a channel-forming peptide

A 16-residue polypeptide model with the sequence acetyl-YALSLAATLLKEAASL-OH was derived by rational de novo peptide design. The designed sequence consists of amino acid residues with high propensity to adopt an alpha helical conformation, and sequential order was arranged to produce an amphipathic surface. The designed sequence was chemically synthesized using a solid-phase method and the polypeptide was purified by reverse-phase liquid chromatography. Molecular mass analysis by electro-spray ionization mass spectroscopy confirmed the correct designed sequence. Structural characterization by circular dichroism spectroscopy demonstrated that the peptide adopts the expected alpha helical conformation in 50% acetonitrile solution. Liposome binding assay using Small Unilamellar Vesicle (SUV) showed a marked release of entrapped glucose by interaction between the lipid membrane and the tested peptide. The channel-forming activity of the peptide was revealed by a planar lipid bilayer experiment. An analysis of the conducting current at various applied potentials suggested that the peptide forms a cationic ion channel with an intrinsic conductance of 188 pS. These results demonstrate that a simple rational de novo design can be successfully employed to create short peptides with desired structures and functions.     

Identification of an alpha(3)beta(1) integrin recognition sequence in thrombospondin-1.

A synthetic peptide containing amino acid residues 190-201 of thrombospondin-1 (TSP1) promoted adhesion of MDA-MB-435 breast carcinoma cells when immobilized and inhibited adhesion of the same cells to TSP1 when added in solution. Adhesion to this peptide was enhanced by a beta(1) integrin-activating antibody, Mn(2+), and insulin-like growth factor I and was inhibited by an alpha(3)beta(1) integrin function-blocking antibody. The soluble peptide inhibited adhesion of cells to the immobilized TSP1 peptide or spreading on intact TSP1 but at the same concentrations did not inhibit attachment or spreading on type IV collagen or fibronectin. Substitution of several residues in the TSP1 peptide with Ala residues abolished or diminished the inhibitory activity of the peptide in solution, but only substitution of Arg-198 completely inactivated the adhesive activity of the immobilized peptide. The essential residues for activity of the peptide as a soluble inhibitor are Asn-196, Val-197, and Arg-198, but flanking residues enhance the inhibitory activity of this core sequence, either by altering the conformation of the active sequence or by interacting with the integrin. This functional sequence is conserved in all known mammalian TSP1 sequences and in TSP1 from Xenopus laevis. The TSP1 peptide also inhibited adhesion of MDA-MB-435 cells to the laminin-1 peptide GD6, which contains a potential integrin-recognition sequence Asn-Leu-Arg and is derived from a similar position in a pentraxin module. Adhesion studies using recombinant TSP1 fragments also localized beta1 integrin-dependent adhesion to residues 175-242 of this region, which contain the active sequence.     

The amino acid sequence of the activation peptide of bovine pro-carboxypeptidase A

The amino acid sequence of the activation peptide of bovine pro-carboxypeptidase A subunit I has been determined by automated Edman degradation of the cyanogen bromide fractions derived from the precursor protein. The activation peptide contains 94 amino acid residues in a unique sequence which precedes directly the amino-terminal alanine residue of carboxypeptidase A alpha. A notable feature of the activation peptide is the presence of acidic amino acid residues immediately preceding the site of activation. The amino acid sequence of the activation peptide of bovine pro-carboxypeptidase A shows extensive similarity to those of the corresponding porcine and rat enzymes.     

Localization of the pyridoxal phosphate binding site at the COOH-terminal region of erythrocyte band 3 protein

A human erythrocyte Band 3 peptide, affinity labeled with pyridoxal phosphate, was purified by a combination of gel permeation and reverse-phase high performance liquid chromatography. The amino acid sequence of the transmembrane peptide was determined by sequencing subfragments of the peptide obtained from lysyl endopeptidase and staphylococcal proteinase V8 digestions. When a peptide containing the COOH-terminal of human erythrocyte Band 3 was also purified and sequenced, the affinity-labeled peptide was found to be located close to the COOH-terminal of Band 3, where it could be aligned with amino acid residues 852-927 of a murine erythrocyte Band 3, deduced from a nucleotide sequence of a cDNA clone (Kopito, R. R., and Lodish, H. F. (1985) Nature 316, 234-238). The amino acid sequence of the COOH-terminal region was highly homologous to that of murine Band 3. As a result, the sequence of the COOH-terminal peptide of Band 3 was established as follows. (Formula: see text). The pyridoxal phosphate binding site was identified as Lys-18 which corresponded to Lys-869 of the deduced sequence. It appears that the COOH-terminal region of Band 3 constitutes at least a part of the active center for anion transport in human erythrocyte membranes.     

A bradykinin-potentiating peptide (peptide K12) isolated from the venom of Egyptian scorpion Buthus occitanus

A nontoxic peptide with bradykinin-potentiating activity was isolated from the dialyzed venom of the scorpion Buthus occitanus by reverse-phase high performance liquid chromatography (RP-HPLC). The pharmacological activity of the peptide was bioassayed by its ability to potentiate added bradykinin (BK) on the isolated guinea pig ileum as well as the isolated rat uterus for contraction. Moreover, the peptide potentiates in vivo the depressor effect of BK on arterial blood pressure in the normotensive anesthetized rat. Chemical characterization of the peptide was also performed. The amino acid composition of the peptide showed 21 amino acid residues per molecule including three proline residues. The amino acid sequence of the purified peptide was confirmed by mass spectrometry. Either N- or C-terminal ends were free. The sequence does not show a homology with bradykinin-potentiating peptides isolated from either scorpion or snake venoms. Furthermore, we did not find a significant sequence homology between the sequence of the isolated peptide and any of proteins or peptides in GenPro or NBRF data banks. The peptide also inhibited angiotensin-converting enzyme (ACE), and could not serve as substrate for the enzyme. It could be concluded that the mechanism of bradykinin-potentiating peptide (BPP) activity may be due to ACE inhibition.     

Cloning of a cDNA encoding porcine brain natriuretic peptide

Complimentary DNA (cDNA) clones encoding porcine brain natriuretic peptide (BNP) were isolated from a porcine atrial cDNA library. The longest of the cDNA clones (1507 nucleotides) apparently originated from an unprocessed messenger RNA, since the nucleotide sequence encoding BNP-26 was interrupted by an intron of 554 nucleotides. A partial cDNA clone representing processed BNP mRNA was prepared by polymerase chain reaction. A comparison of the sequence of these two cDNAs reveals the presence of an additional intron within the sequence encoding the BNP precursor. The identification of these introns suggests that the BNP gene structure differs from the atrial natriuretic peptide gene in the location of intron 2. BNP mRNA encodes a propeptide of 131 amino acids, including a signal peptide domain (25 amino acids) and a prohormone domain (106 amino acids). Like atrial natriuretic peptide, the bioactive BNP sequence is localized at the carboxyl terminus of the prohormone. Although the carboxyl-terminal peptide sequences of porcine atrial natriuretic peptide and BNP are well conserved, there is relatively little homology within their propeptide regions.     

Solubility improvement of an anthrax toxin peptide inhibitor by rational amino acid randomization

We previously described a potent anthrax toxin inhibitor, based on a phage-library-selected peptide sequence, synthesized as a tetra-branched molecule on a lysine core and further modified for improvement of activity [Pini et al., Biochem. J., 2006, 395, 157]. This branched peptide had very low solubility because of several hydrophobic residues in the peptide sequence. This complicated molecule purification and manufacturing. Here we report a rational modification of the peptide sequence, obtained by construction and selection of several mini libraries of branched peptides, containing sequences randomized in non crucial positions of the original peptide. Mini libraries were screened for solubility and inhibitory activity. This procedure enabled us to obtain a new peptide with a better solubility and identical inhibitory activity.     

Dominance of an alternative CLIP sequence in the celiac disease associated HLA-DQ2 molecule

During assembly, HLA class II molecules associate with the invariant chain. As the result, the peptide-binding groove is occupied by an invariant chain peptide termed CLIP (class-II-associated invariant chain peptide; sequence MRMATPLLM). By mass spectrometry, we have now characterized peptides that are naturally present in HLA-DQ2. This analysis revealed that 22 variants of Ii-derived peptides are associated with HLA-DQ2. Strikingly, the large majority of those do not contain the conventional CLIP sequence MRMATPLLM, but instead a peptide that partially overlaps with CLIP, sequence TPLLMQALPM. Peptide binding studies indicate that this alternative CLIP peptide has superior HLA-DQ2 binding properties compared to the conventional CLIP and that the minimal nine-amino-acid binding core consists of the sequence PLLMQALPM, findings that could be corroborated by molecular simulation. The alternative CLIP peptide was also found to be present in HLA-DQ2 molecules isolated from human thymus. Moreover, the alternative CLIP peptide was also found in association with HLA-DQ8. Together, these results indicate that HLA-DQ2 and HLA-DQ8 associate with an alternative CLIP sequence, a property that may relate to the strong association between HLA-DQ molecules and human autoimmune diseases.     

Primary structure of a base non-specific ribonuclease from Rhizopus niveus

The primary structure of a base non-specific ribonuclease from Rhizopus niveus (RNase Rh) was determined by nucleotide sequence analysis of the DNA fragment encoding RNase Rh gene including signal peptide sequence, and amino acid sequence analysis of the peptide obtained from RNase Rh and RNase Rh' (a protease-modified RNase Rh created during the course of purification). The sequence determined was: MKAVLALATLIGSTLASSCSSTA LSCSNSANSDTCCSPEYGLVVLNMQWAPGYGPANAFTLHGLWPDKCSGAYAPSGGCDSN RASSSIASVIKSKDSSLYNSMLTYWPSNQGNNNVFWSHEWSKHGTCVSTYDPDCYDNYE EGEDIVDYFQKAMDLRSQYNVYKAFSSNGITPGGTYTATEMQSAIESYFGAKAKIDCSSG TLSDVALYFYVRGRDTYVITDALSTGSCSGDVEYPTK (the sequence of signal peptide is underlined). The sequence indicates that the homology with the sequence of RNase T2 from A. oryzae with the same base specificity is about 42% and that the sequences around the two histidine residues which are supposed to be involved in the active site are fairly conserved.     

Purification and sequence of a non-opioid peptide derived from ovine proenkephalin: implications for possible species specific processing

A non-enkephalin containing pentadeca peptide derived from ovine adrenal proenkephalin has been purified and sequenced. The sequence of the peptide is: Phe-Ala-Glu-Pro-Leu-Pro-Ser-Glu-Glu-Glu-Gly-Glu-Ser-Tyr-Ser (preproenkephalin 237-251) representing the amino portion of peptide B (preproenkephalin 237-268). The sequence is identical to bovine preproenkephalin 237-251, differing from the corresponding human sequence at positions 240 and 244. This peptide can be generated by a processing event common to other opioid peptides and is present in chromaffin granules in significant amounts. The presence of this peptide in substantial quantities suggests a possible difference in proenkephalin processing between the bovine and ovine adrenal medulla.     

Characterization of lamprey fibrinopeptides

1. Lamprey fibrinopeptide B is a relatively large peptide made up of about 40 amino acid residues. The peptide is highly electronegative, containing a large number of aspartic acid residues and a tyrosine O-sulphate residue. 2. The amino acid sequence of the first 18 residues from the N-terminal end of fibrinopeptide B has been established. The C-terminal ends with the sequence Val-Arg. Fibrino-peptide B is released by both lamprey and bovine thrombins. 3. Lamprey fibrino-peptide A is a short peptide containing only eight residues. The proposed amino acid sequence is: Asp-Asp-Ser-Ile/Leu-Asp-Ser-Leu/Ile-ArgThis peptide is released by lamprey thrombin but not by bovine thrombin.     

Sequence determination by MALDI-TOF mass spectrometry of an insecticidal lentil peptide of the PA1b type

Mature seeds of lentil (Lens culinaris Medik.) were previously reported to contain an insecticidal cysteine-rich peptide, likely of the albumin-1 subunit b type. The purpose of this work was to determine the amino acid sequence of this insecticidal lentil peptide in an Eston lentil extract by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), after reduction of the disulfide bridges, alkylation of the cysteine residues and hydrolysis by pronase, trypsin, chymotrypsin and endoproteinase Asp-N. Sequences of key fragments were supported by monoisotopic mass measurements and by sequence ions from collision-induced dissociation (CID) experiments with a MALDI-TOF/TOF analyzer (MS/MS analysis). The new 37 amino acid sequence revealed strong similarities to a histidine-containing pea PA1b peptide and to soybean leginsulins but with a unique segment of RSSA in the middle. The lentil PA1b peptide sequence agreed completely with that derived from a L. culinaris genomic DNA sequence.