Isolation, biological and chemical characterization, and synthesis of a neurotensin-related hexapeptide from chicken intestine

A new biologically active peptide of the neurotensin (NT) family, shown previously to cross-react in a COOH-terminal-directed radioimmunoassay for bovine NT, has been isolated from extracts of chicken intestine and identified as H-Lys-Asn-Pro-Tyr-Ile-Leu-OH, which is identical with the biologically active COOH-terminal half of NT except for the amino acid substitutions Lys/Arg and Asn/Arg. It is proposed that this peptide be referred to as Lys8, Asn9, NT8-13 (LANT-6). Synthetic material prepared with this amino acid sequence using the Merrifield technique was immunochemically, chromatographically, and biologically indistinguishable from the native peptide. In contrast to chicken NT which induced hypotension, hyperglycemia, increased vascular permeability, and cyanosis when injected intravenously into anesthetized rats, synthetic LANT-6 brought about primarily a hypertensive response and had little ability to promote hyperglycemia, increased vascular permeability, and cyanosis. In rats pretreated with the alpha-blocker phentolamine and in adrenalectomized rats, the hypertensive response to LANT-6 was blocked, suggesting that adrenal catecholamines mediated this effect. These findings suggest that LANT-6, a natural variant of NT with a different spectrum of biologic activity, may be a NT-related messenger peptide with a different function(s).     

Immunochemical characterization of neurotensin-like peptides in chicken

Using radioimmunoassay and 3 region specific antisera toward bovine neurotensin (NT), the NT-like peptides in chicken have been shown to differ from NT but to strongly resemble its COOH-terminal region. Three substances were identified, one of which resembled NT biologically and appeared to share 7 or 8 of its COOH-terminal residues. The two other peptides were smaller than NT but seemed to possess 4–6 residue homologies with it. Tissue distribution studies indicated that the chicken pancreas and thymus had unusually high levels of this material (>200 fold that in rat) and that the 3 substances were distributed differently in tissues. Chromatographic studies showed that the peptides obtained from brain, intestine, thymus, and pancreas were similar. These results, demonstrating evolutionary conservation of the COOH-terminal region of NT, are in keeping with the known importance of this region for biological activity. These findings also suggest the existence of an NT-family of peptides serving multiple biological roles.     

The site at which 4-iodoacetamidosalicylate reacts with glutamate dehydrogenases

1. Bovine, porcine and chicken liver glutamate dehydrogenases were irreversibly inhibited by a tenfold excess of radioactive 4-iodoacetamidosalicylic acid at pH7.5. 2. Inhibition was accompanied by the covalent incorporation of 1.1 mol of labelled inhibitor/mol of polypeptide chain. Acid hydrolysis yielded N^ε-carboxymethyl-lysine as the sole labelled amino acid. No labelled S-carboxymethylcysteine was recovered from the bovine or porcine enzymes. 3. The labelled bovine enzyme was hydrolysed with trypsin. The radioactivity was found at lysine-126 in a peptide comprising residues 119–130 of the sequence. 4. The amino acid compositions of the tryptic peptides containing labelled lysine from the porcine and chicken enzymes were similar to that of the bovine peptide.     

Porcine liver low Mr phosphotyrosine protein phosphatase: The amino acid sequence

Porcine low M_r phosphotyrosine protein phosphatase has been purified and the complete amino acid sequence has been determined. Both enzymic and chemical cleavages are used to obtain protein fragments. FAB mass spectrometry and enzymic subdigestion followed by Edman degradation have been used to determine the structure of the NH₂-terminal acylated tryptic peptide. The enzyme consists of 157 amino acid residues, is acetylated at the NH₂-terminus, and has arginine as COOH-terminal residue. It shows kinetic parameters very similar to other known low Mr PTPases. This PTPase is strongly inhibited by pyridoxal 5′-phosphate (K=21ΜM) like the low Mr PTPases from bovine liver, rat liver (AcP2 isoenzyme), and human erythrocyte (Bslow isoenzyme). The comparison of the 40–73 sequence with the corresponding sequence of other low Mr PTPases from different sources demonstrates that this isoform is highly homologous to the isoforms mentioned above, and shows a lower homology degree with respect to rat AcP1 and human Bfast isoforms. A classification of low M_r PTPase isoforms based on the type-specific sequence and on the sensitivity to pyridoxal 5?-phosphate inhibition has been proposed.     

Molecular cloning and SNP association analysis of chicken PMCH gene

The pre-melanin-concentrating hormone (PMCH) gene is an important gene functionally concerning the regulations of body fat content, feeding behavior and energy balance. In this study, the full-length cDNA of chicken PMCH gene was amplified by SMART RACE method. The single nucleotide polymorphisms (SNPs) in the PMCH gene were screened by comparative sequence analysis. The obtained non-synonymous coding SNPs (ncSNPs) were designed for genotyping firstly. Its effects on growth, carcass characteristics and meat quality traits were investigated employing the F₂ resource population of Gushi chicken crossed with Anak broiler by AluI CRS-PCR–RFLP. Our results indicated that the cDNA of chicken PMCH shared 67.25 and 66.47 % homology with that of human and bovine PMCH, respectively. The deduced amino acid sequence of chicken PMCH (163 amino acids) were 52.07 and 50.89 % identical to those of human and bovine PMCH, respectively. The PMCH protein sequence is predicted to have several functional domains, including pro-MCH, CSP, IL7, XPGI and some low complexity sequence. It has 8 phosphorylation sites and no signal peptide sequence. gga-miR-18a, gga-miR-18b, gga-miR-499 microRNA targeting site was predicted in the 3′ untranslated region of chicken PMCH mRNA. In addition, a total of seven SNPs including an ncSNP and a synonymous coding SNP, were identified in the PMCH gene. The ncSNP c.81 A > T was found to be in moderate polymorphic state (polymorphic index = 0.365), and the frequencies for genotype AA, AB and BB were 0.3648, 0.4682 and 0.1670, respectively. Significant associations between the locus and shear force of breast and leg were observed. This polymorphic site may serve as a useful target for the marker assisted selection of the growth and meat quality traits in chicken.     

The cellular immune response to synthetic peptides containing sequences known to be heptenic in performic acid-oxidized ferredoxin from Clostridium pasteurianum

The NH₂-terminal heptapeptide and the COOH-terminal pentapeptide of performic acid-oxidized ferredoxin from Clostridium pasteurianum have been shown to encompass the two major haptenic regions of this molecule. These peptides were conjugated to succinylated bovine serum albumin (S-BSA) to yield an immunologically bivalent hapten-carrier conjugate (N + C-S-BSA). Peptides were also synthesized which contained the NH₂-terminal and COOH-terminal haptenic peptides linked by a bridge of five amino acids (N-5-C), these two peptides linked by 10 amino acids (N-10-C), and one containing two COOH-terminal peptides linked by 12 amino acids (C-12-C). The ability of these preparations to elicit various immunological responses was tested. In O-Fd-sensitized guinea pigs, N + C-S-BSA, N-5-C, and N-10-C elicited immediate and delayed skin reactions; N-5-C and N-10-C inhibited the migration of macrophages; N + C-S-BSA and N-10-C stimulated the proliferation of lymphocytes from these sensitized animals, however, N-5-C and C-12-C did not. In animals sensitized to N + C-S-BSA, skin reactions were elicited by O-Fd, S-BSA, and the NH₂-and COOH-terminal peptides alone. In these animals, lymphocyte proliferation was stimulated significantly by either O-FD or S-BSA. The N-5-C peptide was found to be nonimmunogenic by the schedule used here. However, the N-10-C peptide was found to be strongly immunogenic, and, in animals sensitized to N-10-C, skin reactions and MIF were elicited by N-10-C and 0-Fd, and lymphocyte proliferation was stimulated by N-10-C and O-Fd, but not by C-12-C. The implications of these results in relation to the bicellular mechanism of the immune response are discussed.     

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.     

The carboxy-terminal amino acid sequence of rabbit liver fructose 1,6-bisphosphatase

A peptide derived from the COOH-terminus of rabbit liver fructose 1,6-bisphosphatase (Fru-P₂ase, EC 3.1.3.11) has been isolated and its amino acid sequence determined. The COOH-terminus is lysine, but some preparations contain COOH-terminal alanine or lysyl lysine. This region of the protein appears to be susceptible to modification by the action of an endogenous peptidyldipeptidase.     

On the substrate specificity of cathepsins B1 and B2 including a new fluorogenic substrate for cathepsin B1.

Cathepsin B1 from bovine spleen exhibited its greatest rates of hydrolysis on peptide β-naphthylamide (βNA) derivatives containing paired basic residues, i.e., Cbz-Arg-Arg-βNA, t-Boc-Lys-Lys-βNA, and t-Boc-Lys-Arg-βNA. Internal peptide bonds were not attacked. At its pH 6.5 optimum, cathepsin B1 hydrolyzed Cbz-Arg-Arg-βNA (K_m 0.18 mM) 64 times faster than Bz-DL-Arg-βNA (K_m 3.3 mM or 1.6 mM for the L isomer) and was therefore chosen to replace the latter as a more soluble and sensitive substrate for the assay of cathepsin B1. Although cathepsin B2 had no action on the β-naphthylamide substrates, it did manifest carboxypeptidase activity by attacking COOH-terminal residues exposed by the action of cathepsin B1. At its pH 5.0 optimum, cathepsin B2 behaved as a SH-dependent, non-specific carboxypeptidase by releasing COOH-terminal amino acids from a variety of Cbz-Gly-X substrates and polypeptides such as glucagon, Val-Leu-Ser-Glu-Gly, and penta-lysine.     

Deoxyribose-5-P aldolase: subunit structure and composition of active site lysine region

Deoxyribose-5-P aldolase, a Class I aldolase, from Salmonella typhimurium has a molecular weight of 57,000 and is composed of two subunits of 28,500 molecular weight. Evidence from fingerprint analysis of tryptic digests and carboxypeptidase digestion suggests that the two subunits are identical. The COOH-terminal tyrosine residue which is removed by carboxypeptidase digestion appears to be necessary for catalytic activity but not for substrate binding. A tryptic peptide containing the “active site” lysyl residue modified by acetaldehyde has been purified and the following amino acid composition determined: (Ala₃, Gly₃, Thr₃, Asx₂, Ser, Ile, Phe, 1N-Lys)-Lys.     

The difference in the carboxy-terminal sequence is responsible for the difference in the activity of chicken and rat liver fructose-2,6-bisphosphatase

The fructose-2,6-bisphosphatase domain of the bifunctional chicken liver enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase shares approximately 95% amino acid sequence homology with that of the rat enzyme. However, these two enzymes are significantly different in their phosphatase activities. In this report, we show that the COOH-terminal 25 amino acids of the two enzymes are responsible for the different enzymatic activities. Although these 25 amino acids are not required for the phosphatase activity, their removal diminishes the differences in the activities between the two enzymes. In addition, two chimeric molecules (one consisting of the catalytic core of the chicken bisphosphatase domain and the rat COOH-terminal 25 amino acids, and the other consisting of most of the intact chicken enzyme and the rat COOH-terminal 25 amino acids) showed the same kinetic properties as the rat enzyme. Furthermore, substitution of the residues Pro456Pro457Ala458 of the chicken enzyme with GluAlaGlu, the corresponding sequence in the rat liver enzyme, yields a chicken enzyme that behaves like the rat enzyme. These results demonstrate that the different bisphosphatase activities of the chicken and rat liver bifunctional enzymes can be attributed to the differences in their COOH-terminal amino acid sequences, particularly the three residues.     

Insights from the analysis of a predicted model of gp63 in Leishmania donovani

Leishmaniasis is a protozoal disease of human that occurs in most parts of the world. By considering the progress of bioinformatics in molecular modeling, major surface glycoprotein of Leishmania donovani (gp63) structure was modeled using homology modeling with high accuracy based on the X-ray crystal structure of the Leishmania major gp63 as a template, and then analyzed 3D structure of gp63 which can reveal exact facts about its structure and interaction. The objective of this study was to find folding and three dimensional structure of the gp63 as potent antigen for human. In this project, we applied the theory of evolution method, including comparative modeling and threading. This study presented a simple protocol for rapid and precise finding 3D structure of gp63 and investigation of its structural properties. The translated amino acid sequence showed that Leishmania donovani gp63 contains 590 amino acids precursor protein consisting of an NH₂-terminal signal peptide of 39 amino acids for membrane targeting, a pro region of 48 amino acids, the mature protein of 478 amino acids containing glycosylation and putative catalytic sites, and a COOH-terminal signal peptide of 25 amino acids for GPI attachment. Based on our model, the protein consists of three domains: the N-terminal, central and C-terminal domains. Additionally, these results could guide future structure-function analyses of gp63 protein.     

On the terminal homologation of physiologically active peptides as a means of increasing stability in human serum--neurotensin, opiorphin, B27-KK10 epitope, NPY

The terminal homologation by CH₂ insertion into the peptides mentioned in the title is described. This involves replacement of the N‐terminal amino acid residue by a β²‐ and of the C‐terminal amino acid residue by a β³‐homo‐amino acid moiety (β²hXaa and β³hXaa, resp.; Fig. 1). In this way, the structure of the peptide chain from the N‐terminal to the C‐terminal stereogenic center is identical, and the modified peptide is protected against cleavage by exopeptidases (Figs. 2 and 3). Neurotensin (NT; 1 ) and its C‐terminal fragment NT(8–13) are ligands of the G‐protein‐coupled receptors (GPCR) NT1, NT2, NT3, and NT analogs are promising tools to be used in cancer diagnostics and therapy. The affinities of homologated NT analogs, 2b – 2e , for NT1 and NT2 receptors were determined by using cell homogenates and tumor tissues (Table 1); in the latter experiments, the affinities for the NT1 receptor are more or less the same as those of NT (0.5–1.3 vs. 0.6 nM ). At the same time, one of the homologated NT analogs, 2c , survives in human plasma for 7 days at 37° (Fig. 6). An NMR analysis of NT(8–13) (Tables 2 and 4, and Fig. 8) reveals that this N‐terminal NT fragment folds to a turn in CD₃OH. – In the case of the human analgesic opiorphin ( 3a ), a pentapeptide, and of the HIV‐derived B27‐KK10 ( 4a ), a decapeptide, terminal homologation (→ 3b and 4b , resp.) led to a 7‐ and 70‐fold half‐life increase in plasma (Fig. 9). With N‐terminally homologated NPY, 5c , we were not able to determine serum stability; the peptide consisting of 36 amino acid residues is subject to cleavage by endopetidases. Three of the homologated compounds, 2b, 2c , and 5c , were shown to be agonists (Fig. 7 and 11). A comparison of terminal homologation with other stability‐increasing terminal modifications of peptides is performed (Fig. 5), and possible applications of the neurotensin analogs, described herein, are discussed.     

Bacillus subtilis aminopeptidase: Specificity toward amino acid amides,dipeptides, and oligopeptides

Bacillus subtilis aminopeptidase hydrolyzed amino acid amides with a specificity similar to that determined using amino acyl-β-naphthylamides, but at much greater catalytic rates. Neutral and basic amino acid amides were the best substrates. A series of Leu and Lys NH₂-terminal dipeptides hydrolyzed by Co²⁺-activated aminopeptidase showed that the $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ ratios for the Lys substrates were fourfold greater than the corresponding Leu substrates and that catalytic differences reflected the identity of COOH terminal residues. Greatest catalytic rates were obtained when aromatic residues were in the COOH terminal position of the substrate (Trp, Tyr, Phe); but, significant hydrolysis was achieved when aliphatic residues were COOH-terminal in the dipeptide. The Co²⁺-activated enzyme would not hydrolyze peptide bonds composed of the imide nitrogen of Pro, thus, bradykinin was not a substrate. However, the Co²⁺-activated enzyme removed sequentially the first four residues from eledoisin-related peptide and the A chain of bovine insulin.     

Alignment of the peptides derived from acid-catalyzed cleavage of an aspartylprolyl bond in the major internal structural polypeptide of avian retroviruses

The major internal structural polypeptide (p27) of Rous sarcoma virus (RSV), and the analogous polypeptide (P27(0)) OF Rous-associated virus-O (RAV-O), an endogenous virus released spontaneously by some chicken cells) have been cleaved selectively at a single aspartylprolyl peptide bond to yield two fragments. The NH2- and COOH-terminal amino acid sequences of p27 and p27(0) and their mild acid-cleavage fragments have been determined. These results show the existence of an identical cleavage site and a similar NH2- and COOH-terminal amino acid sequence in both the polypeptides. Furthermore they indicate that the difference in the molecular weights of p27 and p27(0) results from an insertion of amino acids in the COOH-terminal peptide of p27(0) rather than a shift in the scission site of the precursor molecule.     

Positive Charges Determine the Topology and Functionality of the Transmembrane Domain in the Chloroplastic Outer Envelope Protein Toc34

The chloroplastic outer envelope protein Toc34 is inserted into the membrane by a COOH-terminal membrane anchor domain in the orientation N_cyto-C_in. The insertion is independent of ATP and a cleavable transit sequence. The cytosolic domain of Toc34 does not influence the insertion process and can be replaced by a different hydrophilic reporter peptide. Inversion of the COOH-terminal, 45-residue segment, including the membrane anchor domain (Toc34Cinv), resulted in an inverted topology of the protein, i.e., N_in-C_cyto. A mutual exchange of the charged amino acid residues NH₂- and COOH-proximal of the hydrophobic α-helix indicates that a double-positive charge at the cytosolic side of the transmembrane α-helix is the sole determinant for its topology. When the inverted COOH-terminal segment was fused to the chloroplastic precursor of the ribulose-1,5-bisphosphate carboxylase small subunit (pS34Cinv), it engaged the transit sequence–dependent import pathway. The inverted peptide domain of Toc34 functions as a stop transfer signal and is released out of the outer envelope protein translocation machinery into the lipid phase. Simultaneously, the NH₂-terminal part of the hybrid precursor remained engaged in the inner envelope protein translocon, which could be reversed by the removal of ATP, demonstrating that only an energy-dependent force but no further ionic interactions kept the precursor in the import machinery.     

Cloning, Characterization, and Chromosomal Localization of Human Supervillin (SVIL)

Supervillin is a 205-kDa F-actin binding protein originally isolated from bovine neutrophils. This protein is tightly associated with both actin filaments and plasma membranes, suggesting that it forms a high-affinity link between the actin cytoskeleton and the membrane. Human supervillin cDNAs cloned from normal human kidney and from the cervical carcinoma HeLa S3 predict a bipartite structure with three potential nuclear localization signals in the NH₂-terminus and three potential actin-binding sequences in the COOH-terminus. In fact, throughout its length, the COOH-terminal half of supervillin is similar to segments 2–6 plus the COOH-terminal “headpiece” of villin, an actin-binding protein in intestinal microvilli. A comparison of the bovine and human sequences indicates that supervillin is highly conserved at the amino acid level, with 79.2% identity of the NH₂-terminus and conservation of three of the four nuclear localization signals found in bovine supervillin. The COOH-terminus is even more highly conserved, with 95.1% amino acid identity overall and 100% conservation of the villin-like headpiece. Supervillin mRNAs are expressed in all human tissues tested, but are most abundant in muscle, bone marrow, thyroid gland, and salivary gland; comparatively little message is found in brain. Human supervillin mRNA is ∼7.5 kb; this message is especially abundant in HeLa S3 cervical carcinoma, SW480 adenocarcinoma, and A549 lung carcinoma cell lines. The human supervillin gene (SVIL) is localized to a single chromosomal locus at 10p11.2, a region that is deleted in some prostate tumors.     

Complete amino acid sequence of bovine neurophysin-I. A major secretory product of the posterior pituitary

Bovine neurophysin-I (bNP-I) is the first neurophysin protein which contains histidine and possesses an acidic COOH-terminal segment for which the complete amino acid sequence is presented: NH2-Ala-Val-Leu-Asp-Leu-Asp-Val-Arg-Thr-Cys-Leu-Pro-Cys-Gly-Pro-Gly-Gly-Lys-Gly-Arg-Cys-Phe-Gly-Pro-Ser-Ile-Cys-Cys-Gly-Asp-Glu-Leu-Gly-Cys-Phe-Val-Gly-Thr-Ala-Glu-Ala-Leu-Arg- Cys-Gln-Glu-Glu-Asn-Tyr-Leu-Pro-Ser-Pro-Cys-Gln-SerGly-Gln-Lys-Pro-Cys-Gly-Ser- Gly-Gly-Arg-Cys-Ala-Ala-Ala-Gly-Ile-Cys-Cys-Ser-Pro-Asp-Gly-Cys-His-Glu-Asp-Pro-Ala-Cys-Asp-Pro-Glu-Ala-Ala-Phe-Ser-Leu-COOH. Determination of the structure was greatly facilitated by new procedures used for the isolation of bNP-I and of its tryptic peptide fragments. bNP-I isolated from freshly frozen bovine posterior pituitaries is composed of 93 residues, but some preparations contain neurophysin protein with NH2- and COOH-terminal truncated sequences. bNP-I differs from bovine neurophysin-II, the second major neurophysin of cow, in 20 residue positions, and several of the differences cannot be accounted for by single nucleotide replacements in the genes coding for these two neurophysin proteins. The results reported in this study support our earlier hypothesis that neurophysin-gene duplication preceded species divergence.     

Characterization of β-defensin prepropeptide mRNA from chicken and turkey bone marrow

Four avian β-defensin prepropeptide cDNA sequences [gallinacins: Gal 1 (synonym CHP 1, chicken heterophil peptide 1), and Gal 2; turkey heterophil peptides: THP 1 and THP 2] were amplified from chicken or turkey bone marrow mRNA samples, respectively. Partial chicken β-defensin cDNA sequences were obtained using degenerate primers based on chicken peptide sequences (Gal 1/CHP 1 and Gal 2). The complete cDNA sequences of the chicken β-defensins were then determined by designing specific intrapeptidal primers, from the newly acquired sequence, and pairing one primer with a specific poly A primer tail sequence (3' end) and the other primer with an adapter primer in a 5' rapid amplification of cDNA ends (RACE) reaction. The two, turkey β-defensins were amplified from turkey marrow using primers designed from chicken β-defensin preproregions. The complete amino acid sequences for the prepropeptides were deduced for all four avian β-defensins. Previously, only partial mature peptide sequences for the turkey β-defensins and complete mature peptide sequences for the chicken β-defensins were known. All sequences obtained translated accurately to complete and partial amino acid sequences reported for β-defensins purified from chicken and turkey heterophil granules except for one additional amino acid for Gal 1/CHP 1. The four deduced β-defensin proregions lack the long, negatively charged propiece reported in classical defensin proregions. These regions are thought to stabilize and inactivate the positively charged mature peptide and target the propeptide to the storage granule. Instead, these β-defensin proregions are shorter and similar to storage granule-free β-defensins proregions reported for bovine tracheal antimicrobial peptide (TAP) and lingual antimicrobial peptide (LAP). These are the first prepropeptide β-defensins from leukocyte granules to be completely characterized.