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PubMed1994

Location of PHM/VIP mRNA in human gastrointestinal tract detected by in situ hybridization.

The expression of the gene for vasoactive intestinal polypeptide (VIP) and peptide histidine methionine (PHM) in the human gastrointestinal tract was studied by in situ hybridization and Northern blotting for PHM/VIP mRNA and immunocytochemistry using specific antisera against the bioactive peptides PHM and VIP. In the colon sigmoideum, antisera against all five putative processing products of the VIP precursor (prepro-VIP) were used, namely prepro-VIP 22-79, PHM, prepro-VIP 111-122, VIP and prepro-VIP 156-170. Furthermore, RNA extracted from various regions of the gastrointestinal tract was examined by Northern blots and hybridization to a VIP-cDNA probe. Throughout the gastrointestinal tract, PHM/VIP mRNA was found in neurons only. Using single- or double-staining methods, we demonstrated both PHM/VIP mRNA and the corresponding peptides PHM and VIP in the neurons. In the sigmoideum, the single-staining methods were extended to investigate whether the neurons simultaneously contained PHM/VIP mRNA and each of the five prepro-VIP-derived peptides. Only one major band of PHM/VIP mRNA (1.9 kb) was found by Northern blotting in the tissue of the gastrointestinal tract.

Adult↗

PubMed1994

Neuropeptide expression and processing as revealed by direct matrix-assisted laser desorption ionization mass spectrometry of single neurons.

Neuropeptides were directly detected in single identified neurons and the neurohemal area of peptidergic (neuroendocrine) systems in the Lymnaea brain by using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). The samples were placed in matrix solution and ruptured to allow mixing of cell contents with the matrix solution. After formation of matrix crystals, the analytes were analyzed by MALDI-MS. It was surprising that clean mass spectra were produced, displaying extreme sensitivity of detection. In one of the neuroendocrine systems studied, we could demonstrate for the first time, by comparing the peptide patterns of soma and of neurohemal axon terminals, that processing of the complex prohormone expressed in this system occurs entirely in the soma. In the other system studied, novel peptides could be detected in addition to peptides previously identified by conventional molecular biological and peptide chemical methods. Thus, complex peptide processing and expression patterns could be predicted that were not detected in earlier studies using conventional methods. As the first MALDI-MS study of direct peptide fingerprinting in the single neuron, these experiments demonstrate that MALDI-MS forms a new and valuable approach to the study of the synthesis and expression of bioactive peptides, with potential application to single-cell studies in vertebrates, including humans.

Animals↗

PubMed1994

Uterine endocrinology and paracrinology: insulin-like growth factor binding protein-1 and placental protein 14 revisited.

A large number of proteins and peptides have been identified in the endometrium where they are likely to exert local biological effects. These substances may be enzymes, their inhibitors, proteinases, proteinase inhibitors, hormones, or bioactive peptides with diverse functions. Endometrial function and embryo-endometrial interactions require synchronized actions between endocrine and local factors. As examples of local factors recent studies on the insulin-like growth factor (IGF) system and placental protein 14 (PP14) are reviewed. IGF binding protein-1 (IGFBP-1) and PP14 are products of the secretory phase endometrium: IGFBP-1 is produced by decidualized stromal cells and PP14 by glandular epithelial cells. IGFBP-1 may inhibit the action of IGFs at the endometrial-trophoblastic interphase, and it may also have a role in stromal-epithelial interaction. PP14 has immunosuppressive properties, and recent findings indicate that it may play a part in the fertilization process by inhibiting binding of spermatozoa to the zona.

Carrier Proteins↗

PubMed1997

Enhancing endogenous effects of natriuretic peptides: inhibitors of neutral endopeptidase (EC.3.4.24.11) and phosphodiesterase.

Because diuretic drugs remain the main treatment for disorders of sodium and water metabolism, the quest for improved diuretic and natriuretic agents continues in the hope of achieving fewer side effects and a more rational basis in pathophysiology. One aim has been to enhance endogenous diuretic and natriuretic activity by selective manipulation of atrial natriuretic peptide and related compounds. The first approach has been to inhibit degradation of these peptides using inhibitors of their main catabolic enzyme, neutral endopeptidase, and to offset any antagonistic effect of the renin-angiotensin system by combination with an angiotensin-converting enzyme inhibitor. The second and more recent approach has been to inhibit breakdown of the second messenger of atrial natriuretic peptide, cGMP, using phosphodiesterase inhibitors. As yet, neutral endopeptidase inhibition has not advanced successfully beyond animal experimentation and phosphodiesterase inhibition is still in its infancy. Both strategies suffer from the problem that, on the one hand, neutral endopeptidase metabolizes a variety of bioactive peptides, including endothelin, and it is not possible to develop inhibitors that will be selective for a given peptide; whereas, on the other hand, there are several phosphodiesterase isoforms metabolizing cGMP and cAMP, both second messengers for many different bioactive compounds, and selective inhibitors are still under development.

Animals↗

PubMed1999

Egg-laying hormone peptides in the aplysiidae family.

The neuropeptidergic bag cells of the marine mollusc Aplysia californica are involved in the egg-laying behavior of the animal. These neurosecretory cells synthesize an egg-laying hormone (ELH) precursor protein, yielding multiple bioactive peptides, including ELH, several bag cell peptides (BCP) and acidic peptide (AP). While immunohistochemical studies have involved a number of species, homologous peptides have been biochemically characterized in relatively few Aplysiidae species. In this study, a combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) and electrospray ionization Fourier transform ion cyclotron resonance MS is used to characterize and compare the ELH peptides from related opisthobranch molluscs including Aplysia vaccaria and Phyllaplysia taylori. The peptide profiles of bag cells from these two Aplysiidae species are similar to that of A. californica bag cells. In an effort to characterize further several of these peptides, peptides from multiple groups of cells of each species were extracted, and microbore liquid chromatography was used to separate and isolate them. Several MS-based sequencing approaches are applied to obtain the primary structures of bag cell peptides and ELH. Our studies reveal that (&agr;)-BCPs are 100 % conserved across all species studied. In addition, the complete sequences of (&egr;)-BCP and ELH of A. vaccaria were determined. They show a high degree of homology to their counterparts in A. californica, with only a few amino acid residue substitutions.

Amino Acid Sequence↗

PubMed1995

[Molecular biology in cardiovascular medicine].

Molecular biology has given a strong impact on cardiovascular medicine, not only clinical but also laboratory medicine. cDNA cloning and analysis of gene structures encoding functional proteins or peptides in the cardiovascular system provided insight into molecular mechanisms underlying heart diseases or atherosclerosis. Nucleotide or amino acid sequences for these molecules are useful information for manufacturing bioactive peptides or developing immunoassays. These cDNA clones or genes are useful molecular tools diagnosing patients with high risks for cardiovascular diseases. Recent advances in this field have been made in identification of point mutations of many genes important for cardiovascular functions, including LDL receptors in familial hyperlipidemia, cardiac myosin heavy chain genes in familial hypertrophic cardiomyopathy, mitochondrial DNA in mitochondria cardiomyopathy, or dystrophin genes in secondary cardiomyopathy. Molecular probes are further used in detection of microorganisms causing myocarditis. These progresses that have been made in laboratories of basic researchers will be introduced into clinical medicine and expand the field the laboratory medicine is involved.

Cardiovascular Diseases↗

PubMed1991

Processing-independent analysis (PIA)--a new diagnostic tool.

Posttranslational processing is an important phase of the expression of most eucaryotic genes in terms of functional proteins. Among these, secretory proteins and peptides are of particular interest for clinical chemists, since diagnostic measurements of circulating proteins and peptides constitute a major discipline in clinical chemistry. The posttranslational covalent maturation of secretory proteins and peptides involves multiple enzymatic modifications of the corresponding proproteins along the intracellular secretory pathway. During the eighties, an increasing amount of evidence has indicated that sick secretory cells fail to process their secretory products normally. The diseased cells therefore fail to process their secretory products normally. The diseased cells therefore release also incompletely processed precursors and processing-intermediates. In order to measure the degree of disease, assays that measure proteins and peptides independent of the degree of processing are therefore desirable. We have now designed a new analytical principle, according to which secretory proteins, peptides and their precursors can be accurately quantitated irrespective of the degree of processing. This principle, named processing-independent analysis (PIA), is generally applicable to all cellular synthesized substances. The principle has been applied to and developed first for a well-defined secretory peptide system, progastrin and its products. Using this model, the results obtained so far confirm the diagnostic superiority of processing-independent analysis in comparison with conventional assays for bioactive peptides.

Amino Acid Sequence↗

PubMed1988

Secretion of somatostatin by Saccharomyces cerevisiae. Correct proteolytic processing of pro-alpha-factor-somatostatin hybrids requires the products of the KEX2 and STE13 genes.

Somatostatin is a 14-amino-acid peptide hormone that is proteolytically excised from its precursor, prosomatostatin, by the action of a paired-basic-specific protease. Yeast (Saccharomyces cerevisiae Mat alpha) synthesizes an analogous peptide hormone precursor, pro-alpha-factor, which is proteolytically processed by at least two separate proteases, the products of the KEX2 and STE13 genes, to generate the mature bioactive peptide. Expression in yeast of recombinant DNAs encoding hybrids between the proregion of alpha-factor and somatostatin results in proteolytic processing of the chimeric precursors and secretion of mature somatostatin. To determine if the chimeras were processed by the same enzymes that cleave endogenous pro-alpha-factor, the hybrid DNAs were introduced into kex2 and ste13 mutants, and the secreted proteins were analyzed. Expression of the pro-alpha-factor-somatostatin hybrids in kex2 mutant yeast resulted in secretion of a high molecular weight hyperglycosylated precursor. No mature somatostatin was secreted, and there was no proteolytic cleavage at the Lys-Arg processing site. Similarly, in ste13 yeast, only somatostatin molecules containing the (Glu-Ala)3 spacer peptide at the amino terminus were secreted. Our results demonstrate that in yeast processing mutants, the behavior of the chimeric precursors with respect to proteolytic processing was exactly as that of endogenous pro-alpha-factor. We conclude that the same enzymes that generate mature alpha-factor proteolytically process hybrid precursors. This suggests that structural domains of the proregion rather than the mature peptide are recognized by the processing proteases.

Amino Acid Sequence↗

PubMed1990

Peptides generated from C-reactive protein by a neutrophil membrane protease. Amino acid sequence and effects of peptides on neutrophil oxidative metabolism and chemotaxis.

We have recently provided evidence that C-reactive protein (CRP) could act as an up-regulatable substrate for membrane-associated neutrophil serine protease(s). The resultant degradation of CRP yielded small soluble bioactive peptides that inhibit many of the proinflammatory functions of activated neutrophils and could oppose the tissue destructive potential of these cells. We report on the reverse phase HPLC separation of the small TCA-soluble peptides obtained when CRP is degraded with nonstimulated or PMA-stimulated neutrophils and purified neutrophil membranes. The amino acid sequence of seven peptides isolated from the CRP digest has been ascertained and synthetic peptides homologous to these sequences have been synthesized. Three of the synthetic peptides corresponding to residues 201-206 (CRP-III), 83-90 (CRP-IV), and 77-82 (CRP-V) of the intact protein were identified to significantly inhibit superoxide production from activated neutrophils at 50 microM whereas CRP-III and CRP-V in addition inhibited neutrophil chemotaxis at this concentration. These peptides act additively and their action likely involves the signal transduction pathways for neutrophil activation.

Amino Acid Sequence↗

PubMed1999

Antihypertensive peptides derived from milk proteins.

This paper reviews the angiotensin I-converting enzyme inhibitory peptides originated from milk proteins. Focus was put on the peptides derived from milk casein by the action of some proteolytic enzymes and fermented products by lactic acid bacteria. Some of the angiotensin I-converting enzyme inhibitory peptides exhibit significant antihypertensive effects in spontaneously hypertensive rats. However, there were some antihypertensive peptides with low inhibitory activity of this enzyme. Key factors needed for the peptide to demonstrate the antihypertensive effects are discussed. Fermented milk, which has inhibitory activity of the enzyme, showed the reduction of blood pressure of hypertensive subjects. The possibility of the bioactive peptides for functional foods are also discussed.

Amino Acid Sequence↗

PubMed1999

Spleen antibacterial peptides: high levels of PR-39 and presence of two forms of NK-lysin.

Antibacterial peptides were isolated from porcine spleen by acetic acid extraction, ion exchange chromatography and reverse-phase high-performance liquid chromatography. C-terminal ladder sequence analysis of a bioactive peptide with matrix-assisted laser desorption/ionization mass spectrometry after digestion with carboxypeptidases P and Y showed that it is identical to the antibacterial proline/arginine-rich intestinal peptide PR-39. It is present at high levels in granulocytes of the spleen, and peptides with C-terminal proline amide and internal adjacent Pro residues can be analyzed with this method. In addition, two forms of NK-lysin (NKL) were found. One, NKLi, is identical to that isolated from pig intestine, and the other, NKLbw, to a mature peptide deduced from a clone from a porcine bone marrow cDNA library.

Amino Acid Sequence↗

PubMed2006

The Medicago truncatula small protein proteome and peptidome.

The small protein and native peptide component of plant tissues is a neglected area of proteomic studies. We have used fractionation techniques for denatured and nondenatured protein preparations combined with 2-D LC tandem mass spectrometry to examine the sequences of small proteins and peptides in four tissues of the model legume, Medicago truncatula: the root tip and root of germinating seedlings, nitrogen fixing nodules, and young leaves. The isolation and fractionation strategies successfully enriched the small protein and native peptide content of the samples. Eighty-one small M. truncatula proteins and native peptides were identified. Most samples were dominated by ribosomal and histone proteins, and leaf samples possessed photosynthesis-related proteins. Secreted proteins such as lipid transfer proteins were common to several tissues. Twenty-four hours after germination, the roots and root tip tissues possessed several "seed-specific" and late-embryogenesis proteins. We conclude that these proteins are present in cells prior to germination and that they are subsequently used as a nutritional source for the young tissues. Native UV absorbing peptides were detected in very low molecular weight fractions and sequenced. Each peptide shared C-terminal residues and showed homology to the seed storage protein legumin. The strategies used here would be suitable for combining bioassays and mass spectrometry to identify bioactive peptides in the M. truncatula peptidome.

Amino Acid Sequence↗

PubMed1992

Alpha 2-macroglobulin, a multifunctional binding protein with targeting characteristics.

Alpha 2-macroglobulin (alpha 2M) and related proteins share the function of binding host or foreign peptides and particles, thereby serving as humoral defense barriers against pathogens in the plasma and tissues of vertebrates. In human alpha 2M, several reactive sites including high-affinity sites for zinc, transglutaminase cross-linking sites, and reactive sites derived from the activated thiol ester can mediate reversible or irreversible capture of proteins of diverse biological functions. Alpha 2M interacts and captures virtually any proteinase whether self or foreign, suggesting a function as a unique "panproteinase inhibitor." Activation of alpha 2M generates novel binding sites, which mediate complex formation with cytokines and other peptides. Direct evidence of physical association of cytokines with activated alpha 2M indicated its role as biological response modifier in cell cultures. A mechanism commonly referred to as "clearance of activated alpha 2M" involves Ca(2+)-dependent binding to a specific cell surface receptor, a member of the low-density lipoprotein receptor supergene family, that mediates cellular uptake by endocytosis and delivery to endosomes and lysosomes. The peptide binding function of alpha 2M, therefore, may also be viewed as a mechanism that allows targeting of biologically active peptides to different cell types expressing the alpha 2M receptor. Internalized complexes may be dispatched into different pathways of endocytic/lysosomal pathways in a cell type-specific manner. In addition, bioactive peptides bound to alpha 2M may dissociate in the process of intracellular ligand sorting, thereby modulating cell function, or remain bound and share the catabolic fate of alpha 2M. The diversified and probably programmed binding functions of alpha 2M indicate that in addition to its role in trapping proteinases, it has other biological activities that remain to be fully defined. That alpha 2M may function as a binding and carrier protein with targeting characteristics is predicted from 1) the known functions of alpha 2M, and 2) the similarity of the fate of alpha 2M with proteins whose significance in targeting and intracellular trafficking has been studied in more detail.

Animals↗

PubMed1986

Genes encoding mammalian neuroendocrine peptides: strategies toward their identification and analysis.

During the last five years, cDNAs and genes encoding more than 20 neuroendocrine peptides have been cloned. The information gleaned from these genes has been largely structural to this point, and illustrates the great versatility of neuroendocrine genes in generating diverse sets of bioactive peptides. An ongoing contribution of the application of recombinant DNA techniques to the neuroendocrine system promises to be the identification of novel peptides; it will require a concerted effort to determine their physiological functions. The application of recombinant DNA to the study of expression and regulation of neuroendocrine genes is still in its infancy, but a variety of newly emerging techniques promise to be useful for these approaches. Although we have considered the application of recombinant DNA only to the cloning and analysis of neuroendocrine peptide genes, molecular biology will clearly also have a major future impact in investigating aspects of other molecules involved in neuropeptide action, such as processing enzymes and peptide receptors.

Animals↗

PubMed2003

Development of intramolecularly quenched fluorescent peptides as substrates of angiotensin-converting enzyme 2.

Angiotensin-converting enzyme 2 (ACE2 or ACEH) is a novel angiotensin-converting enzyme-related carboxypeptidase that cleaves a single amino acid from angiotensin I, des-Arg bradykinin, and many other bioactive peptides. Using des-Arg bradykinin as a template, we designed a series of intramolecularly quenched fluorogenic peptide substrates for ACE2. The general structure of the substrates was F-X-Q, in which F was the fluorescent group, Abz, Q was the quenching group (either Phe(NO(2)) or Tyr(NO(2))), and X was the intervening peptide. These substrates were selectively cleaved by recombinant human ACE2, as shown by MS and HPLC. Quenching efficiency increased as the peptide sequence was shortened from 8 to 3 aa, and also when Tyr(NO(2)) was used as a quenching group instead of Phe(NO(2)). Two of the optimized substrates, TBC5180 and TBC5182, produced a signal:noise ratio of better than 20 when hydrolyzed by ACE2. Kinetic measurements with ACE2 were as follows: TBC5180, K(m)=58 microM and k(cat)/K(m)=1.3x10(5)M(-1)s(-1); TBC5182, K(m)=23 microM and k(cat)/K(m)=3.5 x 10(4)M(-1)s(-1). Thus, based on hydrolysis rate, TBC5180 was a better substrate than TBC5182. However, TBC5180 was also hydrolyzed by ACE, whereas TBC5182 was not cleaved, suggesting that TBC5182 was a selective for ACE2. We conclude that these two peptides can be used as fluorescent substrates for high-throughput screening for selective inhibitors of ACE2 enzyme.

Angiotensin-Converting Enzyme 2↗

PubMed2002

Preparation and characterization of microparticles containing peptide produced by a novel process: spray freezing into liquid.

The objective of this study is to evaluate excipient type on the physicochemical properties of insulin microparticles produced by spray freezing into liquid (SFL). A novel process was developed to produce microparticles containing bioactive peptides and proteins. The microparticles were formed by atomization of an aqueous feed solution containing insulin beneath the surface of a cryogenic liquid (e.g. liquid nitrogen). In this study, bovine insulin was dissolved in deionized water alone or with tyloxapol, lactose or trehalose. The aqueous solution was sprayed directly into liquid nitrogen through a polyetheretherketone capillary nozzle under high pressure to form frozen microparticles. Lyophilization was used to sublime the ice. The SFL insulin powder was characterized by different techniques, including X-ray diffraction, reverse-phase high pressure liquid chromatography, size exclusion chromatography, scanning electron microscopy (SEM), particle size distribution and surface area. The mean diameter of the insulin microparticles was 5-7 microm. SEM revealed that the microparticles were highly porous, and the morphology of the microparticles was influenced by the excipient type. The total surface area of the insulin microparticles ranged from 20 to 40 m(2)/g, and the magnitude depended on the specific composition and total solids content of the aqueous feed solution. X-ray diffraction results indicated lack of crystallinity. No change in the level of the degradation product, A-21 desamido insulin, was found in the SFL insulin samples processed alone or with trehalose or tyloxapol. Similarly, no change in formation of high molecular weight transformation products (e.g. covalent insulin dimer) was detected in the samples processed with excipients. The results demonstrated that SFL is a feasible technique for forming porous microparticles containing insulin. The physicochemical properties of insulin were preserved by the SFL technique.

Calorimetry, Differential Scanning↗

PubMed2001

Marked differences between metalloproteases meprin A and B in substrate and peptide bond specificity.

Meprin A and B are highly regulated, secreted, and cell-surface metalloendopeptidases that are abundantly expressed in the kidney and intestine. Meprin oligomers consist of evolutionarily related alpha and/or beta subunits. The work herein was carried out to identify bioactive peptides and proteins that are susceptible to hydrolysis by mouse meprins and kinetically characterize the hydrolysis. Gastrin-releasing peptide fragment 14-27 and gastrin 17, regulatory molecules of the gastrointestinal tract, were found to be the best peptide substrates for meprin A and B, respectively. Peptide libraries and a variety of naturally occurring peptides revealed that the meprin beta subunit has a clear preference for acidic amino acids in the P1 and P1' sites of substrates. The meprin alpha subunit selected for small (e.g. serine, alanine) or hydrophobic (e.g. phenylalanine) residues in the P1 and P1' sites, and proline was the most preferred amino acid at the P2' position. Thus, although the meprin alpha and beta subunits share 55% amino acid identity within the protease domain and are normally localized at the same tissue cell surfaces, they have very different substrate and peptide bond specificities indicating different functions. Homology models of the mouse meprin alpha and beta protease domains, based on the astacin crystal structure, revealed active site differences that can account for the marked differences in substrate specificity of the two subunits.

Amino Acid Sequence↗

PubMed1991

Investigation of the structural requirements for peptide precursor processing in AtT-20 cells using site-directed mutagenesis of proadrenocorticotropin/endorphin.

Mouse pro-ACTH/endorphin (or POMC) contains in its sequence each of the four possible pairs of basic amino acids recognized as potential cleavage sites in the production of bioactive peptides from higher mol wt precursors: KR (lysine-arginine), RR, RK, and KK. To examine the structural requirements for processing and routing in one region of pro-ACTH/endorphin, a reporter mutation was introduced into the mouse pro-ACTH/endorphin cDNA; a methionine residue was mutated to an isoleucine residue to allow biosynthetic double labeling with [3H]Ile and [35S]Met. Analysis of stable cell lines expressing the reporter cDNA indicated that this mutation did not affect processing or secretion. Therefore, additional mutations were introduced on the reporter background to investigate important structural features of the precursor. First, the tripeptide signal for N-linked glycosylation in the N-terminal glycopeptide (Asn65,Ser66,Ser67) was disrupted by the conservative substitution of asparagine65 with a glutamine residue. Secondly, O-glycosylation was prevented by substitution of threonine45 with an alanine residue. Finally, lysine50 was mutated to an arginine residue, transforming the RK doublet preceding the gamma 3MSH sequence into an RR doublet. The results show that the enzymatic machinery of AtT-20 cells fails to cleave efficiently at the Arg-Lys (RK) site even after elimination of any possible structural hindrance by carbohydrate side-chains. Elimination of O-linked oligosaccharides to the N-terminal side of gamma 3MSH did not allow cleavage at the RK site, and elimination of N-linked oligosaccharides did not alter the processing and routing of pro-ACTH/endorphin in AtT-20 cells. However, mutation of the RK sequence to RR allowed extensive cleavage regardless of the occurrence of O- or N-glycosylation.

Adrenocorticotropic Hormone↗