Biomedical subjects
Publications and source records attributed to H P Bennett.
The granulin/epithelin protein motif has an unusual structure consisting of a parallel stack of beta-hairpins stapled together by six disulphide bonds. The new structure also contains a folding subdomain shared by small toxins, protease inhibitors as well as the EGF-like protein modules.
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Rates of stool output are reduced when cooked rice is incorporated into oral rehydration solutions. We found that a fraction extracted from rice inhibited the response of intestinal epithelial crypt cells to adenosine 3'5'-cyclic monophosphate, a major intracellular mediator of secretion. This response to rice was seen as an inhibition of cell shrinkage and of chloride efflux in fresh suspended guinea pig crypt cells. The active fraction was of low molecular weight (< 1.5 kDa), hydrophobic, and not a peptide or glycoprotein. It may be chloride-channel blocker.
Parathyroid hormone (PTH), an 84-amino acid peptide, is the major regulator of blood calcium homeostasis. Its mRNA, in addition to encoding the mature peptide, also encodes a "pre" sequence of 25 amino acids and a basic "pro" hexapeptide. To assess which of the subtilisin-like prohormone convertases can process proPTH to PTH we coinfected cells with a vaccinia virus construct expressing human preproPTH and vaccinia virus constructs expressing furin, PC1 or PC2. BSC-40 cells, having a constitutive secretory pathway, and GH4C1 cells, having a regulated secretory pathway, were used. PTH biosynthetic products in cell extracts and media were purified by high performance liquid chromatography, identified by radioimmunoassay, and unambiguously defined as either proPTH or PTH by ion-spray mass spectrometry. In both cell types, furin was the most effective in processing proPTH to PTH. In all cases only PTH was released into the medium. In addition, partially purified furin and PC1 were tested for their ability to appropriately cleave a tridecapeptide spanning the prohormone cleavage site found in proPTH. Here too furin was much more effective at cleaving at the correct site. Northern blot analysis and in situ hybridization showed that furin and preproPTH mRNA are co-expressed in the parathyroid, whereas PC1, PC2, and PC5 are not and PACE4 is expressed only at very low levels. Taken together these studies strongly suggest that furin is the enzyme responsible for the physiological processing of proPTH to PTH.
Guinea-pig ACTH has been found to be distinct from other mammalian ACTHs in having an alanine for proline substitution at position 24 and in having superagonist aldosterone-stimulating activity relative to synthetic ACTH(1-24) in an isolated rat glomerulosa cell bioassay. We have purified ACTH from extracts of guinea-pig anterior pituitary and confirmed its unusual structural characteristics by amino acid analysis and mass spectrometry. Using isolated rat adrenal fasciculata-reticularis and glomerulosa cell bioassays, guinea pig ACTH was found to have similar activity to that of human ACTH with respect to corticosterone- and aldosterone-stimulating activity, in terms of maximal steroid output but was slightly more potent in terms of the concentration which elicited half-maximal steroid secretion. Under the assay conditions used, guinea-pig ACTH appeared not to be a superagonist as previously suggested. Various biosynthetic derivatives of guinea-pig pro-opiomelanocortin were identified by amino acid analysis and mass spectrometry. Joining peptide, a major product of pro-opiomelanocortin processing, was found in extracts of both anterior and neurointermediate lobes of the pituitary. Post-translational modification of other products of intermediate lobe processing were observed. N- and O-acetylation of alpha-melanotropin, partial O-phosphorylation of corticotropin-like intermediate lobe peptide and carboxyl-terminal amidation of beta-melanotropin were identified.
A new class of low-molecular-weight cysteine-rich regulatory growth factors, designated granulins, has been isolated from hematopoietic tissues of a teleost fish (Cyprinus carpio) and structurally characterized. Granulin-1, the predominant form found in carp spleen, was used to raise polyclonal antibodies in rabbits and to establish a radioimmunoassay. This permitted preliminary tissue distribution studies of granulin-1 to be undertaken in carp (Cyprinus carpio) and goldfish (Carassius auratus). Granulin-1 immunoreactivity was found in the melanomacrophage centers of the spleen and head kidney. Carp tissues anatomically involved in the first line of defense against infection, including skin, gills, gut, and also heart, showed intense granulin-1 immunoreactive staining within presumptive macrophage cells. Granulin-1 immunoreactive macrophages prepared from goldfish spleen and head kidney adhered to glass slides, actively phagocytosed carbon particles, and contained granulin-1 immunoreactivity as well as abundant endogenous peroxidase activity. This study demonstrates that granulin-1 is synthesized and stored in macrophages/monocytes of spleen, head kidney, and peripheral tissues of teleost fish.
beta-Endorphin (beta-endo) (1-31) is the active opioid peptide product of pro-opiomelanocortin processing. Further post-translational modifications of beta-endo(1-31) yield beta-endo(1-27), (1-26) and their acetylated forms which are considered to be opiate receptor antagonists. Mechanistically, alteration in opiatergic properties is likely to result in the loss of a number of physiological functions including reproductive capacity. The purpose of this study was to determine whether there are changes in the way beta-endo neurones process the peptide with age in female C57BL/6J mice. Pooled extracts of arcuate nucleus (ARC) and preoptic area (POA) of 3- to 4-month-old normally cycling (4-5 days at dioestrus), 12- to 13-month-old irregularly cycling (5-7 days at dioestrus), 23- to 24-month-old acyclic (in persistent dioestrus) animals were subjected to reversed-phase HPLC (n = 4 experiments). Column fractions were assayed for beta-endo-like-immunoreactivity by sequence-specific RIAs. The opiate receptor active as well as opiate receptor antagonist forms of beta-endo were present in both ARC and POA at all three age groups although their ratios varied. beta-Endo(1-31), the active opiate, was the predominant form in young animals. At middle age there was a threefold (P < 0.05, ANOVA) increase in the antagonist forms of beta-endo and this was associated with a significant (P < 0.05, ANOVA) increase in the ratio of antagonist to active forms. This was accompanied by a trend toward an increase in acetylated forms of beta-endo in middle-aged mice. HPLC profiles from hypothalami of old animals more closely resembled those of young females. The increase in the antagonist forms of beta-endo at middle age may contribute to a decline of opiatergic influences in the female C57BL/6J mouse and suggest a mechanism whereby alterations in opiate influence over gonadotrophin control may occur.
A simple scheme was developed for the rapid purification of antimicrobial peptides from the skin of Xenopus laevis. An extract of the frog skin was prepared using an acidic medium designed to maximize the solubilization of low-molecular-weight peptides. This extract was subjected to an enrichment procedure using C18 Sep Pak cartridges to yield a salt-free fraction, devoid of high-molecular-weight proteins. This fraction was in turn subjected to heparin affinity high-performance liquid chromatography on a Shodex AF-Pak column. All the antibacterial activity bound to the column and could be eluted using a linear gradient of increasing sodium chloride concentration. Antibacterial activity emerged from the column in fractions corresponding to a sodium chloride concentration of 0.45 M. Reversed-phase high-performance liquid chromatography resolved this material into a series of compounds which could be readily characterized using a combination of amino acid analysis and ion-spray mass spectrometry. Each peptide was found to be antimicrobial and each was positively identified as belonging to a family of amphipathic helix-forming peptides characterized by other investigators. Listed in their order of elution from the reversed-phase column the peptides were magainin 2, magainin 1, peptide-glycine-leucine amide, xenopsin precursor fragment, levitide precursor fragment, and a mixture of fragments derived from the caerulein precursor. These peptides owe their antimicrobial properties to a predeliction to forming amphipathic alpha-helical structures when associated with lipid membranes.(ABSTRACT TRUNCATED AT 250 WORDS)
The NH2-terminal domain of pro-opiomelanocortin, designated as the 16-kDa fragment, is highly conserved throughout the vertebrate family and is likely therefore to have an important functional role. Bovine 16-kDa fragment is a 77- residue glycopeptide, which has been found to be glycosylated at threonine 45 and asparagine 65. Available evidence suggests that glycoforms lacking glycans at the O-linked site are processed in the intermediate pituitary at -Arg49-Lys50- to give the residue 1-49 amino-terminal peptide and a carboxyl-terminal glycopeptide referred to as Lys1 gamma 3-melanotropin. Glycoforms carrying O-glycans remain unprocessed in the intermediate pituitary. Thus O-glycosylation is likely to play an important role in controlling the fate of the NH2-terminal portion of pro-opiomelanocortin, thereby affecting the biological events that are influenced by peptides and glycopeptides derived from this domain. In a recent study (Siciliano, R. A., Morris, H. R., McDowell, R. A., Azadi, P., Rogers, M. E., Bennett, H. P. J., and Dell, A. (1993) Glycobiology 3, 225-239), we sequenced the N-glycans attached to Asn-65 of bovine 16-kDa fragment and demonstrated that the acidic components contain, in addition to neutral antennae, a single SO4-4GalNAc beta 1-4GlcNAc beta 1- antenna, which is characteristic of the pituitary glycohormone N-glycans (Baenziger, J. U., and Green, E. D. (1988) Biochim. Biophys. Acta 947, 287-306). We now report the structural characterization of the O-linked oligosaccharides found in bovine 16-kDa fragment. The major component, which constitutes about 80% of the O-glycan population, is a novel sulfated tetrasaccharide, which carries the same sulfated epitope as the N-glycans. This is the first time that the SO4-4GalNAc beta 1-4GlcNAc beta 1- moiety has been observed in O-glycans, and it raises the interesting possibility that the beta-N-acetylgalactosaminyltransferase responsible for the addition of N-acetylgalactosamine to the pituitary glycohormones (Smith, P. L., and Baenziger, J. U. (1988) Science, 242, 930-933) might be capable of glycosylating both N- and O-linked acceptors.
Granulins are cysteine-rich polypeptides purified from human and rat hematopoietic cells and structurally related to the epithelin family of growth modulatory factors. A prototypic form of granulin was isolated from the hematopoietic organs of teleost fish (Belcourt, D., and Bennett, H.P.J. (1988) J. Cell Biol. 107, 629 (abstr.)). This study reports the structure of three granulins purified from the spleen and head kidney of the carp (Cyprinus carpio). Ion-spray mass spectrometric analysis of granulin-1, -2, and -3 corroborated the observed primary structures and demonstrated that each 57-residue-peptide was monomeric in nature with all cysteines linked via intramolecular disulfide bridges. A comparison of the carp granulin sequences demonstrates that granulins 2 and 3 are most closely related with sequence variations occurring primarily toward the amino terminus. A rabbit polyclonal antibody was raised against carp granulin-1 to develop a radioimmunoassay for this peptide, which showed no significant cross-reactivity with granulin-2 and -3. The distribution of carp granulin-1 was studied by screening purified tissue extracts for immunoreactivity using reversed-phase high performance liquid chromatography. A single form of immunoreactive granulin-1 was identified in all carp tissues studied including spleen, head kidney, heart, skin, gills, and gut. These studies have established that members of the granulin/epithelin family are found in a lower vertebrate and may serve important growth modulatory functions throughout the vertebrate kingdom.
The joining peptide (JP) portion of pro-opiomelanocortin extracted from mouse pituitary glands has previously been shown to exist in one major and several distinct minor forms (Bennett, H. P. J. (1986) Peptides 7, 614-622). We now confirm the heterogeneous nature of JP extracted from mouse neurointermediate pituitary glands and show that similar forms of JP are also to be found in extracts of porcine pituitary glands. Three forms of porcine JP (pJP-A, pJP-B, and pJP-C) were purified from whole porcine pituitary glands using reversed-phase high performance liquid chromatography methods. The three structural variants constitute approximately 10, 75, and 15%, respectively, of the total JP observed. Mass spectrometric analysis revealed that pJP-C was 18 mass units smaller than pJP-B, which is consistent with the formation of a succinimide structure at the aspartyl 16-glycine 17 peptide bond. Such symmetrical imide structures are known to hydrolyze at physiological pH to yield a mixture of the original alpha-aspartyl peptides and isomerized isoaspartyl peptides. We were able to show that pJP-A was the isomerized isoaspartyl form by demonstrating that pJP-A but not pJP-B was a substrate for the protein carboxyl methyltransferase enzyme (L-isoaspartyl/D-aspartyl protein methyltransferase; EC 2.1.1.77) purified from bovine erythrocytes. This cytosolic enzyme is known to preferentially methylate L-isoaspartyl residues within model substrates. Control experiments in which JP was incubated in the acidic medium used to extract the pituitary tissue showed that the isoforms of pJP are not artifacts of peptide purification. Furthermore, we have isolated the isoforms of pJP at levels which are 100 times greater than would be expected for a spontaneous reaction. We conclude that the formation of the aspartimide form of JP appears to be a facilitated process, possibly occurring as a result of conformation constraints dictated by the structure of pro-opiomelanocortin, or an as yet uncharacterized post-translational event.
The N-terminal glycopeptide of pro-opiomelanocortin (POMC), designated as the 16K fragment, is highly conserved throughout vertebrates from amphibians to mammals and is likely therefore to have an important functional role. In this paper, we report the first structural characterization of N-glycans attached to asparagine-65 of a 16K glycopeptide. The 16K fragment was isolated from bovine pituitaries and the N-glycans were analysed using fast atom bombardment mass spectrometry together with sugar and linkage analysis. Sulphated-N-acetylgalactosamine-capped antennae, typical of the pituitary glycohormones, were present in the major acidic components. The POMC oligosaccharides are distinct from those of the pituitary glycohormones because the sulphate is exclusively located on the 3-arm of biantennary structures and, in addition, a significant proportion of the molecules carry the Lewis x epitope. It is probable that these differences reflect the absence of a tripeptide motif in POMC which fully conforms to the criteria previously defined for the recognition sequence for the N-acetylgalactosamine transferase that is specific for the pituitary glycohormones [Smith and Baenziger (1992) Proc. Natl. Acad. Sci. USA, 89, 329-333]. It remains to be seen whether the Lewis x epitope is involved in selectin-mediated events, but previous studies suggest that the sulphated moieties are unlikely to play a major role in clearance. The Lewis x epitope is also present in the neutral N-linked oligosaccharides, together with a variety of other antennae including a rarely found fucosylated GalNAc-GlcNAc structure.
It was observed in the course of other studies that rat fetal lung extracts inhibited proliferation of fetal lung cells in culture. The purpose of the present study was to isolate and characterize this cytostatic factor. It was found that fetal lungs contained a 16 kDa cytostatic factor and its concentration was twofold greater in fetal lungs of diabetic rats compared with control rats. This fetal lung cytostatic protein (FLCP) was purified by reversed-phase, heparin-affinity and gel filtration high-performance liquid chromatography and SDS-PAGE. The purified protein was electroblotted onto polyvinylidene difluoride membrane and subjected to sequence analysis. The amino-terminal sequence of this fetal lung cytostatic protein was P E P A K S A P A P X K G I G K Q X X K A X X K A ... and showed significant homology with histone H2B; however, the amino acid composition of FLCP suggested that it may be structurally distinct from histone H2B. Ion-spray mass spectrometry suggested that FLCP was made up of at least two species of the protein with molecular weights of 13,776.1 and 14,007.3 and was different from the molecular weight of rat histone H2B predicted by its cDNA sequence. The concentration of FLCP, based on amino acid compositions, was 0.32 nmol/g and 0.83 nmol/g wet fetal lung from non-diabetic and diabetic rats respectively. These findings suggest that the fetal rat lung produces a regulatory factor bearing considerable homology with but possibly different from histone H2B and that fetal lung immaturity during diabetic pregnancy might be contributed to by an increase in this factor.
The nucleotide sequence of a novel peptide from a rat Leydig cell hypercalcemic tumor H-500 was determined. This cDNA encodes a peptide of 93 amino acids and contains a heparin binding domain similar to histone 2-B. Northern blot analysis showed tissue specific expression of this peptide mRNA.
Seven cationic, cystine-rich peptides of 29 to 32 amino acid residues have been purified from extracts of rat bone marrow (R-1, R-1a, R-1b, R-2, R-3, R-4 and R-5). Structural analysis clearly indicated that all seven peptides belong to the corticostatin/defensin family of leukocyte-derived peptides known to participate in oxygen-independent killing of phagocytosed bacteria. For R-1 to R-5, six cysteine residues were found at characteristic and highly conserved positions. R-1a and R-1b were partially characterized and appear to be structural variants of R-1. Aside from the conserved cysteines, there is a remarkable degree of structural diversity evident within the sequences of those members of the corticostatin/defensin family characterized so far. The structures of the peptides that we have purified can be compared directly with the sequences obtained for rat defensins isolated from extracts of peritoneal neutrophils (Lehrer, Ganz and Selsted, Cell, 64 (1991) 229-230). Some discrepancies are apparent which can be explained in terms of proteolytic cleavage of several of these peptides at both amino- and carboxyl-termini. The corticostatins owe their bioactivity to their ability to compete with corticotropin for occupancy of the corticotropin receptor (Zhu, Hu, Mulay, Esch, Shimasaki and Solomon, Proc. Natl. Acad. Sci. USA, 85 (1988) 592-596). The potency of these peptides can be expressed in terms of their capacity to inhibit the steroidogenic response of isolated rat adrenocrotical cells half-maximally stimulated by corticotropin (i.e., at the ED50 concentration for corticotropin in this assay, namely 33 pM). In this assay, the rat peptides R-1, R-2 and R-3 were shown to be inactive. In contrast, the more cationic peptides R-4 and R-5 were found to inhibit steroidogenesis. R-4 was somewhat less active than rabbit corticostatin (IC50 25 nM) showing an IC50 value of 50 nM. R-5 appeared to be significantly less potent than R-4. The lower yield of R-5 precluded an accurate estimate of the corticostatic potency of this peptide. R-4 differs in structure from R-5 in having an arginine to serine substitution at position 7. It can be concluded that an arginine at this position accounts, at least in part, for the corticostatic activity of R-4.(ABSTRACT TRUNCATED AT 400 WORDS)
A Guard-PAK precolumn system was used for the reversed-phase liquid chromatography purification of a small, synthetic radiolabeled opioid peptide, FK 33-824 (D-Ala2, methyl-phe4, Met (O)ol5 enkephalin) (FK). This procedure involves trace enrichment of iodinated peptide onto the precolumn while iodination reagents are not retained. Radioactive contamination of high-performance liquid chromatography columns and injectors is thus avoided. Precolumn chromatography has sufficient resolving power to separate not only labeled from unlabeled peptide but also mono- from di-iodinated peptide. Purified 125I-labeled FK (estimated specific activity 85.9-153.7 Ci/mmol) showed high specific binding to mouse corpus striatum, neocortex, cingulate cortex, nucleus accumbens septi, diagonal band of Broca, nucleus medialis septi, area preopticus magnocellularis, and the nucleus of the caudate/putamen. Radioligand binding was inhibited by both antagonists (naloxone and naltrexone); and agonists D-Ala2, N-methyl-phe4, gly-ol5-enkephalin [DAGO]; FK; and beta-endorphin at all concentrations tested (1 x 10(-8) to 1 x 10(-4) M). Adrenocorticotropin hormone (ACTH) did not block ligand binding at any concentration tested. Distribution of mu opioid receptors was analyzed by light microscopic autoradiography. Sections incubated with 125I-labeled FK in the presence of agonists and antagonists demonstrated decreasing ligand binding with increasing doses of competitor. ACTH did not block ligand binding at any concentration tested. HPLC analyses of ligand which had been iodinated 1.5 half lives before the date of the experiment demonstrated a single peak similar to that of freshly iodinated ligand. Similar binding kinetics and autoradiographic labeling patterns were observed as compared to those obtained with freshly iodinated peptide.(ABSTRACT TRUNCATED AT 250 WORDS)
Three corticostatic peptides (GP-CS1, GP-CS2 and GP-CS3) were purified from extracts of guinea pig bone marrow. Each was identified on the basis of their ability to inhibit the secretion of corticosterone by isolated rat adrenal cells stimulated by ACTH. GP-CS1 and GP-CS2 were found to be 31 residues in length, rich in arginine and to have six cysteines typical of the corticostatin/defensin family of peptides previously purified from phagocytic cells of the immune system. GP-CS1 was found to be identical to GP-CS2 except for having a leucine at position 21 instead of isoleucine. GP-CS3 was also found to be rich in arginine and cysteine but structurally distinct from the other peptides. A combination of endoprotease mapping, ion-spray mass spectrometry and gas-phase sequencing revealed that GP-CS3 was a novel homo-dimer consisting of two 13 amino acid residue subunits cross-linked through eight cysteines in an anti-parellel configuration.
The effect of Ca2+ on the extent and pattern of processing of pro-opiomelanocortin and an N-terminal fragment by a purified pituitary secretory vesicle, soluble aspartic endoprotease, was studied. Ca2+ stimulated the first cleavage of pro-opiomelanocortin by pro-opiomelanocortin-converting enzyme to yield 21-23 kDa adrenocorticotropin and beta-lipotropin, but its effect was minimal. The production of adrenocorticotropin from the 21-23 kDa intermediate was stimulated approximately 2.3-fold in the presence of 10 mM Ca2+, and processing of beta-lipotropin to beta-endorphin was stimulated about 1.3-1.4-fold by 5-10 mM Ca2+. The production of gamma-melanotropin-immunoreactive material from bovine N-pro-opiomelanocortin(1-77) was stimulated approximately 1.3-fold at both 100 microM and 1.5-2.0 mM Ca2+. Further characterization of the gamma-melanotropin-immunoreactive material by HPLC demonstrated that the major products were gamma 3-[Lys]melanotropin and gamma 3-melanotropin at both Ca2+ concentrations. These results indicate that pro-opiomelanocortin-converting enzyme is stimulated by Ca2+.