Rat T-cell lines specific to a nonimmunodominant determinant of a retinal protein (IRBP) produce uveoretinitis and pinealitis

Rat lymphocyte lines were established, with specificity toward two synthetic peptides derived from the interphotoreceptor retinoid-binding protein (IRBP), which specifically localizes in the retina and pineal gland. One of the peptides, R4, is immunopathogenic, producing experimental autoimmune uveoretinitis (EAU) and pinealitis (EAP) in immunized rats, while the other peptide, R3, exhibits no detectable immunopathogenicity in rats. The cell lines carry surface markers specific for the helper/inducer subset of T-lymphocytes. When tested by the proliferation assay, the line cells demonstrated major histocompatibility-restricted vigorous responses against the immunizing (homologous) peptide, but failed to recognize the intact IRBP molecule. This finding is in line with other data indicating that peptides R3 and R4 are nonimmunodominant determinants of IRBP for the Lewis rat. Yet, the cell lines specific for R4 were highly immunopathogenic, producing EAU and EAP in naive rats at numbers as low as 0.25 x 10(6), with histopathological changes similar to those induced by active immunization with this peptide. The immunological capacity of the cell lines was further demonstrated by the finding that spleen cells from recipient rats of these lines responded well against the homologous peptides. The uniqueness of this system, in which lymphocytes specific toward a nondominant determinant are immunopathogenic, is underscored and the possible mechanisms of disease induction are discussed.     

Identification of a structural site on acetylcholinesterase that promotes neurite outgrowth and binds laminin-1 and collagen IV

The cell adhesion and neurite outgrowth-promoting function of acetylcholinesterase has been localised to the area of the peripheral anionic site. In order to precisely determine the site involved, we used synthetic peptides representing sequences of the peripheral anionic site and its surrounds, and investigated their binding to a panel of monoclonal antibodies that inhibit cell adhesion/neurite outgrowth and/or to recognise the peripheral anionic site. Binding to laminin-1 and collagen IV was also investigated. A relationship between recognition of the sequence 37-50, representing a surface loop adjacent to the peripheral anionic site, and the degree of inhibition of cell adhesion was observed; both laminin-1 and collagen IV also bound this loop with high affinity. Neurite outgrowth on coverslips coated with this peptide was similar to those coated with acetylcholinesterase itself. Adhesion-inhibiting antibodies also recognised the omega loop 69-96, as did laminin-1 and collagen IV. Laminin also bound the sequences 55-66 and 340-353, recognised by the antibodies to varying degrees, but collagen did not. All these peptides were able to promote neurite outgrowth to some degree. No binding to the amyloid-binding omega loop 275-304 by the ligands was observed, nor did the antibodies recognise this consistently. No relationship was observed between the degree of inhibition of acetylcholinesterase and inhibition of neurite outgrowth by the antibodies from which we conclude that the neurite outgrowth function is non-cholinergic. In conclusion, we have identified a specific conformational structure on acetylcholinesterase, comprising adjacent surface loops between residues 37-50 and 69-96, with additional involvement of the sequences 55-66 and 340-353, that mediates cell adhesion and neurite outgrowth.     

Des-Leu angiotensin I: biosynthesis and drinking response

The crude rat and bovine synaptosomal lysate from brain can hydrolyze angiotensin I (AI) to des-Leu angiotensin I (AI-dL) and no further. This cytosolic enzyme has a specificity for angiotensin-related sequences, R-His-Pro-Phe-His-Leu and therefore named angiotensin-related carboxypeptidase (ARC). These studies led to the biosynthesis and purification of AI-dL in order to determine if it can provoke a drinking response. This nonapeptide is a potent dipsogen when injected into the cerebroventricles of rats. The drinking response probably requires a second hydrolysis to angiotensin II (AII) since both captopril and saralasin can inhibit this response.     

Effect of precipitated morphine withdrawal on post-translational processing of prothyrotropin releasing hormone (proTRH) in the ventrolateral column of the midbrain periaqueductal gray

We have demonstrated that during opiate withdrawal, preprothyrotropin releasing hormone (preproTRH) mRNA is increased in neurons of the midbrain periaqueductal gray matter (PAG) while the concentration of TRH remained unaltered, suggesting that the processing of proTRH may be different in this region of the brain. The aim of the present study was to determine which of the proTRH-derived peptides are affected by opiate withdrawal in the PAG. These changes were compared to other TRH-containing areas such as the hypothalamic paraventricular nucleus (PVN), median eminence (ME) and the lateral hypothalamus (LH). Control and morphine-treated rats 24 h following naltrexone-precipitated withdrawal were decapitated and the brain microdissected. Pooled samples from each animal group were acid extracted, and peptides were electrophoretically separated then analyzed by specific radioimmunoassay. Opiate withdrawal caused a significant change in the level of some post-translational processing products derived from the TRH precursor. In the PAG, opiate withdrawal resulted in an accumulation of the intervening preproTRH(83-106) peptide from the N-terminal side of the prohormone, while the levels of the C-terminal preproTRH(208-285) peptide were reduced, with no change in preproTRH(25-50) or TRH, itself, as compared to control animals. Immunohistochemical analysis also showed significant increases in cellular preproTRH(83-106) peptide immunolabeling in the PAG. Opiate withdrawal in the lateral hypothalamus, unlike from the PAG, was accompanied by an increase in the concentration of TRH. In addition, western blot analysis showed that during opiate withdrawal, the mature form of the prohormone convertase 2 (PC2) increased only in PAG as compared with their respective controls. Thus, these results demonstrate a region-specific regulation of TRH prohormone processing in the brain, which may engage PC2, further suggesting a role for specific proTRH-derived peptides in the manifestations of opiate withdrawal.     

Synthesis and screening of a random dimeric peptide library using the one-bead-one-dimer combinatorial approach

Large combinatorial libraries of random peptides have been used for a variety of applications that include analysis of protein-protein interactions, epitope mapping, and drug targeting. The major obstacle in screening these libraries is the loss of specific but low affinity binding peptides during washing steps. Loss of these specific binders often results in isolation of peptides that bind nonspecifically to components used in the selection process. Previously, it has been demonstrated that dimerizing or multimerizing a peptide can remarkably improve its binding kinetics by 10- to 1000-fold due to an avidity effect. To take advantage of this observation, we constructed a random library of 12 amino acid dimeric peptides on polyethylene glycol acrylamide (PEGA) beads by modifying the 'one-bead-one-compound' approach. The chemical synthesis of 100,000 peptides as dimers can be problematic due to steric and aggregation effects and the presence of many peptide sequences that are difficult to synthesize. We have designed a method, described in detail here, to minimize the problems inherent in the synthesis of a dimeric library by modifying the existing 'split and pool' synthetic method. Using this approach the dimeric library was used to isolate a series of peptides that bound selectively to epithelial cancer cells. One peptide with the amino acid sequence QMARIPKRLARH bound as a dimer to prostate cancer cells spiked into the blood but did not bind to circulating hematopoeitic cells. The monomeric form of this peptide, however, did not bind well to the same LNCaP cell line. These data demonstrate that "hits" obtained from such a 'one-bead-one-dimer' library can be used directly for the final application or used as leads for construction of second generation libraries.     

Superoxide production in human neutrophils is enhanced by treatment with transmembrane peptides derived from human formyl peptide receptor

Formyl peptide receptor (FPR) mediates a number of important host defense functions. Although studies have been performed on the ligand binding site of FPR, FPR dynamic behavior such as receptor dimerization on the cell surface remains unknown. Recently, peptides derived from the transmembrane (TM) domains of GPCRs were shown to disrupt dimer formation by receptors and to result in specific regulation of receptor function. To reveal the function of FPR TM domains, hFPRTM peptides derived from FPR were synthesized, and their biological activities were evaluated with human neutrophils. Synthetic peptides did not exhibit agonistic or antagonistic activity toward superoxide anion production. However, Neutrophils treated with hFPRTM4 produced 4-fold superoxide anion compared with untreated cells when stimulated with FPR agonist fMLP. Short peptide fragments derived from the fourth TM region of FPR did not enhance superoxide anion production, which suggests that hFPRTM4 did not behave as a ligand. CD and fluorescence spectra suggested that hFPRTM peptides were inserted into the membrane. The addition of hFPRTM4 increased the intracellular calcium concentration, which meant the peptide activated some membrane protein on the cell surface. The present study suggests that the fourth TM domain of FPR has a function related to a priming effect.     

Hydrolysis and transport of proline-containing peptides in renal brush-border membrane vesicles from dipeptidyl peptidase IV-positive and dipeptidyl peptidase IV-negative rat strains

In this investigation, we have demonstrated that the renal brush-border membrane of Fischer 344 rats from the Japanese Charles River Inc. specifically lacks dipeptidyl peptidase IV (DPP IV) activity, whereas the renal brush-border membrane of Fischer 344 rats from three different sources within the United States possesses normal levels of DPP IV activity. Comparison of the brush-border proteins between Charles River (U.S.A.) Fischer 344 rats (DPP IV positive) and Japanese Charles River Fischer 344 rats (DPP IV negative) revealed that a protein band (Mr = 100,000), apparently identical with DPP IV, was absent in the membranes from Japanese Charles River Fischer 344 rats. We examined the handling of radiolabeled beta-casomorphin fragment 1-5 (Tyr-Pro-[3H]Phe-Pro-Gly), a specific substrate for DPP IV, in renal brush-border membrane vesicles isolated from DPP IV-positive and DPP IV-negative rats. Although the membrane vesicles from DPP IV-positive rats were able to hydrolyze the pentapeptide to di- and tripeptides with the subsequent active transport of these products via the H+ gradient-dependent peptide transport system, the membrane vesicles from DPP IV-negative rats failed to hydrolyze the pentapeptide and hence lacked the ability to transport the radiolabel actively from the parent peptide. The H+ gradient-dependent glycyl-sarcosine uptake and the Na+ gradient-dependent proline uptake, however, were normal in DPP IV-negative rats. Urine analysis revealed that the DPP IV-negative rats excreted proline- and hydroxyproline-containing peptides in significantly increased amounts in their urine compared with control rats. Furthermore, following intravenous administration of Tyr-Pro-Phe-Pro-NH2, a peptide that is exclusively hydrolyzed by DPP IV, urinary excretion of the peptide in the intact form was many-fold greater in DPP IV-negative rats than in control rats. These data provide conclusive evidence for the obligatory role of DPP IV in the renal handling of proline (and hydroxyproline)-containing peptides.     

VGF Changes during the Estrous Cycle: A Novel Endocrine Role for TLQP Peptides?

Although the VGF derived peptide TLQP-21 stimulates gonadotropin-releasing hormone (GnRH) and gonadotropin secretion, available data on VGF peptides and reproduction are limited. We used antibodies specific for the two ends of the VGF precursor, and for two VGF derived peptides namely TLQP and PGH, to be used in immunohistochemistry and enzyme-linked immunosorbent assay complemented with gel chromatography. In cycling female rats, VGF C-/N-terminus and PGH peptide antibodies selectively labelled neurones containing either GnRH, or kisspeptin (VGF N-terminus only), pituitary gonadotrophs and lactotrophs, or oocytes (PGH peptides only). Conversely, TLQP peptides were restricted to somatostatin neurones, gonadotrophs, and ovarian granulosa, interstitial and theca cells. TLQP levels were highest, especially in plasma and ovary, with several molecular forms shown in chromatography including one compatible with TLQP-21. Among the cycle phases, TLQP levels were higher during metestrus-diestrus in median eminence and pituitary, while increased in the ovary and decreased in plasma during proestrus. VGF N- and C-terminus peptides also showed modulations over the estrous cycle, in median eminence, pituitary and plasma, while PGH peptides did not. In ovariectomised rats, plasmatic TLQP peptide levels showed distinct reduction suggestive of a major origin from the ovary, while the estrogen-progesterone treatment modulated VGF C-terminus and TLQP peptides in the hypothalamus-pituitary complex. In in vitro hypothalamus, TLQP-21 stimulated release of growth hormone releasing hormone but not of somatostatin. In conclusion, various VGF peptides may regulate the hypothalamus-pituitary complex via specific neuroendocrine mechanisms while TLQP peptides may act at further, multiple levels via endocrine mechanisms involving the ovary.     

Effect of indomethacin and propranolol on the blood pressure and renin response to atriopeptin III in conscious rats

The effect of prostaglandin synthesis inhibition and of beta-adrenoceptor blockade on the blood pressure and renin response to the synthetic atrial natriuretic peptide atriopeptin III was assessed in unanesthetized normotensive rats. This peptide was infused i.v. for 30 min at a rate of 1 microgram/min in rats pretreated either with indomethacin (5 mg i.v.) or propranolol (1 mg i.v.). The blood pressure reducing effect of atriopeptin III was attenuated neither by indomethacin nor by propranolol. Atriopeptin III per se did not modify plasma renin activity. Both the administration of indomethacin and of propranolol had a suppressing effect on renin release during atriopeptin III infusion. These data suggest that the vasodilating properties of atrial natriuretic peptides do not depend in the conscious normotensive rats on the production of prostaglandins. They also provide evidence that during infusion of such peptides, both prostaglandins and beta-adrenergic mechanisms are still involved in the regulation of renin secretion.     

Construction of peptides with nucleobase amino acids: design and synthesis of the nucleobase-conjugated peptides derived from HIV-1 Rev and their binding properties to HIV-1 RRE RNA

In order to develop a novel molecule that recognizes a specific structure of RNA, we have attempted to design peptides having L-alpha-amino acids with a nucleobase at the side chain (nucleobase amino acid (NBA)), expecting that the function of a nucleobase which can specifically recognize a base in RNA is regulated in a peptide conformation. In this study, to demonstrate the applicability of the NBA units in the peptide to RNA recognition, we designed and synthesized a variety of NBA-conjugated peptides, derived from HIV-1 Rev. Circular dichroism study revealed that the conjugation of the Rev peptide with an NBA unit did not disturb the peptide conformation. RNA-binding affinities of the designed peptides with RRE IIB RNA were dependent on the structure of the nucleobase moieties in the peptides. The peptide having the cytosine NBA at the position of the Asn40 site in the Rev showed a higher binding ability for RRE IIB RNA, despite the diminishing the Asn40 function. Furthermore, the peptide having the guanine NBA at the position of the Arg44 site, which is the most important residue for the RNA binding in the Rev, bound to RRE IIB RNA in an ability similar to Rev34-50 with native sequence. These results demonstrate that an appropriate NBA unit in the peptide plays an important role in the RNA binding with a specific contact such as hydrogen bonding, and the interaction between the nucleobase in the peptide and the base in the RNA can enhance the RNA-binding affinity and specificity.     

Quantitative analysis of peptides from myelin basic protein binding to the MHC class II protein,I-Au,which confers susceptibility to experimental allergic encephalomyelitis.

BACKGROUND: An important issue in autoimmune diseases mediated by T cells, such as experimental allergic encephalomyelitis (EAE), is the affinity of the disease-inducing determinants for MHC class II proteins. Tolerance, either due to clonal deletion or anergy induction, is thought to require high-affinity interactions between peptides and MHC molecules. Low-affinity binding is compatible with the hypothesis that breaking tolerance to self proteins does not have to occur for onset of disease. In contrast, a high-affinity interaction implies that an event leading to a breakdown of tolerance is central to the autoimmune process. MATERIALS AND METHODS: Detergent-solubilized and affinity-purified I-Au was incubated with varying concentrations of a set of peptides from myelin basic protein and a biotinylated peptide agonist. The specific complexes were separated from excess peptide by capture on antibody-coated plates, and the affinity of the peptides was measured by adding europium-labeled streptavidin and measuring the resultant fluorescence. RESULTS: The immunodominant and encephalitogenic determinant, Ac 1-11, was shown to bind to I-Au relatively poorly (IC50 = 100 microM), demonstrating that in this protein, immunodominance did not correlate with high-affinity binding. In contrast with the natural sequence, the ability of shorter analogs to induce EAE did correlate with their apparent affinity. CONCLUSIONS: The dominance of the natural determinant does not arise from a high-affinity interaction with the MHC class II molecule. This suggests that other mechanisms are operative and that the specific T cell for this peptide/MHC ligand is of high affinity.     

Minimum length of an idiotypic peptide and a model for its binding to a major histocompatibility complex class II molecule.

We have defined the minimum length of a synthetic peptide which can activate I-Ed-restricted BALB/c T cell clones specific for a mutated self-antigen: an idiotope on the syngeneic lambda 2315 immunoglobulin light chain. A peptide comprising residues 91-101 of the lambda 2315 sequence had full stimulatory potency. Surprisingly, a peptide analogue in which His97 was deleted was almost fully active. Truncated, deleted or substituted peptide analogues did not distinguish between seven T cell clones that use different alpha/beta T cell receptors. The 91-101 region in the lambda 2315 light chain does not form an amphipathic helix even though such a helix has been suggested to be important for T cell epitopes. Further, a motif proposed by Rothbard and Taylor as being common to T cell immunogenic peptides is not necessary for the lambda 2315 idiotypic peptide. Comparison with seven other I-Ed-restricted peptides revealed that the peptides are generally positively charged and have two basic amino acids clustered around the centre. On the basis of a model of the class II molecule peptide binding site, we suggest that these positively charged residues may interact with the negatively charged residues at positions 114(Glu) and 155(Asp) of the E beta d chain.     

Anti-sense peptide recognition of sense peptides: direct quantitative characterization with the ribonuclease S-peptide system using analytical high-performance affinity chromatography

The ability of peptides coded by the anti-sense strand of DNA to interact specifically with peptides coded by the sense strand has been evaluated. The sense peptide examined, ribonuclease S-peptide, was immobilized on a coated silica affinity chromatographic matrix. Anti-sense peptides were synthesized on the basis of the anti-sense DNA sequence for the S-peptide region in native pancreatic ribonuclease A. The interaction of synthetic anti-sense peptides with sense peptide was quantitated from the degree of retardation during chromatographic elution on the sense peptide affinity matrix in buffers with and without soluble competing sense peptide. Sense/anti-sense peptide interactions were found to occur with significant affinities with each of two anti-sense 20-residue peptides of opposite amino-to-carboxyl orientations and to weaken progressively with decreasing length of anti-sense peptide. The substantial chromatographic retardation of anti-sense peptides was specific, since it decreased as expected with increasing concentration of the soluble competing S-peptide, could not be mimicked by the elution of several control peptides (including S-peptide itself) on the S-peptide matrix, and did not occur with a blank chromatographic matrix (no S-peptide attached). The stoichiometry of anti-sense peptide binding to immobilized sense peptide was found to be far greater than 1:1, and at least 4-5:1, for the two 20-mer anti-sense peptides. In sum, the analytical affinity chromatographic experiments have established quantitatively that anti-sense peptide binding to sense peptides occurs in the ribonuclease S-peptide case and have identified some structural elements that govern these interactions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhancing the affinity of SEB-binding peptides by repeating their sequence

The utilization of peptide ligands in biosensors and bioassays is dependent on achieving high affinity of these peptides toward their targets. In a previous report, we identified 12-mer peptides that could selectively bind to Staphylococcal enterotoxin B (SEB) using a phage-display library. In this study, we explore for new modification approaches to enhance the affinity of two different SEB-binding peptides. In order to identify the binding regions of selected peptides, the charged residues and the ones, critical for the structure of peptide, were replaced with alanine. However, a specific binding region could not be suggested as all mutant peptides have lost their affinities toward SEB completely. The modifications for the affinity enhancement were done by repeating the 12-mer peptide sequences. A 10-fold increase was observed in the binding affinity of one of the two-repeated peptides, while this modification did not affect the affinity of the other tested peptide. The peptide, with enhanced affinity, was further modified as three repeats; however the affinity of the peptide decreased. The structural basis of the affinity difference between modified peptides was examined by molecular dynamics simulation. The results showed that the conformational differences hold the key for affinity of peptides modified by repeating the sequence. This high affinity peptide with increased affinity is a promising molecular recognition agent to be used in the detection of SEB to be utilized in biosensing systems.     

Fluorescence histochemistry of peptide hormone-producing cells: Observations on the phenanthrenequinone method for the demonstration of arginine residues

Summary Phenanthrenequinone is a sensitive and specific fluorescence histochemical reagent for monosubstituted guanidines. It is probable that it selectively demonstrates the guanidino group of arginine residues of proteins and peptides. Phenanthrenequinone induces moderate to intense fluorescence in gastric chief cells, parenchymal cells of the pancreas, and certain peptide hormone-producing cell types such as the GH cells of the adenohypophysis and the glucagon cells of the pancreatic islets. Similar fluorescence spectra were obtained from an arginine-containing peptide in a histochemical model and from the GH cells of the adenohypophysis following exposure to phenanthrenequinone. We conclude that the cells demonstrated with this reagent store peptides or proteins rich in arginine.     

Epitope prediction and confirmation for the human androgen receptor: generation of monoclonal antibodies for multi-assay performance following the synthetic peptide strategy.

The human androgen receptor (hAR) is an important regulatory protein particularly in male sexual differentiation. The investigation of hAR functionality has been hampered by the lack of AR specific monoclonal antibodies recognizing the functional domains of the receptor. Therefore production of high affinity mono-specific polyclonal (PAbs) and monoclonal antibodies (MAbs) directed to the hAR was initiated following the synthetic peptide (SP) strategy. Five hAR specific peptides were selected on the basis of their predicted antigenic properties avoiding homology with other steroid hormone receptors. Peptide specific polyclonal antisera were obtained following selected immunization protocols. Mono-specific polyclonal antibody responses were elicited to all peptides in mice and rabbits. Crossreactivity of the peptide specific antisera with the native hAR in various biochemical assays was observed with two out of five peptides. Peptide SP61 (hAR residues 301-320) was used for the generation site-directed MAbs specific for the hAR. Specificity for the hAR was established by immunoprecipitation, immune-complex density gradient centrifugation and immunohistochemistry on human prostate tissue sections. The multi-assay performance of the selected high affinity antibodies proved the usefulness of the straight forward peptide approach and opens a wide field of possible biochemical and physiological investigations into questions related to androgen action.