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PubMed1992

Plasma concentrations and comparisons of brain natriuretic peptide and atrial natriuretic peptide in normal subjects, cardiac transplant recipients and patients with dialysis-independent or dialysis-dependent chronic renal failure.

1. We have developed a radioimmunoassay for the measurement of immunoreactive brain natriuretic peptide (1-32) in human plasma. Simultaneous measurements of atrial natriuretic peptide have also been carried out to allow for direct comparison between circulating brain natriuretic peptide and atrial natriuretic peptide. Plasma levels of immunoreactive brain natriuretic peptide (means +/- SEM) were 1.1 +/- 0.1 pmol/l in 36 normal healthy subjects and were significantly elevated in cardiac transplant recipients (18.8 +/- 3.9 pmol/l, n = 12) and in patients with dialysis-independent (8.8 +/- 1.5 pmol/l, n = 11) or dialysis-dependent (41.6 +/- 8.8 pmol/l, n = 14) chronic renal failure. Similarly, in these groups of patients plasma levels of atrial natriuretic peptide were also significantly raised when compared with those in the group of normal healthy subjects. 2. The plasma level of atrial natriuretic peptide was significantly higher than that of brain natriuretic peptide in normal subjects and in patients with dialysis-independent chronic renal failure, with ratios (atrial natriuretic peptide/brain natriuretic peptide) of 2.8 +/- 0.2 and 2.2 +/- 0.3, respectively. However, in both cardiac transplant recipients and patients on dialysis plasma levels of atrial natriuretic peptide and brain natriuretic peptide were similar, with ratios of 1.3 +/- 0.2 and 1.0 +/- 0.1, respectively, in these two groups. 3. Plasma levels of brain natriuretic peptide and atrial natriuretic peptide were significantly correlated in the healthy subjects and within each group of patients. When all groups were taken together, there was an overall correlation of 0.90 (P < 0.001, n = 73).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1996

Peptides isolated from random peptide libraries on phage elicit a neutralizing anti-HIV-1 response: analysis of immunological mimicry.

Peptides binding to a murine, human immunodeficiency virus type 1 (HIV-1) neutralizing monoclonal antibody (F58/H3) were isolated from two random peptide libraries expressed on the surface of phage. The antibody was originally elicited by immunization with HIV-1 envelope protein gp120LAI, and has previously been shown to interact with the -I-GPGRA- motif of the V3 loop. The peptide libraries consisted of nine or 15 random amino acid residues flanked by two cysteines, and fused to the amino terminal end of the cpIII protein on the filamentous phage. Selection of specific peptides was carried out in three rounds, with decreasing antibody concentration. An expected peptide motif -GPGRA-, a similar segment, -GPAR-, and two unrelated motifs -FRLLG- and -WRM/ALG- were selected. Binding of antibody was tested both to synthetic peptides in solution, and the corresponding peptide on phage. The GPXR motifs bound in both formats, while the FRLLG bound antibody only when present on the phage The reactivity of peptides on phage was highly dependent on an intact disulphide bond between the cysteines flanking the peptide. The molecular mimicry of the found motifs was tested by immunizing mice and rabbits with conjugated synthetic peptides or peptide on phage. In mice, peptide-specific antisera were raised, but no reactivity to the whole protein (gp120) was detected. In rabbits, however, this was accomplished with the -GPGRA- containing peptide when present on phage. In addition, this antisera precipitated virus particles, and neutralized HIV-1SF2 virus in vitro.

Animals↗
PubMed2003

Antimicrobial peptides from hylid and ranin frogs originated from a 150-million-year-old ancestral precursor with a conserved signal peptide but a hypermutable antimicrobial domain.

The dermal glands of frogs produce antimicrobial peptides that protect the skin against noxious microorganisms and assist in wound repair. The sequences of these peptides are very dissimilar, both within and between species, so that the 5000 living anuran frogs may produce approximately 100 000 different antimicrobial peptides. The antimicrobial peptides of South American hylid frogs are derived from precursors, the preprodermaseptins, whose signal peptides and intervening sequences are remarkably conserved, but their C-terminal domains are markedly diverse, resulting in mature peptides with different lengths, sequences and antimicrobial spectra. We have used the extreme conservation in the preproregion of preprodermaseptin transcripts to identify new members of this family in Australian and South American hylids. All these peptides are cationic, amphipathic and alpha-helical. They killed a broad spectrum of microorganisms and acted in synergy. 42 preprodermaseptin gene sequences from 10 species of hylid and ranin frogs were analyzed in the context of their phylogeny and biogeography and of geophysical models for the fragmentation of Gondwana to examine the strategy that these frogs have evolved to generate an enormous array of peptide antibiotics. The hyperdivergence of modern antimicrobial peptides and the number of peptides per species result from repeated duplications of a approximately 150-million-year-old ancestral gene and accelerated mutations of the mature peptide domain, probably involving a mutagenic, error-prone, DNA polymerase similar to Escherichia coli Pol V. The presence of antimicrobial peptides with such different structures and spectra of action represents the successful evolution of multidrug defense by providing frogs with maximum protection against infectious microbes and minimizing the chance of microorganisms developing resistance to individual peptides. The hypermutation of the antimicrobial domain by a targeted mutagenic polymerase that can generate many sequence changes in a few steps may have a selective survival value when frogs colonizing a new ecological niche encounter different microbial predators.

Amino Acid Sequence↗
PubMed2000

Long-acting natriuretic peptide, vessel dilator, and kaliuretic peptide enhance the urinary excretion rate of beta2-microglobulin.

The atrial natriuretic peptide (ANP) gene synthesizes a 126-amino acid (aa) prohormone from which four peptide hormones are derived. These 4 peptide hormones consisting of aa 1 to 30 (ie, long-acting natriuretic peptide [LANP]), aa 31 to 67 (vessel dilator), aa 79 to 98 (kaliuretic peptide), and aa 99 to 126 (ie, ANP) have diuretic, natriuretic, and/or kaliuretic properties. ANP has been reported to have its natriuretic and protein-excreting effects via both the proximal and distal tubules, but where in the kidney the other three peptide hormones have their natriuretic and/or diuretic effects is unknown. Further, it has never been investigated as to whether these three other peptide hormones enhance protein excretion. The present investigation was designed to determine (1) if these atrial peptides enhance protein excretion and (2) if their effects involve the proximal tubules of healthy humans by examining the excretion rate of beta2-microglobulin, a marker of proximal tubule function. Twenty-four healthy human subjects were studied following the infusion of 100 ng/kg body weight/min for 60 minutes of each of the respective peptides. LANP enhanced the excretion rate of beta2-microglobulin 2-fold within 20 minutes of beginning its infusion (P < .05) and was 2.5-fold higher than the preinfusion excretion rate at the end of the infusion. The excretion rate of beta2-microglobulin continued to be significantly (P < .01) increased for 3 hours after cessation of the LANP infusion, with the maximal excretion rate (ie, 3.8-fold increase) at 2.5 hours after stopping the infusion. Vessel dilator showed a more marked enhancement of beta2-microglobulin during its infusion, with the excretion rate increasing 2.5-fold at 20 minutes, and was increased 4-fold (P < .01) at the end of the infusion. With cessation of the vessel dilator infusion, the excretion rate of beta2-microglobulin decreased but was still elevated 2-fold (P < .05) 3 hours after stopping the infusion. Kaliuretic peptide enhanced the beta2-microglobulin excretion rate a maximal 3-fold, which occurred at the end of its infusion. The beta2-microglobulin excretion secondary to kaliuretic peptide remained 2-fold (P < .05) above baseline during the 3-hour postinfusion period. These peptide hormones similarly enhanced the albumin and total protein excretion rates 2- to 4-fold. These results indicate that LANP, vessel dilator, and kaliuretic peptide each (1) enhance protein excretion in healthy humans and (2) inhibit proximal tubular protein reabsorption.

Adult↗
PubMed1996

Crystallographic analysis of endogenous peptides associated with HLA-DR1 suggests a common, polyproline II-like conformation for bound peptides.

The structure of the human major histocompatibility complex (MHC) class II molecule HLA-DR1 derived from the human lymphoblastoid cell line LG-2 has been determined in a complex with the Staphylococcus aureus enterotoxin B superantigen. The HLA-DR1 molecule contains a mixture of endogenous peptides derived from cellular or serum proteins bound in the antigen-binding site, which copurify with the class II molecule. Continuous electron density for 13 amino acid residues is observed in the MHC peptide-binding site, suggesting that this is the core length of peptide that forms common interactions with the MHC molecule. Electron density is also observed for side chains of the endogenous peptides. The electron density corresponding to peptide side chains that interact with the DR1-binding site is more clearly defined than the electron density that extends out of the binding site. The regions of the endogenous peptides that interact with DRI are therefore either more restricted in conformation or sequence than the peptide side chains or amino acids that project out of the peptide-binding site. The hydrogen-bond interactions and conformation of a peptide model built into the electron density are similar to other HLA-DR-peptide structures. The bound peptides assume a regular conformation that is similar to a polyproline type II helix. The side-chain pockets and conserved asparagine residues of the DR1 molecule are well-positioned to interact with peptides in the polyproline type II conformation and may restrict the range of acceptable peptide conformations.

Amino Acid Sequence↗
PubMed1985

Effects of synthetic model peptides resembling the extension peptides of mitochondrial enzyme precursors on import of the precursors into mitochondria.

One common and characteristic feature of the extension peptides of mitochondrial enzyme precursors is the presence of repeating short stretches of uncharged amino acids linked by basic amino acids. We synthesized several model peptides having this particular feature of the extension peptides. The peptides contained arginine or lysine as a basic amino acid residue linking sequences of two to four residues of leucine and alanine. We examined the effects of the peptides on the import of the precursors of two mitochondrial enzymes, cytochrome P-450(SCC) and adrenodoxin, and found that the peptides were generally inhibitory to the import of the precursors into mitochondria. The effective concentrations of some of the inhibitory peptides were as low as a few microM. The peptides containing lysine instead of arginine had an essentially similar inhibitory effect on the import. The peptides did not inhibit the binding of pre-P450(SCC) to the surface of mitochondria. The synthetic model peptides uncoupled oxidative phosphorylation of mitochondria prepared from either rat liver or bovine adrenal cortex, and induced leakage of enzymes from the inner compartments of mitochondria. However, the synthetic model peptides did not solubilize membrane-bound enzymes from mitochondria, suggesting that their effect on the membranes is different from that of detergents. The synthetic model peptides seem to bind to the membranes causing significant perturbation in the membrane structure, which is possibly related to the functions of the particular common sequence found in the extension peptides of mitochondrial enzyme precursors.

Adrenodoxin↗
PubMed2006

Composition effect on peptide interaction with lipids and bacteria: variants of C3a peptide CNY21.

The effect of peptide hydrophobicity and charge on peptide interaction with model lipid bilayers was investigated for the C3a-derived peptide CNY21 by fluorescence spectroscopy, circular dichroism, ellipsometry, z-potential, and photon correlation spectroscopy measurements. For both zwitterionic and anionic liposomes, the membrane-disruptive potency for CNY21 variants increased with increasing net positive charge and mean hydrophobicity and was completely lost on elimination of all peptide positive charges. Analogous effects of elimination of the peptide positive net charge in particular were found regarding bacteria killing for both Pseudomonas aeruginosa and Bacillus subtilis. The peptides, characterized by moderate helix content both in buffer and when attached to the liposomes, displayed high adsorption for the net positively charged peptide variants, whereas adsorption was non-measurable for the uncharged peptide. That electrostatically driven adsorption represents the main driving force for membrane disruption in lipid systems was also demonstrated by a drastic reduction in both liposome leakage and peptide adsorption with increasing ionic strength, and this salt inactivation can be partly avoided by increasing the peptide hydrophobicity. This increased electrolyte resistance translates also to a higher antibacterial effect for the hydrophobically modified variant at high salt concentration. Overall, our findings demonstrate the importance of the peptide adsorption and resulting peptide interfacial density for membrane-disruptive effects of these peptides.

Adsorption↗
PubMed2002

Peptide and non-peptide HIV fusion inhibitors.

Fusion of the HIV envelope with the target cell membrane is a critical step of HIV entry into the target cell. The HIV envelope glycoprotein gp41 plays an important role in the fusion of viral and target cell membranes and serves as an attractive target for development of HIV fusion inhibitors. The extracellular domain of gp41 contains three important functional regions, i.e. fusion peptide (FP), N- and C-terminal heptad repeats (NHR and CHR, respectively). The FP region is composed of hydrophobic, glycine-rich residues that are essential for the initial penetration of the target cell membrane. NHR and CHR regions consist of hydrophobic residues, which have the tendency to form alpha-helical coiled coils. During the process of fusion of HIV or HIV-infected cells with uninfected cells, FP inserts into the target cell membrane and subsequently the NHR and CHR regions change conformations and associate with each other to form a fusion-active gp41 core. Peptides derived from NHR and CHR regions, designated N- and C-peptides, respectively, have potent inhibitory activity against HIV fusion by binding to the CHR and NHR regions, respectively, to prevent the formation of the fusion-active gp41 core. C-peptide may also bind to FP, thereby blocking its insertion into the target cell membrane. One of the C-peptides, T-20, which is in the phase III clinical trials, has potent in vivo activity against HIV infection and is expected to become the first peptide HIV fusion inhibitory drug in the near future. However, this peptide HIV fusion inhibitor lacks oral availability and is sensitive to the proteolytic digestion. Therefore, it is essential to develop small molecular non-peptide HIV fusion inhibitors having a mechanism of action similar to the C-peptides. One of the approaches in identifying the inhibitors is to use an immunological assay to screen chemical libraries for the compounds that potentially block the interaction between the NHR and CHR regions to form a fusion-active gp41 core. In combination with computer-aided molecular docking techniques, the first active non-peptide HIV fusion inhibitor targeting gp41, ADS-J1, was identified. Other potential candidates of non-peptide HIV fusion inhibitors have also been identified using different approaches. It is expected that both peptide and non-peptide HIV fusion inhibitors will be developed as new classes of anti-HIV drugs, which will be used alone or in combination with HIV reverse transcriptase and protease inhibitors, for the treatment of HIV infection and AIDS.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Estimation of insulin secretion rates from C-peptide levels. Comparison of individual and standard kinetic parameters for C-peptide clearance.

Insulin secretion rates can be accurately estimated from plasma C-peptide levels with a two-compartment model for C-peptide distribution and degradation. In previous studies, the kinetic parameters of C-peptide clearance were derived in each subject from the decay curve observed after bolus intravenous injection of biosynthetic human C-peptide. To determine whether standard parameters for C-peptide clearance could be defined and used to calculate insulin secretion without obtaining a decay curve in each subject, we analyzed 200 decay curves of biosynthetic human C-peptide obtained in normal, obese, and non-insulin-dependent diabetes mellitus subjects studied in our laboratory. This analysis showed that the volume of distribution and kinetic parameters of C-peptide distribution and metabolism vary by less than 30% in a population highly heterogeneous in terms of age, sex, degree of obesity, and degree of glucose tolerance. The volume of distribution correlated with the degree of obesity as quantified by body surface area (BSA). This dependence of C-peptide distribution volume on BSA was more marked in men than in women. The long half-life was slightly longer in elderly subjects than in younger adults. When effects of BSA, sex, and age were taken into account, the parameters of C-peptide kinetics were very similar in normal, obese, and diabetic subjects. Based on these findings, a simple procedure to derive standard parameters for C-peptide clearance taking into account degree of obesity, sex, and age was defined. These standard parameters resulted in estimations of mean insulin secretion rates, which differed in each subject by only 10-12% from those obtained with individual parameters. The approach of using standard rather than individual parameters did not systematically underestimate or overestimate insulin secretion so that group values for the fasting secretion rate, the mean 24-h secretion rate, and the number and the amplitude of secretory pulses obtained with standard parameters differed by only 1-2% from the values obtained with individual parameters. Furthermore, the accuracy of measurements based on standard parameters was not different from that associated with replicate determinations of the parameters of C-peptide clearance in the same subject. We conclude that it is possible to estimate insulin secretion rates from plasma C-peptide levels with standard parameters for C-peptide clearance rather than individually derived parameters without significant loss of accuracy.

Adult↗
PubMed2001

Peptide and major histocompatibility complex-specific breaking of humoral tolerance to native insulin with the B9-23 peptide in diabetes-prone and normal mice.

NOD mice spontaneously develop anti-insulin autoantibodies and diabetes. A dominant peptide recognized by T-cell clones from NOD mice is insulin B-chain peptide B9-23. When administered subcutaneously to NOD mice, this peptide decreases the development of diabetes. In this study, we evaluated the autoantibody response to native insulin after administration of the B9-23 peptide. In NOD mice, administration of the B9-23 peptide in incomplete Freund's adjuvant enhanced their insulin autoantibody response with a higher level and longer persistence. Induction of insulin autoantibodies with the B9-23 peptide was observed in non-diabetes-prone BALB/c mice and NOR mice within 2 weeks of administration, but this was not observed in C57BL/6 mice. A series of A-chain, other B-chain, and proinsulin peptides did not induce insulin autoantibodies. Induced anti-insulin autoantibodies could not be absorbed with the peptide alone but could be absorbed with native insulin. The B13-23 peptide (one of two identified epitopes within B9-23) when administered to BALB/c mice, induced autoantibodies, whereas peptide B9-16 did not. Induction of autoantibodies mapped to the major histocompatibility complex (MHC) rather than to the background genes. Both splenocytes with I-A(d)/I-E(d) or I-A(g7)/I-E(null) presented the B9-23 peptide to NOD islet-derived T-cell clones. Finally, administration of the B9-23 peptide to BALB/c mice, even without adjuvant, could induce insulin autoantibodies. Our results indicate that B-cell tolerance to intact insulin is readily broken with the presentation of the B9-23 insulin peptide, depending on the host's specific MHC.

Animals↗
PubMed2005

The H-2Kk MHC peptide-binding groove anchors the backbone of an octameric antigenic peptide in an unprecedented mode.

A wealth of data has accumulated on the structure of mouse MHC class I (MHCI) molecules encoded by the H-2(b) and H-2(d) haplotypes. In contrast, there is a dearth of structural data regarding H-2(k)-encoded molecules. Therefore, the structures of H-2K(k) complexed to an octameric peptide from influenza A virus (HA(259-266)) and to a nonameric peptide from SV40 (SV40(560-568)) have been determined by x-ray crystallography at 2.5 and 3.0 A resolutions, respectively. The structure of the H-2K(k)-HA(259-266) complex reveals that residues located on the floor of the peptide-binding groove contact directly the backbone of the octameric peptide and force it to lie deep within the H-2K(k) groove. This unprecedented mode of peptide binding occurs despite the presence of bulky residues in the middle of the floor of the H-2K(k) peptide-binding groove. As a result, the Calpha atoms of peptide residues P5 and P6 are more buried than the corresponding residues of H-2K(b)-bound octapeptides, making them even less accessible to TCR contact. When bound to H-2K(k), the backbone of the SV40(560-568) nonapeptide bulges out of the peptide-binding groove and adopts a conformation reminiscent of that observed for peptides bound to H-2L(d). This structural convergence occurs despite the totally different architectures of the H-2L(d) and H-2K(k) peptide-binding grooves. Therefore, these two H-2K(k)-peptide complexes provide insights into the mechanisms through which MHC polymorphism outside primary peptide pockets influences the conformation of the bound peptides and have implications for TCR recognition and vaccine design.

Animals↗
PubMed1992

Peptide binding to HLA-A2 and HLA-B27 isolated from Escherichia coli. Reconstitution of HLA-A2 and HLA-B27 heavy chain/beta 2-microglobulin complexes requires specific peptides.

The specificity of peptide binding by human leukocyte antigen (HLA) class I molecules was investigated in a cell-free direct-binding assay. Peptides were assessed for binding to HLA-A2 and HLA-B27 by measuring the formation of heterotrimeric HLA complexes that consisted of iodinated beta 2-microglobulin, HLA heavy chain fragments isolated from the Escherichia coli cytoplasm, and peptide. In this system, no detectable HLA heavy chain-beta 2-microglobulin complexes were formed unless appropriate peptides were intentionally added to the reconstitution solution. Analysis with monoclonal antibodies demonstrated that these heterotrimeric complexes were correctly folded. Five nonhomologous peptides, known to form complexes with HLA-A2 or HLA-B27 from T-cell functional studies, were tested for their capacity to bind to HLA-A2 and HLA-B27 using the reconstitution assay. Four of the peptides bound to the appropriate class I molecule only. One peptide and some (but not all) substitution analogs of it bound to both HLA-A2 and HLA-B27. The effect of peptide length on binding to HLA-B27 was studied, and it was found that the optimal length was 9 or 10 amino acid residues; however, one peptide that bound to HLA-B27 was 15 amino acids long. All peptides that bound to HLA-B27 in the direct-binding assay also competed with antigenic peptides for binding to HLA-B27 on the surface of intact cells, as determined by a standard cytotoxic T-lymphocyte functional assay. Thus, we conclude that HLA-A2 and HLA-B27 bind distinct but partially overlapping sets of peptides and that, at least in vitro, the assembly of HLA heavy chain-beta 2-microglobulin complexes requires specific peptides.

Amino Acid Sequence↗
PubMed1988

Evidence for the phosphorylation of a proenkephalin-derived peptide, peptide B.

The potential phosphorylation of a proenkephalin-derived peptide, Peptide B, was investigated in primary cultures of bovine adrenal chromaffin cells and fresh adrenal medullary tissue. Cultures were labeled with [32P]phosphate for 24 h and extracts subjected to immunoprecipitation using affinity-purified anti-serum directed against the carboxyl terminus of Peptide B. A 4.6-kDa-labeled peptide was observed in autoradiograms of immunoprecipitates separated by sodium dodecyl sulfate-polyacrylamide electrophoresis; this peptide was not observed when excess antigen was present during the immunoprecipitation. Radioimmunoassay of extracts prepared from adrenal medullary tissue and separated by isoelectric focusing revealed the presence of four isoelectric forms of Peptide B-immunoreactive peptides; these peptides also exhibited Met-enkephalin-Arg-Phe immunoreactivity. The isoelectric points of these peptides (4.5, 4.3, 4.1, and 3.9) were consistent with the predicted pI values for phosphorylated derivatives of Peptide B. Treatment of samples with alkaline phosphatase prior to isoelectric focusing resulted in the conversion of the more acidic forms to the least acidic form. The presence of phosphate in the more acidic peaks was additionally verified by isoelectric focusing of 32P-labeled immunoprecipitates; the pI values of the radioactive peptides corresponded precisely to the peaks of immunoreactivity. In adrenal medullary tissue, the relative contributions of the various phosphorylated species to the total Peptide B immunoreactivity were as follows: unphosphorylated form, 13%; singly phosphorylated, 31%; doubly phosphorylated, 37%; and triply phosphorylated, 17%. Thus more than 85% of the Peptide B molecules present in the bovine adrenal medulla are phosphorylated.

Adrenal Medulla↗
PubMed1986

Amidation of joining peptide, a major pro-ACTH/endorphin-derived product peptide.

Based on sequence data, rat and mouse pro-adrenocorticotropin (ACTH)/endorphin could give rise to joining peptide, a short acidic peptide that could terminate with a glutamic acid alpha-amide. Rat and mouse pituitary cells were found to cleave the pro-ACTH/endorphin precursor at an -Arg-Arg- site to produce primarily joining peptide-sized material. The amounts of joining peptide were approximately equimolar to the other major pro-ACTH/endorphin-derived products. Using antisera specific for the COOH-terminal modifications of joining peptide and three analytical approaches which separate amidated from glycine-extended forms of joining peptide, it was found that most of the joining peptide in murine anterior and intermediate pituitary was amidated. Identification of the amidated and glycine-extended forms of joining peptide was confirmed by amino acid analysis of the purified molecules. When anterior pituitary corticotrope tumor cells were grown in culture medium lacking ascorbate, there was no detectable ascorbate in the cells; nevertheless, a significant fraction of the joining peptide produced was alpha-amidated, indicating that production of alpha-amidated product was not totally dependent on ascorbate. The amidation state of the joining peptide produced by mouse corticotrope tumor cells was responsive to added ascorbate. Cells grown in medium containing ascorbic acid at the levels found in plasma concentrated the ascorbate to the levels normally found in pituitary tissue, and nearly all of the joining peptide produced was alpha-amidated. The amidation state of secreted joining peptide mirrored the amidation state of the joining peptide in the cells.

Amides↗
PubMed1995

Genetic evidence for difference between intracellular and extracellular peptides in influenza A matrix peptide-specific CTL recognition.

During the course of extensive mutagenesis of HLA-A2.1, we examined influenza A matrix peptide (FMP)-specific CTL recognition of HMy2.C1R (C1R) cells expressing mutant HLA-A2.1 molecules, sensitized with synthetic peptide, FMP 58-66, (exogenous peptide), or infected with influenza A virus (endogenous peptide). Most mutants showed equivalent presentation of exogenous and endogenous peptides to FMP-specific CTL. However, five of the mutants differed in this property. Two of the five mutants, F9L and T134K, present exogenous peptide to FMP-specific CTL, but fail to present endogenous peptide to CTL. Western blot analysis using anti-matrix protein Ab indicates that the matrix protein is expressed in these mutants after infection with virus. Interestingly, transfection of these two mutants with a minigene encoding FMP 58-66 results in efficient lysis by FMP-specific CTL. Peptide-binding assays demonstrate that the two mutations dramatically decrease the binding of FMP. However, these mutants bind FMP as well as wild type in the presence of exogenously added human beta 2-m, suggesting that the lower affinity for beta 2-m leads to the inability to present endogenous peptide. The remaining three mutants, Y27N, Q32K, and S132C, fail to present exogenous peptide, but present endogenous peptide to FMP-specific CTL. Pulse-chase analyses followed by endoglycosidase-H treatment show that the rate of maturation and processing of the five mutant HLA-A2 molecules in C1R cells is identical to that of wild type. Overall, this study suggests that the assembly and subsequent recognition of endogenous peptide differs from that of exogenous peptide.

Amino Acid Sequence↗
PubMed1997

Alternative peptide binding motifs of Qa-2 class Ib molecules define rules for binding of self and nonself peptides.

Studies of naturally processed peptides eluted from membrane-bound and soluble isoforms of murine class Ib Qa-2 molecules determined several features of these ligands, such as the conserved nonameric length and the preferred usage of specific residues at four to six of nine peptide positions. The structural information derived from these studies proved insufficient to distinguish between two interpretations: 1) that Qa-2 are peptide receptors of higher stringency than ordinary class I molecules, and 2) that Qa-2 molecules, like classical class I Ags, bind diverse arrays of peptides. We have addressed this issue by a systematic analysis of peptide residues involved in the binding of membrane-bound Qa-2 molecule, MQ9b. The optimal binding of synthetic peptides in vitro occurs at neutral pH. Two dominant anchors are required for peptide binding to MQ9b: His at position 7 and a hydrophobic residue, Leu, Ile, or Phe, at position 9. In addition, one or two auxiliary anchors participate in binding. The identity and the position of the auxiliary anchors differ from peptide to peptide, suggesting that the binding motifs defined from pool sequencing are composed of many superimposed alternative motifs present in individual peptides. The number of anchors used by Qa-2 peptides is similar to that found in ligands of classical class I Ags. Consequently, the Qa-2 are predicted to bind large repertoires of self and nonself peptides. In support of this interpretation we demonstrate that MQ9b binds strongly 5 of 17 motif-positive, pathogen-derived synthetic peptides.

Animals↗
PubMed2002

Translocating proline-rich peptides from the antimicrobial peptide bactenecin 7.

The intracellular delivery of most peptides, proteins, and nucleotides to the cytoplasm and nucleus is impeded by the cell membrane. To allow simplified, noninvasive delivery of attached cargo, cell-permeant peptides that are either highly cationic or hydrophobic have been utilized. Because cell-permeable peptides share half of the structural features of antimicrobial peptides containing clusters of charge and hydrophobic residues, we have explored antimicrobial peptides as templates for designing cell-permeant peptides. We prepared synthetic fragments of Bac 7, an antimicrobial peptide with four 14-residue repeats from the bactenecin family. The dual functions of cell permeability and antimicrobial activity of Bac 7 were colocalized at the N-terminal 24 residues of Bac 7. In general, long fragments of Bac(1-24) containing both regions were bactericidal and cell-permeable, whereas short fragments with only a cationic or hydrophobic region were cell-permeant without the attendant microbicidal activity when measured in a fluorescence quantitation assay and by confocal microscopy. In addition, the highly cationic fragments were capable of traversing the cell membrane and residing within the nucleus. A common characteristic shared by the cell-permeant Bac(1-24) fragments, irrespective of their number of charged cationic amino acids, is their high proline content. A 10-residue proline-rich peptide with two arginine residues was capable of delivering a noncovalently linked protein into cells. Thus, the proline-rich peptides represent a potentially new class of cell-permeant peptides for intracellular delivery of protein cargo. Furthermore, our results suggest that antimicrobial peptides may represent a rich source of templates for designing cell-permeant peptides.

Amino Acid Sequence↗
PubMed1989

Multiple gastrin-releasing peptide gene-associated peptides are produced by a human small cell lung cancer line.

Products of the gastrin-releasing peptide gene were isolated from culture medium supernatant of a small cell lung cancer line, NCI-H345, by several (high performance liquid chromatography) HPLC steps. The column eluates were monitored by immunoassay and absorbance profiles. Gastrin-releasing peptide was identified in HPLC eluates by a specific radioimmunoassay. Two carboxyl-terminal gastrin-releasing peptide gene-associated peptides were identified by a radioimmunoassay specific for their predicted carboxyl terminus. The amino termini of these two peptides were determined by microsequence analysis. The shorter peptide was revealed to be a fragment of the larger peptide. Expression of an alternate mRNA was shown by isolation and characterization of a novel tetradecapeptide. Amino acid analysis, microsequence analysis, and mass spectral analysis confirmed that the structure was Ser-Leu-Leu-Gln-Val-Leu-Asn-Val-Lys-Glu-Gly-Thr-Pro-Ser. This peptide represents the carboxyl terminus of a peptide resulting from alternate processing of gastrin releasing peptide mRNA. This mRNA contains a 19-base deletion, creating a frame shift. A radioiodinated synthetic analog of this peptide (Tyr-Leu-Val-Asp-Ser-Leu-Leu-Gln-Val-Leu-Asn-Val-Lys-Glu-Gly-Thr-Pro-Ser ) bound specifically to a small cell cancer line with high affinity, suggesting possible biological activity of the isolated peptide.

Amino Acid Sequence↗