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PubMed2004

Synthesis of an immunosuppressant SQAG9 and determination of the binding peptide by T7 phage display.

SQAG9, a new class of immunosuppressive sulfoquinovosylacylglycerol, and its biotinylated derivatives have been synthesized. A T7 Phage library, composed of random cDNA fragments from Drosophila melanogaster, displayed a possible binding peptide of 14 amino acids. The immobilized synthetic peptide on a sensor chip showed a dissociation constant of K(D)=1.5 x 10(-6) against SQAG9 in a surface plasmon resonance experiment.

Animals↗

PubMed1999

An IgA-binding peptide derived from a streptococcal surface protein.

Surface proteins that bind to the Fc part of human IgA are expressed by many strains of Streptococcus pyogenes, a major human pathogen. Studies of these proteins have been complicated by their size and by their ability to bind human plasma proteins other than IgA. Here, we describe a synthetic 50-residue peptide, derived from streptococcal protein Sir22, that binds human IgA but not any of the other plasma proteins known to bind to Sir22. The peptide binds serum IgA and secretory IgA and binds IgA of both subclasses. Evidence is presented that the peptide folds correctly both in solution and when it is immobilized and that it readily renatures after denaturation. Together, these data indicate that the peptide corresponds to a protein domain that binds IgA with high specificity. This is the first report of an IgA-binding domain that retains its properties in isolated form.

Bacterial Outer Membrane Proteins↗

PubMed1991

Fluorescence analysis of calmodulin mutants containing tryptophan: conformational changes induced by calmodulin-binding peptides from myosin light chain kinase and protein kinase II.

Peptide-induced conformational changes in five isofunctional mutants of calmodulin (CaM), each bearing a single tryptophan residue either at the seventh position of each of the four calcium-binding loops (i.e., amino acids 26, 62, 99, and 135) or in the central helix (amino acid 81) were studied by using fluorescence spectroscopy. The peptides RS20F and RS20CK correspond to CaM-binding amino acid sequence segments of either nonmuscle myosin light chain kinase (nmMLCK) or calmodulin-dependent protein kinase II (CaMPK-II), respectively. Both steady-state and time-resolved fluorescence data were collected from the various peptide-CaM complexes. Steady-state fluorescence intensity measurements indicated that, in the presence of an excess of calcium, both peptides bind to the calmodulin mutants with a 1:1 stoichiometry. The tryptophans located in loops I and IV exhibited red-shifted emission maxima (356 nm), high quantum yields (0.3), and long average lifetimes (6 ns). They responded in a similar manner to peptide binding, by only slight changes in their fluorescence features. In contrast, the fluorescence intensity of the tryptophans in loops II and III decreased markedly, and their fluorescence spectrum was blue-shifted upon peptide binding. Analysis of the tryptophan fluorescence decay of the last mentioned calmodulins supports a model in which the equilibrium between two (Trp-99) or three (Trp-62) states of these tryptophan residues, each characterized by a different lifetime, was altered toward the blue-shifted short lifetime component upon peptide binding. Taken together, these data provide new evidence that both lobes of calmodulin are involved in peptide binding. Both peptides induced similar changes in the fluorescence properties of the tryptophan residues located in the calcium-binding loops, with the exception of calmodulin with Trp-135. For this last mentioned calmodulin, slight differences were observed. Tryptophan in the central helix responded differently to RS20F and RS20CK binding. RS20F binding induced a red-shift in the emission maximum of Trp-81 while RS20CK induced a blue-shift. The quenching rate of Trp-81 by iodide was slightly reduced upon RS20CK binding, while RS20F induced a 2-fold increase. These results provide evidence that the environment of Trp-81 is different in each case and are, therefore, consistent with the hypothesis that the central helix can play a differential role in the recognition of, or response to, CaM-binding structures.

Amino Acid Sequence↗

PubMed1988

Adrenoferredoxin-binding peptide of NADPH-adrenoferredoxin reductase.

The amino-acid sequence at the adrenoferredoxin-binding site of NADPH-adrenoferredoxin reductase ferredoxin:NADP+ oxidoreductase, EC 1.18.1.2) from bovine adrenocortical mitochondria was investigated chemically. NADPH-adrenoferredoxin reductase has an essential lysine residue at the adrenoferredoxin-binding site. A polypeptide at the adrenoferredoxin-binding site was isolated by high-pressure liquid chromatography from NADPH-adrenoferredoxin reductase modified with pyridoxal 5'-phosphate and cleaved with cyanogen bromide. The amino-acid sequence of the adrenoferredoxin-binding peptide was identified. The peptide accounted for 95% of the sugar content of the NADPH-adrenoferredoxin reductase.

Adrenal Cortex↗

PubMed2003

Selection of RRE RNA binding peptides using a kanamycin antitermination assay.

The arginine-rich domains of several RNA-binding proteins have been shown to bind their cognate RNAs with high affinities and specificities as isolated peptides, adopting different conformations within different complexes. The sequence simplicity and structural diversity of the arginine-rich motif has made it a good framework for constructing combinatorial libraries and identifying novel RNA-binding peptides, including those targeted to the HIV Rev response element (RRE). Here we describe a modified transcription antitermination reporter assay engineered with kanamycin resistance that enables larger in vivo screens (approximately 10(9) sequences) than previously possible. We show that the assay detects only specific RNA-protein complexes, and that binders are enriched at least 300-fold per round of selection. We screened a large peptide library in which amino acids with charged, polar, and small side chains were randomly distributed within a polyarginine framework and identified a set of high affinity RRE-binding peptides. Most contain glutamine at one particular peptide position, and the best peptides display significantly higher antitermination activities than Rev or other previously described high-affinity RRE-binding peptides. The kanamycin antitermination (KAN) assay should be useful for screening relatively large libraries and thereby facilitate identification of novel RNA binders.

Biological Assay↗

PubMed1997

[Binding-peptide motifs of HLA class II molecules susceptible to autoimmune diseases].

Recent advances in knowledge of crystal structures of MHC class II molecules has advanced understanding of the molecular basis for interactions between peptides and HLA class II molecules. Polymorphism of HLA class II molecules influences structures of peptides bound to HLA class II molecules. To elucidate mechanisms for statistical association between particular HLA class II alleles and susceptibility to autoimmune diseases, it is important to identify self peptides presented by disease-susceptible HLA class II molecules and triggering disease-causative autoreactive T cells. In this study, we tried to identify self-peptides triggering autoimmune diseases including rheumatoid arthritis, insulin autoimmune syndrome, insulin dependent diabetes mellitus and infant-onset myasthenia gravis. Susceptibility to all of these diseases in the Japanese population are known to be strongly associated with particular HLA-DR-DQ haplotypes unique to Asians, and clinical features of some of these diseases are different between Caucasians and Asians including Japanese. We investigated differences in binding-peptide motifs between disease susceptible and non-susceptible HLA class II molecules and predicted candidates of autoimmune self-peptides carrying binding-motifs to disease-susceptible HLA class II molecules. Indeed the major epitope for insulin-autoreactive CD4+ T cell was successfully identified by this strategy. We also found heterogeneity in immunogenetic background between Western type and Asian type of multiple sclerosis. Our data indicated that our strategy is useful to identify autoimmune self-peptides, and it is suggested that not only disease-susceptible HLA class II but also self-peptides causing diseases are different between Caucasians and Asians. These differences may well correlate to different clinical manifestations of diseases between the two ethnic groups.

Amino Acid Sequence↗

PubMed1995

Localization of calcitonin gene-related peptide binding sites in the eye of different species.

The localization of calcitonin gene-related peptide (CGRP) binding sites in the eye of monkey, pig, cat and guinea pig was studied by autoradiography. Specific binding of CGRP was found in ciliary muscle, ciliary processes and limbal conjunctiva in all tested species. Furthermore, specific binding sites of CGRP was found in the choroidea of monkey, pig and guinea pig, in the iris of pig, cat and guinea pig, in the retina of pig and in the anterior chamber angle of cat. The number of specific binding sites varied depending on the tissue and species. The present study shows that there are specific binding sites of CGRP in the eye of monkey, pig, cat and guinea pig. CGRP binding sites found in vascular system of ciliary body, choroidea and iris further demonstrates the role of CGRP as a vasoregulatory peptide. Binding sites in the ciliary muscle, in the limbal conjunctiva and in the chamber angle area may indicate a role in the regulation of ciliary muscle tone, epithelial cell regeneration and aqueous humour outflow.

Animals↗

PubMed1993

Immunogenic peptides bind to class II MHC molecules in an early lysosomal compartment.

Exogenous protein Ag are processed within endocytic compartments to produce peptides that bind to class II MHC (MHC-II) molecules for presentation to T cells. We have now identified a subcellular compartment in which immunogenic peptides bind to MHC-II as a subset of high density lysosomes. Immunoelectron microscopy of whole cells and dense Percoll gradient subcellular fractions showed early tubulovesicular lysosomes with high levels of MHC-II. Typical mature lysosomes contained less MHC-II. Pulse-chase biosynthetic labeling of macrophages followed by immunoprecipitation of MHC-II from dense lysosomal fractions showed that MHC-II molecules targeted efficiently to lysosomes after biosynthesis. Moreover, lysosomal MHC-II molecules were rapidly loaded with immunogenic peptide (as detected by T cells) soon after exposure of macrophages to Ag and before similar expression of peptide-MHC-II complexes on the plasma membrane; this loading was blocked at 18 degrees C. We propose that nascent MHC-II molecules target to early tubulovesicular lysosomes and bind immunogenic peptides therein; the resulting peptide-MHC-II complexes are then transported to the plasma membrane.

Animals↗

PubMed1998

Cell separation based on the reversible interaction between calmodulin and a calmodulin-binding peptide.

A cell separation system based on the calcium-dependent interaction of calmodulin (CM) with a calmodulin-binding peptide (CBP) has been developed. The prototype of this system utilizes an indirect method to label the target cell population. Cells are first labeled with a primary monoclonal antibody directed to a specific cell surface antigen, then with a secondary affinity reagent, consisting of a polyclonal goat anti-mouse IgG (GAM-IgG) that has been cross-linked to a CBP derived from the sequence of the rabbit skeletal muscle myosin light chain kinase. In the presence of Ca2+, the CBP on the cells labeled with GAM-IgG-CBP binds to biotinylated calmodulin (CM-Biotin) with high affinity. The target cells are then captured with a solid-phase streptavidin. The unbound non-target cells are washed away and the immobilized target cells are released by chelating Ca2+ with EGTA. The specificity of the GAM-IgG-CBP and CM-Biotin and the feasibility of using this system to separate cells was demonstrated using the KG-1 human acute myelogenous leukemia cell line. KG-1 cells were fractionated on the basis of cell surface expression of HLA-DR. The cell selection reagents and the cell separation process did not affect KG-1 cell viability while cells selected by this procedure were 90% pure with a yield of 75%. This cell separation system also was used for rare cell isolation from normal human peripheral blood mononuclear cells. T cells expressing the Vbeta5 T cell receptor, which represent < 5% of the unfractionated cells, were isolated with 89% viability, 72% purity, 80% yield, and retained the ability to respond to activation signals as measured by blast transformation. The results from this study show that a cell selection system based on the reversible interaction between CM and a CBP can be applied to gently and efficiently isolate cells from a heterogeneous starting population that are free of the solid matrix without exposure to the stresses of mechanical or enzymatic release.

Biotin↗

PubMed1994

The peptide-binding specificity of HLA-B27 subtype (B*2705) analyzed by the use of polyalanine model peptides.

Model peptides have been used to quantitate the effect on HLA-B*2705 binding of the spacing between primary anchor residues, the type of amino acid accepted in the P9 anchor position, and the type of amino acid accepted in the "secondary anchor positions" P3 and P7. Peptide binding was measured by the HLA class I alpha-chain-refolding assay. The results obtained show that (a) Among the model peptides differing in the spacing between anchor residues, the nonamer with Arg in P2 and Lys in P9 (R2, K9) has the maximum binding with B*2705 molecule. The decamer, with an extra Ala inserted between Arg and Lys (R2, K10), has much lower binding, and still lower binding is observed for the octamer, where an Ala is removed (R2, K8). (b) Besides the "classic" Lys and Arg, several other aminoacids such as Tyr, Leu, Ala, and Gln can be accepted in P9, but with significant differences in binding affinity. (c) Different amino acids in P3 have an influence on peptide binding. Trp and Phe have a favorable influence, whereas Lys and Val appear to hinder the binding. Some variations are seen also for different amino acids in P7.

Amino Acid Sequence↗

PubMed1995

Stimulation of T cells by antigen-presenting cells is kinetically controlled by antigenic peptide binding to major histocompatibility complex class II molecules.

Activation of CD4+ T cells by antigenic peptide involves the interaction of major histocompatibility complex (MHC) class II-peptide complexes on the surface of antigen-presenting cells (APCs) with T-cell receptors. This report describes the kinetics of T-cell triggering by exogenous antigenic peptides in the presence of APCs. A rapid specific increase in extracellular acidification rate is observed within minutes upon exposure of A.E7 T cells (restricted for IEk and moth cytochrome c peptide containing residues 88-103) and 4R3.9 T cells (restricted for IAk and myelin basic protein peptide containing residues 1-14 [AcMBP-(1-14)]) to their cognate peptides in the presence of CH27 cells bearing both IAk and IEk MHC class II molecules. Pretreatment of cloned T cells, but not APCs, with herbimycin A resulted in complete inhibition of triggering events, indicating that the acidification response is mediated by T-cell second messenger pathways. This rapid assay for 4R3.9 T-cell stimulation showed increased T-cell triggering activity for AcMBP-(1-14)-A4 and MBP-(1-14)-M4 peptides compared to the native AcMBP-(1-14)-K4. By using the previously determined kinetic constants for MBP-(1-14)-A4 reactions with IAk, it is possible to show that at the lowest peptide concentrations the kinetics of T-cell triggering are limited by the kinetics of the peptide binding to MHC class II molecules.

Amino Acid Sequence↗

PubMed2004

Characterization of the Alzheimer's disease-associated CLAC protein and identification of an amyloid beta-peptide-binding site.

Amyloid beta-peptide (Abeta) deposition into amyloid plaques is one of the invariant neuropathological features of Alzheimer's disease. Other proteins co-deposit with Abeta in plaques, and one recently identified amyloid-associated protein is the collagen-like Alzheimer amyloid plaque component CLAC. It is not known how CLAC deposition affects Abeta plaque genesis and the progress of the disease. Here, we studied the in vitro properties of CLAC purified from a mammalian expression system. CLAC displays features characteristic of a collagen protein, e.g. it forms a partly protease-resistant triple-helical structure, exhibits an intermediate affinity for heparin, and is glycosylated. Purified CLAC was also used to investigate the interaction between CLAC and Abeta. Using a solid-phase binding assay, we show that CLAC bound with a similar affinity to aggregates formed by Abeta-(1-40) and Abeta-(1-42) and that the interaction was impaired by increasing salt concentrations. An 8-residue-long sequence located in non-collagenous domain 2 of CLAC was found to be crucial for the interaction with Abeta. These findings may be useful for future therapeutic interventions aimed at finding compounds that modulate the binding of CLAC to Abeta deposits.

Alzheimer Disease↗

PubMed2006

Peptide-binding motifs for the I-Ad MHC class II molecule: alternate pH-dependent binding behavior.

The ability of peptides to form stable complexes with MHC class II molecules expressed in the host determines their ability to recruit CD4 T cells during an immune response. In this study, we sought to define the features of the antigenic peptides that control their kinetic stability with I-A(d) because of the diversity of peptides that this molecule is known to present. Peptide dissociation assays indicated that each pocket of I-A(d) displays exquisite sensitivity to side chain structure, size, and charge. Most surprising were results related to the P1 pocket, which has been difficult to define by conventional competition assays. Our studies revealed a considerable degree of specificity in the P1 pocket but also an unexpected degree of structural flexibility. Amino acids with neutral side chains such as Met and the alternatively negatively charged Glu are both highly favored at P1. Interestingly, these two options at the P1 pocket in I-A(d) display dramatically different pH-dependent interactions with the class II molecule. These findings are discussed in the context of a structural model to explain these data and in light of the immunological implications of pH-dependent behavior of class II-peptide complexes in acidic endosomal compartments, where DM-catalyzed loading of class II molecules takes place, and at the neutral pH of the APC cell surface, where class II-peptide complexes promote activation of CD4 T cells.

Amino Acid Motifs↗

PubMed2005

A new prostate carcinoma binding peptide (DUP-1) for tumor imaging and therapy.

PURPOSE: Prostate carcinomas belong to the most widespread tumors, and their number is increasing. Imaging modalities used for diagnosis, such as ultrasound, computed tomography, and positron emission tomography, often produce poor results. Radiolabeled peptides with high sensitivity and specificity for prostate cancer would be a desirable tool for tumor diagnosis and treatment. EXPERIMENTAL DESIGN: We used phage display and the prostate-specific membrane antigen-negative cell line DU-145 to identify a peptide. The isolated DUP-1 was tested in vitro for its binding specificity, kinetics, and affinity. Internalization of the peptide was evaluated with confocal microscopy. The tumor accumulation in a nude mouse model was analyzed with 131I-labeled DUP-1 in PC-3 and DU-145 prostate tumors as well as in the rat prostate tumor model AT-1. RESULTS: The synthesized peptide showed rapid binding kinetics peaking at 10 minutes. It shows specific binding to prostate carcinoma cells but low binding affinity to nontumor cells. Peptide binding is competed with unlabeled DUP-1, and a time-dependent internalization into DU-145 cells was shown. Biodistribution studies of DUP-1 in nude mice with s.c. transplanted DU-145 and PC-3 tumors showed a tumor accumulation of 5% and 7% injected dose per gram, and bound peptide could not be removed by perfusion. The rat prostate tumor model showed an increase of radioactivity in the prostate tumor up to 300% in comparison with normal prostate tissue. CONCLUSIONS: DUP-1 holds promise as a lead peptide structure applicable in the development of new diagnostic tracers or anticancer agents that specifically target prostate carcinoma.

Animals↗

PubMed1998

Crucial role of N-terminal residue of binding peptides in recognition of the monoclonal antibody specific for the peptide-HLA-B5, -B35 complex.

The monoclonal antibody (mAb) 4D12 specific for the HLA-B5, -B35 cross-reacting group (CREG) bound to a fraction of HLA-B*3501 and HLA-B*5101 molecules carrying self-peptides. Analysis of the binding of mAb 4D12 to HLA-B*3501 and -B*5101 molecules pulsed with chemically synthesized peptides revealed that this mAb recognizes a restricted number of peptides and that P1 of the bound peptides critically influences its binding. The 4D12 mAb bound only to HLA-B*3501 molecules carrying peptides with Asn, Asp, Glu, Ser, and Val at P1. Analysis using an HLA-B*3501 crystallographic model suggested that 4D12 may recognize the side chain of the P1 residue that is pointing to the solvent. On the other hand, 4D12 bound only to HLA-B*5101 molecules carrying peptides with Asn or Asp at P1, suggesting that the 4D12 epitope formed by Glu, Ser, or Val at P1 and the A-pocket was changed by the substitution of His for Tyr at residue 171 of HLA-B*3501 molecules. This was confirmed by testing the binding of mAb 4D12 to HLA-B*3501 mutant molecules at residue 171 carrying these peptides. These results together suggest that the conformation of the A-pocket and its hydrogen bound network with the P1 residue is also critical for the binding of mAb 4D12. The present study shows the molecular basis of the specificity of 4D12 for the peptide-HLA class I complex.

Amino Acid Sequence↗

PubMed2006

Hyaluronic acid binding peptides prevent experimental staphylococcal wound infection.

Staphylococcus aureus is a major cause of surgical wound infections. The development of mechanisms of antimicrobial resistance by this and other bacterial pathogens has prompted the search for new approaches to treat infectious diseases. Hyaluronic acid binding peptides have been shown to modulate cellular trafficking during host responses and were assessed for their ability to treat and possibly prevent experimental surgical wound infections caused by S. aureus. Treatment with these peptides was highly efficacious in reducing the number of S. aureus cells at the wound site and ameliorated the inflammatory host response associated with these infections. These data suggest a novel approach for the treatment and prophylaxis of staphylococcal wound infections in the clinical setting.

Amino Acid Sequence↗

PubMed1996

Selection of RNA-binding peptides in vivo.

Many priniciples of sequence-specific DNA recognition have been established over the past decade, largely from structural studies of protein-DNA and drug-DNA complexes. On the basis of these principles, it has been possible to design or select variants of known structural motifs, including zinc-fingers and minor groove-binding drugs, that bind desired sequences. Here we describe a strategy, based on transcriptional termination in bacteria, to identify specific RNA-binding peptides using the arginine-rich RNA-binding motif as a framework. Peptides were isolated from two combinatorial libraries that bind tightly and specifically to the Rev response element of HIV. It appears that alpha-helical peptides resembling Rev were selected from one library whereas new peptides that probably do not form helices were selected from the other, suggesting that the arginine-rich motif may be a particularly versatile framework for recognizing RNA structures.

Amino Acid Sequence↗

PubMed2002

Kinetic studies of calcium and cardiac troponin I peptide binding to human cardiac troponin C using NMR spectroscopy.

Ca2+ and human cardiac troponin I (cTnI) peptide binding to human cardiac troponin C (cTnC) have been investigated with the use of 2D [1H,15N] HSQC NMR spectroscopy. The spectral intensity, chemical shift, and line-shape changes were analyzed to obtain the dissociation ( K(D)) and off-rate ( k(off)) constants at 30 degrees C. The results show that sites III and IV exhibit 100-fold higher Ca2+ affinity than site II ( K(D(III,IV)) approximately 0.2 microM, K(D(II)) approximately 20 microM), but site II is partially occupied before sites III and IV are saturated. The addition of the first two equivalents of Ca2+ saturates 90% of sites III and IV and 20% of site II. This suggests that the Ca2+ occupancy of all three sites may contribute to the Ca2+-dependent regulation in muscle contraction. We have determined a k(off) of 5000 s(-1) for site II Ca2+ dissociation at 30 degrees C. Such a rapid off-rate had not been previously measured. Three cTnI peptides, cTnI(34-71), cTnI(128-147), and cTnI(147-163), were titrated to Ca2+-saturated cTnC. In each case, the binding occurs with a 1:1 stoichiometry. The determined K(D) and k(off) values are 1 microM and 5 s(-1) for cTnI(34-71), 78+/-10 microM and 5000 s(-1) for cTnI(128-147), and 150+/-10 microM and 5000 s(-1) for cTnI(147-163), respectively. Thus, the dissociation of Ca2+ from site II and cTnI(128-147) and cTnI(147-163) from cTnC are rapid enough to be involved in the contraction/relaxation cycle of cardiac muscle, while that of cTnI(34-71) from cTnC may be too slow for this process.

Calcium↗