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PubMed2003

Influence of peptide conformation on oligosaccharide binding characteristics--a study using apamin-based chimeric peptide.

Interactions between proteins and heparin play a crucial role in most of the cellular process. Unraveling the forces that govern the formation of these complexes is vital for understanding the specificities involved in these biomolecular events. In the present study, a detailed analysis has been undertaken to evaluate the effect(s) of peptide conformation on heparin-binding, using a chimeric peptide, apaK6--a chimera of a highly stable neurotoxic peptide from honey-bee venom and a de novo designed lysine-rich peptide. The dissociation constants of these peptide-heparin complexes were found to be in the submicromolar range. Comparison of the results obtained from the titration of the disulfide-reduced and disulfide-intact chimeric peptide with various sulfated oligosaccharides, derived from heparin, suggest that the initial structure of the peptide has pronounced effect on the binding affinity, binding modes and also on binding preferences. The results of this study indicate that the heparin-binding specificity of an isolated peptide and that exhibited by the same peptide when present in a globular protein could be significantly different, especially if the isolated peptide undergoes conformational change(s) upon binding to the sulfated oligosaccharides. In addition, such dependency of the binding specificity on the preformed structures could be utilized for the design of high-affinity and sequence-specific heparin-binding polypeptides.

Apamin↗
PubMed2005

Natriuretic peptides cause relaxation of human and guinea-pig gallbladder muscle through interaction with natriuretic peptide receptor-B.

Atrial natriuretic peptide (ANP) binding sites have been demonstrated in the guinea-pig gallbladder muscle with unclear function. To investigate effects of natriuretic peptides in the gallbladder, we measured relaxation of isolated human and guinea-pig gallbladder strips caused by natriuretic peptides, including C-type natriuretic peptide (CNP), brain natriuretic peptide (BNP) and ANP, as well as des[Gln18, Ser19, Gly20, Leu21, Gly22]ANP(4-23) amide (cANP(4-23)), a selective natriuretic peptide receptor-C (NPR-C) agonist. Results in the human gallbladder were similar to those in the guinea-pig gallbladder. CNP, BNP, ANP and cANP(4-23) alone did not cause contraction or relaxation in resting gallbladder strips. However, in carbachol or endothelin-1-contracted strips, CNP caused moderate, sustained and concentration-dependent relaxation. The relaxation was not affected by tetrodotoxin or atropine in endothelin-1-contracted gallbladder strips and not by tetrodotoxin in carbachol-contracted strips. These indicate a direct effect of CNP on the gallbladder muscle. The relative potencies for natriuretic peptides to cause relaxation were CNP>>BNP> or = ANP. cANP(4-23) did not cause relaxation. These indicate the existence of the natriuretic peptide receptor-B (NPR-B) mediating the relaxation. Taken together, these results demonstrate that natriuretic peptides cause relaxation of human and guinea-pig gallbladder muscle through interaction with the natriuretic peptide receptor-B.

Animals↗
PubMed1997

Differential effects of ambient blood glucose level and degree of obesity on basal serum C-peptide level and the C-peptide response to glucose and glucagon in non-insulin-dependent diabetes mellitus.

We student basal, glucose- and glucagon-induced insulin secretion in non-insulin diabetes mellitus (NIDDM) patients in relation to body mass index (BMI) and fasting serum glucose (FBS) level. A total of 46 NIDDM patients and 22 control subjects with varying degrees of BMI and FBS were given 100 g of oral glucose and 1 mg of intravenous glucagon on separate days. C-peptide response to glucose, but not basal serum C-peptide and C-peptide response to glucagon, was significantly lower in NIDDM than in controls (P < 0.001). FBS was inversely correlated with C-peptide response to glucose in NIDDM patients (r = -0.67, P < 0.001), but not with basal C-peptide level and C-peptide response to glucagon. On the other hand, BMI was positively correlated with basal serum C-peptide level both in NIDDM (r = 0.60, P < 0.001) and in control subjects (r = 0.74, P < 0.001). In 15 poorly controlled NIDDM patients, the tests were repeated after insulin treatment for 10-14 days. C-peptide response to glucose significantly increase, but not to a level in control subjects, after glycemic control. Basal serum C-peptide level and the C-peptide response to glucagon decreased after glycemic control to significantly lower levels than those in the baseline and those in control subjects. These results suggest that beta cell secretory reserve is reduced in moderate to severe NIDDM patients.

Administration, Oral↗
PubMed2000

Transfection using synthetic peptides: comparison of three DNA-compacting peptides and effect of centrifugation.

Positively charged peptides have been shown to allow efficient transfection in vitro, especially when mixed with lipids. We have compared the ability of three positively charged peptides both to compact DNA and to increase the transfection efficiency of the cationic lipid DOTAP. The peptides are: a polymer of 17 lysines (pK17), YKAWK8WK (peptide K8) and SPKRSPKRSPKR (peptide P2). Peptides pK17 and K8 compact DNA efficiently in a gel retardation assay and protect DNA efficiently against DNase I degradation. Peptide P2, on the other hand, interacts weakly with DNA and provides poor protection. In order to compare their transfection efficiency, the three peptides were mixed with DNA (plasmid pEGFP-N1) at different charge ratios (+/-) and DOTAP (at a charge ratio of 2). The transfection efficiency was measured by FACS analysis at different times post-transfection. With NIH-3T3 cells, peptide P2 provides the highest transfection efficiency (about 40%), when compared with peptides pK17 (29%) and K8 (31%) and DOTAP alone (21%) under optimal conditions. Finally, we showed that centrifugation of the complexes onto the cells increased the transfection efficiency by a factor 1.5 to 2 with the various cell lines tested (ECV, primary human keratinocyte, CFT-2, NT-1).

3T3 Cells↗
PubMed1998

A hydrophobic peptide (VAP-peptide) of the silkworm, Bombyx mori: structure, expression and an enhancing function of diapause hormone activity.

We have recently identified a unique lipophilic peptide (VAP-peptide) with diapause egg inducing activity in the silkworm, Bombyx mori (Imai et al., 1996). The cloning and sequencing of cDNA encoding VAP-peptide have demonstrated that the deduced amino acid sequence consisted of 84 amino acid residues, from which the mature VAP-peptide of 68 amino acid residues was released by cleaving a signal sequence. Searches of the GenBank data base revealed no significant sequence similarity to other proteins including diapause hormone (DH). VAP-peptide gene was selectively expressed just before and at adult eclosion in the head and the thorax not in the abdomen. By a Western blot analysis, VAP-peptide was also localized in the head and the thorax of adults. The purified recombinant VAP-peptide could not induce diapause eggs even when injected at a high dose of 10 nmol/pupa. Whereas, injection of a mixture of VAP-peptide and DH clearly decreased a half-maximum dose (ED50 value) and a threshold dose (TD value) of DH, and these values decreased according to increasing molar ratios of VAP-peptide to DH. Thus, the VAP-peptide is concluded to be an endogenous protein acting as a potent enhancer of DH activity through interaction with DH.

Amino Acid Sequence↗
PubMed2000

Selective gas-phase cleavage at the peptide bond C-terminal to aspartic acid in fixed-charge derivatives of Asp-containing peptides.

This study focuses on the molecular level interpretation of the selective gas-phase cleavage at aspartic acid residues (Asp) in protonated peptides. A phi3P+CH2C(=O)group (phi = 2,4,6-trimethoxyphenyl) is attached to the N-terminal nitrogen of the selected peptides LDIFSDF and LDIFSDFR, via solid-phase synthesis, to "mimic" the tightly held charge of a protonated arginine (Arg) residue. Collision-induced dissociation in a quadrupole ion trap instrument and surface-induced dissociation in a dual quadrupole instrument were performed for electrospray-generated ions of the fixed-charge peptide derivatives. Selective cleavages at Asp-Xxx are observed for those ions with charge provided only by the fixed charge or for those with a fixed charge and one Arg plus one added proton. This supports a previously proposed mechanism which suggests that the cleavages at Asp-Xxx, initiated by the acidic hydrogen of the Asp residue, become significant when ionizing protons are strongly bound by Arg in the protonated peptides. It is clear that the fixed charge is indeed serving as a "mimic" of protonated Arg and that a protonated Arg side chain is not required to interact with the Asp to induce cleavage at Asp-Xxx. When the number of protons exceeds the number of Arg in a peptide containing Arg and Asp, nonselective cleavages occur. The fragmentation efficiency of the peptides is consistent with the idea that these nonselective cleavages are promoted by a mobile proton. The peptide with a fixed charge and one added proton, [phi3P+CH2C(=O)-LDIFSDF + H]2+, fragments much more efficiently than the corresponding peptide with a fixed charge, an Arg and one added proton, [phi3P+CH2C(=O)-LDIFSDFR + H]2+; both of these fragment more efficiently than the peptide with a fixed charge and no added proton, phi3P+CH2C(=O)-LDIFSDF. MS/MS/MS (i.e., MS3) experimental results for bn ions formed at Asp-Xxx from phi3P+CH2C(=O)-LDIFSDF and its H/D exchange derivative, phi3P+CH2C(=O)-LDIFSDF-d11, are consistent with the bn ions formed at Asp-Xxx having a succinic anhydride cyclic structure. MS/MS experiments were also carried out for phi3P+CH2C(=O)-AAAA, a peptide derivative containing active hydrogens only at amide nitrogens plus the C-terminus, and its active H/D exchange product, phi3P+CH2C(=O)-AAAA-d5. The results show that a hydrogen originally located at an amide nitrogen is transferred away in the formation of a cyclic charge remote b ion.

Aspartic Acid↗
PubMed2005

Coumarin tags for analysis of peptides by MALDI-TOF MS and MS/MS. 2. Alexa Fluor 350 tag for increased peptide and protein Identification by LC-MALDI-TOF/TOF MS.

The goal of this study was the development of N-terminal tags to improve peptide identification using high-throughput MALDI-TOF/TOF MS. Part 1 of the study was focused on the influence of derivatization on the intensities of MALDI-TOF MS signals of peptides. In part 2, various derivatization approaches for the improvement of peptide fragmentation efficiency in MALDI-TOF/TOF MS are explored. We demonstrate that permanent cation tags, while significantly improving signal intensity in the MS mode, lead to severe suppression of MS/MS fragmentation, making these tags unsuitable for high-throughput MALDI-TOF/TOF MS analysis. In the present work, it was found that labeling with Alexa Fluor 350, a coumarin tag containing a sulfo group, along with guanidation of epsilon-amino groups of Lys, could enhance unimolecular fragmentation of peptides with the formation of a high-intensity y-ion series, while the peptide intensities in the MS mode were not severely affected. LC-MALDI-TOF/TOF MS analysis of tryptic peptides from the SCX fractions of an E. coli lysate revealed improved peptide scores, a doubling of the total number of peptides, and a 30% increase in the number of proteins identified, as a result of labeling. Furthermore, by combining the data from native and labeled samples, confidence in correct identification was increased, as many proteins were identified by different peptides in the native and labeled data sets. Additionally, derivatization was found not to impair chromatographic behavior of peptides. All these factors suggest that labeling with Alexa Fluor 350 is a promising approach to the high-throughput LC-MALDI-TOF/TOF MS analysis of proteomic samples.

Acetates↗
PubMed2006

Improved peptide elution time prediction for reversed-phase liquid chromatography-MS by incorporating peptide sequence information.

We describe an improved artificial neural network (ANN)-based method for predicting peptide retention times in reversed-phase liquid chromatography. In addition to the peptide amino acid composition, this study investigated several other peptide descriptors to improve the predictive capability, such as peptide length, sequence, hydrophobicity and hydrophobic moment, and nearest-neighbor amino acid, as well as peptide predicted structural configurations (i.e., helix, sheet, coil). An ANN architecture that consisted of 1052 input nodes, 24 hidden nodes, and 1 output node was used to fully consider the amino acid residue sequence in each peptide. The network was trained using approximately 345,000 nonredundant peptides identified from a total of 12,059 LC-MS/MS analyses of more than 20 different organisms, and the predictive capability of the model was tested using 1303 confidently identified peptides that were not included in the training set. The model demonstrated an average elution time precision of approximately 1.5% and was able to distinguish among isomeric peptides based upon the inclusion of peptide sequence information. The prediction power represents a significant improvement over our earlier report (Petritis, K.; Kangas, L. J.; Ferguson, P. L.; Anderson, G. A.; Pasa-Tolic, L.; Lipton, M. S.; Auberry, K. J.; Strittmatter, E. F.; Shen, Y.; Zhao, R.; Smith, R. D. Anal. Chem. 2003, 75, 1039-1048) and other previously reported models.

Amino Acid Sequence↗
PubMed2001

EPR spectroscopic studies of the reduction of chromium(VI) by methanol in the presence of peptides. Formation of long-lived chromium(V) peptide complexes.

The synthesis and characterization of the first Cr(V) complexes with non-sulfur-containing peptides, which may mimic the chemistry of the intermediates in the formation of Cr-induced peptide-DNA cross-links in vivo, are reported. The reduction of Cr(VI) with methanol in the presence of a number of non-sulfur-containing peptides produced relatively stable Cr(V)-peptide complexes, which were characterized by EPR spectroscopy and electrospray mass spectrometry. The reaction of Cr(VI) with methanol alone (in the absence of peptide ligands) resulted in the formation of two Cr(V)-methanol intermediates, with giso values of 1.9765 and 1.9687. The methanol reduction of Cr(VI) in the presence of the glycine peptides, triglycine, tetraglycine, and pentaglycine resulted in the formation of both Cr(V)-methanol and Cr(V)-peptide intermediates, while only the Cr(V)-peptide complexes were detected in the reactions with the alanine peptides trialanine, tetraalanine, and pentaalanine. Similar EPR signals were observed for all of the Cr(V)-peptide complexes with giso values between approximately 1.986 and approximately 1.979, and AN values of (2.1-2.6) x 10(-4) cm-1.

Alanine↗
PubMed2005

MaP peptides: programming the self-assembly of peptide-based mesoscopic matrices.

We describe an approach that utilizes nonlinear peptides to direct the assembly of previously reported Self-Assembling Fibers (SAFs). The SAF system comprises two complementary linear peptides, SAF-p1 and SAF-p2a, which combine to form exclusively linear, nonbranched fibers. The Matrix-Programming (MaP) peptides described herein are based on these peptides: they comprise two or three half-peptide blocks derived from the SAF peptides, which are conjugated via dendritic hubs. Different MaP peptides coassembled with the standard SAF peptides to form specific structures, such as hyperbranched networks, polygonal matrices, and regularly segmented and terminated fibers. The role of each half-peptide block in dictating the different features has been elucidated. This provides a strong basis for designing new peptide-based nanostructured materials from the bottom up.

Circular Dichroism↗
PubMed2005

Room-temperature Wurtzite ZnS nanocrystal growth on Zn finger-like peptide nanotubes by controlling their unfolding peptide structures.

ZnS nanocrystal, a class of wide-gap semiconductors, has shown interesting optical, electrical, and optoelectric properties via quantum confinement. For those applications, phase controls of ZnS nanocrystals and nanowires were critical to tune their physical properties to the appropriate ones. The wurtzite ZnS nanocrystal growth at room temperature is the useful fabrication; however, the most stable ZnS structure in nanoscale is the zinc blende (cubic) structure, and scientists have just begun exploring the room-temperature synthesis of the wurtzite (hexagonal) structure of ZnS nanocrystals. In this report, we applied the Zn finger-like peptides as templates to control the phase of ZnS nanocrystals to the wurtzite structure at room temperature. The peptide nanotubes, consisting of a 20 amino acids (VAL-CYS-ALA-THR-CYS-GLU-GLN-ILE-ALA-ASP-SER-GLN-HIS-ARG-SER-HIS-ARG-GLN-MET-VAL, M1 peptide) synthesized based on the peptide motif of the Influenza Virus Matrix Protein M1, could grow the wurtzite ZnS nanocrystals on the nanotube templates in solution. In the M1 protein, the unfolding process of the helical peptide motif via pH change creates a linker region between N- and C-terminated helical domains that contains a Zn finger-like Cys2His2 motif. Because the higher pH increases the uptake of Zn ions in the Cys2His2 motif of the M1 peptide by unfolding more helical domains, the pH change can essentially control the size and the number of the nucleation sites in the M1 peptides to grow ZnS nanocrystals with desired phases. Here we optimized the nucleation sites in the M1 peptides by unfolding them via pH change to obtain highly monodisperse and crystalline wurtzite ZnS nanocrystals on the template nanotubes at room temperature. This type of peptide-induced biomineralization technique will provide a clean and reproducible method to produce semiconductor nanotubes due to its efficient nanocrystal formation, and the band gaps of resulting nanotubes can also be tuned simply by phase control of ZnS nanocrystal coatings via the optimization of the unfolding peptide structures.

Crystallization↗
PubMed2006

HLA-DM targets the hydrogen bond between the histidine at position beta81 and peptide to dissociate HLA-DR-peptide complexes.

The peptide editor HLA-DM (DM) mediates exchange of peptides bound to major histocompatibility (MHC) class II molecules during antigen processing; however, the mechanism by which DM displaces peptides remains unclear. Here we generated a soluble mutant HLA-DR1 with a histidine-to-asparagine substitution at position 81 of the beta-chain (DR1betaH81N) to perturb an important hydrogen bond between MHC class II and peptide. Peptide-DR1betaH81N complexes dissociated at rates similar to the dissociation rates of DM-induced peptide-wild-type DR1, and DM did not enhance the dissociation of peptide-DR1betaH81N complexes. Reintroduction of an appropriate hydrogen bond (DR1betaH81N betaV85H) restored DM-mediated peptide dissociation. Thus, DR1betaH81N might represent a 'post-DM effect' conformation. We suggest that DM may mediate peptide dissociation by a 'hit-and-run' mechanism that results in conformational changes in MHC class II molecules and disruption of hydrogen bonds between betaHis81 and bound peptide.

Antigen Presentation↗
PubMed1998

Linear and cyclic peptides as substrates and modulators of P-glycoprotein: peptide binding and effects on drug transport and accumulation.

One cause of multidrug resistance (MDR) in human cancers is the overexpression of the P-glycoprotein multidrug transporter, a member of the ABC superfamily of membrane proteins. Natural products and chemotherapeutic drugs are pumped out of the cell by P-glycoprotein in an ATP-dependent fashion. There is growing evidence that many hydrophobic peptides are also P-glycoprotein substrates. With the use of a fluorescence-quenching assay, we have shown that some linear and cyclic hydrophobic peptides interact with P-glycoprotein, whereas others do not. The measured values of the quenching constant, Kq, for interaction of peptides with P-glycoprotein ranged from 200 nM for cyclosporine A to 138 microM for the tripeptide N-acetyl-leucyl-leucyl-norleucinal. Peptides that interacted with P-glycoprotein in the fluorescence assay also blocked colchicine transport into plasma membrane vesicles from MDR cells. The values of Dm, the peptide concentration causing 50% inhibition of drug uptake, were highly correlated with the values of Kq, over three orders of magnitude. The P-glycoprotein ATPase stimulation/inhibition profile of the peptides was not helpful in making a quantitative assessment of the ability of a peptide to interact with P-glycoprotein or to block drug transport. Some hydrophobic peptides were able to restore accumulation in MDR cells of the chemotherapeutic drug daunorubicin and the fluorescent dye rhodamine 123 to the levels observed in the drug-sensitive parent. Peptides that interacted with P-glycoprotein also displayed a relatively low overall toxicity to intact MDR cells, and inhibited drug transport at concentrations below the toxic range. Hydrophobic peptides should be given serious consideration for development as clinical chemosensitizing agents.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Preparation of functionally active cell-permeable peptides by single-step ligation of two peptide modules.

Noninvasive cellular import of synthetic peptides can be accomplished by incorporating a hydrophobic, membrane-permeable sequence (MPS). Herein, we describe a facile method that expedites synthesis of biologically active, cell-permeable peptides by site-specific ligation of two free peptide modules: one bearing a functional sequence and the second bearing a MPS. A nonpeptide thiazolidino linkage between the two modules is produced by ligation of the COOH-terminal aldehyde on the MPS and the NH2-terminal 1, 2-amino thiol moiety on the functional sequence. This thiazolidine ligation approach is performed with stoichiometric amounts of fully unprotected MPS and functional peptide in an aqueous buffered solution, eliminating the need for additional chemical manipulation and purification prior to use in bioassays. Two different MPSs were interchangeably combined with two different functional sequences to generate two sets of hybrid peptides. One set of hybrid peptides, carrying the cytoplasmic cell adhesion regulatory domain of the human integrin beta3, inhibited adhesion of human erythroleukemia cells to fibrinogen-coated surfaces. A second set of hybrid peptides, carrying the nuclear localization sequence of the transcription factor NF-kappaB, inhibited nuclear import of transcription factors NF-kappaB, activator protein 1, and nuclear factor of activated T cells in agonist-stimulated Jurkat T lymphocytes. In each assay, these nonamide bond hybrids were found to be functionally comparable to peptides prepared by the conventional method. Cumulatively, this new ligation approach provides an easy and rapid method for engineering of functional, cell-permeable peptides and demonstrates the potential for synthesis of cell-permeable peptide libraries designed to block intracellular protein-protein interactions.

Amino Acid Sequence↗
PubMed1999

Amyloid beta peptides do not form peptide-derived free radicals spontaneously, but can enhance metal-catalyzed oxidation of hydroxylamines to nitroxides.

Amyloid beta (Abeta) peptides play an important role in the pathogenesis of Alzheimer's disease. Free radical generation by Abeta peptides was suggested to be a key mechanism of their neurotoxicity. Reports that neurotoxic free radicals derived from Abeta-(1-40) and Abeta-(25-35) peptides react with the spin trap N-tert-butyl-alpha-phenylnitrone (PBN) to form a PBN/.Abeta peptide radical adduct with a specific triplet ESR signal assert that the peptide itself was the source of free radicals. We now report that three Abeta peptides, Abeta-(1-40), Abeta-(25-35), and Abeta-(40-1), do not yield radical adducts with PBN from the Oklahoma Medical Research Foundation (OMRF). In contrast to OMRF PBN, incubation of Sigma PBN in phosphate buffer without Abeta peptides produced a three-line ESR spectrum. It was shown that this nitroxide is di-tert-butylnitroxide and is formed in the Sigma PBN solution as a result of transition metal-catalyzed auto-oxidation of the respective hydroxylamine present as an impurity in the Sigma PBN. Under some conditions, incubation of PBN from Sigma with Abeta-(1-40) or Abeta-(25-35) can stimulate the formation of di-tert-butylnitroxide. It was shown that Abeta peptides enhanced oxidation of cyclic hydroxylamine 1-hydroxy-4-oxo-2,2,6, 6-tetramethylpiperidine (TEMPONE-H), which was strongly inhibited by the treatment of phosphate buffer with Chelex-100. It was shown that ferric and cupric ions are effective oxidants of TEMPONE-H. The data obtained allow us to conclude that under some conditions toxic Abeta peptides Abeta-(1-40) and Abeta-(25-35) enhance metal-catalyzed oxidation of hydroxylamine derivatives, but do not spontaneously form peptide-derived free radicals.

Amyloid beta-Peptides↗
PubMed2000

Thermodynamic stability of HLA-B*2705. Peptide complexes. Effect of peptide and major histocompatibility complex protein mutations.

Designing synthetic vaccines from class I major histocompatibility complex (MHC)-binding antigenic peptides requires not only knowledge of the binding affinity of the designed peptide but also predicting the stability of the formed MHC-peptide complex. In order to better investigate structure-stability relationships, we have determined by circular dichroism spectroscopy the thermal stability of a class I MHC protein, HLA-B*2705, in complex with a set of 39 singly substituted peptide analogues. The influence of two anchoring side chains (P3 and P9) was studied by peptide mutation and appropriate site-directed mutagenesis of the HLA-B*2705 binding groove. The side chain at P9 is clearly the one that contributes the most to the thermal stability of the MHC-peptide complexes, as destabilization up to 25 degrees C are obtained after P9 mutation. Interestingly, structure-stability relationships do not fully mirror structure-binding relationships. As important as the C-terminal side chain are the terminal ammonium and carboxylate groups. Removal of a single H-bond between HLA-B27 and the terminal peptide moieties results in thermal destabilization up to 10 degrees C. Depending on the bound peptide and the location of the deleted H-bond, the decrease in the thermal stability of the corresponding complex is quantitatively different. The present study suggests that any peptidic amino acid at positions 3 and 9 promotes refolding of the B27-peptide complex. Once the complex is formed, the C-terminal side chain seems to play an important role for maintaining a stable complex.

Base Sequence↗
PubMedDate not supplied

Molecular and cellular effects of C-peptide--new perspectives on an old peptide.

New results present C-peptide as a biologically active peptide hormone in its own right. Although C-peptide is formed from proinsulin and cosecreted with insulin, it is a separate entity with biochemical and physiological characteristics that differ from those of insulin. There is direct evidence of stereospecific binding of C-peptide to a cell surface receptor, which is different from those for insulin and other related hormones. The C-peptide binding site is most likely a G-protein-coupled receptor. The association constant for C-peptide binding is approximately 3 x 10(9) M(-1). Saturation of the binding occurs already at a concentration of about 1 nM, which explains why C-peptide effects are not observed in healthy subjects. Binding of C-peptide results in activation of Ca2+ and MAPK-dependent pathways and stimulation of Na+,K(+)-ATPase and eNOS activities. The latter 2 enzymes are both deficient in several tissues in type 1 diabetes. There is some evidence that C-peptide, and insulin may interact synergistically on the insulin signaling pathway. Clinical evidence suggests that replacement of C-peptide, together with regular insulin therapy, may be beneficial in patients with type 1 diabetes and serve to retard or prevent the development of long-term complications.

Amino Acid Sequence↗
PubMed1991

Probing the accessibility of peptide in an MHC class II protein--foreign peptide complex.

The peptide antigen OVA323-339 forms a highly stable complex with the I-Ad molecule which when reconstituted into lipid bilayers can stimulate the release of interleukin 2 (IL-2) by specific T cell hybrids. To probe the accessibility of various regions of the peptide in the I-Ad binding site, six analogues of OVA323-339, each with a unique site for fluorescein labeling, were synthesized. OVA peptides modified with fluorescein at the N terminus position 323 and at the epsilon-amino group of lysines at positions 328, 329, 330, 331 and 336 all bind to the I-Ad molecule. These substitutions include four amino acids previously identified as representing a structural motif common to MHC binding peptides. The fluorescein hapten on each analogue was fully accessible to quenching by anti-fluorescein antibody after binding of the peptide to the MHC class II protein, indicating that these regions of the peptide are exposed in the MHC binding site. These data suggest that the MHC class II peptide binding site is remarkably permissive with respect to tolerating bulky substitutions in the peptide antigen. The data further suggest that either the central region (328-331) of the peptide OVA323-339 is oriented in MHC class II binding site such that all of its side chains are exposed, or that the binding of the peptide is conformationally flexible allowing reorientation of the bulky substituent to the outside of the binding site in each case studied.

Amino Acid Sequence↗