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J Gariépy

Publications and source records attributed to J Gariépy.

At least 37 records · Page 2Linked to original sources
PubMed1993

The Escherichia coli heat-stable enterotoxin is a long-lived superagonist of guanylin.

The mechanism by which bacterial heat-stable enterotoxins (ST I STA) cause diarrhea in humans and animals has been linked to the activation of an intestinal membrane-bound guanylate cyclase. Guanylin, a recently discovered rat intestinal peptide, is homologous in structure to ST I and can activate guanylate cyclase present on the human colonic carcinoma cell line T84. To directly test the mechanistic association of guanylate cyclase activation with diarrhea, we synthesized guanylin and a guanylin analog termed N9P10 guanylin and compared their biological activities with those of a synthetic ST I analog, termed ST Ib(6-18). We report that guanylin is able to inhibit the binding of a radiolabeled ST I analog to rat intestinal cells but causes diarrhea in infant mice only at doses at least 4 orders of magnitude higher than that of ST Ib(6-18). In contrast, N9P10 guanylin was enterotoxic in mice at much lower doses than guanylin but proved to be a weaker inhibitor of radiolabeled ST I than guanylin in the receptor binding assay. The pattern of guanylate cyclase activation observed for ST Ib(6-18) and the two guanylin analogs parallels the results observed in the receptor binding assay rather than those observed in the diarrheal assay. Treatment of guanylin with chymotrypsin or lumenal fluid derived from newborn mouse intestines resulted in a rapid loss of binding activity. Together, these results suggest that ST I enterotoxins may represent a class of long-lived superagonists of guanylin.

Amino Acid Sequence↗
PubMed1991

Structural characterization of functionally important regions of the Escherichia coli heat-stable enterotoxin STIb.

The biological properties of the Escherichia coli enterotoxin STIb (STA-3, STh) reside in a 13 amino acid C-terminal domain, abbreviated STIb(6-18). This tridecapeptide contains six cysteine residues involved in three intramolecular disulfide bridges. The solution structure of STIb(6-18) has been modeled as a series of three consecutive reverse turns [Gariépy et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 83, 8907-8911]. Synthetic tridecapeptide analogues of STIb(6-18) with single amino acid substitutions at non-cysteine sites, as well as a truncated decapeptide lacking one of the three disulfide bridges, were prepared in order to examine the relationship between primary sequence and biological activity. The relative affinity of each analogue for intestinal cell receptors only partially correlates with their dose-dependent ability to cause diarrhea in suckling mice, suggesting that subsaturation doses of the enterotoxin with respect to receptor occupancy on intestinal cells may be sufficient to cause diarrhea. Two substitutions in the central-turn region of the molecule, namely, Asn12----Ala and Ala14----D-Ala, resulted in a large decrease or loss of receptor binding activity as compared to native STIb(6-18), pointing out the functional importance of this region. Analogues containing replacements at other sites showed moderate to slight reductions in biological activity. In particular, residues in the C-terminal region appear to be less important for activity, although their presence remains essential, since a truncated analogue missing the last three amino acids is inactive.(ABSTRACT TRUNCATED AT 250 WORDS)

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↗
PubMed1990

Dictyostelium myosin II heavy-chain kinase A is activated by autophosphorylation: studies with Dictyostelium myosin II and synthetic peptides.

One of the major sites phosphorylated on the Dictyostelium myosin II heavy chain by the Dictyostelium myosin II heavy-chain kinase A (MHCK A) is Thr-2029. Two synthetic peptides based on the sequence of the Dictyostelium myosin II heavy chain around Thr-2029 have been synthesized: MH-1 (residues 2020-2035; RKKFGESEKTKTKEFL-amide) and MH-2 (residues 2024-2035). Both peptides are substrates for MHCK A and are phosphorylated to a level of 1 mol of phosphate/mol. Tryptic digests indicate that the peptides are phosphorylated on the threonine corresponding to Thr-2029. When assays are initiated by the addition of MHCK A, the rate of phosphate incorporation into the peptides increases progressively for 4-6 min. The increasing activity of MHCK A over this time period is a result of autophosphorylation. Although each 130-kDa subunit of MHCK A can incorporate up to 10 phosphate molecules, 3 molecules of phosphate per subunit are sufficient to completely activate the kinase. Autophosphorylated MHCK A displays Vmax values of 2.2 and 0.6 mumol.min-1.mg-1 and Km values of 100 and 1200 microM with peptides MH-1 and MH-2, respectively. Unphosphorylated MHCK A displays a 50-fold lower Vmax with MH-1 but only a 2-fold greater Km. In the presence of Dictyostelium myosin II, the rate of autophosphorylation of MHCK A is increased 4-fold. If assays are performed at 4 degrees C (to slow the rate of MHCK A autophosphorylation), autophosphorylation can be shown to increase the activity of MHCK A with myosin II.

Amino Acid Sequence↗
PubMed1987

Importance of disulfide bridges in the structure and activity of Escherichia coli enterotoxin ST1b.

A 13-amino acid sequence of the Escherichia coli heat-stable enterotoxin ST1b encodes its receptor-binding and diarrheal functions. This sequence includes six cysteines involved in three intramolecular disulfide bridges. To determine the importance of disulfide bridges to the biological activity of ST1b, we synthesized 15 analogues of the tridecapeptide representing all possible replacements of two of the six cysteines by alanines. Only 2 analogues--namely, A6,11ST1b-(6-18) and A10,18ST1b(6-18)--could inhibit the binding of a radiolabeled analogue of ST1b to rat intestinal cells. The purified peptides were, respectively, 4200 and 130 times less effective as inhibitors than ST1b(6-18), the sequence that includes all six cysteines. In addition, both peptides produce diarrhea when given orally to suckling mice. These analogues share in common only two cysteines (Cys-7 and Cys-15), suggesting that four cysteines, two of which are Cys-7 and Cys-15, are necessary for activity. A pattern of disulfide linkages is proposed where Cys-7 is paired to Cys-15, Cys-6 to Cys-11, and Cys-10 to Cys-18, the preceding disulfide bridges being ranked in descending order of importance in terms of their respective contribution to the activity of the enterotoxin. Using this disulfide bridge arrangement and constraints derived from NMR spectroscopy, we propose a folding pattern for the toxic domain of ST1b.

Animals↗
PubMed1986

Structure of the toxic domain of the Escherichia coli heat-stable enterotoxin ST I.

Active fragments of the heat-stable enterotoxin ST I of Escherichia coli were chemically synthesized with the sequence Cys-Cys-Glu-Leu-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys-(Tyr) and studied by proton (1H NMR) and carbon-13 (13C NMR) nuclear magnetic resonance spectroscopy as a function of pH and temperature. All of the nonexchangeable protons in the 1H NMR spectrum were assigned. Although all amide protons were present at temperatures below 25 degrees C and and pH values below 6, some of the resonances are broad and could not be assigned. The temperature dependence of these broad resonances indicates a change in conformation that is localized in the N-terminus. Other amide protons disappear at higher temperatures owing to chemical exchange with the solvent. Sufficient resonance assignments can be made at high and low temperatures to permit structural conclusions to be made. The chemical shifts of the alpha-carbon protons indicate the presence of substantial structure, which was further defined with the observed pattern of nuclear Overhauser enhancements (NOEs), coupling constants, and exchange rates. The NMR data identify a turn from Ala-14 to Cys-18. A second likely turn is centered around the proline residue. An interresidue NOE between the alpha-carbon protons of Asn-12 and Gly-17 indicates that the molecule folds back on itself. The NMR information is sufficient to define the structure of the C-terminal region of ST I. Manual model building then indicated that one arrangement of the three disulfides is particularly compatible with the NMR data and van der Waals constraints. A model incorporating the disulfide arrangement proposed by Houghten and his co-workers [Houghten, R.A., Ostresh, J.M., & Klipstein, F.A. (1984) Eur. J. Biochem. 145, 157-162] and the NMR constraints was derived with the programs PROTO [Frayman, F. (1985) Ph.D. Thesis, Northwestern University] and NOEMOT [Lane, A.N., Lefévre, J.-F., & Jardetsky, O. (1986) Biochim. Biophys. Acta 867, 45-56].

Amides↗
PubMed1986

Design of a photoreactive analogue of the Escherichia coli heat-stable enterotoxin STIb: use in identifying its receptor on rat brush border membranes.

The Escherichia coli heat-stable enterotoxin, STIb was prepared by solid-phase peptide synthesis and purified to homogeneity by high-pressure liquid chromatography. This analogue was iodinated and shown to bind specifically to rat intestinal membranes. The radiolabeled peptide was derivatized at the amino terminus with the photoreactive heterobifunctional crosslinking agent N-hydroxysuccinimidyl p-benzoylbenzoate. This photoreactive probe also exhibited binding specificity. It was mixed with rat intestinal brush border membranes and photolyzed in the presence or absence of excess unlabeled STIb. Polyacrylamide gel electrophoresis performed in the presence of sodium dodecyl sulfate and 2-mercaptoethanol indicated that the peptide probe was crosslinked specifically to two molecular species of 57 and 75 kDa. One or both of these molecules appear to constitute the enterotoxin receptor or to be in close proximity to it.

Animals↗
PubMed1986

Peptide antisera as sequence-specific probes of protein conformational transitions: calmodulin exhibits calcium-dependent changes in antigenicity.

Local changes in conformation between the calcium-saturated and calcium-free forms of calmodulin were monitored using antisera to four peptides corresponding to three helical regions of the calcium-saturated protein. The N-terminal helix was monitored using antiserum to residues 9-19, calmodulin-(9-19); the C-terminal helix using antiserum to residues 141-148, calmodulin-(141-148); and the long central helix with antisera to residues 68-79 and 80-92, calmodulin-(68-79) and -(80-92). Crossreactivities of peptide antisera with calmodulin (either in the presence or absence of calcium) were determined using solution-phase and solid-phase immunoassays. When examined by the fluid-phase assay, all four peptides elicited antibody that precipitated radiolabeled apocalmodulin but not the calcium-saturated form of the protein. Similarly, when calmodulin was immobilized on a solid-support, only the calcium-free form readily bound the antibodies to calmodulin-(80-92) and -(141-148). In addition, the crossreactivity of antiserum to calmodulin-(68-79) with calcium-saturated calmodulin in solid phase was reduced by approximately equal to 40% relative to reactivity with apocalmodulin. According to the x-ray crystal structure of Ca2+-saturated calmodulin and the antigenic reactivity of calmodulin for the peptide antisera in the absence of calcium, the regions of the protein monitored by these antisera are exposed to the surface in both conformational states and probably accessible to specific antibodies. The apparent preference of peptide antibodies for one conformation of the molecule suggests that changes in the conformation of calmodulin occur in cognate sequences that are transformed by calcium from antigenic, flexible structures to less antigenic, relatively helical structures. Peptide antibodies may be employed as sequence-specific reporter molecules to monitor local conformational changes providing the cognate sequence is sterically accessible to antibody in both states but antigenic in only one.

Amino Acid Sequence↗
PubMed1985

Nuclear magnetic resonance determination of metal-proton distances in a synthetic calcium binding site of rabbit skeletal troponin C.

The binding of gadolinium to a synthetic peptide of 13 amino acid residues representing the calcium binding loop of site 3 of rabbit skeletal troponin C [AcSTnC(103-115)amide] has been studied by using proton nuclear magnetic resonance (1H NMR) spectroscopy. In particular, the proton line broadening and enhanced spin-lattice relaxation have been used to determine proton-metal ion distances for several assigned nuclei in the peptide-metal ion complex. These distances have been used in conjunction with other constraints and a distance algorithm procedure to demonstrate that the structure of the peptide-metal complex as shown by 1H NMR is consistent with the structure of the EF calcium binding loop in the X-ray structure of parvalbumin but that the available 1H NMR distances do not uniquely define the solution structure.

Animals↗
PubMed1985

T-cell activation by peptide antigen: effect of peptide sequence and method of antigen presentation.

A series of synthetic peptide analogues of a determinant recognized by the ovalbumin-specific, I-Ad-restricted, T-cell hybridoma 3DO-54.8 were synthesized. The resulting peptides were tested for activation of 3DO-54.8 cells by using glutaraldehyde-fixed cells as well as reconstituted membranes as antigen-presenting surfaces. The results show that the minimum epitope for activation of this T cell is between 7 and 11 amino acids in length. This region includes two important histidine residues. The order of preference of the various peptide analogues was the same regardless of the method of antigen presentation. However, the amount of peptide required for T-cell activation was considerably higher when reconstituted membranes, rather than fixed cells, were used as antigen-presenting surfaces.

Animals↗
PubMed1983

Primary sequence analysis and folding behavior of EF hands in relation to the mechanism of action of troponin C and calmodulin.

The primary sequence of EF hands encodes for elements of secondary structure which includes the presence of hydrophobic and charged domains in the helical regions of these sites. The hydrophobic and charged surfaces located in the N-terminal region of EF hands offer a potential site of interaction with complimentary surfaces on target proteins. Although the binding of calcium to the EF hands of calmodulin and troponin C may lead to a local exposure of these domains, it is the tertiary structure of these proteins that probably dictates the extent to which these domains are exposed and the selectively of these proteins for target proteins.

Amino Acid Sequence↗
PubMed1983

Lanthanide-induced peptide folding: variations in lanthanide affinity and induced peptide conformation.

The present work has demonstrated the utility of the diamagnetic lanthanides lutetium and lanthanum as metal binding probes for a synthetic 13-residue fragment representing calcium binding site 3 of rabbit skeletal troponin C (residues 103-115). The peptide conformation induced by these metals was monitored by the proton magnetic resonance at 270 MHz. The peptide affinity for these rare earths is 50-400 times higher than that for calcium (KLu3+, 1.3 X 10(4) M-1; KLa3+, 1.1 X 10(5) M-1; KCa2+, 3 X 10(2) M-1) which is related to the change in cation charge from 2+ to 3+. The peptide conformation induced by the presence of La3+ generates a different 1H NMR spectrum than the one observed for the lutetium-saturated peptide. Thus, it appears that these metals do not fold the peptide into exactly the same conformation. The resonance shifts observed during the Lu3+ titration are much smaller than those seen in the case of La3+ addition. The fact that lutetium binds less tightly than lanthanum to the peptide may be linked directly or indirectly to the difference in ionic radius between these metals (Lu3+, 0.86 A; La3+, 1.03 A). This may in turn indicate that the peptide primary sequence encodes for some aspects of metal ion specificity. The 1H NMR results also demonstrate that glycine-108 adopts a restricted geometry in the absence of metal such that its two alpha-carbon protons are in different environments which are further affected by the addition of either metal. These observations support the concept that geometric constraints arising from the particular peptide folding pattern near this residue correlate with the highly conserved nature of this site of the EF hand. This position remains occupied by glycine in most EF hand domains with the exception of known distorted calcium binding sites present in intestine calcium binding proteins and S-100.

Amino Acid Sequence↗
PubMed1983

Interaction of neuroleptic drugs with a synthetic calcium-binding peptide analog of site III of rabbit skeletal troponin C. Phenothiazine selective binding.

The effects of nine drugs on the CD spectra of a synthetic calcium binding analog of site III of rabbit skeletal troponin C, can generally be divided into 3 groups: (1) that consisting of haloperidol, benperidol, molindone and promethazine, had no effect on the CD spectrum or calcium sensitivity of the apopeptide; (2) that composed of structurally rigid thioxanthenes, induced CD-detectable structural change in the apopeptide but prevented Ca2+-induced structural change; (3) that consisting of chlorpromazine, trifluoperazine and fluphenazine, induced structural change in the peptide but had no effect on the Ca2+-induced structural change.

Animals↗
PubMed1983

Localization of a trifluoperazine binding site on troponin C.

Trifluoperazine (TFP) was shown to interact with the cyanogen bromide fragment 9 (CB9) (residues 84-135) of rabbit skeletal troponin C and with a synthetic peptide representing the N-terminal region of CB9. The phenothiazine did not affect the calcium binding property of CB9 as observed by proton magnetic resonance and circular dichroism spectroscopies. The calculated calcium binding constants for CB9 in the presence and absence of trifluoperazine were identical (KCa2+ = 1.3 X 10(5) M-1). Localization of the trifluoperazine binding site was achieved by analyzing the 1H NMR spectrum of CB9 and of a synthetic fragment corresponding to residues 90-104 of CB9. Drug-induced shifting and broadening of the ring protons of phenylalanine residues and the methyl resonances of alanine, leucine, and isoleucine residues suggest that the segment 95-102 is in close proximity to the phenothiazine aromatic region. The neighboring negative side chains in the peptide sequence also suggest that the single positive charge present on the piperazine nitrogens of trifluoperazine may interact with them and sterically block a region of interaction of calmodulin (CaM) and troponin C (TnC) with modulated proteins such as phosphodiesterase. Primary sequence analysis of CaM and troponin C reveals that a homologous hydrophobic region to site 3 is also found in the N-terminal region of site 1 of both calcium binding proteins. Binding of TFP to CB9 occurs both in the presence and absence of calcium since the hydrophobic region in these small fragments is completely accessible to TFP whether calcium is present or not. The dissociation constant of the drug to apoCB9 (8 microM) was obtained by ellipticity measurements at 222 nm and was comparable to the 5 microM value obtained by Levin and Weiss [Levin, R. M., & Weiss, B. (1978) Biochim. Biophys. Acta 540, 197-204] for calcium-saturated rabbit skeletal troponin C.

Amino Acids↗
PubMed1981

Calcium-induced protein folding. Structure-affinity relationships in synthetic analogs of the helix-loop-helix calcium binding unit.

Three peptide analogs of the helix-loop-helix Ca2+ binding unit, 21-, 26-, and 34-residues in length, similar in sequence to rabbit skeletal troponin C site III have been prepared by the solid-phase method. The CD spectra of the 21-residue fragment indicated very little secondary structure in aqueous medium in the absence of Ca2+. Addition of Ca2+ increased the secondary structure of the peptide but the KCa was very weak, 3.1 x 10(2) M-1. The same peptide in hydrophobic medium in the absence of Ca2+ had considerable secondary structure and the KCa value increased considerably, 3.5 x 10(5) M-1. The 26-residue peptide, containing 5 more residues on the NH2 terminus of the 21-residue peptide, showed slightly more secondary structure in aqueous medium in the absence of Ca2+. Addition of Ca2+ to this peptide raised the amount of secondary structure in the metal ion-peptide complex and resulted in a higher KCa value, 3.8 x 10(4) M-1. By assuming that the COOH-terminal region of the 26-residue peptide-metal ion complex assumes a structure similar to that of the 21-residue peptide-metal ion complex, one is able to assign the increase in structure to the NH2-terminal side of the Ca2+-binding loop. Hydrophobic medium further increased the secondary structure of this peptide and also increased the KCa value to 4.5 x 10(5) M-1, a value similar to that obtained for the 21-residue peptide. The 34-residue peptide contained a further 8 amino acid residues on the NH2 terminus of the 26-residue peptide. This peptide had considerable secondary structure in aqueous medium which increased in the presence of Ca2+. The peptide has a reasonable affinity for Ca2+ in aqueous medium, KCa = 2.6 x 10(5) M-1. Again, a hydrophobic medium increased both the amount of secondary structure and the Ca2+ affinity constant, KCa = 9.2 x 10(5) M-1. A model of Ca2+-induced folding of the three peptides under different conditions is described and results obtained from this model are used to describe Ca2+ binding to the four Ca2+ binding units in rabbit skeletal troponin C.

Animals↗
PubMedDate not supplied

The use of Shiga-like toxin 1 in cancer therapy.

The ribosome-inactivating protein, Shiga-like toxin-1 (SLT-1, SLT-I, Verotoxin 1, VT1) targets cells that express the glycolipid globotriaosylceramide (CD77) on their surface. The frequent occurrence of SLT-1 receptors on tumor cells derived from patients with hematological cancers (follicular lymphoma, multiple myeloma, chronic lymphocytic leukemia) and their absence on human CD34(+) hematopoietic stem cells suggest the ex vivo use of Shiga-like toxin-1 in purging CD77(+) tumor cells from autologous stem cell transplants. SLT-1 receptors are also commonly expressed on breast cancer, ovarian cancer and astrocytoma cells. In particular, the sensitivity of astrocytoma cell lines to this toxin provides an opportunity for using SLT-1 in vivo in the context of treating patients afflicted by this common form of brain tumor. Finally, the known structural features of SLT-1 allow one to contemplate altering its receptor specificity in an effort to target CD77(-) tumor cell populations.

Antineoplastic Agents↗
PubMedDate not supplied

Rapid synthesis and introduction of a protected EDTA-like group during the solid-phase assembly of peptides.

A simple two-step procedure is reported for the synthesis of a tert-butyl ester protected form of an EDTA-like bifunctional chelating agent. This reagent can be easily introduced on any available amino group during the assembly of peptides on solid-phase supports. Using the model tetradecapeptide OVA(323-336), we have introduced an EDTA group at the N-terminus of this T-cell epitope and confirmed that the EDTA group is present on the molecule, can chelate metals, and does not affect the biological activity of the peptide.

Antigens↗