Interaction of Cholera Toxin B-subunit with Human T-lymphocytes.

In this work, 125I-labeled cholera toxin B-subunit (CT-B) (specific activity 98 Ci/mmol) was prepared, and its high-affinity binding to human blood T-lymphocytes (Kd = 3.3 nM) was determined. The binding of the 125I-labeled CT-B was inhibited by unlabeled interferon-α2 (IFN-α2), thymosin-α1 (TM-α1), and by the synthetic peptide LKEKK, which corresponds to sequences 16-20 of human TM-α1 and 131-135 of IFN-α2 (Ki 0.8, 1.2, and 1.6 nM, respectively), but was not inhibited by the unlabeled synthetic peptide KKEKL with inverted sequence (Ki > 1 µM). In the concentration range of 10-1000 nM, both CT-B and peptide LKEKK dose-dependently increased the activity of soluble guanylate cyclase (sGC) but did not affect the activity of membrane-bound guanylate cyclase. The KKEKL peptide tested in parallel did not affect sGC activity. Thus, the CT-B and peptide LKEKK binding to a common receptor on the surface of T-lymphocytes leads to an increase in sGC activity.

Thermodynamic, conformational and functional properties of the human C1q globular heads in the intact C1q molecule in solution.

Thermodynamic. conformational and functional properties of the human C1q globular heads (hgC1q) were studied with the experimental approaches, which allow investigating these properties in the intact hC1q molecule in solution. Surprisingly, the scanning calorimetry data reveal a low level of cooperativity of interactions between the hgC1q A, B and C domains even at a neutral pH area. Ionization of His residues due to acidification of the medium at the pH range from 6 to 5 or the chemical modification of His residues completely abolishes the cooperative interactions between the domains without significant effect on their conformation. The thermodynamic data provide evidence that the hgC1q module is composed of three structurally independent A, B and C globular domains characterized by the practically identical thermal stability and very similar enthalpy of melting. The spectroscopic studies and modification with 2-oxy-5-nitrobenzylbromide (ONBB) indicate that Trp residues in the hgC1q A and C domains are accessible to the solvent that has been confirmed by the hgC1q crystal structure solved and refined to 1.9 A. The modification of Trp residues significantly affects the complement-dependent cytotoxicity without noticeable effect on the hC1q conformation. These data provide evidence that Trp residues are the components of immunoglobulin-binding sites both in the hgC1q A and C domains.

Synthetic peptide VKGFY and its cyclic analog stimulate macrophage bactericidal activity through non-opioid beta-endorphin receptors.

We synthesized linear and cyclic pentapeptides corresponding to the sequence 369-373 of human immunoglobulin G heavy chain--VKGFY (referred to as pentarphin and cyclopentarphin, respectively). The effect of pentarphin and cyclopentarphin on phagocytosis of Salmonella typhimurium virulent 415 strainbacteria by mouse peritoneal macrophages in vitro was studied. Control experiments showed that macrophages actively captured these bacteria, but did not digest them: the captured microbes were viable and continued to proliferate inside the phagocytes; within 12 h all macrophage monolayer was destroyed (incomplete phagocytosis). If 1 nM pentarphin or cyclopentarphin was added to the cultivation medium, macrophage bactericidal activity was significantly increased and they digested all captured microorganisms within 6 h (complete phagocytosis). To study the receptor binding properties of pentarphin and cyclopentarphin we prepared (125)I-labeled pentarphin (179 Ci/mmol specific activity). The binding of (125)I-labeled pentarphin to mouse peritoneal macrophages was high-affinity (K(d) = 3.6 +/- 0.3 nM) and saturable. Studies on binding specificity revealed that this binding was insensitive to naloxone and [Met(5)]enkephalin, but completely inhibited by unlabeled cyclopentarphin (K(i) = 2.6 +/- 0.3 nM), immunorphin (K(i) = 3.2 +/- 0.3 nM), and beta-endorphin (K(i) = 2.8 +/- 0.2 nM). Thus, the effects of pentarphin and cyclopentarphin on macrophages are mediated by naloxone-insensitive receptors common for pentarphin, cyclopentarphin, immunorphin, and beta-endorphin.

Donor strand complementation mechanism in the biogenesis of non-pilus systems.

The F1 antigen of Yersinia pestis belongs to a class of non-pilus adhesins assembled via a classical chaperone-usher pathway. Such pathways consist of PapD-like chaperones that bind subunits and pilot them to the outer membrane usher, where they are assembled into surface structures. In a recombinant Escherichia coli model system, chaperone-subunit (Caf1M:Caf1n) complexes accumulate in the periplasm. Three independent methods showed that these complexes are rod- or coil-shaped linear arrays of Caf1 subunits capped at one end by a single copy of Caf1M chaperone. Deletion and point mutagenesis identified an N-terminal donor strand region of Caf1 that was essential for polymerization in vitro, in the periplasm and at the cell surface, but not for chaperone-subunit interaction. Partial protease digestion of periplasmic complexes revealed that this region becomes buried upon formation of Caf1:Caf1 contacts. These results show that, despite the capsule-like appearance of F1 antigen, the basic structure is assembled as a linear array of subunits held together by intersubunit donor strand complementation. This example shows that strikingly different architectures can be achieved by the same general principle of donor strand complementation and suggests that a similar basic polymer organization will be shared by all surface structures assembled by classical chaperone-usher pathways.

beta-Endorphin-like peptide SLTCLVKGFY is a selective agonist of nonopioid beta-endorphin receptor.

It has been found that beta-endorphin (beta-END) and a synthetic beta-END-like decapeptide Ser-Leu-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr (termed immunorphin, IMN) corresponding to the sequence 364-373 of human IgG heavy chain stimulate Con A-induced proliferation of T lymphocytes from the blood of healthy donors. [Met(5)]enkephalin ([Met(5)]ENK) and an antagonist of opioid receptors naloxone (NAL) tested in parallel were not active. The stimulating effect of beta-END and IMN on T lymphocyte proliferation was not inhibited by NAL. Studies on receptor binding of (125)I-labeled IMN to T lymphocytes revealed that it binds with high affinity to NAL-insensitive binding sites (K(d) = 7.0 +/- 0.3 nM). Unlabeled beta-END completely inhibited the specific binding of (125)I-labeled IMN to NAL-insensitive binding sites on T lymphocytes (K(i) = 1.1 +/- 0.2 nM). Thus, beta-END and IMN bind to common NAL-insensitive binding sites on T lymphocytes and enhance Con A-induced proliferation of these cells.

Secretion of recombinant proteins via the chaperone/usher pathway in Escherichia coli.

F1 antigen (Caf1) of Yersinia pestis is assembled via the Caf1M chaperone/Caf1A usher pathway. We investigated the ability of this assembly system to facilitate secretion of full-length heterologous proteins fused to the Caf1 subunit in Escherichia coli. Despite correct processing of a chimeric protein composed of a modified Caf1 signal peptide, mature human interleukin-1beta (hIL-1beta), and mature Caf1, the processed product (hIL-1beta:Caf1) remained insoluble. Coexpression of this chimera with a functional Caf1M chaperone led to the accumulation of soluble hIL-1beta:Caf1 in the periplasm. Soluble hIL-1beta:Caf1 reacted with monoclonal antibodies directed against structural epitopes of hIL-1beta. The results indicate that Caf1M-induced release of hIL-1beta:Caf1 from the inner membrane promotes folding of the hIL-1beta domain. Similar results were obtained with the fusion of Caf1 to hIL-1beta receptor antagonist or to human granulocyte-macrophage colony-stimulating factor. Following coexpression of the hIL-1beta:Caf1 precursor with both the Caf1M chaperone and Caf1A outer membrane protein, hIL-1beta:Caf1 could be detected on the cell surface of E. coli. These results demonstrate for the first time the potential application of the chaperone/usher secretion pathway in the transport of subunits with large heterogeneous N-terminal fusions. This represents a novel means for the delivery of correctly folded heterologous proteins to the periplasm and cell surface as either polymers or cleavable monomeric domains.

An extended hydrophobic interactive surface of Yersinia pestis Caf1M chaperone is essential for subunit binding and F1 capsule assembly.

A single polypeptide subunit, Caf1, polymerizes to form a dense, poorly defined structure (F1 capsule) on the surface of Yersinia pestis. The caf-encoded assembly components belong to the chaperone-usher protein family involved in the assembly of composite adhesive pili, but the Caf1M chaperone itself belongs to a distinct subfamily. One unique feature of this subfamily is the possession of a long, variable sequence between the F1 beta-strand and the G1 subunit binding beta-strand (FGL; F1 beta-strand to G1 beta-strand long). Deletion and insertion mutations confirmed that the FGL sequence was not essential for folding of the protein but was absolutely essential for function. Site-specific mutagenesis of individual residues identified Val-126, in particular, together with Val-128 as critical residues for the formation of a stable subunit-chaperone complex and the promotion of surface assembly. Differential effects on periplasmic polymerization of the subunit were also observed with different mutants. Together with the G1 strand, the FGL sequence has the potential to form an interactive surface of five alternating hydrophobic residues on Caf1M chaperone as well as in seven of the 10 other members of the FGL subfamily. Mutation of the absolutely conserved Arg-20 to Ser led to drastic reduction in Caf1 binding and surface assembled polymer. Thus, although Caf1M-Caf1 subunit binding almost certainly involves the basic principle of donor strand complementation elucidated for the PapD-PapK complex, a key feature unique to the chaperones of this subfamily would appear to be capping via high-affinity binding of an extended hydrophobic surface on the respective single subunits.

Synthetic beta-endorphin-like peptide immunorphin binds to non-opioid receptors for beta-endorphin on T lymphocytes.

The synthetic decapeptide H-SLTCLVKGFY-OH (termed immunorphin) corresponding to the sequence 364-373 of the CH3 domain of human immunoglobulin G heavy chain was found to compete with [125I]beta-endorphin for high-affinity receptors on T lymphocytes from the blood of healthy donors (K(i) = 0.6 nM). Besides immunorphin, its synthetic fragments H-Val-Lys-Gly-Phe-Tyr-OH (K(i) = 15 nM), H-Leu-Val-Lys-Gly-Phe-Tyr-OH (K(i) = 8.0 nM), H-Cys-Leu-Val-Lys-Gly-Phe-Tyr-OH (K(i) = 3.4 nM), H-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr-OH (K(i) = 2.2 nM), H-Leu-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr-OH (K(i) = 1.0 nM) possessed the ability to inhibit specific binding of [125I]beta-endorphin to T lymphocytes. Tests of the specificity of the receptors revealed that they are not sensitive to naloxone and Met-enkephalin, i.e. they are not opioid receptors. K(d) values characterizing the specific binding of 125I- labeled immunorphin and its fragment H-Val-Lys-Gly-Phe-Tyr-OH to the receptors have been determined to be 7.4 nM and 36.3 nM, respectively.

Influence of synthetic peptide corresponding to the ACTH-like sequence of human immunoglobulin G1 on activity of murine thymocytes and peritoneal macrophages.

The purpose of the present study was to investigate properties and mechanism of action of the synthetic adrenocorticotropin (ACTH)-like peptide VKKPGSSVKV, corresponding to the sequence 11-20 of the variable part of human immunoglobulin G1 (IgG1) heavy chain. The ACTH-like peptide was shown to act as an immunosuppressive agent in vitro: it inhibits the blast transformation of mouse thymocytes and reduces the spontaneous motility of mouse peritoneal macrophages as well as their bactericidal activity against Salmonella typhimurium 415 virulent strain bacteria. High affinity receptors for the ACTH-like peptide were found on thymocytes and macrophages and shown to be at the same time the receptors for ACTH. The kinetic characteristics of the ACTH-like peptide and 125I-labeled ACTH (13-24) (ACTH 'address segment') specific binding to the receptors were determined. It was found that the ACTH-like peptide binding to the receptors on target cells is accompanied by an increase in both adenylate cyclase activity and intracellular cAMP content.

Influence of synthetic peptide corresponding to the ACTH-like sequence of human immunoglobulin G1 on proliferation of lymphoblastoid cells.

Influence of the ACTH-like peptide H-Val-Lys-Lys-Pro-Gly-Ser-Ser-Val-Lys-Val-OH corresponding to the sequence 11-20 of the variable part of human immunoglobulin G1 (IgG1) heavy chain on growth of MT-4 human T-lymphoblastoid cell line was investigated. It was found that the ACTH-like peptide at concentration range 10(-11) -10(-7) M inhibits the proliferation of MT-4 cells. Labeled ACTH 'address segment' [(125)I]ACTH (13-24) was used to establish that MT-4 cells express specific receptors for ACTH (K(d) = 97 pM). The ACTH-like peptide and human ACTH (but not IgG1 heavy chain) were shown to compete with [(125)I]ACTH (13-24) for binding to these receptors (K(i1) = 0.38 nM and K(i2) = 0.34 nM).

Study of immunosuppressive activity of a synthetic decapeptide corresponding to an ACTH-like sequence of human immunoglobulin G1.

The synthetic ACTH-like decapeptide H-Val-Lys-Lys-Pro-Gly- Ser-Ser-Val-Lys-Val-OH, corresponding to amino acid residues 11-20 of the variable part of the human IgG1 heavy chain (referred to as immunocortin) was found to have an immunosuppressive effect on cells in vitro: it inhibits blast transformation of mouse thymocytes and reduces spontaneous motility of mouse peritoneal macrophages as well as their bactericidal activity against the virulent bacterial strain Salmonella typhimurium 415. Tritium-labeled immunocortin binds with high affinity to ACTH receptors on thymocytes and macrophages (Kd 2. 1 and 2.5 nM, respectively) and activates adenylate cyclase in these cells. Thus, the interaction of immunocortin with the target cell includes the following main steps: binding to the receptor, activation of adenylate cyclase, and elevation of the intracellular content of cAMP.

Structural and functional significance of the FGL sequence of the periplasmic chaperone Caf1M of Yersinia pestis.

The periplasmic molecular chaperone Caf1M of Yersinia pestis is a typical representative of a subfamily of specific chaperones involved in assembly of surface adhesins with a very simple structure. One characteristic feature of this Caf1M-like subfamily is possession of an extended, variable sequence (termed FGL) between the F1 and subunit binding G1 beta-strands. In contrast, FGS subfamily members, characterized by PapD, have a short F1-G1 loop and are involved in assembly of complex pili. To elucidate the structural and functional significance of the FGL sequence, a mutant Caf1M molecule (dCaf1M), in which the 27 amino acid residues between the F1 and G1 beta-strands had been deleted, was constructed. Expression of the mutated caf1M in Escherichia coli resulted in accumulation of high levels of dCaf1M. The far-UV circular dichroism spectra of the mutant and wild-type proteins were indistinguishable and exhibited practically the same temperature and pH dependencies. Thus, the FGL sequence of Caf1M clearly does not contribute significantly to the stability of the protein conformation. Preferential cleavage of Caf1M by trypsin at Lys-119 confirmed surface exposure of this part of the FGL sequence in the isolated chaperone and periplasmic chaperone-subunit complex. There was no evidence of surface-localized Caf1 subunit in the presence of the Caf1A outer membrane protein and dCaf1M. In contrast to Caf1M, dCaf1M was not able to form a stable complex with Caf1 nor could it protect the subunit from proteolytic degradation in vivo. This demonstration that the FGL sequence is required for stable chaperone-subunit interaction, but not for folding of a stable chaperone, provides a sound basis for future detailed molecular analyses of the FGL subfamily of chaperones.

Thiol/disulfide exchange between small heat shock protein 25 and glutathione.

Murine small heat shock protein 25 (Hsp25) carries a single Cys-residue at position 141 of its amino acid sequence. In glutathione redox buffers, Hsp25 equilibrates between reduced protein (PSH), mixed disulfide (PSSG) and protein dimer (PSSP) forms. At highly oxidative conditions, native Hsp25 predominantly forms PSSP while denatured Hsp25 forms PSSG. Conversion of PSSP to PSSG correlates with urea and temperature denaturation of tertiary and/or quaternary structure of Hsp25. At pH 7.5, 25 degreesC, the second-order rate constant for the formation of PSSP in the reaction of native PSH with GSSG is 20.1+/-1.4 M-1 min-1. This is approximately 3-fold lower than the reaction velocity of GSSG with a typical, unhindered thiol of pKa 8.6. At redox equilibrium, the fractions of PSSP, PSSG, and PSH depend on the concentration of GSH and less on the ratio [GSH]/[GSSG] (R). At a constant R, the fractions of PSSG and PSH species depend similarly on GSH concentration, being approximately equal in glutathione redox buffers with low R. It is concluded that in oligomeric complexes, Hsp25 subunits in vitro form stable dimers, in which the reacting -SH groups are in a proximity to form intersubunit disulfide bonds. Within a reaction of one of these -SH groups with GSSG, steric hindrances and electrostatic repulsion complicate penetration of another reduced or oxidized glutathione molecule to the reaction site.

Analogous conformations of both binding and effector regions in cyclosporin A, FK506 and rapamycin.

Three immunosuppressant drugs, cyclosporin A, FK506 and rapamycin were compared in their three-dimensional structures by computer modelling. The pairwise comparisons of cyclosporin A, FK506 and rapamycin show two structurally common fragments. One fragment is Mle9-Bmt1 region in cyclosporin A, C22-O5 region in FK506 and C29-O5 region in rapamycin. Another fragment is Mle4-Mle6 region in cyclosporin A and C14-C21 regions in FK506 and rapamycin. The correspondence of the structurally analogous regions with the regions which are involved in the interactions with peptidyl-prolyl cis/trans isomerases and calcineurin or FKBP-rapamycin-associated protein is discussed.

Influence of L-glutamic acid on binding of interleukin-1beta, tumour necrosis factor-alpha and interleukin-6 to HL-60 cells.

To elucidate the mechanism of differentiating activity on L-Glu to HL-60 cells, its influence on binding of human recombinant interleukine-1beta (rHuIL-1beta), tumour necrosis factor-alpha (rHuTNF-alpha) and interleukine-6 (IL-6) by HL-60 cells was studied. It was established, that L-Glu converted the high affinity binding of [125I]rHuIL-1beta (Kd = 0.32 nM) and [125I]rHuIL-6 (Kd = 0.075 nM) to HL-60 cells into low affinity (corresponding Kd values -13.3 and 2.1 nM) at concentration 0.1 microM. The preincubation for an hour of HL-60 cells with 0.1 microM L-Glu was shown to result in an increase of affinity and number of [125I]rHuTNF-alpha binding sites. Thus, L-Glu decreases the sensitivity of the HL-60 cells to IL-1beta and IL-6 and increases TNF-alpha binding at concentration 0.1 microM.

Investigation of the cooperative structure of Fc fragments from myeloma immunoglobulin G.

The cooperative structure of Fc fragments prepared from myeloma human IgG1 was studied using scanning microcalorimetry and fluorescence at pH 4.2-8.0. It was shown that the first to be melted are CH2 domains whose interaction with each other is rather weak, while that with CH3 domains is strong. Then CH3 domains which form a single cooperative block are melted. The data for the structure of the Fc fragment in solution agree with the X-ray data according to which the interaction between CH2 domains is mediated by the carbohydrate moiety while the two CH3 domains are strongly associated. The presence of intensive CH2-CH3 interaction is a distinctive feature of the state of the Fc fragment in the given pH region as compared to that at pH <4.1 [Tischenko, V. M., et al. (1982) Eur. J. Biochem. 126, 517-521; Ryazantsev, S., et al. (1990) Eur. J. Biochem. 190, 393-399]. First, cis interactions greatly increase the free energy of the native structure stabilization in CH2 domains. Second, they decrease this energy for CH3 domains when compared to the state of the latter at pH 3.8 or within the Fc' fragment (the dimer of CH3 domains). The temperature and enthalpy of melting of CH2 domains coincide in all the samples studied despite heterogeneity of the carbohydrate moiety. Thus, it may be postulated that the conservative part of CH2 domains makes a cardinal contribution to the interaction of these domains with the carbohydrate moiety.

Molecular models of two competitive inhibitors, IL-2delta2 and IL-2delta3, generated by alternative splicing of human interleukin-2.

Molecular models of IL-2delta2 and IL-2delta3, two alternative splice variants of human IL-2 without exon 2 and 3, respectively, are described. These alternative splice variants attract particular interest as potential competitive inhibitors of the cytokine. Tertiary structure of IL-2 consists of four-helix bundle including helices A, B, C and D and a beta-pleated sheet. Exon 2 encodes the A-B loop (Asn30-Lys49 residues) linking helices A and B running in one direction. Rotation of the helix A around putative centre during the construction of IL-2delta2 model have not produced any significant changes in the hydrophobic core of IL-2 molecule. However, a large hole was formed on the surface of IL-2delta2 molecule instead of A-B loop in IL-2 fold. A high affinity IL-2 receptor is formed by combination of alpha, beta, and gamma(c) chains. Comparison of the model of the receptor bound IL-2 with the model of IL-2delta2 has shown that their beta-chain binding sites have minimum differences as distinct from alpha and gamma(c) chain-binding sites. Exon 3 encodes Ala50-Lys97 fragment which forms helices B and C with their short connecting loop. Model IL-2delta3 consists of helices A and D and long linking loop. This loop was composed of A-B and C-D loops which run in opposite directions in IL-2 structure and contain beta-strands making a beta-pleated sheet. Conformation of the linking loop relatively to helices A and D was stabilized by creation of a disulphide bond between cysteines 105 and 125. In addition, the hydrophobic residues of beta-sheet interact with the hydrophobic surface of A-D helical complex and close the latter from contacts with solution. Comparison of the model of IL-2 bound to receptor with IL-2delta3 model has shown that absence of helices B and C in IL-2delta3 model results in insignificant conformational changes only in residues interacting with gamma(c) chain of the receptor. The beta/gamma(c) heterodimer is an intermediate affinity receptor of IL-2. Most likely, both IL-2delta2 and IL-2delta3 are naturally occurring IL-2 antagonists since they keep the ability of binding with an intermediate affinity receptor of this cytokine and fail to engage the alpha chain of its high affinity receptor.

Molecular model of an alternative splice variant of human IL-4, IL-4 delta 2, a naturally occurring inhibitor of IL-4-stimulated T cell proliferation.

The molecular model of IL-4 delta 2, a naturally occurring splice variant of human IL-4 with exons 1, 3, and 4 in an open reading frame, is described. The second exon codes the main part of the long loop AB connected the helices A and B in parallel superposition. Therefore the hydrophobic core and the native fold of the rest part of IL-4 delta 2 molecule could be preserved without any significant changes only in the case of revolution of the helix A relative to other helices. In the result, the dominated a left-handed four-helix bundle structure of IL-4 with an up-up-down-down structural pattern is transformed to the IL-4 delta 2 structure with a down-up-down-down structural pattern.

Influence of the conserved disulphide bond, exposed to the putative binding pocket, on the structure and function of the immunoglobulin-like molecular chaperone Caf1M of Yersinia pestis.

The Yersinia pestis protein Caf1M is a typical representative of a subfamily of periplasmic molecular chaperones with characteristic structural and functional features, one of which is the location of two conserved cysteine residues close to the putative binding pocket. We show that these residues form a disulphide bond, the reduction and alkylation of which significantly increases the dissociation constant of the Caf1M-Caf1 (where Caf 1 is a polypeptide subunit of the capsule) complex [from a Kd of (4.77+/-0.50)x10(-9) M for the intact protein to one of (3.68+/-0.68)x10(-8) M for the modified protein]. The importance of the disulphide bond for the formation of functional Caf1M in vivo was demonstrated using an Escherichia coli dsbA mutant carrying the Y. pestis f1 operon. In accordance with the CD and fluorescence measurements, the disulphide bond is not important for maintenance of the overall structure of the Caf1M molecule, but would appear to affect the fine structural properties of the subunit binding site. A three-dimensional model of the Caf1M-Caf1 complex was designed based on the published crystal structure of PapD (a chaperone required for Pap pili assembly) complexed with a peptide corresponding to the C-terminus of the papG subunit. In the model the disulphide bond is in close proximity to the invariant Caf1M Arg-23 and Lys-142 residues that are assumed to anchor the C-terminal group of the subunit. The importance of this characteristic disulphide bond for the orchestration of the binding site and subunit binding, as well as for the folding of the protein in vivo, is likely to be a common feature of this subfamily of Caf1M-like chaperones. A possible model for the role of the disulphide bond in Caf1 assembly is discussed.