Peptide analogs of thymopentin distinguish distinct thymopoietin receptor specificities on two human T cell lines

Thymopoietin, a polypeptide hormone of the thymus, and the synthetic pentapeptide thymopentin, corresponding to thymopoietin32-36, both induced elevations of intracellular cyclic GMP in two human T cell lines, CEM and MOLT-4. In contrast, the closely related polypeptide thysplenin, which differs from thymopoietin at position 34, induced intracellular cyclic GMP elevation in MOLT-4 but not in CEM. We synthesized a series of penta- and tetrapeptide analogs of amino acids 32-36 of human thymopoietin and thysplenin, and now show that distinct patterns of activity can be obtained in these small peptides, with selectivity for cyclic GMP elevation in MOLT-4 alone or CEM alone. This suggests that the thymopoietin receptors (TPR) on these two human T cell lines are distinguishable by their differing ligand specificities, and we have termed them alpha TPR and beta TPR for CEM and MOLT-4 receptors, respectively.     

Structural and dynamic properties of thymopoietin mimetics

We propose a hypothesis that the T-cell receptor is a possible target of thymic hormones. We modelled the conformational dynamics of thymopentin and its structural variants in solution, as well as the interactions of these short peptides with the proposed molecular target. Thymopentin is a five-amino-acid fragment of the thymic hormone thymopoietin (residues 32 to 36) that reproduces the immunomodulatory activity of the complete hormone. Using molecular dynamics and flexible docking methods, we demonstrated high-affinity binding of thymopentin and its prospective mimetics with the T-cell receptor. The calculated biological activity spectra of thymopentin and its two promising modifications can be used in immunomodulatory activity screenings with live systems.     

Structural requirements for the biological activity of thymopentin analogs

Thymopentin, the synthetic pentapeptide Arg-Lys-Asp-Val-Tyr, corresponds to residues 32-36 of the thymic hormone thymopoietin. Thymopentin, like thymopoietin, induces intracellular cGMP elevations in the human T-cell line, CEM. Thymopentin also displaces radiolabeled thymopoietin from a receptor glycoprotein prepared from CEM cells, provided that a nonapeptide corresponding to thymopoietin is added to block thymopoietin binding to an additional binding site. Twenty nine analogs with single position substitutions were synthesized by solid-phase or classical solution synthesis, and are evaluated in these assays. All analogs that were active gave positive effects in both assays. A number of substitutions were tolerated at positions 2, 4, and 5, but there was an absolute requirement for L- or D-Arg at position 1 and L- or D-Asp at position 3 to maintain biological activity.     

Arg-Lys-Asp-Val-Tyr (thymopentin) accelerates the cholinergic-induced inactivation (desensitization) of reconstituted nicotinic receptor

1. The thymic hormone thymopoietin blocks neuromuscular transmission and was proposed (Goldstein, 1974) as a modulator of synaptic conductivity. 2. The cholinergic-induced inactivation of nicotinic receptor reconstituted into asolectin lipid vesicles was studied in the presence and in the absence of thymopentin, a synthetic pentapeptide corresponding to positions 32-36 of thymopoietin. 3. The present data show that thymopentin accelerates desensitization of the nicotinic acetylcholine receptor, supporting the aforementioned physiological role proposed for thymopoietin. 4. They also suggest that the hormone itself and/or a yet unidentified hormine-derived peptide fragment may act as an endogenous ligand for nicotinic acetylcholine receptor desensitization.     

Short in vitro half-life of thymopoietin32--36 pentapeptide in human plasma.

Thymopoietin32--36 (TP5) is a synthetic pentapeptide that has the biological activity of its parent molecule, the 49 amino acid thymic hormone thymopoietin. Tritiated thymopoietin32--36 (3 /-TP5) was prepared by reductive tritiation of dibromotyrosyl-TP5. The stability of 3 H-TP5 in human plasma was studied by analyzing samples by thin-layer chromatography at different time points and quantitating the radioactivity associated with TP5 (Arg-Lys-Asp-Val-Tyr) and its tyrosyl-containing breakdown products (Lys-Asp-Val-Tyr, Asp-Val-Tyr, Val-Tyr, Tyr). In plasma (but not in saline) the pentapeptide was rapidly degraded (apparent t1/2 approximately 30 seconds) with the corresponding appearance of radioactivity associated with the other tyrosyl-containing reference compounds. These data imply that the pentapeptide, which is active in vivo, may rapidly trigger changes in responsive cells; sustained circulating levels may not be required for activity.     

Structural similarity and functional activity of molecular fragments of staphylococcal enterotoxin B and thymopoietin II

Structural similarity between Staphylococcus enterotoxin B and a thymus hormone, thymopoietin, has been revealed concerning thymopentin, one of active centres of thymopoietin. Fragments of enterotoxin B homologous to thymopoietin and their analogues were synthesized their immunological activity studied. Acetyl derivative of the fragment 225-229 of enterotoxin B increased infectional resistance and stimulated delayed type of hypersensitivity reaction.     

Biologically active analogs of thymopentin with enhanced enzymatic stability

Thymopentin, a synthetic pentapeptide fragment of thymopoietin (residues 32–36, Arg-Lys-Asp-Val-Tyr) is biologically active but susceptible to proteolytic digestion. Analogs were synthesized and studied for biological activity and susceptibility to peptidases. Amino acid changes were incorporated at positions known to not affect activity adversely and N-terminal acetylation and C-terminal amidation were used to increase resistance to proteolytic degradation by exopeptidases. Ac-Pro²-TP5-NH₂ and Aib²-TP5-NH₂ retained activity and were shown to exhibit a high degree of stability when incubated in human serum.     

Truncated atrial natriuretic peptide analogs. Comparison between receptor binding and stimulation of cyclic GMP accumulation in cultured vascular smooth muscle cells

A series of truncated atrial natriuretic peptide analogs were examined as a means of defining the structural requirements for receptor occupancy and stimulation of cyclic GMP accumulation in bovine aortic smooth muscle cells. It was determined that deletion of amino acids from the carboxyl and/or amino termini of the peptides diminished their ability to increase cyclic GMP levels. Deletion of amino acids from the carboxyl terminus had the greatest effect, and atrial natriuretic peptide analogs lacking the carboxyl-terminal phenylalanyl-arginyl-tyrosine tripeptide were 100-1000-fold less active than parent compounds in stimulating intracellular cyclic GMP accumulation. In marked contrast to the cyclic GMP effects, deletion of amino- and/or carboxyl-terminal amino acids had only minor effects on the affinity of the peptides for specific smooth muscle cell-associated receptors. Peptide analogs lacking the phenylalanyl-arginyl-tyrosine tripeptide bound to receptors with an affinity only 1.1-5-fold weaker than the parent compounds. Thus, there was no correlation between apparent receptor binding affinity of atrial natriuretic peptide analogs and potency of these same peptides for stimulating intracellular cyclic GMP accumulation. Furthermore, analogs that bound to receptors and failed to elicit significant cyclic GMP responses did not antagonize or modulate increases in cyclic GMP induced by parent compounds. These data are most consistent with the existence of multiple subpopulations of atrial natriuretic peptide receptors on aortic smooth muscle cells.     

C-terminal fragments of thymopoietin favor formation of F-actin bundles: Electron microscopic data

Electron microscopy showed that PEP₃₃, a synthetic peptide corresponding to the C-terminal part of the thymic hormone thymopoietin, favors bundling of F-actin filaments in the presence of 0.1 M KCl. The structure of PEP₃₃ aggregates located within bundles between actin filaments is very similar to the structure of aggregates visible in preparations of pure PEP₃₃. No changes were observed in the structure of G-actin in the presence of PEP₃₃. A similar though weaker bundling effect was also detected for PEP₅, or thymopentin, a fragment of PEP₃₃. This peptide favors the formation of bundles of actin filaments of small size. The possible role of aggregation of actin filaments under the action of thymopoietin peptides is discussed in the light of the fact that the systematic release of thymopoietin from thymus leads to a phenomenon characteristic of the serious neuromuscular disease myasthenia gravis.     

Immunohistochemical localization of thymopoietin with an antiserum to synthetic Cys-thymopoietin28-39

Thymopoietin-containing cells in the thymus were identified immunohistochemically using murine antiserum generated by immunization with synthetic Cys-thymopoietin28-39 (Cys-TP28-39). human thymopoietin, This antiserum, previously shown to react with both bovine and human thymopoietin, gave reactivity restricted to cortical and medullary epithelial cells of bovine and human thymus. Monoclonal antibodies with reactivity restricted to native bovine thymopoietin did not react with tissue sections of bovine thymus; most likely the epitopes recognized by monoclonal antibodies are not expressed on the inactive precursor forms of thymopoietin within thymic epithelial cells.     

Adrenergic stimulation of membrane protein phosphorylation in human erythrocytes.

Adrenergic modification of membrane protein phosphorylation was studied in intact human erythrocytes. Micromolar norepinephrine increased 32P incorporation into Band 2 by 70%, and into Band 3 by 40%. Phosphorylation levels observed with a series of specific agonists and antagonists suggest that an alpha-adrenergic receptor is involved in this effect. The mechanism of linkage between this receptor and protein phosphorylation does not appear to involve modulation of intracellular concentrations of ATP, cyclic AMP, or cyclic GMP.     

Adrenergic stimulation of membrane protein phosphorylation in human erythrocytes

Adrenergic modification of membrane protein phosphorylation was studied in intact human erythrocytes. Micromolar norepinephrine increased ³²P incorporation into Band 2 by 70%, and into Band 3 by 40%. Phosphorylation levels observed with a series of specific agonists and antagonists suggest that an α-adrenergic receptor is involved in this effect. The mechanism of linkage between this receptor and protein phosphorylation does not appear to involve modulation of intracellular concentrations of ATP, cyclic AMP, or cyclic GMP.     

Cyclic hexapeptides derived from the human thymopoietin III.

Six diastereoisomeric cyclic hexapeptides of the partial sequence 39-44 of human thymopoietin III in which each residue was replaced with the D-amino acid were synthesized. In contrast to expectations, the yields from cyclization were not influenced by the location of the D-residue.     

The amino acid sequence of thymopoietin II.

The amino acid sequence of bovine thymopoietin II is presented. This T cell differentiating hormone of the thymus is a single 49 amino acid polypeptide chain of 5562 daltons. There is microheterogeneity at the C terminus with approximately two thirds of the molecules lacking the C terminal arginine found on the remaining molecules. Determination of the primary structure of thymopoietin II was facilitated by a long automated sequenator run on thymopoietin II coupled to 2-isothiocyanonaphthalene-4,8-disulfonic acid (NITC), tryptic cleavage of maleated thymopoietin II to yield the overlapping C terminal peptide, and efficient manual sequencing of this peptide using benzene extractions to minimize extractive losses of peptide.     

The C-terminal fragment of thymopoietin forms F-actin bundles: electron microscopic data

It was shown by electron microscopy that PEP33 a synthetic C-terminal peptide of the thymus hormone thymopoietin, formed bundles of actin 'filaments in the presence of 0.1 M KCl. The structure of PEP33 aggregates localizated in the bundles between actin filaments is very similar to that of aggregates observed in samples of pure PEP33. No changes were revealed in the structure of G-actin in the presence of PEP33. A similar, but a weaker bundling effect of thymopentin (PEP5) was also found. It forms bundles of actin filaments of small size. Further studies can shed light on the physiological importance of actin filament aggregation with the peptides of thymopoietin, the systematic release of which from the thymus produces the phenomena characteristic for the serious neuromuscular disease myasthenia gravis.     

Effect of thymopentin on the production of cytokines, heat shock proteins, and NF-κB signaling proteins

In vivo effects of thymopentin, an active fragment of the naturally occurring thymic hormone thymopoietin, on the production of cytokines, nitric oxide, heat shock proteins, and signaling proteins NF-κB, phNF-κB, and IκB-α in lymphoid cells of male NMRI mice was studied. Activation of production of several cytokines (IL-1α, IL-2, IL-6, IL-10, and IFN-γ), nitric oxide, and heat shock proteins (HSP70 and HSP90) was observed in peritoneal macrophages and spleen lymphocytes of mice that received intraperitoneal injections of thymopentin (15μg per 100 g body weight). Thymopentin apparently produces stress-like rather than damaging effects. A probable action mechanism of this hormone is activation of the NF-κB signaling pathway, which is most pronounced at the NF-κB phosphorylation stage.     

Phosphatidic acid mimics the muscarinic action of acetylcholine in cultured bovine chromaffin cells

In cultured bovine chromaffin cells, acetylcholine as well as muscarine stimulated the 32Pi incorporation into phosphatidic acid, induced the efflux of 45Ca2+ from prelabelled cells, and, in parallel, elevated intracellular cyclic GMP content. Phosphatidic acid added to the medium also stimulated the efflux of 45Ca2+ and the synthesis of cyclic GMP in the cells in the same fashion as muscarinic agents, whereas it did not induce the secretion of catecholamines indicating that the effect of phosphatidic acid is specific to muscarinic action. The result supports the hypothesis that phosphatidic acid produced during phosphatidylinositol turnover is linked to the regulation mechanism of Ca2+ mobilization and cyclic GMP synthesis by muscarinic stimulation.     

Thymopoietin enhances the allogeneic response and cyclic GMP levels of mouse peripheral, thymus-derived lymphocytes.

The action of the purified thymic factor, thymopoietin, on populations of post-thymic lymphocytes has been studied. Thymopoietin, at concentrations as low as 1.5 ng/ml, uniquely enhanced the proliferative response of peripheral T cells from lymph node and spleen to allogeneic stimulation. Enhancement of the allogeneic response (MLR) was not produced by several polypeptide hormones, including insulin, ACTH, HCG, or Ubiquitin. Treatment of spleen cells with anti-Thy-1 antiserum almost completely abolished the MLR. Thymopoietin's stimulatory effects could not reverse this. Thymopoietin treatment of Thy-1+-enriched spleen cell populations enhanced the MLR even when thymopoietin was removed as early as 2 min after incubation with responding cells. The interaction of thymopoietin with peripheral Thy-1+ cell populations produced a rapid and transient rise in cyclic GMP levels and slightly decreased cyclic AMP levels. These results suggest that thymopoietin interacts with one or more Thy-1+ subpopulations and that this interaction involves early changes in cyclic nucleotide metabolism.     

An aminoboronic acid derivative inhibits thymopentin metabolism by mucosal membrane aminopeptidases

Thymopentin is a pentapeptide with immunomodulatory activity. Transmucosal delivery may offer advantages over other routes, but published data have shown relatively poor efficacy when dosed nasally. Metabolism of thymopentin by the rat nasal mucosa and the effects of an aminoboronic acid aminopeptidase inhibitor, boroleucine, were evaluated. Thymopentin concentrations in a solution perfused through the isolated nasal cavity decayed with a first-order half-life of 12 minutes. Thymopentin was metabolized primarily by aminopeptidases, based on the amount of tetrapeptide metabolite formed. In the presence of boroleucine, at an inhibitor/substrate molar concentration ratio of 0.015/1, the degradation half-life was prolonged to 37 minutes. Appearance of the tetrapeptide metabolite of aminopeptidases was delayed. Boroleucine and other aminoboronic acid peptidase inhibitors may be useful for improving thymopentin delivery.