[Hyp3]-tuftsin ([Hyp3]-TU) synthesis and biological activity.

[Hyp3]-tuftsin (Thr-Lys-Hyp-Arg) has been synthesized by the liquid-phase method. In biological investigations performed on rats antinociceptive and diuretic effects have been determined. It has been suggested that the presence of hydroxyl substituent in pyrrolidine ring of proline slightly modifies antinociceptive TU effect and is responsible for the increased diuretic [Hyp3]-TU activity.

The characteristics of purified HL60 tuftsin receptors.

The purification and characteristics of purified HL60 tuftsin receptors are described. Purification was accomplished by affinity chromatography similar to that described earlier, wherein a tuftsin analog Thr-Lys-Pro-Pro-Arg, is covalently linked at the N alpha group to a solid support. The receptor consists presumably of two subunits approximately 66 KDa and 57 KDa. The dissociation constant of the receptor complex is 4.7 X 10(-8) M with 5 X 10(4) receptors per cell. It can form oligomers with an Mr of about 560 KDa suggesting an octomeric structure, assuming the same number of each subunit is associated.

Covalent peptide transfer to cell membrane proteins (peptidyl transferase).

HL60 cells, rabbit peritoneal granulocytes or membrane preparations of these cells incorporate radioactivity when reacted with the radioactive peptide tuftsin [3H Pro3]-Thr-Lys-Pro-Arg. The radioactivity which is not diminished by repeated treatments with TCA and NaOH, is covalently bound to a membrane acceptor protein of 100 kDa. The peptide is not displaced by large concentrations of its constituent amino acids. The acceptor protein is resolved into one labeled peak by gel filtration on Sephadex G-200, Sephacryl S-300 and by SDS-PAGE. Digestion by trypsin and chymotrypsin results in the production of smaller fragments.

Tuftsin: its chemistry, biology, and clinical potential.

Tuftsin is a tetrapeptide, Thr-Lys-Pro-Arg, which resides in the Fc-domain of the heavy chain of immunoglobulin G. The peptide originates from a specific fraction of the parent protein through enzymatic processing. Tuftsin possesses a broad spectrum of activities related primarily to the immune system function and exerts on phagocytic cells, notably on macrophages. These include potentiation of various cell functions such as phagocytosis, motility, immunogenic response, and bactericidal and tumoricidal activities. The features of tuftsin, coupled with its low toxicity, make the peptide an attractive candidate for immunotherapy. Tuftsin's capacity to augment cellular activation is mediated by specific receptors that were identified, characterized, and recently isolated from rabbit peritoneal granulocytes. Tuftsin has been chemically synthesized by a variety of techniques, some of which are adequate for large-scale preparations. A multitude of analogs have also been synthesized and extensively studied for structure-function relationships.

Tuftsin stimulates growth of HL60 cells.

Many functions of monocyte/macrophage and granulocyte are activated by tuftsin; principally phagocytosis, motility, immunogenic stimulation, antibacterial and antineoplastic activities. Here it is shown that tuftsin stimulates HL60 growth to twice the control rate. The uptake of [3H]uridine and [3H]leucine in a pulse of 30 min was also double that of the control. The uptake of thymidine was not stimulated.

Tuftsin induced tumor necrosis activity.

Tuftsin induced tumor necrosis activity was investigated. The activity was found in mice serum several days after i.p. injection of tuftsin. Further experiments with adhering peritoneal and spleen cells indicated that macrophages were the source of the observed activity. The same effect was observed when promyelocytic leukemia cells (HL60) were stimulated with different concentrations of the peptide. These showed yet another possible mechanism for tuftsin antineoplastic activity.

Isolation and subunit composition of tuftsin receptor.

Tuftsin (Thr-Lys-Pro-Arg) receptor was purified to apparent homogeneity by affinity chromatography, using a pentapeptide analog (Thr-Lys-Pro-Pro-Arg) that binds the receptor more than 4 times as avidly as tuftsin. The analog was covalently linked to a solid support (Affi-Gel 10). Rabbit peritoneal granulocyte membrane solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate was applied to the affinity column, the column was washed with 0.1 M ammonium carbonate (pH 7.9) and 0.1 M ammonium acetate (pH 5), and bound material was eluted with 20 nM tuftsin or pentapeptide. The eluate was concentrated and subjected to gel filtration; this yielded one major peak of [3H]tuftsin binding activity corresponding to approximately 500 kDa and a minor peak at approximately 250 kDa. Rechromatography of either peak resulted in the appearance of the same major and minor peaks. NaDodSO4/PAGE of the affinity-purified material under nonreducing conditions showed only two silver-staining bands. Electroblotting followed by [3H]tuftsin overlay and fluorography showed two adjacent radioactive bands corresponding in mobility to the silver-stained bands. Under reducing conditions, NaDodSO4/PAGE yielded molecular mass values 62 kDa and 52 kDa for the two tuftsin receptor subunits. Electron microscopy revealed a homogeneous population of spherical molecules with diameters of 104 A.

Suppression of Friend virus-induced leukaemia in mice by tuftsin.

A significant decrease in mortality was observed when 25 micrograms of the tetrapeptide tuftsin was given to DBA/2J mice 5 days before infection with Friend leukaemia virus (FLV). The same effect was observed when tuftsin was given 5 days before and twice a week for 3 weeks after FLV infection. No effect was observed when the same amount of tuftsin was given 1 day before infection. A 5 micrograms dose of tuftsin given 5 days before and twice-weekly for 3 weeks had no effect on leukaemia induced by FLV infection. These findings showed that time and dosage were critical to the protective effect of tuftsin against virus-induced leukaemia.

Tuftsin (Thr-Lys-Pro-Arg), a natural modulator of macrophage activity: further studies.

Tuftsin, Thr-Lys-Pro-Arg, that activates macrophage functions, binds to specific receptors on these cells. The receptor capacity to bind tuftsin is diminished by prior treatment of the cells with dithiothreitol. Adherent mouse peritoneal macrophages bind tuftsin to a far less extent than non-adherent macrophages. Michaelis constant (Km) of tuftsin for phagocytic stimulation of macrophages is 111 eta M. The half maximal binding concentration of tuftsin by these cells is 117 eta M. These are similar values and indicate that full occupancy of the receptors by tuftsin is a necessary prerequisite for maximal phagocytosis.

The similarity between tuftsin (Thr-Lys-Pro-Arg) receptors and tuftsin antibody: a case of induced molecular mimicry.

The characteristics of binding of tuftsin (Thr-Lys-Pro-Arg) and several of its synthetic analogues, to specific membrane receptors on rabbit polymorphonuclear granulocytes, were compared with the binding characteristics of rabbit specific antibody to tuftsin. [3H-Arg4]-tuftsin was used as the principal ligand. Six analogues were studied. Two of these, Thr-Lys-Pro and Ala-Lys-tuftsin-Glu-Ala3, showed no binding affinity either to receptor or antibody. Ala-Lys-tuftsin showed less binding than tuftsin to both acceptors. Three showed stronger binding than tuftsin. The order of binding among these was tuftsin ( [Glu]2-tuftsin ( [Pro-Pro3]-tuftsin (tuftsinyltuftsin. This same order of binding was found with both receptor and antibody.

The antineoplastic effects of tuftsin and tuftsinyltuftsin on B16/5B melanoma and L1210 cells.

The intraperitoneal (i.p.) injection of tuftsin, Thr-Lys-Pro-Arg, into C57BL/6 mice that were injected with B16/5B melanoma cells, resulted in a considerable suppression and elimination of solid tumor growth. While 100% of control animals exhibited tumor growth, 38% of the treated animals failed to show tumor formation for the duration of the experiment, 60-80 days. The octapeptide, tuftsinyltuftsin, was effective at 3 ng per mouse as was a dose of 2 and 20 micrograms per mouse. In each case there was a significant number of mice free of tumors. The octapeptide was also quite effective against L1210 cells resulting in the survival of 35-40% of the treated animals. The lethal effect of increased superoxide, O X 2, production by tuftsin treatment may explain the antineoplastic effect of the tetrapeptide. This may result not only from higher concentrations of O X 2 but also from the potentially lethal effects of H2O2 and OH X radical, both of which are products of O X 2 metabolism.

Tuftsin, a natural activator of phagocyte cells: an overview.

The recognition that a small oligopeptide was responsible for the full stimulation effect of specific cytophilic gamma-globulin on blood neutrophils arose from a study of the kinetics of phagocytosis. These were unusual in that the stimulation was short lived and that preincubation of the phagocyte with the gamma-globulin rendered the latter inactive. The oligopeptide was isolated, its structure determined (Thr-Lys-Pro-Arg) and synthesized. The discovery of human mutants with tuftsin deficiency exhibiting signs and symptoms of frequent severe infection further emphasized the specific biological function of the tetrapeptide. The mutant peptide was isolated, sequenced (Thr-Glu-Pro-Arg), and synthesized. Further studies showed that tuftsin requires two enzymes for its liberation from the parent carrier gamma-globulin. One enzyme is in the spleen that cleaves distal to the arginine end, and the other, on the outer side of the plasma membrane, cleaves proximal to the threonine residue. The tetrapeptide tuftsin stimulates all functions of phagocytic cells: phagocytosis, pinocytosis, motility, immunogenic activity including processing of the antigen and augmentation of the number of antibody-forming cells, bactericidal activity, and, above all, tumoricidal activity. The latter has been shown by several laboratories.

Tuftsin, a natural activator of phagocytic functions including tumoricidal activity.

Some of the properties of the tetrapeptide tuftsin, Thr-Lys-Pro-Arg, are discussed. We describe three phases of tuftsin activation of the macrophage. Tuftsinyltuftsin, the octapeptide Thr-Lys-Pro-Arg-Thr-Lys-Pro-Arg, was synthesized with a view of minimizing the formation of Lys-Pro-Arg, from tuftsin by tissue aminopeptidases. The tripeptide is a tuftsin inhibitor. The octapeptide proved to be quite effective in prolonging the life of syngeneic mice injected with L1210 leukemia cells. Its effect in our laboratory, was considerably better than we could obtain with tuftsin. A simple method for purifying tuftsin by high performance liquid chromatography is described using 0.75% trifluoroacetic acid in water. The tuftsin sequence Thr-Lys-Pro-Arg is present in P12 protein of Rausher murine leukemia virus. A close analog Thr-Arg-Pro-Lys appears in yet another virus protein the haemagglutinin of influenza virus. A second close analog Thr-Arg-Pro-Arg forms the penultimate carboxyterminal of a pancreatic polypeptide found in human and several animals.

Biochemical aspects of tuftsin deficiency syndrome.

From work reported so far it is possible to draw certain conclusions namely, that Tuftsin, Thr-Lys-Pro-Arg, is a biologically functional entity. The presence of congenital familial deficiency reinforces this conclusion. The fact that these patients suffer from repeated infections points at an in vivo system that parallels the in vitro studies showing tuftsin stimulation of the phagocytic activity of the tissue macrophage and blood granulocyte. Such stimulation occurs at hormonal concentrations; (half maximal at 100 M). Furthermore, tuftsin enchances pinocytosis, as it does phagocytosis, only in phagocytic cells. It stimulates the motility of these cells as well as their longevity. Tuftsin stimulates the hexosemonophosphate shunt and, presumably through the formation of active oxygen-derived compounds, augments the bactericidal as well as the tumoricidal activity of the macrophage. There are highly specific receptors on the cell membrane of phagocytic cells. The structure of tuftsin cannot be altered without producing inactive and/or inhibitory analogs, an exception being the interchange of lysine and arginine. The release of tuftsin from carrier leukokinin requires two enzymes, one of which is on the outer membrane of the phagocyte and the other in the spleen. The absence of the latter explains the deficiency observed after the abrogation of splenic function for whatever cause.

Nuclear magnetic resonance studies on the structure of the tetrapeptide tuftsin, L-threonyl-L-lysyl-L-prolyl-L-arginine, and its pentapeptide analogue L-threonyl-L-lysyl-L-prolyl-L-prolyl-L-arginine.

Nuclear magnetic resonance spectroscopy has been used to investigate the solution conformation of tuftsin, threonyllysylprolylarginine, as well as a pentapeptide inhibitor of tuftsin, threonyllysylprolylprolylarginine. Both proton and carbon-13 studies were performed. In water, neither peptide gives evidence of a preferred conformation. In dimethyl-d6 sulfoxide, tuftsin appears to prefer a particular conformation, but the inhibitor does not. The conformation of tuftsin is one in which the amide NH proton of arginine is solvent shielded. The conformation does not, however, appear to be such that a normal 4 leads to 1 beta turn exists.

Evidence for a tyrosine residue at the active site of phosphoglucomutase and its interaction with vanadate.

The rate of transfer of [32P]phosphate from [32P]-labeled phosphoglucomutase (alpha-D-glucose-1,6-bisphosphate:alpha-D-glucose-1-phosphate phosphotransferase, EC 2.7.5.1) to glucose increases dramatically between pH 8.5 and 10.5 with a half maximal rate at pH 9.8. This suggests the participation of a residue containing an ionizable group with a pK close to 10. The inhibition of enzyme activity obtained with tyrosine-derivatizing reactions--iodination, nitration, acetylation, and diazo coupling--is strongly indicative of tyrosine participation. Thiol reagents, p-hydroxymercuribenzoate and ethyleneimine, were without effect. Vanadate and arsenate augmented the transfer reaction 200- and 2.5-fold, respectively, and lowered the pH optimum of the reaction.

The clinical and physiological aspects of tuftsin deficiency syndromes exhibiting defective phagocytosis.

The tetrapeptide tuftsin (Thr-Lys-Pro-Arg) stimulates phagocytosis by blood neutrophilic granulocytes and tissue macrophages in a highly specific manner. Tuftsin is cleaved off the carrier gamma-globulin molecule as the free active form by two enzymes. One of these is in the spleen and the other on the outer membrane of the phagocyte. Congenital tuftsin deficiency usually arises when the peptide is mutated to an inactive peptide. The acquired type occurs if the spleen function is curtailed by removal or disease. Tuftsin deficiency is manifested by severe recurrent infections involving primarily the skin, lymph nodes and lungs. Therapy is limited to gamma-globulin injection along with appropriate chemotherapy.