13C nuclear magnetic resonance and circular dichroism studies of the tuftsin conformation in water

13C-NMR and circular dichroic (CD) spectra of tuftsin and its analogues are discussed in connection with our hypothesis that the beta-turn is the biologically active conformation of tuftsin. The changes in CD spectra evoked by an increase in pH are interpreted as a demonstration of the increasing amount of beta-turn conformers in solution. Configurational changes in successive residues of tuftsin showed that residues 2 and 3 of the peptide chain are important for the tuftsin conformation.     

The phagocytosis stimulating peptide tuftsin: Further look into structure-function relationships

Summary Sixteen new analogs of the phagocytosis-stimulating peptide tuftsin have been synthesized. The biological activities of these synthetic peptides, in which either the C-terminal or both C- and N-terminals are chemically altered, were evaluated by studying their effects on the phagocytosis of heat-killed yeasts and on the reduction of the dye nitroblue tetrazolium by normal human polymorphonuclear leukocytes. The results demonstrate that the integrity of the guanidine side chain of arginine at position four of tuftsin is crucial for maximal activity. Modification, even in side chain length, of the guanidine leads to decreasing activity. Preservation of the positive charge of position four of tuftsin yields analogs possessing considerable activity. Simultaneous alterations of both C- and N-terminal results in diminishing activites. The results of this study are discussed in relation to the structural features of tuftsin. It appears that interaction between the carboxyl of Arg⁴ and the amino group of Thr¹ which would indicate a specific conformation such as a 4 1 -turn are not favored.     

Receptor-mediated endocytosis of tuftsin by macrophage cells

A fluorescent analog of the phagocytosis stimulating peptide tuftsin was prepared by coupling tetramethyl rhodamine isothiocyanate to a C-terminal elongated derivative of tuftsin. This analog, Thr-Lys-Pro-Arg-Gly-Lys(N epsilon-tetramethyl rhodamine)-OH, was used to visualize tuftsin receptors on mice macrophage cells by fluorescent image intensification. Fluorescent labelling was carried out at 37 degrees C, using a concentration of 200 nM and 2 microM of the fluorescent tuftsin derivative. The formation of peptide-receptor clusters and their subsequent internalization, as discerned by image intensification, were rapid processes, 5 min and 5-30 min, respectively. Preincubation of macrophages with tuftsin for various time intervals, followed by quantification of the tuftsin receptor using radiolabelled tuftsin, suggest that tuftsin receptors are initially increased in amount (5-7 min) and subsequently reduced (after 10-15 min) as judged by sites available for tritiated tuftsin. The binding studies are rather complementary to the fluorescence observations and support the assumption that the tuftsin receptor on the membrane of the mice macrophage cell is rapidly mobilized.     

Salt effects on peptide conformers: a dielectric study of tuftsin.

Four 1-ns molecular dynamics computer simulations of tuftsin, Thr-Lys-Pro-Arg, are analyzed: (1) cis tuftsin in water, (2) trans tuftsin in water, (3) cis tuftsin in 1 M NaCl, and (4) trans tuftsin in 1 M NaCl. Independently of the salt concentration, the trans conformer has a higher dielectric constant than the cis conformer because the former exhibits a more widely distributed charge distribution in space. Independently of the peptide conformation, the presence of salt reduces the dielectric constants of both the peptide and the solvating water molecules because ions, on binding, restrict the motion of other atoms. In contrast to the dielectric constants, neither the peptide conformation nor the salt concentration shows a significant influence on the dielectric relaxation time of water molecules.     

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.     

Tuftsin: A hormone-like tetrapeptide with antimicrobial and antitumor activities

A specific fraction of immunoglobulin G binds to polymorphonuclear neutrophils and stimulates their phagocytic activity. This phagocytosis-stimulating activity resides solely in a small peptide termed tuftsin, of the sequence Thr-Lys-Pro-Arg, which has been isolated from the leukophilic immunoglobulin G fraction. The physiological significance of tuftsin has been demonstrated in splenectomized patients and patients with a congenital tuftsin abnormality, in whom the low levels of tuftsin in sera (measurable by radioimmunoassay) coincides with a high incidence of infection. Tuftsin has also been shown to enhance bactericidal activity in addition to phagocytosis. Its biological activities appear to be mediated via specific tuftsin receptors which have been found on macrophages, monocytes and granulocytes. In addition, tuftsin possesses chemotactic, migration-enhancing and mitogenic properties for leukocytes and has recently been shown to enhance their anti-tumor activity $\text{in}$$\text{vitro}$ as well as $\text{in}$$\text{vivo}$. Other known activities of tuftsin include effects on the activity of the hexose monophosphate shunt, on the concentrations of intracellular cyclic nucleotides and on the efflux of Ca²⁺ in leukocytes. Tuftsin has been chemically synthesized in various laboratories using different procedures and also is available commercially. The above features of tuftsin plus the expected low toxicity of this peptide make tuftsin a very attractive agent for immunotherapy against infection and cancer. However, a great deal of caution needs to be exercised when using tuftsin due to inhibitory contaminants found in certain commercial preparations.     

Enhancement of phagocytosis - a newly found activity of substance P residing in its N-terminal tetrapeptide sequence

The undecapeptide Substance P stimulates phagocytosis by mouse macrophages and human polymorphonuclear leukocytes. The activity of Substance P resides in its N-terminal tetrapeptide protion. Substance P and its N-terminal tetrapeptide are as active as tuftsin in their phagocytosis-stimulating activity and compete with tuftsin for its binding sites. The phagocytosis-enhancing activity of Substance P may play a role in inflammatory processes of neural origin where the involvement of the peptide has been implicated.     

Proteolysis of human C-reactive protein produces peptides with potent immunomodulating activity

We have studied the ability of human C-reactive protein to modulate the immune response in vitro. Whereas native C-reactive protein did not induce phagocytic leukocytes to chemotax or to produce superoxide, treatment of purified C-reactive protein with human neutrophil-derived acid proteases produced substances with potent effects on leukocyte function. Close examination of the primary structure of human C-reactive protein revealed three regions evenly distributed throughout the protein each of which contain peptide sequences closely resembling the amino acid sequence of the immunomodulator peptide tuftsin, Thr-Lys-Pro-Arg. We have synthesized the three peptides which include Thr-Lys-Pro-Leu ([Leu4]tuftsin), Gly-Lys-Pro-Arg ([Gly1]tuftsin), and Thr-Lys-Pro-Gln ([Gln4]tuftsin) and assayed them for biological activity. The three synthetic peptides were found to stimulate phagocytic leukocytes to chemotax, produce superoxide, and induce mononuclear cells to produce interleukin 1 in vitro at concentrations similar to those concentrations required for tuftsin to induce these phenomena. These results support a potentially important role for C-reactive protein as a possible immunomodulator during inflammation.     

Specific translocation of tuftsin (Thr-Lys-Pro-Arg), a natural immunomodulating peptide, into the nuclei of human monocytes

To delineate the mechanism of growth and differentiation activities of tuftsin (Thr-Lys-Pro-Arg), we examined the translocation of tuftsin after internalization by the target cells. We found using two independent techniques, fluorescence microscopy and autoradiography, that while in human polymorphonuclear leukocytes (terminally differentiated cells) the peptide remains in the cytoplasmic compartment, in monocytes it translocates to the nucleus. The ability of tuftsin to directly interact with DNA was documented by a large increase in the melting point of bovine DNA in the presence of tuftsin. It is suggested that the translocation, processing and action of tuftsin may depend on the differentiation state and/or on the type of effector cells. Also, tuftsin has the capacity to interact directly with DNA and, therefore, may have a potential for affecting gene activity.     

Antitumor effect of tuftsin

Summary Tuftsin, a physiological tetrapeptide derived from the Fc region of leukophilic IgG possesses a variety of immunopotentiating properties including the ability to act as an immunotherapeutic agent against the experimental tumors, L 1210 leukemia and Cloudman S-91 melanoma. Although the mechanism of action of tuftsin in vivo is not known, several types of leukocytes have been shown to become cytotoxic effector cells following activation with tuftsin. These cells presently include macrophages, natural killer cells, and granulocytes. The possibility that tuftsin can also activate other types of effector cells have not been ruled out. We feel this small peptide has a high potential (largely unrecognized) as an antitumor immunopotentiating agent. It is naturally occurring in man and appears to be relatively non-toxic. Its exact sequence (Thr-lys-Pro-Arg) is known and it can be chemically synthesized. Methods are also available to monitor the levels of tuftsin in body fluids. These properties along with its ability to control infectious disease make this agent one of the more promising immunopotentiators.     

An extracellular 5'-nucleotidase with both monoesterase and diesterase activity from Micrococcus sodonensis.

Bovine γ-globulin was separated into four fractions by chromatography on cellulose phosphate. The chromatographic distribution was similar to that reported for human and dog γ-globulin. More than 80% of a nonspecific phagocytosis stimulating factor (leucokinin) present in the serum was isolated in γ-globulin fraction IV. Bocine red blood cells and polymorphonuclear leukocytes bind γ-globulin without appreciable selectivity for any of the four chromatographic fractions, but they do selectively bind the phagocytosis stimulating factor. Splenectomy caused no observable change in either the chromatographic distribution or phagocytosis stimulating activity of bovine serum γ-globulin. The tetrapeptide tuftsin stimulates phagocytosis by bovine neutrophiles, but on a molar basis the activity of tuftsin was only 10% that of the phagocytosis stimulating factor. If the factor exerts its effect, as has been proposed, by having a phagocytosis stimulating peptide cleaved from it by an enzyme on the leukocyte membrane, that peptide must differ in structure from tuftsin. This conclusion is supported by the inability of trypsin to liberate an active peptide from bovine serum.     

Excitatory effect of tuftsin tetrapeptide on the activity of white rats

Studies on male white rats have shown that tuftsin (trelys-pro-arg) enhances the locomotion in animals as disclosed by a series of the behavioral tests. The effect is dose-dependent: a dose of 50 microgram/kg did not change any of the test parameters, while that of 150 microgram/kg induced a short-term elevation of locomotion measured with an "Animeks". Administration of tuftsin in a dose of 300 microgram/kg led to the enhancement of the animals' locomotion as measured with the "Animeks", to the increased "open field" running time and to the reduced latent period of the reaction during the training in a T-shaped maze. Also, this dose of the peptide relaxed the reactions associated with fear. It is assumed that the effects observed are consequent on the stimulant action of tuftsin on the body of white rats.     

Tuftsin,Thr-Lys-Pro-Arg

Summary Tuftsin, a natural occurring tetrapeptide, has been found to exhibit several biological activities connected with immune system function. Although little is known about tuftsin's biogenesis, much information has been gleaned about its structure-function relationships, which have shown that several features of the molecule are essential for expression of full biological activity. Furthermore, specific receptor sites for tuftsin have been found to exist exclusively on phagocytic cells. Research indicates that tuftsin binding to target cells effect intracellular calcium and cyclic nucleotide levels. Implication of these facts on tuftsin's mode of action are discussed.Basic peptidic segments resembling tuftsin are found in a variety of regulatory peptides. Questions are, therefore, raised as to the biospecificity and cross-reactivity of these sequences. Substance P, one such peptide, which binds with and activates tuftsin receptors, is described.In light of tuftsin's therapeutic potential, assays for its determination have been introduced. When applied to analyze human blood serum of normal as well as of various pathological origins, direct correlation was found between tuftsin levels and susceptibility to bacterial infections.     

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.     

Polypeptide modification and cross-linking by oxidized 3-hydroxykynurenine

3-Hydroxykynurenine (3OHKyn) is present in the mammalian lens as a UV filter and is formed from kynurenine in the tryptophan metabolic pathway. 3OHKyn is a readily autoxidized o-aminophenol which binds to proteins in vitro. The lens, particularly its central region, the nucleus, becomes increasingly oxidized with age. Under such conditions, the oxidation products of 3OHKyn may bind to lens proteins and contribute to nuclear cataract formation. The purpose of this study was to determine the structures of in vitro reaction products of 3OHKyn with model peptides as a general model for 3OHKyn modification of proteins. 3OHKyn was incubated with the dipeptide glycylglycine (GG) and the tetrapeptide tuftsin (sequence TKPR) under oxidizing conditions, and the reaction products were characterized by a variety of spectroscopic techniques. The major 3OHKyn-GG reaction product involves formation of a benzimidazole moiety between the GG N-terminus and the oxidized amino and/or phenol groups of 3OHKyn. In contrast, tuftsin, which has an N-terminal threonine, forms predominantly a cross-linked dimer with oxidized 3OHKyn. This product is analogous in structure to the dimeric reaction product, quinilinobenzoxamine, formed between oxidized 3OHKyn and glycyllysine [Aquilina, J. A., et al. (1999) Biochemistry 38, 11455-11464], which contains a benzoxazole moiety. The identification of a tuftsin dimer suggests that 3OHKyn can react with any peptide having a free alpha-amino group, via a general side chain elimination mechanism. The identification of both benzimidazole and benzoxazole adducts in peptides with a free N-terminus suggests that peptide amino groups can react initially at either the aromatic amino or hydroxyl group of oxidized 3OHKyn. The proportion of each adduct may change, however, depending on the amino acid sequence at the N-terminus.     

Glyco-tuftsin derivatives modulate interleukin-1 and tumor necrosis factor production

Six Thr1 (O-glyco)-derivatives of the "phagocytosis stimulating peptide" tuftsin, H-Thr-Lys-Pro-Arg-OH and the N-glycosylated undecapeptide H-Thr-Lys-Pro-Arg-Glu-Gln-Gln-Tyr-Asn(beta-D-GlcNAc)-Ser-Thr-OH, which correspond to the "tuftsin-region" at the Fc-domain of immunoglobulin G (amino acid residues 289-299), were evaluated in comparison with tuftsin and rigin, H-Gly-Gln-Pro-Arg-OH, for their capacity to evoke the release of interleukin-1 and tumor necrosis factor from mouse peritoneal macrophages and from human monocytes. Several glycosylated tuftsin derivatives were found to modulate, in a rather dose-dependent manner, the release of the two cytokines from both cell types.     

Tuftsin binds neuropilin-1 through a sequence similar to that encoded by exon 8 of vascular endothelial growth factor

Tuftsin, Thr-Lys-Pro-Arg (TKPR), is an immunostimulatory peptide with reported nervous system effects as well. We unexpectedly found that tuftsin and a higher affinity antagonist, TKPPR, bind selectively to neuropilin-1 and block vascular endothelial growth factor (VEGF) binding to that receptor. Dimeric and tetrameric forms of TKPPR had greatly increased affinity for neuropilin-1 based on competition binding experiments. On endothelial cells tetrameric TKPPR inhibited the VEGF(165)-induced autophosphorylation of vascular endothelial growth factor receptor-2 (VEGFR-2) even though it did not directly inhibit VEGF binding to VEGFR-2. Homology between exon 8 of VEGF and TKPPR suggests that the sequence coded for by exon 8 may stabilize VEGF binding to neuropilin-1 to facilitate signaling through VEGFR-2. Given the overlap between processes involving neuropilin-1 and tuftsin, we propose that at least some of the previously reported effects of tuftsin are mediated through neuropilin-1.     

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.     

Purification and properties of a bovine brain thyrotropin-releasing-factor deamidase. A post-proline cleaving enzyme of limited specificity

A bovine brain thyrotropin-releasing-factor (thyroliberin) deamidase has been purified 1100-fold to apparent homogeneity. Molecular weight estimates by gel filtration and sodium dodecylsulfate gel electrophoresis indicate that the enzyme consists of a single polypeptide chain of molecular weight of about 62 000-65 000. The enzyme is inactivated by sulfhydryl blocking agents. Serine proteinase inhibitors, phenylmethanesulfonyl fluoride and benzamidine, have no effect. Besides thyroliberin, the enzyme hydrolyzes peptide bonds involving the carboxyl group of proline residues in luliberin, tuftsin, angiotensin II, melanotropin, and neurotensin. Oxytocin, vasopressin, and bradykinin are not cleaved; they are, however, strong competitive inhibitors of thyroliberin deamidation. The specificity studies indicate that the enzyme is a "post-proline cleaving enzyme" which hydrolyzes peptides of the general structure, Yaa-Pro-Xaa, in which Xaa = amino acid, peptide, or amide (not Pro), and Yaa = N-blocked basic amino acid or a peptide sequence in which the C-terminal residue (i.e. the residue prior to Pro) is a basic amino acid such as His, Lys, or Arg. The enzyme is compared to other post-proline cleaving enzymes.