Tuftsin-THF-gamma 2 chimeric peptides: potential novel immunomodulators.

Synthesis of two chimeric peptides composed of tuftsin and thymic humoral factor-gamma 2 (THF-gamma 2) conjugates was accomplished. Our goal was the generation of novel immunomodulators. Initially, we demonstrate an IL-6 inducing activity of the phagocytic cells stimulant, tuftsin, on murine macrophages. This activity was documented only in the presence of antigen, either KLH or lysozyme. The augmentation was dose dependent, with optimal activity at a concentration of 200 and 20 nM, respectively. The chimeric peptides, either H2N-tuftsin-THF-gamma 2-OH or H2N-THF-gamma 2-tuftsin-OH, were also evaluated in the IL-6 system in the presence of the more potent antigen, KLH. The IL-6 inducing effect was maintained, although maximal activity appeared only at a concentration an order of magnitude greater than that of tuftsin. The chimeric peptides were further tested in an assay evaluating enhancement in murine bone marrow myeloid colony formation, a system in which THF-gamma 2, a T cell stimulant, has an established beneficial effect. The compounds were found to be inactive at the 25-200 ng/ml (14-112 nM) concentration range evaluated. Finally, the chimeric peptides were tested in a combined macrophages-T cells assay, i.e. antigen presentation, in which H2N-tuftsin-THF-gamma 2-OH was found to be more active than either parent peptide, thus representing a possible therapeutic agent.

Single-step bulk purification of synthetic tetrapeptide tuftsin by high performance liquid chromatography.

A protocol on reversed phase high performance liquid chromatogrpahy (RP-HPLC) using water-acetonitrile gradients under lesser stringent conditions, has been devised to obtain highly purified tuftsin in bulk amount. The protocol was also tested for two different preparations of tuftsin to yield identical quality of peptide.

A comparative study of [Leu1]Tuftsin and tuftsin, a natural phagocytosis-stimulating peptide.

1. [Leu1]tuftsin was reported to have greater phagocytosis-stimulating activity than tuftsin (Thr-Lys-Pro-Arg). 2. However, a study on inactivation of tuftsin by polymorphonuclear leukocytes (PMNs) demonstrated that leucine aminopeptidase, an ecto-enzyme, located on PMN surface was responsible for this mechanism. 3. Since leucine aminopeptidase is known to cleave Leu more easily than Thr at the N-terminal position of peptides, this suggested to us that [Leu1]tuftsin might then be inactivated by PMNs more easily than tuftsin, and thus this analog might be less active than tuftsin. 4. In addition, many tuftsin preparations used in earlier studies were not fully active, as high-performance liquid chromatography was not available to separate out many contaminating diastereomers. 5. In view of this, we have synthesized and purified [Leu1]tuftsin and compared its phagocytosis-stimulating activity with tuftsin. 6. Our results indicate that [Leu1]tuftsin is not as active as tuftsin in stimulating phagocytosis.

Receptor-mediated internalization of tuftsin by human polymorphonuclear leukocytes.

A high-performance liquid chromatography (HPLC) purified fluorescein-labeled analogue of tuftsin was prepared, which retains the full biological activity of the native molecule. Characterization of the derivatization site by amino acid analysis, N-terminal cleavage, and dansylation revealed a monofluorescinated derivative at the alpha-amino terminus. Binding of the fluorescent tuftsin to living polymorphonuclear leukocytes (PMN) was observed by means of video intensification microscopy. At 37 degrees C, diffuse membrane fluorescence was seen initially, followed by rapid aggregation and internalization. The latter was demonstrated by saltation of intracellular fluorescent aggregates. These processes are temperature-dependent and rely on specific binding to the tuftsin receptor.

Specific isolation of biologically-active peptides by means of immobilized anhydrotrypsin and anhydrochymotrypsin.

Anhydrotrypsin, a derivative of bovine trypsin, immobilized on Sepharose tightly adsorbs various peptides containing L-arginine at the carboxyl termini, such as bradykinin and tuftsin. These peptides correspond to the specific products of the action of trypsin-like enzymes. Native trypsin immobilized on Sepharose does not show such strong affinity. Fragment 2, a peptide with 41 amino acid residues, which has been released together with bradykinin from bovine high-molecular-weight kininogen by the action of plasm kallikrein, is also adsorbed on the immobilized anhydrotrypsin. When only the carboxyl-terminal arginine is removed with carboxy-peptidase B, however, the peptide loses its adsorptive ability. Immobilized anhydrochymotrypsin, on the other hand, exerts specific affinity for the peptides which correspond to the products of chymotrypsin. These results suggest that the anhydro-derivatives of serine-proteseas in general may be of great use in the affinity chromatography of respective series of various naturally occurring peptides.

Defective phagocytosis due to deficiencies involving the tetrapeptide tuftsin.

A description of the symptoms and causes of tuftsin deficiency is presented. Two major causes which give rise to a deficiency of the tetrapeptide (thr-lys-pro-arg) are discussed. One is familial tuftsin deficiency syndrome that results from an inherited mutation involving the tetrapeptide. All patients give a history of repeated infection with variable severity. One parent, father or mother, is deficient and occasionally other siblings may have the disease. The other type of deficiency is the result of loss of splenic function whether it is due to surgical removal of the spleen and to infarction or infiltration of the organ. A method for the assay of tuftsin activity is described.