Philip Gottlieb

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

Abstract 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.

Specific binding sites for the phagocytosis stimulating peptide tuftsin on human polymorphonuclear leukocytes and monocytes

Abstract Highly purified and biologically active [3H]tuftsin (specific activity 9 Ci/mmol) was synthesized and its binding to several types of human circulating blood cells was studied at 22°C. The binding to polymorphonuclear leukocytes and to monocytes was found to be specific, fast, saturable and reversible. Values for the dissociation constants (KD) were derived from equilibrium experiments and are 130 and 125 nM, respectively. The number of binding sites is approximately 50,000 and 100,000 per cell, respectively. Under the same experimental conditions lymphocytes exhibited only a threshold binding capacity for [3H]tuftsin whereas erythrocytes revealed no detectable binding.

Tuftsin, Thr-Lys-Pro-Arg

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.

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.

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

SummarySixteen 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 β-turn are not favored.

Peptide fragments from the tuftsin containing domain of immunoglobulin G synthesis and biological activity

Peptides corresponding to sequences of the Fc-portion of immunoglobulin G (IgG) surrounding and containing the tuftsin molecule were synthesized. The compounds were assayed for their ability to compete with [3H-Arg4]tuftsin in binding to mouse peritoneal macrophages and to stimulate the cells capacity to phagocytize. Despite the sensitivity that tuftsin has demonstrated to various chemical modifications and structural alterations which usually cause reduction or total loss of biological activity, IgG-related analogs possess potent tuftsin-like activity. The activity is not caused by enzymatic breakdown and release of tuftsin. The fact that the elongated tuftsin analogs can specifically be attached to and activate macrophages may indicate a possible connection between Fc and tuftsins receptors.

The Tuftsin Receptors

Publisher Summary This chapter reviews the findings relevant to the tuftsin receptor focusing on: the distribution in various body cells, specificity of the tuftsin-receptor interaction and its parameters, membrane and cellular events that proceed following peptide–cell encounter, and the receptors nature. Tuftsin is a humoral basic tetrapeptide, of the sequence L-threonyl-L-lysyl-L-prolyl-L-arginine, which has been found to manifest various biological activities associated with the reticuloendothelial and immune system function. Tuftsin has a wide range of activities that it exerts on the phagocytic cells, the polymorphonuclear leukocyte, the monocyte, and the macrophage, from human as well as from other mammalian species. Structure–function studies with numerous synthetic analogs of tuftsin have shown that the unique structural integrity of the peptide must be maintained to achieve full biological potency. Chemical alterations of the tuftsin molecule that are introduced into the main peptide chain or at the side chains lead to a reduction in the activity and sometimes to tuftsins inhibitors.

Substrate-mediated channeling of a chemical reagent to the active site of cAMP-dependent protein kinase

The specific affinity between enzymes and their physiological ligands (substrates, coenzymes) has been widely used for achieving selective labeling of enzyme active sites. This approach, most commonly referred to as ‘affinity labeling’ [I] has given rise to several variations and refinements, e.g., the introduction of transition-state analogs and mechanism-based irreversible inhibitors. In affinity labeling reagents, the biorecognition elements (which target the reagent to the active site) and the chemically reactive group (which anchors the reagent at the active site) are both part of the same molecule, We describe here a case in which a carefully designed peptide substrate is used to preferentially channel a separate group-specific reagent to one (kinetically characterized) sulfhydryl group at the active site of CAMP-dependent protein kinase .

Tuftsin binding to various macrophage hybridomas.

Macrophages participate in a wide variety of immunological processes, including phagocytosis, immune response through antigen presentation and lymphocyte activation, tumoricidal, and bactericidal activities. Despite their seemingly common cellular origin, macrophages do not consist of a homogeneous population. Macrophage subsets differ in morphology, expression of membrane markers, enzyme content, and biological activities. The effect of tuftsin on the above biological activities has been and currently is the subject of investigation in a number of laboratories. These include stimulation of phagocytosis,’** enhanced bactericidal’ and antitumor activities both in vivo and in vitro;bs and enhancement of antigen presentation capacity.* Although much information has been gleaned concerning the activation of macrophages by tuftsin through specific cellular receptors?” little is known about the mode by which it is capable of stimulating the multitude of activities associated with tuftsin-macrophage interaction. In order to further understand the specific controlling mechanisms of the many cellular immune responses in general, and the tuftsin effect in particular, separation and purification of macrophage subsets become an imperative step. The vast variety of macrophages and the limitations of current separation methods has impeded the study of molecular mechanisms that regulate the above specific cellular activities. One approach to circumvent these problems is the development of macrophage cell lines by means of hybridization technology. The “immortalization” of various types of macrophages may offer experimental systems that are most suitable for analyzing controlling mechanisms of macrophages. We report the characterization of macrophage hybridomas. This will be correlated with the availability of tuftsin receptors. A number of implications about tuftsin activation will be discussed.