Shigeharu Nagasawa

Structural and Functional Properties of Complement-activating Protein M161Ag, a Mycoplasma fermentans Gene Product That Induces Cytokine Production by Human Monocytes

Human malignant cells are targeted by homologous complement C3b if they express M161Ag, a 43-kDa protein with C3-activating property. cDNA of M161Ag cloned from human leukemia cell lines predicted M161Ag as a novel secretory protein comprised of 428 amino acids including 5 amino acids encoded by TGA codons (Matsumoto M., Takeda, J., Inoue, N., Hara, T., Hatanaka, M., Takahashi, K., Nagasawa, S., Akedo, H., and Seya, T. (1997) Nat. Med. 3, 1266–1270), although the origin of this gene was obscure. Here we clarified this point through genomic and biochemical analysis: 1) 5′-UT and genomic sequences represented the prokaryote promoter and ribosomal binding site; 2) the TGA codons in M161Ag cDNA were translated not into selenocysteines but into tryptophans; 3) M161Ag anchored onto the membrane secondary to its N-terminal palmitoylation like prokaryote lipoproteins; 4) genomic and cDNA clones of M161Ag were highly homologous to Mycoplasma fermentans gene encoding P48, a monocytic differentiation/activation factor, recently released in the data base, although the resultant proteins were different in the amino acid sequences. Additionally, purified soluble M161Ag efficiently provoked IL-1β, tumor necrosis factor α, and IL-6 like P48, and further IL-10 and IL-12 in human peripheral blood monocytes. Thus, M161Ag originates from M. fermentans, and latently infectedM. fermentans allows human cells to produce M161Ag. The liberated protein serves as a potent modulator of innate and cellular immune responses via its complement-activating and cytokine-producing activities.

Conversion of bovine prekallikrein to kallikrein. Evidence of limited proteolysis of prekallikrein by bovine hageman factor (factor XII)

Hageman factor, an intrinsic trigger of the sequence of proenzyme-enzyme transformations leading to blood coagulation [ 1,2], has been found to trigger off liberation of hypotensive polypeptides called plasma kinins [3]. We recently obtained evidence using highly purified preparations that Hageman factor activates prekallikrein directly to kailikrein [4]. The activation was found to be of a catalytic nature [5], but no information has been obtained on the role of Hageman factor in the transformation of prekallikrein to kallikrein. This paper reports evidence indicating that the conversion of prekallikrein to kallikrein by Hageman factor may be accompanied by limited proteolysis of the precursor molecule.

Purification and Properties of Bradykininogen and of the Bradykinin-Releasing and -Destroying Enzymes in Snake Venom

The arginine ester hydrolase in the venom of Agkistrodon halys blomhoffii (“Mamushi” in Japanese) was separated into three enzymatic entities: the “bradykinin-releasing,” “clotting,” and “capillary permeability-increasing” enzymes. The latter two enzymes were purified to physicochemically homogeneous states (Sato et al., 1965), and the bradykinin-releasing enzyme described in this paper containedno “clotting” or “capillary permeability-increasing” enzyme, and no bradykinin-destroying enzyme. The properties of the bradykinin-releasing enzyme of the venom were similar to those of pancreatic kallikrein (Werle and Trautschold, 1963). The authors also purified bovine bradykininogen, and from the results of Sanger’s DNP method, exopeptidase digestion methods, and the release of bradykinin by the venom bradykinin-releasing enzyme, it was concluded that bradykinin is not located at either end of the polypeptide chain of bradykininogen.

The N-terminal sequence of the light chain derivative of bovine plasmin.

Abstract Activation of bovine plasminogen by urokinase occurred by limited proteolysis of peptide bonds resulting in the formation of active plasmin. Electrophoresis of reduced plasmin on sodium dodecylsulfate (SDS)-polyacrylamide gel revealed the two-chain structure of bovine plasmin. The heavy and light chains were separated by dialysis of reduced and carboxymethylated (RCM)-plasmin against deionized water. By the Edman procedure, the N-terminal sequence of the light chain was concluded to be Ile-Val-Gly-Gly-, which was homologous to that of bovine trypsin.

Molecular remodelling of human CD46 for xenotransplantation: designing a potent complement regulator without measles virus receptor activity

In pig‐to‐human discordant xenotransplantation, human complement (C) is a major barrier to long survival of xenografts. The current idea on how to cope with this barrier is that human complement regulatory proteins are forcibly expressed on xenografts to serve as safeguards against host C‐induced hyperacute rejection of xenografts. Co‐expression of decay‐accelerating factor (DAF) (CD55) and membrane cofactor protein (MCP) (CD46) would be the first choice for this trial, because most of the human cells are protected from C‐mediated damage by two different modes with these two kinds of C‐regulators. Many problems have arisen, however, for MCP expression on grafts. (i) MCP acts as a measles virus receptor, which may function to render donor pigs measles virus (MV) sensitive. (ii) MCP signals immune suppression which causes devastation of the recipients immune responses. (iii) MCP exerts relatively low self‐protective activity against C compared with other cofactors; development of more efficient forms is desirable. (iv) Grafts with a high expression level of MCP are difficult to produce. In this study, we made a number of cDNA constructs of MCP, expressed them on swine endothelial cell lines, and tested cell‐protective potency and MV susceptibility. The short consensus repeat 1 (SCR1)‐deleted MCP with glycosyl phosphatidylinositol (GPI)‐anchored form (Δ1MCP‐PI) of MCP was found to be most suitable for the purpose of overcoming these problems. However, it was also found that MV induces two modes of cytopathic effect (CPE) on swine endothelial cells, either MCP‐dependent or ‐independent. Here, we discuss these two points which will be raised through study of MCP‐transgenic animals.

Evidence for the location of methionyl-lysyl-bradykinin moieties at the carboxyl terminus and inside of bovine kininogen-I.

Abstract The location of the kinin moiety in high molecular weight (HMW) bovine kininogen (kininogen-I), a sensitive substrate for serum kallikrein, was investigated. Treatment with carboxypeptidase B destroyed about one-half of the initial kinin-yielding ability of kininogen-I, and selective cleavage of the methionyl peptide bonds in kininogen-I with cyanogen bromide liberated free kallidin (lysyl-bradykinin) and an inactive kallidin-containing peptide. These results show that there is a methionyl-lysyl-bradykinin sequence located both at the carboxyl terminus and inside of the polypeptide chain of bovine kininogen-I.

A Novel Negative Regulator Molecule, Cho‐1, Is Involved in the Cytotoxicity by Human Natural Killer Cells but Not in Cytotoxic T Lymphocytes

We previously reported the cytotoxic negative regulatory molecule, Cho‐1, that was expressed on the cell surface of rat fetal fibroblast cells in the cytotoxicity by natural killer (NK) cells. This molecule was IFN‐γ‐inducible, but appeared to be different from MHC class I. It was expressed on NK‐resistant cells but not on NK‐sensitive murine target cells such as YAC‐1. In this paper, first we determined whether Cho‐1 could also act as the negative regulatory molecule in a human NK‐resistant HEPM line. Our data strongly suggested that Cho‐1 could act as such a negative regulatory molecule in human NK cytotoxicity. The immunoprecipitates made with HEPM cell lysate and anti‐MHC class I monoclonal antibody (mAb) did not react against anti‐Cho‐1 mAb, indicating that Cho‐1 was different from MHC class I. Second, an assessment was made as to whether or not this molecule is involved in the cytotoxicity of CD8 (+) cytotoxic T lymphocytes (CTL) against human autologous tumor cells. The data indicated that although this cell surface molecule was expressed on certain tumor lines, it was not involved in the cytotoxic mechanism of CTL. Thus, Cho‐1 appeared to be the novel regulatory molecule in the NK cytotoxic mechanism.

Protein Components which Relate to the Kinin Releasing System in Bovine Plasma

Blood plasma contains several discrete systems consisting of the sequential conversion of proenzymes into active enzymes, in which their activations lead to a hypotension due to the liberation of vasoactive polypeptide termed kinin, and to thrombosis and hemorrhage due to the formation and lysis of fibrin clot. From physiological point of view, it is of interest how is the kallikrein-kinin system to be concerted with the other enzyme systems, such as blood coagulation and fibrinolysis. Recently, it was suggested that Hageman factor, which is known to have triggering action for the intrinsic coagulation and fibrinolysis systems, also pull the trigger for the activation of kallikreinkinin system. To clarify the above relations, it is extremely important to isolate the pure components, which concern to the liberation of kinin, and to elucidate their interrelations, reconstructing the kinin liberating systems in vitro.