Yoshiyuki Kanai

Molecular cloning of nucleobindin, a novel DNA-binding protein that contains both a signal peptide and a leucine zipper structure

We have previously reported that KML1-7 cells cloned from a lupus-prone MRL/l mouse produced a soluble factor that preferentially expanded anti-DNA antibody production across the H-2 barrier. We purified this factor, a 55 kD protein that we termed nucleobindin (Nuc), and obtained its cDNA clone. Although the gene for Nuc encodes a signal peptide and, in fact, Nuc was identified as a secreted protein, Nuc had a DNA-binding property. The putative polypeptide predicted from the cDNA sequence featured a signal peptide, a leucine zipper structure and a basic amino acid-rich region. The DNA-binding property of Nuc was destroyed by deletion of either the leucine zipper structure or the basic amino acid-rich region. The amino acid sequences of Nuc are highly conserved between mouse and human. We discuss the possible role of Nuc in autoimmunity.

Effects of Corticosteroid and 1,24R-Dihydroxy-Vitamin D3 Administration on Lymphoproliferation and Autoimmune Disease in MRL/MP-lpr/lpr Mice

The pharmacological effects of prolonged administration of a corticosteroid, betamethasone, and active vitamin D3 [1,24R-(OH)2D3] on lymphoproliferation and autoimmune disease of MRL/MP-lpr/lpr (MRL/1) mice were examined. Relatively high doses of betamethasone (0.25 mg/kg/day) prevented lymphoproliferation, reduced serum levels of anti-dsDNA, anti-ssDNA, and anti-poly (ADP-ribose) antibodies, and brought about clinical improvement, such as reduced proteinuria and diminution of skin lesions. It is noteworthy that not only did prevention of lymphoproliferation occur, but also recovery of the Lyt-2+ T cell subset in the thymus and the spleen was observed. The administration of 1,24R-(OH)2D3 (0.1 microgram/kg/day) similarly prevented proteinuria, and produced recovery of a Lyt-2+ subset in the thymus.

Naturally-occurring antibodies to poly(ADP-ribose) in patients with systemic lupus erythematosus

POLY(ADP-RIBOSE) is a biopolymer which is synthesised by poly(ADP-ribose) polymerase in cell nuclei1. Its structure and physicochemical characteristics have been reviewed1. Its biological function was suggested to be related to DNA synthesis in nuclei2–4, and there are several reports on its natural occurrence5–9. We reported previously that a specific antibody to poly(ADP-ribose) could be produced in rabbits injected with a complex of poly(ADP-ribose) and methylated bovine serum albumin10. Kidwell and Mage reported on the presence of poly(ADP-ribose) in HeLa cells and the correlation of its content to cell cycles by a radioimmunoassay11. Recently we found that binding activities of the sera of patients with systemic lupus erythematosus (SLE) to 14C-poly(ADP-ribose) were higher than those of the sera of normal individuals, and we suggest that poly(ADP-ribose) as well as antibodies to poly(ADP-ribose) occur naturally in human beings. This paper reports that binding activities of the sera of SLE patients to 14C-poly(ADP-ribose) are attributable to immunoglobulins, mainly to IgG, but not to nonspecific interactions between 14C-poly(ADP-ribose) and IgG; specificity of the binding was verified by inhibition tests using unlabelled poly(ADP-ribose) and 14 related compounds.

Interaction of the protein nucleobindin with Gαi2, as revealed by the yeast two-hybrid system

The heterotrimeric G protein, Gαi2, transduces signals from seven membrane spanning receptors to effectors such as adenylyl cyclase and ion channels. The purpose of this study was to identify these or other cellular proteins that interact with Gαi2 by use of the yeast two‐hybrid system. A human B cell cDNA library was screened by this system using full length Gαi2. Four positive colonies were obtained. Two of the four were identified as nucleobindin, a calcium binding protein and a putative antigen to which anti‐nuclear antibodies are generated in mice with a disorder that resembles systemic lupus erythematosus. Nucleobindin has a leucine zipper, EF hands, and a signal peptide sequence and is thought to localize to the nucleus as well as being secreted. The specificity of intehraction between Gαi2 and nucleobindin was confirmed by an in vitro binding assay using recombinant proteins. Transfection of Gαi2 and nucleobindin in COS cells increased Gαi2 expression relative to cells transfected with Gαi2 and mock vector. Our results indicate that the yeast two‐hybrid system provides a means to identify novel proteins that interact with Gα proteins. Nucleobindin appears to represent one of those proteins.

Studies on anti-poly(adenosine diphosphate ribose) antibody.

Summary Specific antibody against poly(ADP-Rib) was produced in a rabbit by injecting poly(ADP-Rib) mixed with methylated bovine serum albumin. Under standardized conditions, 1 mg of purified anti-poly(ADP-Rib) antibody combined with 400 pmoles (4 μg) of poly(ADP-Rib) and was retained on a millipore filter. The binding of [ 14 C]poly-(ADP-Rib) was not inhibited by poly(A) or other related nucleotides.

Purification of a novel B cell growth and differentiation factor associated with lupus syndrome.

We have previously reported that KML1-7 cells cloned from a lupus-prone MRL/l mice produced a soluble factor that preferentially expanded anti-DNA antibody production across the H-2 barrier. We purified this factor, a 55-kDa protein that we termed nucleobindin (Nuc). Nuc showed not only induction of anti-ssDNA IgG antibody in cultures of B cells from MRL/l mice (greater than 16 weeks old), but also growth activity. Furthermore, antibodies against existing cytokines have so far not been shown to block Nuc activity on these B cells. In view of the fact that Nuc did not boost anti-ssDNA IgG antibody production in cultures of spleen cells of comparable age from MRL/n mice, which develop a mild form of lupus after the age of one year, Nuc may act on pre-activated B cells to help IgG anti-DNA antibody production. Taken together, Nuc is a new kind of growth and differentiation factor associated with lupus syndrome.

A monoclonal anti-double stranded DNA antibody from an autoimmune MRL/Mp-lpr/lpr mouse: specificity and idiotype in serum immunoglobulins

Anti-double stranded (ds) DNA antibodies in the sera of lupus-prone MRL/Mp-lpr/lpr (MRL/l) mice were determined by an enzyme-linked immunosorbent assay in parallel with anti-single stranded (ss) DNA, anti-left-handed Z-DNA and anti-poly(ADP-ribose) antibodies. The serum levels of these antibodies in these mice increased with age, and in particular anti-dsDNA antibodies appeared in mice more than 14 weeks old, along with progressive lymphadenopathy. We therefore established a hybridoma producing monoclonal anti-dsDNA antibody (2C10) from an 8-month-old MRL/l mouse. Monoclonal antibody 2C10 did not react with either poly(dT) or poly(I), which are major cross-reactants with previously reported monoclonal MRL mouse autoantibodies. Antibody 2C10 showed preference for phi X-174 replicative form DNA and calf thymus dsDNA over ssDNA. 2C10 idiotype (Id) was present in the sera of MRL/l mice, but only occasionally at high levels even in the aged mice tested. This result suggested that many Ids with anti-dsDNA antibody activity may contribute to lupus pathogenesis in this strain of mouse.

Studies of nuclear ADP-ribosylation

Abstract Recent studies on poly(ADP-ribose) are reviewed. Poly(ADP-ribose) was shown to have α (1″ → 2′) ribose-ribose bonds. High molecular weight poly(ADP-ribose) with a branched structure was demonstrated. The structure of the branch linkage was determined as 2″-[1′-ribosyl-2″ (1‴-ribosyl)]-adenosine 5′,5″,5‴-tris(phosphate). The known sites of ADP-ribosylation of proteins were summarized. The enzymatic synthesis of poly(ADP-ribose) was classified into three steps, initiation, elongation and branching. Degradation of poly(ADP-ribose) may be mainly performed by α (1″ → 2′)-poly(ADP-ribose) ribohydrolase. Specific antibodies against poly(ADP-ribose) and its monomer, 2′-ribosyl adenosine 5′,5″-bis(phosphate), were obtained and used for quantitative measurement of naturally occurring poly(ADP-ribose). High antibody activity was found in the serum of the patients with systemic lupus erythematosus. Poly(A)·poly(U) could also raise specific antibody against poly(ADP-ribose) in rabbits. Nicotinamide, an inhibitor of poly(ADP-ribose) polymerase, increased sister chromatid exchanges. Among the nicotinamide analogues tested, N ′-methylnicotinamide was the best inducer of differentiation of Friend erythroleukemia cells. Purified poly(ADP-ribose) induced differentiation of mouse myelogenous leukemia cells.

Poly(ADP-ribose) polymerase (PARP) revisited. A new role for an old enzyme: PARP involvement in neurodegeneration and PARP inhibitors as possible neuroprotective agents.

Poly(ADP4bose) polymerase (PAW), discovered in 1966 by Chambon and colleagues,2 is a chromatin binding protein that utilizes the oxidized form of nicotinamide adenine dinucleotide (NAD+) as substrate. This enzyme transfers monomers of adenosine diphosphate ribose from NAD + to chromatin-associated proteins (including PARP itself), or to other molecules of ADP-ribose to form a polymer. PolyADP-ribosylation can, in turn, modify the activities of various proteins. P A W has been shown to be activated by nicks in the DNA molecule and to be involved in DNA plasticity-related phenomena such as DNA repair, carcinogenesis, cell proliferation and gene expre~sion.”~ A unifying concept has been proposed,’ that PAW regulates cellular metabolism in response to DNA damage through modula-

Antigen expression associated with lymph node metastasis in gastric adenocarcinomas

A total of 100 gastric adenocarcinomas, comprising 50 cases with lymph node metastasis and 50 cases without lymph node metastasis, were examined for immunohistochemical reactivity with the monoclonal antibody to urokinase‐type plasminogen activator (u‐PA), Lex related 4C9 antigen, Jun, or to nucleobindin (Nuc). In tumors with lymph node metastasis, 41 (82%) were positive for u‐PA and 28 (56%) were positive for Nuc. In tumors without lymph node metastasis, 26 (52%) were positive for u‐PA and five (10%) were positive for Nuc. The percentage of cases positive for u‐PA or Nuc was significantly higher in tumors with lymph node metastasis than that in tumors without lymph node metastasis (P < 0.01). The expression of u‐PA was found to be significantly correlated with that of Nuc (P < 0.001), mode of infiltrative growth (P < 0.05), depth of invasion (P < 0.01), and grade of lymphatic invasion (P < 0.01). However, the expression of Nuc was found to be significantly correlated with the expression of Jun (P< 0.05), depth of invasion (P < 0.01), and grade of lymphatic invasion (P < 0.001). These results suggest that immunohistochemical examination for the expression of u‐PA and Nuc in tumor cells may help evaluate the potential of adenocarcinomas of the stomach for lymph node metastasis.