The long term goals are:(i) to employ catalytic antibodies to define the fundamental relationships between substrate binding and its catalytic chemical conversion, (ii) to determine whether antibody-mediated catalytic hydrolysis of cell surface antigens, hormones and neurotransmitters is a fundamental pathogenetic mechanism in autoimmune disease, and (ii) to investigate the promise that catalytic anti-peptide antibodies hold as specific anti-viral and anti-tumor therapeutic agents. The specific objectives of the proposed studies are: (i) to demonstrate catalytic hydrolysis of the neurotransmitter VIP by human autoantibodies, (ii) to define the clonotype of the VIP autoantibodies and to purify the VIP- specific antibodies to homogeneity, (iii) to identify the scissile peptide bond(s) and compare the site of hydrolysis with the epitope(s) bound by the antibodies, (iv) to define the sequence specificity of the antibodies based on binding and hydrolysis studies, (v) to measure the frequency of catalytic VIP autoantibodies in defined human subject populations, and to evaluate the bioactivity of fragments produced by antibody-mediated VIP hydrolysis, (vi) to investigate whether the sensitivity of certain peptide bonds to hydrolysis arises from an intrinsic instability of these bonds, (vii) to determine whether known peptidase inhibitors influence antibody- mediated hydrolysis of VIP, (viii) to measure the ability of presumed transition state analogs to bid the antibodies and inhibit hydrolysis of VIP, (ix) to develop human hybridoma cell lines producing monoclonal catalytic anti-VIP antibodies. The experimental design is: (i) purification of mono(125l,Tyr10)-VIP by reverse phase HPLC, (ii) measurement and analysis of mono (125l,Tyr10)-VIP binding by radioimmunoassay and mono(125l,Tyr10)-VIP hydrolysis by trichloracetic acid precipitation and HPLC, (iii) purification of the antibodies by ligand specific affinity chromatography and isoelectric focusing, (iv) covalent crosslinking of antibody:mono(125l,Tyr10) VIP complexes and analysis by two dimensional electrophoresis, (v) localization of antibody binding and hydrolytic activity in gels by novel blotting techniques, (vi) purification of VIP fragments by HPLC and their identification by peptide sequencing and fast atom bombardment-mass spectroscopy, (vii) localization of the antibody binding epitopes by measuring the reactivity of synthetic VIP subsequences in radioimmunoassay, (vii) measurement of antibody specificity using peptides bearing partial sequence identify with VIP in binding and hydrolysis assays, (ix) measurement of binding and catalytic activity of VIP autoantibodies in asthmatics and healthy subjects, (x) investigation of reaction mechanisms by evaluating the instability of peptide bonds, the effect of known inhibitors of the various classes of proteases, on the antibody-mediated hydrolysis, and the reactivity of presumed transition state analogs, by the methods stated above, and (xii)construction of human cell lines making monoclonal catalytic anti-VIP antibody by lymphocyte transformation with Epstein-Barr virus, fusion with myeloma cells, and cloning by limiting dilution techniques.
| Planque, Stephanie; Zhou, Yong-Xin; Nishiyama, Yasuhiro et al. (2003) Autoantibodies to the epidermal growth factor receptor in systemic sclerosis, lupus, and autoimmune mice. FASEB J 17:136-43 |
| Berisha, H I; Bratut, M; Bangale, Yogesh et al. (2002) New evidence for transmitter role of VIP in the airways: impaired relaxation by a catalytic antibody. Pulm Pharmacol Ther 15:121-7 |
| Nishiyama, Yasuhiro; Taguchi, Hiroaki; Luo, Jin-Quan et al. (2002) Covalent reactivity of phosphonate monophenyl esters with serine proteinases: an overlooked feature of presumed transition state analogs. Arch Biochem Biophys 402:281-8 |
| Paul, S; Tramontano, A; Gololobov, G et al. (2001) Phosphonate ester probes for proteolytic antibodies. J Biol Chem 276:28314-20 |
| Li, L; Kalaga, R; Paul, S (2000) Proteolytic components of serum IgG preparations. Clin Exp Immunol 120:261-6 |
| Thiagarajan, P; Dannenbring, R; Matsuura, K et al. (2000) Monoclonal antibody light chain with prothrombinase activity. Biochemistry 39:6459-65 |
| Paul, S; Kalaga, R S; Gololobov, G et al. (2000) Natural catalytic immunity is not restricted to autoantigenic substrates: identification of a human immunodeficiency virus gp 120-cleaving antibody light chain. Appl Biochem Biotechnol 83:71-82; discussion 82-4, 145-53 |
| Gololobov, G; Tramontano, A; Paul, S (2000) Nucleophilic proteolytic antibodies. Appl Biochem Biotechnol 83:221-31; discussion 231-2, 297-3 |
| Gololobov, G; Sun, M; Paul, S (1999) Innate antibody catalysis. Mol Immunol 36:1215-22 |
| Gololobov, G; Noda, Y; Sherman, S et al. (1998) Stabilization of vasoactive intestinal peptide by lipids. J Pharmacol Exp Ther 285:753-8 |
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