HDAC Inhibitors as Epigenetic Regulators

After washing, the amount of bound VH/VHH in each well was detected using mouse anti-E Tag antibody, goat anti-mouse immunoglobulin-horseradish peroxidase conjugate (Southern Biotech), and ABTS substrate. had an FR2 amino acid tetrad of conventional VH,i.e.V42G49L50W52. VHH of one clone (VHH17) neutralized the SNAP25 hydrolytic activity of BoTxA/LC, whereas mouse polyclonal anti-BoTxA/LC did not have such activity. Mimotope sequences of VHH17 matched with the 194206 amino acid residues of BoTxA/LC, which are located near the S1 subsite of the catalytic cleft of the enzyme. Molecular docking revealed that CDR3 of the VHH17 bound to epitope in the toxin enzymatic cleft. Therefore, the BoTxA/LC neutralization by the VHH17 should be due to the VHH insertion into the enzymatic cleft of the toxin, which is usually inaccessible to a conventional antibody molecule. This antibody fragment warrants further development as a therapeutic agent for botulism. Keywords:Antibodies, Enzymes/Inhibitors, Protease/Inhibitor, Proteases/Metalloprotease, Toxins, Toxins/Drugs/Xenobiotics/Botulinum, Affinity, Catalytic Activity, Snap25, Vhh == Introduction == Botulism is a clinical manifestation characterized by generalized flaccid paralysis and respiratory insufficiency, which may be fatal if not treated properly. It is caused mainly by consumption of food contaminated with neurotoxins ofClostridium botulinum(BoTxs).4The BoTxs are zinc-dependent endopeptidases that cleave SNARE proteins used for the exocytosis of the neurotransmitter at the motor nerve end plate (1,2). BoTxs are recognized as the most potent toxic substance of humans with a lethal dose as low as 1 ng/kg body weight (35) and are classified as a category A bio-weapon by the Centers for Disease Control and Prevention (67). Presently, there are seven antigenic types of BoTxs, serotypes AG (35). Among these, serotype A causes the most serious KIT clinical manifestations in humans due to its prolonged localization within the cytoplasm of Moxisylyte hydrochloride the affected neuron (8). The molecular structure of BoTxs has been revealed by crystallography as an A-B toxin (9,10). The two peptides are synthesized as a single polypeptide, which is modified post-translationally to a 150-kDa, di-chain active holotoxin. Each molecule of the holotoxin is composed of an A subunit or light chain (50 kDa), which is linked to a B subunit or heavy chain (100 kDa) by a single disulfide bond. The heavy chain is responsible for receptor binding, internalization, and translocation of the holotoxin into the endosome of cholinergic neurons (11). After an early endosomal exit, the light chain hydrolyzes SNARE proteins such as SNAP25 (for types A, C, and E BoTxs), synaptobrevin (for types B, D, F, and G BoTxs), and Moxisylyte hydrochloride syntaxin (type C BoTx) resulting in the disruption of the neurotransmission process (12,13). A licensed BoTx antagonist is not available. Patients with botulism are treated with animal-derived anti-BoTx antibodies together with supportive measures, such as artificial respiration. There are several drawbacks of using the antitoxin of heterologous species. The animal antibodies often elicit allergic reactions, which may be as serious as fatal anaphylaxis, Moxisylyte hydrochloride as well as an anti-isotype/idiotype response that causes serum sickness (6). Besides, a prolonged immunization process of the donor animals is required before a satisfactory level of the antitoxin is reached. Because of their small size (1520 kDa), high tissue-penetrating efficacy, and relative stability, single domain heavy chains (VHH) from a dromedary (Camelus dromedarius), which are devoid of a variable light chain domain have attractive molecular structures for a potent enzyme/toxin inhibitor (1420). VHH could directly recognize the conformational structure within the pocket of an enzyme active site, which can never be reached by the large sized conventional heavy light chain antibody (2123). In this study, VHH produced from a phage clone derived from a VH/VHH phage display library constructed from immunoglobulin genes of B cells of a nonimmune Arabian camel,C. dromedarius, are used to bind specifically to the catalytic light chain of the type A botulinum neurotoxin and to inhibit the toxin endopeptidase activity. Experimental details and results are reported herein. == EXPERIMENTAL PROCEDURES == == == == == == Production of a Full-length Recombinant Light Chain of Type.