title: A V H H that neutralizes the zinc metalloproteinase activity of botulinum neurotoxin type A.
abstract: The current treatment of botulism is to administer animal-derived antitoxin, which frequently causes severe adverse reactions in the recipients. In this study, a heavy chain antibody fragment (VH/V(H)H) phage display library was constructed by amplification of the immunoglobulin genes of a nonimmune camel, Camelus dromedarius, using primers specific to human VH gene segments. A recombinant light chain of type A botulinum toxin, BoTxA/LC, with zinc endoprotease activity was used in phage bio-panning to select phage clones displaying BoTxA/LC-bound VH/V(H)H. Soluble VH/V(H)H were produced and purified from 10 VH/V(H)H phagemid-transformed E. coli clones. Complementary determining regions (CDRs) and immunoglobulin frameworks (FRs) of the 10 camel VH/V(H)H-deduced amino acid sequences were determined. FR2 sequences of two clones showed a hallmark of camel V(H)H, i.e. (F/Y)(42)E(49)R(50)(G/F)(52). The remaining eight clones had an FR2 amino acid tetrad of conventional VH, i.e. V(42)G(49)L(50)W(52). V(H)H of one clone (V(H)H17) neutralized the SNAP25 hydrolytic activity of BoTxA/LC, whereas mouse polyclonal anti-BoTxA/LC did not have such activity. Mimotope sequences of V(H)H17 matched with the 194-206 amino acid residues of BoTxA/LC, which are located near the S'1 subsite of the catalytic cleft of the enzyme. Molecular docking revealed that CDR3 of the V(H)H17 bound to epitope in the toxin enzymatic cleft. Therefore, the BoTxA/LC neutralization by the V(H)H17 should be due to the V(H)H 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.
authors.affiliation: Departments of Immunology, Bangkok 10700, Thailand.
authors.forename: Jeeraphong
authors.initials: J
authors.lastname: Thanongsaksrikul
authors.affiliation: Parasitology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand; Graduate Program, Faculty of Allied Health Sciences, Thammasat University, Pathumthani 12120, Thailand.
authors.forename: Potjanee
authors.initials: P
authors.lastname: Srimanote
authors.affiliation: Department of Tropical Hygiene, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand.
authors.forename: Santi
authors.initials: S
authors.lastname: Maneewatch
authors.affiliation: Department of Biochemistry, Faculty of Sciences, Kasetsart University, Bangkok 10900, Thailand.
authors.forename: Kiattawee
authors.initials: K
authors.lastname: Choowongkomon
authors.affiliation: Graduate Program, Faculty of Allied Health Sciences, Thammasat University, Pathumthani 12120, Thailand.
authors.forename: Pramuan
authors.initials: P
authors.lastname: Tapchaisri
authors.affiliation: Research Center for Animal Hygiene and Food Safety, Hokkaido 080-8555, Japan.
authors.forename: Sou-Ichi
authors.initials: SI
authors.lastname: Makino
authors.affiliation: Department of Applied Veterinary Medicine and Public Health, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido 080-8555, Japan.
authors.forename: Hisao
authors.initials: H
authors.lastname: Kurazono
authors.affiliation: Parasitology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand. Electronic address: tmwcc@mahidol.ac.th.
authors.forename: Wanpen
authors.initials: W
authors.lastname: Chaicumpa
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry*; D019274:Botulinum Toxins, Type A / Q000378:metabolism*; D002162:Camelus; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000737:chemistry; D008297:Male; D045726:Metalloproteases / Q000737:chemistry*; D051379:Mice; D008813:Mice, Inbred ICR; D008969:Molecular Sequence Data; D019151:Peptide Library; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D015032:Zinc / Q000737:chemistry*
chemical_list: D000906:Antibodies; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D011994:Recombinant Proteins; D045726:Metalloproteases; D019274:Botulinum Toxins, Type A; D015032:Zinc