A novel designed single domain antibody on 3-D structure of ricin A chain remarkably blocked ricin-induced cytotoxicity.
Ricin A chain (RA), an N-glycosidase, is able to fatally disrupt protein synthesis by attacking the Achilles heel of the ribosome RNA (rRNA). As specific immunotoxins, emergence of inhibitors for RA may obtain access to antagonistics against ricin intoxication and contribute to ameliorate the concomitant side effects. Many experimental results showed that the engineered VHs, which possessed solubility, stability, small size and consequently easier to express, purify and manipulate in vitro, were self and long-lived molecules compared to synthetic peptides. In this study, based on the crystal structure of RA, a novel recombinant human single-domain antibody expressing a polypeptide against RA in the CDR3 loop (named rVH(PT)) was obtained using computer-guided molecular design method. Theoretically, rVH(PT) could penetrate deeply into the active cleft of RA and act as a potent antagonist analogue to block the RA-rRNA interaction. Followed results showed that the recombinant VH(PT) was easily expressed of high-yield production and in a partially soluble fusion form in Escherichia coli. In vitro cytotoxicity experiments demonstrated that it possessed remarkable ability to block ricin-induced cytotoxicity. This study highlights the potential of human VH to display biostructure and biofunction of peptides designed on RA functional domain and could be useful in developing new antidotes with potential therapeutic uses to neutralize unintended exposure to ricin.
Shuntao
Wang
W
Institute of Basic Medical Sciences, Department of Molecular Immunology, P.O. Box 130 (3), Taiping Road, Beijing 100850, PR China.
Jiannan
Feng
F
Jianwei
Guo
G
Leiming
Guo
G
Yan
Li
L
Yingxun
Sun
S
Weisong
Qin
Q
Meiru
Hu
H
Gencheng
Han
H
Beifen
Shen
S
D000906:Antibodies / Q000737:chemistry* / Q000276:immunology* / Q000302:isolation & purification / Q000494:pharmacology; D001666:Binding Sites, Antibody / Q000276:immunology; D001667:Binding, Competitive / Q000187:drug effects; D003001:Cloning, Molecular; D003602:Cytotoxicity, Immunologic / Q000187:drug effects*; D015195:Drug Design; D015870:Gene Expression; D006801:Humans; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D012276:Ricin / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000276:immunology* / Q000494:pharmacology*
D000906:Antibodies; D011993:Recombinant Fusion Proteins; D012276:Ricin