MP-4 Contributes to Snake Venom Neutralization by Mucuna pruriens Seeds through an Indirect Antibody-mediated Mechanism
Authors: Chitra Gupta
Deepak T Nair
Ashish Kumar
Dinakar M Salunke
Journal: Journal of Biological Chemistry (2016)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: antibody; crystal structure; humoral response; protease inhibitor; snake venom.
Affiliations: From the Structural Biology Unit, National Institute of Immunology, Aruna Asaf Ali Road, New Delhi 110 067, India, the Regional Centre for Biotechnology, NCR Biotech Science Cluster, 3rd Milestone, Faridabad-Gurgaon Expressway, Faridabad 121 001, India, and., From the Structural Biology Unit, National Institute of Immunology, Aruna Asaf Ali Road, New Delhi 110 067, India., the Regional Centre for Biotechnology, NCR Biotech Science Cluster, 3rd Milestone, Faridabad-Gurgaon Expressway, Faridabad 121 001, India, and ., From the Structural Biology Unit, National Institute of Immunology, Aruna Asaf Ali Road, New Delhi 110 067, India, the Regional Centre for Biotechnology, NCR Biotech Science Cluster, 3rd Milestone, Faridabad-Gurgaon Expressway, Faridabad 121 001, India, and the International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110 067, India .

Abstract:  Mortality due to snakebite is a serious public health problem, and available therapeutics are known to induce debilitating side effects. Traditional medicine suggests that seeds of Mucuna pruriens can provide protection against the effects of snakebite. Our aim is to identify the protein(s) that may be important for snake venom neutralization and elucidate its mechanism of action. To this end, we have identified and purified a protein from M. pruriens, which we have named MP-4. The full-length polypeptide sequence of MP-4 was obtained through N-terminal sequencing of peptide fragments. Sequence analysis suggested that the protein may belong to the Kunitz-type protease inhibitor family and therefore may potentially neutralize the proteases present in snake venom. Using various structural and biochemical tools coupled with in vivo assays, we are able to show that MP-4 does not afford direct protection against snake venom because it is actually a poor inhibitor of serine proteases. Further experiments showed that antibodies generated against MP-4 cross-react with the whole venom and provide protection to mice against Echis carinatus snake venom. This study shows that the MP-4 contributes significantly to the snake venom neutralization activity of M. pruriens seeds through an indirect antibody-mediated mechanism. 

Retracted: No

PubPeer comments:
 2018 expression of concern. http: www.jbc.org content 293 28 11253.short  The publisher of the Journal of Biological Chemistry is issuing an Expression of Concern to inform readers that credible concerns have been raised regarding the crystallography data shown in this paper. The Journal of Biological Chemistry will provide additional information as it becomes available.  , 
 #1 We have redetermined the structure of MP-4, and the new structure obtained at higher resolution shows that all the structural inferences in the JBC paper are correct. More details will follow soon. , 
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 Figure 1A and Figure 1B do not represent independent experiments. Figure 1B shows lanes 3 and 5 from Figure 1A to highlight which proteins could be identified using N-terminal sequencing. , 
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 #3, #5 : Commenters #3 and #5 display no evidence of understanding the experiment. Because they have said so little, it is difficult to decipher their thinking, but it appears that they believe there is a difference between (for example)  30-40% and  40% . If so, this is a misunderstanding. The figures with hyphens (e.g,. 0-30%, 30-40%, etc.) refer to fractionation STEPS. That is,  30-40% refers to the precipitated protein after the solution was adjusted from 30% to 40% ammonium sulfate. Thus, this fraction can be referred to equally as the  30-40% fraction or more simply as the  40% fraction. They are the same -- i.e., they refer to the proteins that precipitate at 40%, and did not previously precipitate at 30%. If you thought they refer to different samples, you were wrong. P.S., in the future, please explicitly describe your reasoning, if you want to be understood. , 
 With reference to Comment #2 please see: Jain A, Kumar A, Shikhi M, Kumar A, Nair DT, Salunke DM. (2020) The structure of MP-4 from Mucuna pruriens at 2.22u2005u00c5 resolution. Acta Crystallogr F Struct Biol Commun. 2020 76:47-57 ,