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Current Research Interests

 Botulinum
Neurotoxin
Biochemistry
Action

biochem faculty ram interest 1
Neuromuscular
junction is the
target site of
botulinum
neurotoxin
(botulinum toxins
are the most deadly
poisonous poison
known in world)





This research group is working in collaboration with SMAST, (School for Marine Science and Technology, New Bedford), UMass Cranberry Station (Wareham) and the UMass Dartmouth main campus. The SMAST laboratory is involved in the Bioremediation and Cranberry research projects while the UMass main campus laboratory is involved in the Botulinum Neurotoxin Research. All laboratories are well equipped with state-of-the-art research instruments. The research environment is challenging and competitive. This research is funded by Federal, State and private funding agencies.



Marine Biochemistry
biochem faculty ram interest 2

Replenishing of the shell-fish stocks in New Bedford Harbor Quahog
Oligomeric
nature of BoNT:

Site-directed
polyclonal
antibodies :

Cranberry Research

biochem faculty ram interest 3

Molecular Mode of Botulinum Neurotoxin Action

            BoNTs (seven serotypes, A-G) are relatively large water-soluble proteins (150 kDa). Each protein has two polypeptide chains: a 100 kDa H chain and a 50 kDa L chain linked through a disulfide bond. BoNTs are very similar to TeNT in their structure and mechanism of action. Therefore, in most respects, the knowledge of their mode of action is interchangeable. Complete primary structures of types A, B, C1, D, E, F and G have been known for several years now. Primary structures of BoNTs have provided two very useful clues to their mechanism of toxic action. One relates to the presence of a zinc-binding motif present in the L chain. The second relates to the presence of a transmembrane segment in the N-terminal half of the H chain. In BoNT/A, hydrophobicity calculations suggested only 1-2 membrane compatible segments whereas hydrophobic moment analysis revealed 5 transmembrane segments. In addition, several amphiphilic (surface-seeking) peptide segments were identified. The presence of several transmembrane/amphiphilic peptide segments is likely to provide a structural basis for the membrane channel activity of the neurotoxin.

         The mode of BoNT action is not well understood at the molecular level. Based on some experimental evidence and analogies with other dichain toxins such as diphtheria, cholera and Pseudomonas exotoxin A, a working model has been proposed. Three major steps (Fig. 3) involved are (i) Extracellular step which involves the binding of the neurotoxin to presynaptic membranes through the C-terminal half of the H chain,  (ii) Internalization and Translocation steps which involve internalization of BoNT through endocytosis, and (iii) Intracellular step that involves biochemical steps leading to the blockage of the neurotransmitter release. Internalization and translocation of BoNT are the least understood steps at the molecular level. Two major questions need to be answered: (i) How can a water soluble protein get integrated into non-polar lipid bilayer for its translocation? It has been proposed in the past that a membrane channel formation is involved in the translocation of BoNT or its L chain into the nerve cell. Our theoretical calculations  have identified possible transmembrane/amphiphilic segments that could be involve in the channel formation. (ii) Is a channel formed by a monomeric unit of BoNT large enough for the L chain translocation? Could the membrane channel involve oligomeric structure of BoNT? One of our hypotheses suggests oligomeric form of the neurotoxin for the formation of the membrane channel. Recently, we have demonstrated existence of oligomeric form of BoNT/A, BoNT/B and BoNT/E and TeNT based on native gel electrophoresis and chemical cross-linking experiments. Our results are consistent with an earlier observation of the presence of  "aggregation" of BoNT/A based on native gel electrophoresis band of 450-600 kDa. Crystal form of BoNT/A is also believed to exist as a dimer.

     Our long term objective is to identify the peptide segments of BoNT and demonstrate their involvement in the formation of membrane channels capable of translocating at least the light (L) chain of BoNT. Comprehensive plan includes demonstration of amphiphilic nature of peptides, structural changes in the neurotoxin and peptides in different pH conditions and upon interaction with lipids, use of peptide specific antibodies to demonstrate involvement of specific peptides in the membrane channel formation using artificial and natural membrane systems for the channel activity assay, use of site-directed mutagenesis to establish involvement of specific amino acid residues in the membrane channel activity, investigation of oligomeric structure of the neurotoxin in different pH conditions and in association with lipids and lipid bilayers, modeling protein structure to predict the membrane channel structure. Our near-future plans include the following.

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Experimental investigation of oligomeric nature of BoNT:

        The involvement of oligomeric structures in the formation of membrane channels by water-soluble bacterial protein toxins is well established. Several of these toxins such as a-toxin of Staphylococcus aureus form oligomeric structures upon binding with membrane whereas others such as cholera and pertussis toxins have oligomeric multi-subunit membrane channel forming domains. However, diphtheria toxin membrane channel domain has not been shown to have oligomeric structure. Thus, the molecular basis of membrane channel formation by water-soluble toxins is perhaps not universal. It may be particularly unique for BoNT as its active subunit (L chain) that needs to be translocated through the channel is larger than any other toxins known, so far. Therefore, the quaternary structures of BoNT could prove to be instrumental in understanding its mode of action, especially in the formation of membrane channels and translocation of the toxic subunit. Our own preliminary studies indicate that BoNTs A, B and E exist in several oligomeric forms, most notably in dimer, trimer, and larger species. We have also suggested possible sites of association in the form of leucine zipper-like structures that are present both on the L and H chains. Further investigations are needed to confirm our preliminary data of the existence of oligomeric neurotoxin structures, effect of low pH and lipids on the oligomeric structures  and to identify the sites of oligomerization.

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Site-directed polyclonal antibodies to specific peptide segments of the neurotoxin involved in membrane channel formation and neurotoxin translocation :

        Using neuromuscular junction preparations, whereas it has been demonstrated that the H chain subunit of the BoNT is involved in translocation of the neurotoxin into neuronal cells, the specific domains of the H chain involved in the translocation process are not known, except for the fact that it is the N-terminal half of the H chain that mediates the membrane channel formation. One site that has recently been identified is a hydrophobic segment located in the N-terminal half of the H chain. However, this segment is perhaps not sufficient for the translocation of the neurotoxin or its L chain. Moreover, the involvement of membrane channel activity itself has been questioned in the past for the translocation of the L chain. Recent reports of membrane channel activity by the L chain further raise questions about membrane channel activity being a total reflection of the translocation process.  It is likely that other domains of the H chain are involved in the translocation process as well in the membrane channel formation. Based on our preliminary studies of BoNT/A H chain ( unpublished), it seems that even the C-terminal half of the H chain plays an important role in the membrane channel activity. Interestingly, polypeptide segments that we have identified as amphiphilic (based on hydrophobic moment calculations)  are  located throughout the entire sequence of the L and H chains, thus opening the possibility of their interaction with the lipid bilayer.

         Therefore, our approach of examining the participation of all the transmembrane/amphiphilic segments located both on the H and L chains of BoNT/A is likely to yield information relevant not only to the understanding of role of the L chain and the two halves of the H chain, but the approach will also identify specific peptide segments involved in the membrane channel activity as well as in the translocation of the L chain. The approach will also provide an opportunity to correlate channel activity with the translocation of the neurotoxin or its L chain.

          To confirm our hypothesis that the predicted amphiphilic segments  participate in the membrane channel formation and neurotoxin translocation, we propose to raise antibodies against small peptide segments (15-20 amino acids each) containing predicted amphiphilic segments of L and H chains. The idea is to raise antibodies which are specific to the amphiphilic segments that are likely to be involved in the integration of the neurotoxin with membrane. Such antibodies will be used to test the role of specific peptide segments of the L and H chains in the L chain translocation. The antibodies raised against specific peptide segments have high accuracy in recognizing their respective segments present in the native proteins. This is unlike monoclonal antibodies which, for all practical purposes, are used to identify only long stretches of proteins, because of the non-availability of small proteolytic peptide fragments of native proteins for identification.  Another advantage of the polyclonal antibodies against peptides is that they have stronger affinity for a given epitope compared with monoclonal antibodies. Moreover, monoclonal antibodies are difficult and expensive to prepare, and it is time consuming to map their epitopes on a protein. Peptide specific antibodies provide a tool to subsequently map the entire polypeptide chain  whereas the antibodies (monoclonal or polyclonal) prepared against the whole proteins recognize only the peptide segments that are antigenic.

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Marine Biochemistry

 We have recently initiated a project on the isolation and characterization of glutathione-S-transferases (GSTs) from marine organisms including oyster, scallops and marine algae. GSTs are known to be xenobiotic detoxifying enzyme, and our long-term goal is use the enzyme as a biomarker for the organic pollution of the ocean environment. An aricle describing our initial results has been published in the Journal of Marine Biotechnology. In recent years, Northeastern Section of the American Chemical Society and UMass Dartmouth Foundation have provided funds for this research.

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Cranberry Research

 We are working on two different projects related to cranberry agricultural crop. One project deals with the detection and identification of Phytophthora cinnamomi which causes root rot in the cranberry plants. We have recently cloned and sequenced the gene for the cinnamomin protein. The other project relates to the involvement of phytochrome in the color development of cranberry fruits.

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