| dc.description.abstract | Ricin toxin’s enzymatic A subunit (RTA) is a 267 amino acid RNA N-glycosidase that depurinates a conserved adenine residue of 28S rRNA, resulting in ribosome arrest and apoptosis. One of the leading subunit vaccine candidates for ricin is RiVax, a two point mutant (V76M, Y80A) of RTA. RiVax has proven to be safe in humans, however, it could not elicit a robust toxin-neutralizing antibody response. In order to redesign a potent ricin subunit vaccine candidate based on RTA, an immunological rationale has to be implemented. Protection against ricin is antibody mediated and hence generating a comprehensive B cell epitope map of ricin toxin would not only help in evaluating future ricin vaccine candidates in humans but also provides an immunological rationale for designing new vaccine candidates. Previous studies have shown that toxin neutralizing antibodies recognized four immunodominant regions on RTA i.e. four epitope clusters (namely cluster I to IV). RTA’s active site is surrounded by cluster III. Surprisingly, mAb IB2, which defines cluster III is the only antibody that recognized this immunodominant region. We previously showed that IB2 binds to helices C and G on the surface of RiVax. In this study, we sought to gain a better understanding of cluster III using a collection (21) of single domain antibodies (VHHs) that are derived from ricin immunized alpacas. To this end, I first produced and characterized RiVax (a safer version which is structurally identical to RTA) for its structural integrity since our main emphasis lies in identifying discontinuous/ conformational epitopes. Based on hydrogen exchange mass spectrometry (HX-MS) studies, VHHS recognized overlapping epitopes with four spatially distinct contact regions i.e. they were grouped into four subclusters (namely 3.1 to 3.4) within cluster III region. Subcluster 3.1 includes helices C and G. Subcluster 3.2 encompasses additionally helix B along with helices C and G. While subcluster 3.3 consists of helices B and G, subcluster 3.4 includes helices C, E and strand h. Of the 21 antibodies that we analyzed, only two, namely V1D3 and V6D4 have shown toxin neutralizing activity (TNA). Both neutralizing antibodies have strong binding affinity (sub nanomolar range) to the toxin and engaged a common secondary structural element, namely helix G as part of their epitope. The second part of my dissertation focusses on fibroblast growth factor-1 (FGF-1), a member of β-trefoil family of proteins. FGFs regulate a number of developmental process including mitogenesis, angiogenesis and homeostasis etc. Due to their wide range of biological activities, FGFs has been of interest in several clinical applications. In particular, wound healing has generated considerable interest. Studies have shown that several polyanions (sulfated and phosphorylated) have increased the thermal stability of FGF-1 by 15-30oC. In this study, we sought to identify the binding sites of the polyanions. In particular, we studied two sulfated (heparin, low MW heparin) and two phosphorylated (phytic acid and ATP) polyanions. Using HX-MS, we examined the local dynamics as well as binding sites of the polyanions. For local dynamics, we identified strand 4 and 5 and the turn connecting them to be most flexible which agrees with previous NMR studies. On the other hand, strands 8, 9 and 10 appear to be more rigid which is consistent with crystallographic B factors as well as local dynamic studies conducted by NMR. Crystal studies have shown that heparin binds to N-terminal Asn18 and to C-terminal Lys105, Tryp107, Lys112, Lys113, Arg119, Pro121, Arg122, Gln127 and Lys128 indicating electrostatic forces as the dominant interactions. Heparin binding as determined by HX-MS is consistent with the crystallography data. We find other polyanions tested bind in a similar manner to heparin, primarily targeting the turns in the lysine rich C-terminal region of FGF-1 along with two distinct N-terminal regions that contains lysines and arginines/ histidines. This confirms the interactions between FGF-1 and polyanions are primary directed by electrostatics. | |
