Ricin is a member of the ribosome-inactivating protein (RIP) family of toxins found throughout the plant and microbial worlds. In its mature form, ricin is a 65 kDa glycoprotein consisting of two subunits, RTA and RTB, joined by a single disulfide bond. RTA (267 amino acids) is an RNA N-glycosidase that mediates the depurination of a universally conserved residue within the Sarcin-ricin loop (SRL) ribosomal RNA element. RTB is a galactose-/N-acetylgalactosamine-specific lectin that facilitates attachment, endocytosis, and intracellular trafficking of ricin in mammalian cells. Following endocytosis, ricin is routed to the trans-Golgi network (TGN) and then shuttled to the endoplasmic reticulum (ER), where RTA is liberated from RTB. RTA is then translocated into the cytosol where it targets ribosomes.
Structurally, RTB contains two homologous globular domains, each divided into three subdomains (α, β, ɣ). While each pair of subdomains (e.g. 1α and 2α) have similar folding topologies and amino acid sequence homology, not all subdomains are productive binding pockets. Only 1α and 2ɣ have specific polar contacts that are pertinent for binding galactosides. Subdomain 1α contains a low-affinity carbohydrate recognition domain (CRD) that binds terminal galactose (Gal), while 2ɣ, the presumed high-affinity binding pocket, recognizes both galactose and N-acetylgalactosamine (GalNAc). Furthermore, mutational studies have demonstrated that altering key contact residues in one or the other site alone does not abrogate lectin activity, suggesting that 1α and 2ɣ CRDs are likely redundant.
Numerous studies of RTB-specific monoclonal Abs (mAbs) have found that the neutralizers likely function through two mechanisms: by inhibiting toxin attachment to cell surfaces (Type I) or by neutralizing via intracellular mechanisms (Type II). SylH3 is considered a Type I MAb which blocks ricin binding to its receptors, while 24B11 is categorized as a Type II neutralizer due to its ability to derail toxin trafficking to the TGN. Despite extensive characterizations, specific epitopes of Type I and Type II mAbs on RTB remain unclear.
The objective of this study was to provide a detailed B-cell epitope map of RTB and identify the crucial regions responsible for in vitro neutralizing activity. To define and localize the regions vulnerable to toxin-neutralizing Abs, I characterized 68 camelid-derived heavy chain only antibodies (VHHs) from an anti-ricin phage display library. Within this panel, 19 RTB and holotoxin specific VHHs neutralized ricin in a dose-dependent manner and were further categorized based on domain specificity. Of these, 11 VHHs targeted domain 1 (RTB-D1), while 8 VHHs bound specifically to domain 2 (RTB-D2). To characterize the VHHs in greater detail, I determined their abilities to inhibit toxin binding using a THP-1 cell attachment assay and a solid phase attachment assay. Moreover, I used surface plasmon resonance to determine their binding affinities to ricin.
In Chapter 3, I localized the epitopes of a panel of potent neutralizing VHHs to RTB-D2. Their epitopes were mapped to the 2ɣ high-affinity binding pocket and localized adjacent to the neutralizing hotspot Cluster II on RTA. Despite targeting the high-affinity binding site, 50% of ricin remained bound on cell surfaces in the presence of potent neutralizer V2C11 and other 2ɣ-specific VHHs. Furthermore, there was no correlation between toxin neutralizing activities (TNAs) and VHHs’ abilities to inhibit ricin binding to cell surfaces. Rather, their TNAs correlated strongly with epitope proximity to Cluster II. Thus, I propose that 2ɣ-specific VHHs must neutralize in a step downstream of toxin attachment. In Chapter 4, I found that 2ɣ-specific VHHs continued to neutralize ricin downstream of toxin attachment. This result was obtained using a Vero cell post-attachment cytotoxicity assay. In this assay, ricin alone was applied to Vero cells at 4oC to allow toxin adherence without toxin internalization. Unbound toxin was removed, cells were treated with VHH and then warmed to 37oC to promote toxin uptake. Furthermore, in collaboration with Oslo University, it was demonstrated in HeLa cells that potent neutralizers such as V2C11 increased dissociation of receptor-bound toxin and decreased total ricin from trafficking into the TGN.
While the previous two chapters focused on VHHs that target RTB-D2, in the following chapters I sought to characterize VHHs that bind RTB-D1. In Chapter 5, I localized three potent toxin attachment inhibiting VHHs to RTB-D1 based on their competition with multiple other D1-specific mAbs. In addition to the three VHHs identified in Chapter 5, 11 additional D1-specific VHHs were characterized in Chapter 6. These 14 VHHs produced a large range of TNAs and inhibited 95% of toxin binding in a solid phase binding assay. Furthermore, their TNAs correlated strongly with their toxin attachment inhibition activity. Epitope mapping experiments localized potent neutralizers such as V11E10 to subdomain 1β, while weak and moderate neutralizers such as V8H2 and V11D1 were mapped to subdomains 1α and 1ɣ. I propose that subdomain 1β is a neutralizing hotspot on RTB-D1 and is associated with mediating toxin attachment.
In summary, I identified two neutralizing hotspots on RTB that were localized to subdomains 1β and 2ɣ. D1-specific toxin-neutralizing correlated strongly with their toxin attachment inhibition activities, while 2ɣ-specific VHHs were most proficient at neutralizing receptor-bound ricin, blocking toxin trafficking at multiple steps after receptor attachment. By elucidating the mechanisms by which D1- and D2-specific VHHs neutralize ricin, I propose that RTB-D1 plays an important role in mediating toxin binding, while RTB-D2 facilitates toxin intracellular trafficking. Knowledge of vulnerable regions on RTB along with Ab neutralization mechanisms can be applied to other AB toxins such as anthrax and cholera toxin, which are of interest for developing therapeutics.
|Commitee:||Pata, Janice, Egan, Christina, Rudolph, Michael, Linhardt, Robert, McDonough, Kathleen|
|School:||State University of New York at Albany|
|School Location:||United States -- New York|
|Source:||DAI-B 82/2(E), Dissertation Abstracts International|
|Subjects:||Biology, Cellular biology, Molecular biology|
|Keywords:||Antibody, Epitope, Neutralizing, Ricin|
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