Abstract
AbstractRicin toxin’s B subunit (RTB) is a multifunctional galactose (Gal)-/N-acetylgalactosamine (GalNac)-specific lectin that promotes efficient uptake and intracellular trafficking of ricin’s ribosome-inactivating subunit (RTA) into mammalian cells. Structurally, RTB consists of two globular domains (RTB-D1, RTB-D2), each divided into three homologous sub-domains (α, β, γ). The two carbohydrate recognition domains (CRDs) are situated on opposite sides of RTB (sub-domains 1α and 2γ) and function non-cooperatively. Previous studies have revealed two distinct classes of toxin-neutralizing, anti-RTB monoclonal antibodies (mAbs). Type I mAbs, exemplified by SylH3, inhibit (∼90%) toxin attachment to cell surfaces, while type II mAbs, epitomized by 24B11, interfere with intracellular toxin transport between the plasma membrane and the trans-Golgi network (TGN). Localizing the epitopes recognized by these two classes of mAbs has proven difficult, in part because of RTB’s duplicative structure. To circumvent this problem, full-length RTB or the two individual domains, RTB-D1 and RTB-D2, were expressed as pIII fusion proteins on the surface of filamentous phage M13 and subsequently used as “bait” in mAb capture assays. The results indicated that SylH3 captured RTB-D1, while 24B11 captured RTB-D2. Analysis of additional toxin-neutralizing and non-neutralizing mAbs along with single chain antibodies (VHHs) known to compete with SylH3 or 24B11 confirmed these domain assignments. These results not only indicate that so-called type I and type II mAbs segregate on the basis of domain specificity, but suggest that RTB’s two domains may contribute to distinct steps in the intoxication pathway.
Publisher
Cold Spring Harbor Laboratory