A novel recombinant immunotoxin with the smallest ribosome‐inactivating protein Luffin P1: T‐cell cytotoxicity and prolongation of allograft survival

Abstract

In the creation of stable tolerance to MHC-incompatible allografts, reducing the large mass of donor-reactive cells via apoptosis is often required. Apoptosis induction by immunotoxins targeting surface molecules specifically presented on donor-reactive cytopathic T effector (Teff) cells is a promising strategy. Traditionally, the toxin moieties are bacterial exotoxins or plant-derived ribosome-inactivating proteins (RIPs) with large molecular size and strong immunogenicity, hence causing the problems of tissue penetration, host immune reaction and quick clearance. We have identified a novel class of small molecule RIPs (<10 kD) from the seeds of the plant Luffa cylindrica. The smallest member of this family, Luffin P1, has a molecular weight of 5226.8 Da, yet possessing a highly potent inhibitory activity on cell-free protein synthesis with IC50 of 0.88 nM. We now report a recombinant hIL-2-Luffin P1 immunotoxin, which strongly inhibited T-cell proliferation in mixed lymphocyte reaction and ConA response with IC50 of 1.8–10 nM. In vivo, hIL-2-Luffin P1 significantly prolonged the survival of major MHC-mismatched skin and kidney allografts in animal models. Thus, we demonstrate for the first time the efficacy of the smallest immunotoxin that could be further combined with other pharmacological and immunological reagents for synergistic control of pathogenic lymphocytes in immune-mediated diseases.

Structure, Immunogenicity, and IgE Cross-Reactivity among Walnut and Peanut Vicilin-Buried Peptides

“…Vicilins are a family of ubiquitous seed storage proteins, which are translated as preproproteins consisting of three conserved regions: a short, hydrophobic signal peptide that is removed upon location to a storage vacuole; an N-terminal, cysteine-rich leader sequence (LS), which is removed by asparaginyl endopeptidase (AEP); and the mature vicilin domain. − Recent studies have identified a new family of peptides, termed vicilin-buried peptides (VBPs) within this LS region. These VBPs are defined by a common cysteine motif (CxxxCx (10–14) CxxxC) and are widely dispersed across most angiosperm families including trees, grasses, nightshades, and legumes, , where they have been implicated in a variety of biological functions including ribosome inactivation, protease inhibition, and antimicrobial defense. − Despite this diversity, the structure of all VBPs that have been solved to date reveals a common α-hairpin fold/motif (also called α-hairpinin) mediated by disulfide bonds between the highly conserved CxxxC motifs, potentially providing a common structural scaffold that can mediate peanut/tree nut cross-reactivity. , …”