A Novel Plant Protein-disulfide Isomerase Involved in the Oxidative Folding of Cystine Knot Defense Proteins

Abstract

We have isolated a protein-disulfide isomerase (PDI) from Oldenlandia affinis (OaPDI), a coffee family (Rubiaceae) plant that accumulates knotted circular proteins called cyclotides. The novel plant PDI appears to be involved in the biosynthesis of cyclotides, since it co-expresses and interacts with the cyclotide precursor protein Oak1. OaPDI exhibits similar isomerase activity but greater chaperone activity than human PDI. Since domain c of OaPDI is predicted to have a neutral pI, we conclude that this domain does not have to be acidic in nature for PDI to be a functional chaperone. Its redox potential of ؊157 ؎ 4 mV supports a role as a functional oxidoreductase in the plant. The mechanism of enzyme-assisted folding of plant cyclotides was investigated by comparing the folding of kalata B1 derivatives in the presence and absence of OaPDI. OaPDI dramatically enhanced the correct oxidative folding of kalata B1 at physiological pH. A detailed investigation of folding intermediates suggested that disulfide isomerization is an important role of the new plant PDI and is an essential step in the production of insecticidal cyclotides.Protein-disulfide isomerase (PDI 3 ; EC 5.3.4.1) is an oxidoreductase enzyme that belongs to the thioredoxin superfamily (1). It has a major role in oxidative folding of polypeptides in the endoplasmic reticulum (ER) of eukaryotic cells and functions as an ER chaperone (2). The exact mechanism of action of PDI is not clear, but it is believed to bind polypeptides through hydrophobic interactions and forms (oxidizes), breaks (reduces), and/or shuffles (isomerizes) disulfide bonds in substrate molecules via a dithiol-disulfide exchange between its active-site CXXC motif and the substrate polypeptide (3).Cyclotides are small disulfide-rich peptides found in plants of the coffee (Rubiaceae) and violet (Violaceae) families (4). They are typically about 30 amino acids in length and have the unique structural features of a cyclic backbone and a knotted arrangement of three-disulfide bonds, referred to as the cyclic cystine knot motif (5). Their compact cyclic cystine knot motif makes them exceptionally resistant to thermal, chemical, or enzymatic degradation (6). Cyclotides exhibit a range of biological activities, including anti-bacterial, cytotoxic, and anti-human immunodeficiency virus activities (7), but their natural function is as plant defense molecules (8, 9). Kalata B1, from the Rubiaceae species Oldenlandia affinis, was the first cyclotide discovered (10), although its macrocyclic structure was not delineated until 1995 (11). So far, the sequences of nearly 100 cyclotides have been reported, and it has been suggested that they may surpass the well known plant defensins in number and diversity (12, 13). Their unique structural framework, range of bioactivities, and sequence diversity make them interesting targets for pharmaceutical applications (14).Cyclotides have a characteristic surface-exposed patch of hydrophobic residues that accounts for their late elution on reverse-phase HPLC ...

Insights into Processing and Cyclization Events Associated with Biosynthesis of the Cyclic Peptide Kalata B1

“…Cyclotides are processed inside the ER where protein-disulfide isomerase helps with the formation of disulfide bonds within the cysteine knot motif (23). Protein-disulfide isomerases are thought to exist in complex with prolylhydroxylases in the ER so the cyclotide precursor would be in close proximity to these proteins when being folded (24).…”

Protein Disulfide Isomerase-2 of Arabidopsis Mediates Protein Folding and Localizes to Both the Secretory Pathway and Nucleus, Where It Interacts with Maternal Effect Embryo Arrest Factor

“…The chloroplast PDI, CYO1, is involved in thylakoid biogenesis in cotyledons, but not leaves (Shimada et al, 2007). In Coffea arabica, a PDI isomerizes disulfide bonds to create circular knotted proteins called cyclotides (Gruber et al, 2007).…”

Do plant cyclotides have potential as immunosuppressant peptides?

“…13 In contrast to non-ribosomal-synthesized plant metabolites, cyclotides are true gene products, and their biosynthesis involves ribosomal precursor synthesis, enzymatic processing, 14,15 and protein-folding events. 16 Since their discovery in the coffee family (Rubiaceae), cyclotides have been extensively studied in the violets (Violaceae) and have recently been found in legumes (Fabaceae). [17][18][19] Although there is an increasing effort to screen plants of different families for the occurrence and distribution of cyclotides, 20 we still lack information on the individual cyclotide content of single plant species, mainly due to difficulties associated with the analytical analysis and the identification of cyclotides.…”