Importance of the Cyclic Cystine Knot Structural Motif for Immunosuppressive Effects of Cyclotides

Abstract

The cyclotide T20K inhibits the proliferation of human immune cells and is currently in clinical trials for multiple sclerosis. Here, we provide novel functional data and mechanistic insights into structure–activity relationships of T20K. Analogs with partial or complete reduction of the cystine knot had loss of function in proliferation experiments. Similarly, an acyclic analog of T20K was inactive in lymphocyte bioassays. The lack of activity of non-native peptide analogs appears to be associated with the ability of cyclotides to interact with and penetrate cell membranes, since cellular uptake studies demonstrated fast fractional transfer only of the native peptide into the cytosol of human immune cells. Therefore, structural differences between cyclic and linear native folded peptides were investigated by NMR to elucidate structure–activity relationships. Acyclic T20K had a less rigid backbone and considerable structural changes in loops 1 and 6 compared to the native cyclic T20K, supporting the idea that the cyclic cystine knot motif is a unique bioactive scaffold. This study provides evidence that this structural motif in cyclotides governs bioactivity, interactions with and transport across biological membranes, and the structural integrity of these peptides. These observations could be useful to understand the structure–activity of other cystine knot proteins due to the structural conservation of the cystine knot motif across evolution and to provide guidance for the design of novel cyclic cysteine-stabilized molecules.

Cyclotides: The next generation in biopesticide development for eco‐friendly agriculture

“…72,83,88,[97][98][99] The CCK motif serves to effectively stabilize the structure of the cyclotide, thereby preventing any potential structural rearrangement that may occur upon interaction with receptors. 100 Some studies have also shown that cyclotides are highly toxic in the early stages of the insect's life cycle, the juvenile phase contributing to great devastation on the flora when they need a lot of nutrients to mature. In H. armigera larvae, the larval response to cyclotideinduced midgut cell damage is to increase the number of basal stem cells, which are responsible for replacement and repair during infancy.…”

Nutritional and Pharmaceutical Applications of Under-Explored Knottin Peptide-Rich Phytomedicines

“…Prominent among them is the nature-inspired T20K kalata B1, originally characterized from the African traditional plant Oldenlandia affinis . This knottin-like cyclotide has demonstrated therapeutic promise in inflammatory-related and autoimmune disorders [ 13 , 120 ]. A linear knottin, bevuTI-I, isolated from the aqueous extracts of Beta vulgaris , showed interesting mammalian enzyme inhibitory activity against trypsin (IC 50 = 471 nM) and human prolyl oligopeptidase (IC 50 = 11 μM), a drug target for neurodegenerative and inflammatory disorders [ 13 ].…”

Cyclic cystine knot and its strong implication on the structure and dynamics of cyclotides

“…In a recent interesting report, the immunosuppressive propensity of a cyclotide T20K and its analogs are explored. The analogs of T20K, with partial or total reduction of the cystine linkages of the CCK motif are found to lose all of their function as shown in human immune cell proliferation experiments 15 …”

“…The analogs of T20K, with partial or total reduction of the cystine linkages of the CCK motif are found to lose all of their function as shown in human immune cell proliferation experiments. 15 In many organisms, the presence of interpenetrating disulfide bond scaffold, the "cystine knot" is noticed, primarily among peptides and proteins which include growth factors and toxins. [16][17][18][19] However, in cyclotides, the knot is specifically placed within a macrocyclic peptide backbone, thereby forming the CCK motif.…”