Serra Aida, Hemu Xinya, Nguyen Giang K T, Nguyen Ngan T K, Sze Siu Kwan, Tam James P
School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, 637551, Singapore.
Sci Rep. 2016 Mar 11;6:23005. doi: 10.1038/srep23005.
Cyclotides are plant cyclic cysteine-rich peptides (CRPs). The cyclic nature is reported to be gene-determined with a precursor containing a cyclization-competent domain which contains an essential C-terminal Asn/Asp (Asx) processing signal recognized by a cyclase. Linear forms of cyclotides are rare and are likely uncyclizable because they lack this essential C-terminal Asx signal (uncyclotide). Here we show that in the cyclotide-producing plant Clitoria ternatea, both cyclic and acyclic products, collectively named cliotides, can be bioprocessed from the same cyclization-competent precursor. Using an improved peptidomic strategy coupled with the novel Asx-specific endopeptidase butelase 2 to linearize cliotides at a biosynthetic ligation site for transcriptomic analysis, we characterized 272 cliotides derived from 38 genes. Several types of post-translational modifications of the processed cyclotides were observed, including deamidation, oxidation, hydroxylation, dehydration, glycosylation, methylation, and truncation. Taken together, our results suggest that cyclotide biosynthesis involves 'fuzzy' processing of precursors into both cyclic and linear forms as well as post-translational modifications to achieve molecular diversity, which is a commonly found trait of natural product biosynthesis.
环肽是富含半胱氨酸的植物环肽（CRPs）。据报道，其环化性质由基因决定，前体含有一个具有环化能力的结构域，该结构域包含一个被环化酶识别的必需的C端Asn/Asp（Asx）加工信号。环肽的线性形式很少见，并且可能由于缺乏这种必需的C端Asx信号（非环肽）而无法环化。在这里，我们表明，在产生环肽的植物蝶豆中，环状和非环状产物（统称为蝶豆肽）都可以从同一个具有环化能力的前体中生物加工得到。使用改进的肽组学策略，结合新型的Asx特异性内肽酶butelase 2，在生物合成连接位点将蝶豆肽线性化以进行转录组分析，我们鉴定了来自38个基因的272种蝶豆肽。观察到加工后的环肽有几种类型的翻译后修饰，包括脱酰胺、氧化、羟基化、脱水、糖基化、甲基化和截短。综上所述，我们的结果表明，环肽生物合成涉及前体“模糊”加工成环状和线性形式以及翻译后修饰以实现分子多样性，这是天然产物生物合成中常见的特征。
