A new family of cystine knot peptides from the seeds of Momordica cochinchinensis
Highlights
► Four new peptides (MCo-3–MCo-6) were characterized from the seeds of Momordica cochinchinensis. ► These peptides form a novel family of M. cochinchinensis ICK peptides. ► Peptide content and concentration of the seeds from China and Vietnam are different.
Introduction
Momordica cochinchinensis (Lour.) Spreng. (Cucurbitaceae) is a Southeast Asian vine whose seeds, roots and leaves have been used as food and in a range of traditional Chinese medicines with applications including the treatment of inflammation and pain [7]. Three trypsin inhibitors (MCoTI-I (M. cochinchinensis trypsin inhibitor-I), MCoTI-II and MCoTI-III) and a chymotrypsin inhibitor have been isolated from the seeds of this plant [5], [23], [31]. Partial sequences of additional trypsin inhibitors have also been reported [34]. These trypsin inhibitors are highly potent, with sub-nanomolar Ki's and are thought to be present for plant defense [1]. The MCoTI peptides from M. cochinchinensis belong to the squash trypsin inhibitor family and have similar sequences and structures to other members of this family isolated from a range of Cucurbitaceae species [16], [21]. Squash trypsin inhibitors are characterized by an inhibitor cystine knot (ICK) motif formed by three disulfide bonds [13], [19]. This structural motif comprises an embedded ring formed by the I–IV, II–V disulfide bonds and their connecting backbone loops, which is penetrated by the III–VI disulfide bond. MCoTI-I and MCoTI-II contain a macrocyclic backbone coupled with the ICK motif and consequently have been classified as part of the cyclotide family [4], [12], [23], whose hallmark is the cyclic cystine knot (CCK) motif [18].
We have previously shown that M. cochinchinensis contains two cytotoxic peptides (MCoCC-1 and MCoCC-2) with sequences unrelated to the MCoTI peptides but of a similar size and having three disulfide bonds. NMR analysis indicated that the peptides also have an ICK motif [23]. However, in contrast to MCoTI-I/II these two peptides do not have a cyclic backbone. MCoCC-1 and MCoCC-2 have been renamed in the current study to MCo-1 and MCo-2 respectively. As part of our broad-ranging investigations into bioactive peptides [20], [27], [28], [29], [30], [36], [37], in the current study we have characterized four new peptides, MCo-3 to MCo-6, from M. cochinchinensis seed extracts obtained from China and Vietnam, and two known squash trypsin inhibitors, MCoTI-I [23] and MCoTI-II [23]. These peptides were screened in several biological assays including trypsin inhibition, antimalaria and cytotoxicity assays.
Section snippets
General experimental procedures
For MALDI-TOF MS analysis, a Voyager DE-STR mass spectrometer (Applied Biosystems) was used and data were collected between 300 and 4000 Da. Nanospray MS/MS experiments were conducted using the QStar Pulsar mass spectrometer (Applied Biosystems) and ion spray voltage was applied between 900 and 1100 V. To analyze fractions using electrospray ionization (ESI), a mass range of 200–2000 was used; the multiply charged ions were then deconvoluted to yield neutral masses. The collision energy for
Peptide characterization
To characterize the peptide profile of M. cochinchinensis [5], [23] we screened seed coats, decoated seeds, stems, fruits, and vines for the presence of novel peptides. Plant material was extracted using 50% aqueous ethanol and the extract was size-fractionated using a Sephadex LH-20 column [9]. The fractions were collected and analyzed using thin layer chromatography [36], [37] and mass spectrometry (MS). Only the decoated seed extract showed evidence of peptides in the 2500–4000 Da range.
Discussion
In this study we have discovered four novel peptides (MCo-3 to MCo-6) that together with MCo-1 and MCo-2 form a new family of ICK peptides. These peptides were isolated and detected from the seeds of M. cochinchinensis from China and Vietnam, respectively, and the sequences were determined using a combination of MS and NMR spectroscopy. Further analysis of the NMR spectra indicated the presence of a cystine knot motif, which is present in a range of peptides from a variety of plants and animals
Acknowledgements
This work was supported by the National Natural Science Foundation of China (U1032602, 91013002, 21202174, 30725048), the China/Australia Special Fund for S & T (NSFC and ARC), the National New Drug Innovation Great Project of China (2011ZX09307-002-02), the National Basic Research Program of China (2009CB522300 and 2012CB017104) and the foundation of Chinese Academy of Sciences (Hundred Talents Program and KSCXZ-EW-R-15). Wen-Jun He is a recipient of a fellowship from the China Scholarship
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These authors contributed equally to this work.