Elsevier

Peptides

Volume 39, January 2013, Pages 29-35
Peptides

A new family of cystine knot peptides from the seeds of Momordica cochinchinensis

https://doi.org/10.1016/j.peptides.2012.09.018Get rights and content

Abstract

Momordica cochinchinensis, a Cucurbitaceae plant commonly found in Southeast Asia, has the unusual property of containing both acyclic and backbone-cyclized trypsin inhibitors with inhibitor cystine knot (ICK) motifs. In the current study we have shown that M. cochinchinensis also contains another family of acyclic ICK peptides. We recently reported two novel peptides from M. cochinchinensis but have now discovered four additional peptides (MCo-3–MCo-6) with related sequences. Together these peptides form a novel family of M. cochinchinensis ICK peptides (MCo-ICK) that do not have sequence homology with other known peptides and are not potent trypsin inhibitors. Otherwise these new peptides MCo-3 to MCo-6 were evaluated for antimalarial activity against Plasmodium falciparum, and cytotoxic activity against the cancer cell line MDA-MB-231. But these peptides were not active.

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.

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