The legumain McPAL1 from Momordica cochinchinensis is a highly stable Asx-specific splicing enzyme.

Legumains, also known as asparaginyl endopeptidases (AEPs), cleave peptide bonds after Asn/Asp (Asx) residues. In plants, certain legumains also have ligase activity that catalyzes biosynthesis of Asx-containing cyclic peptides. An example is the biosynthesis of MCoTI-I/II, a squash family-derived cyclic trypsin inhibitor, which involves splicing to remove the N-terminal prodomain and then N-to-C-terminal cyclization of the mature domain. To identify plant legumains responsible for the maturation of these cyclic peptides, we have isolated and characterized a legumain involved in splicing, McPAL1, from Momordica cochinchinensis (Cucurbitaceae) seeds. Functional studies show that recombinantly expressed McPAL1 displays a pH-dependent, trimodal enzymatic profile. At pH 4 to 6, McPAL1 selectively catalyzed Asp-ligation and Asn-cleavage, but at pH 6.5 to 8, Asn-ligation predominated. With peptide substrates containing N-terminal Asn and C-terminal Asp, such as is found in precursors of MCoTI-I/II, McPAL1 mediates proteolysis at the Asn site and then ligation at the Asp site at pH 5 to 6. Also, McPAL1 is an unusually stable legumain that is tolerant of heat and high pH. Together, our results support that McPAL1 is a splicing legumain at acidic pH that can mediate biosynthesis of MCoTI-I/II. We purport that the high thermal and pH stability of McPAL1 could have applications for protein engineering.

A phylogenetic analysis of Momordica (Cucurbitaceae) in India based on karyo-morphology, nuclear DNA content and rDNA ITS1-5.8S-ITS2 sequences.

The infrageneric delimitation of Momordica, a medicinally important genus of Cucurbitaceae, is ill-defined until date. Momordica chromosomes are extremely small and are difficult to stain and visualize because of the dense cytoplasmic background. We have conducted karyomorphometric analysis by EMA method in five Indian Momordica species, and the nuclear genome sizes were estimated by flow cytometry for the first time. The somatic chromosome numbers ranged from 2n = 18 to 56 in the species. We have resolved previously disputed chromosome numbers in M. cymbalaria and M. dioica as 2n = 18 (lowest) and 2n = 56, respectively. Chromosome counts in the other species were re-confirmed as 2n = 22 in M. charantia, 2n = 28 in M. cochinchinensis and 2n = 56 in M. subangulata. The largest genome size was recorded in M. cymbalaria (3.74 pg 2C-1), while the smallest size (0.72 pg 2C-1) was detected in M. charantia var. charantia. The nuclear genome sizes were analysed in comparison to chromosome numbers and total chromosome lengths of the species. Karyomorphometric indices showed comparable symmetric karyotypes in the species except in M. cymbalaria having tendency towards asymmetry. The UPGMA phenogram and principle component analysis based on nuclear DNA contents and karyomorphometric parameters demonstrated interspecies differences, intraspecific distinction within M. charantia varieties and highlighted distinction of M. cymbalaria. This study was further supported by the rDNA ITS sequence-based phylogenetic analysis which revealed the monophyletic origin of the Indian members of Momordica and clarified the intraspecies relationship among the studied members. As a whole, the study brought out new insights on species diversification within the genus Momordica in India and would benefit further studies on biosystematics and plant breeding programmes.

Purification and functional characterization of recombinant balsamin, a ribosome-inactivating protein from Momordica balsamina.

Balsamin, a type I ribosome-inactivating protein (RIP), has been shown to inhibit HIV-1 replication at the translation step. Our recent studies have shown that balsamin also possess anti-tumor, antibacterial and DNase-like activity, however, the amount of natural balsamin in Momordica balsamina seeds is limited and preclinical studies require large quantities of pure, bioactive balsamin. Therefore, in this study, we cloned the balsamin gene, expressed it in E.coli BL21 (DE3) strain and purified it by nickel affinity chromatography. Functional analysis indicated that balsamin exhibits both RNA N-glycosidase activity, releasing the Endo-fragment from rabbit reticulocyte rRNA, and DNase-like activity, converting the supercoiled form of a plasmid into the linear form in a concentration-dependent manner. Analysis of secondary structure revealed that recombinant balsamin mainly consisted of α-helical and random coiled with minimal turns and ß-sheets. Recombinant balsamin was found to be stable in the temperature range of 20-60 °C and pH range of 6-9. Antimicrobial assays showed that the minimum inhibitory concentrations of recombinant balsamin for various pathogens ranged between 1.56 and 12.5 µg/ml. Heterologous expression and purification of balsamin carries great importance as it provides an alternative approach for large-scale preparation of biologically active recombinant balsamin, which is difficult from its natural source.

The evolution of Momordica cyclic peptides.

Cyclic proteins have evolved for millions of years across all kingdoms of life to confer structural stability over their acyclic counterparts while maintaining intrinsic functional properties. Here, we show that cyclic miniproteins (or peptides) from Momordica (Cucurbitaceae) seeds evolved in species that diverged from an African ancestor around 19 Ma. The ability to achieve head-to-tail cyclization of Momordica cyclic peptides appears to have been acquired through a series of mutations in their acyclic precursor coding sequences following recent and independent gene expansion event(s). Evolutionary analysis of Momordica cyclic peptides reveals sites that are under selection, highlighting residues that are presumably constrained for maintaining their function as potent trypsin inhibitors. Molecular dynamics of Momordica cyclic peptides in complex with trypsin reveals site-specific residues involved in target binding. In a broader context, this study provides a basis for selecting Momordica species to further investigate the biosynthesis of the cyclic peptides and for constructing libraries that may be screened against evolutionarily related serine proteases implicated in human diseases.

Cyclic peptides arising by evolutionary parallelism via asparaginyl-endopeptidase-mediated biosynthesis.

The cyclic miniprotein Momordica cochinchinensis Trypsin Inhibitor II (MCoTI-II) (34 amino acids) is a potent trypsin inhibitor (TI) and a favored scaffold for drug design. We have cloned the corresponding genes and determined that each precursor protein contains a tandem series of cyclic TIs terminating with the more commonly known, and potentially ancestral, acyclic TI. Expression of the precursor protein in Arabidopsis thaliana showed that production of the cyclic TIs, but not the terminal acyclic TI, depends on asparaginyl endopeptidase (AEP) for maturation. The nature of their repetitive sequences and the almost identical structures of emerging TIs suggest these cyclic peptides evolved by internal gene amplification associated with recruitment of AEP for processing between domain repeats. This is the third example of similar AEP-mediated processing of a class of cyclic peptides from unrelated precursor proteins in phylogenetically distant plant families. This suggests that production of cyclic peptides in angiosperms has evolved in parallel using AEP as a constraining evolutionary channel. We believe this is evolutionary evidence that, in addition to its known roles in proteolysis, AEP is especially suited to performing protein cyclization.

De novo transcriptome sequencing of Momordica cochinchinensis to identify genes involved in the carotenoid biosynthesis.

The ripe fruit of Momordica cochinchinensis Spreng, known as gac, is featured by very high carotenoid content. Although this plant might be a good resource for carotenoid metabolic engineering, so far, the genes involved in the carotenoid metabolic pathways in gac were unidentified due to lack of genomic information in the public database. In order to expedite the process of gene discovery, we have undertaken Illumina deep sequencing of mRNA prepared from aril of gac fruit. From 51,446,670 high-quality reads, we obtained 81,404 assembled unigenes with average length of 388 base pairs. At the protein level, gac aril transcripts showed about 81.5% similarity with cucumber proteomes. In addition 17,104 unigenes have been assigned to specific metabolic pathways in Kyoto Encyclopedia of Genes and Genomes, and all of known enzymes involved in terpenoid backbones biosynthetic and carotenoid biosynthetic pathways were also identified in our library. To analyze the relationship between putative carotenoid biosynthesis genes and alteration of carotenoid content during fruit ripening, digital gene expression analysis was performed on three different ripening stages of aril. This study has revealed putative phytoene synthase, 15-cis-phytone desaturase, zeta-carotene desaturase, carotenoid isomerase and lycopene epsilon cyclase might be key factors for controlling carotenoid contents during aril ripening. Taken together, this study has also made availability of a large gene database. This unique information for gac gene discovery would be helpful to facilitate functional studies for improving carotenoid quantities.

Cytotaxonomical analysis of Momordica L. (Cucurbitaceae) species of Indian occurrence.

Somatic chromosome number and detailed karyotype analysis were carried out in six Indian Momordica species viz. M. balsamina, M. charantia, M. cochinchinensis, M. dioica, M. sahyadrica and M. cymbalaria (syn. Luffa cymbalaria; a taxon of controversial taxonomic identity). The somatic chromosome number 2n = 22 was reconfirmed in monoecious species (M. balsamina and M. charantia). Out of four dioecious species, the chromosome number was reconfirmed in M. cochinchinensis (2n = 28), M. dioica (2n = 28) and M. subangulata subsp. renigera (2n = 56), while in M. sahyadrica (2n = 28) somatic chromosome number was reported for the first time. A new chromosome number of 2n = 18 was reported in M. cymbalaria against its previous reports of 2n = 16, 22. The karyotype analysis of all the species revealed significant numerical and structural variations of chromosomes. It was possible to distinguish chromosomes of M. cymbalaria from other Momordica species and also between monoecious and dioecious taxa of the genus. Morphology and crossability among the dioecious species was also studied. Evidence from morphology, crossability, pollen viability and chromosome synapsis suggests a segmental allopolyploid origin for M. subangulata subsp. renigera. The taxonomic status of the controversial taxon M. cymbalaria was also discussed using morphological, karyological and crossability data.

Slowdowns in diversification rates from real phylogenies may not be real.

Studies of diversification patterns often find a slowing in lineage accumulation toward the present. This seemingly pervasive pattern of rate downturns has been taken as evidence for adaptive radiations, density-dependent regulation, and metacommunity species interactions. The significance of rate downturns is evaluated with statistical tests (the gamma statistic and Monte Carlo constant rates (MCCR) test; birth-death likelihood models and Akaike Information Criterion [AIC] scores) that rely on null distributions, which assume that the included species are a random sample of the entire clade. Sampling in real phylogenies, however, often is nonrandom because systematists try to include early-diverging species or representatives of previous intrataxon classifications. We studied the effects of biased sampling, structured sampling, and random sampling by experimentally pruning simulated trees (60 and 150 species) as well as a completely sampled empirical tree (58 species) and then applying the gamma statistic/MCCR test and birth-death likelihood models/AIC scores to assess rate changes. For trees with random species sampling, the true model (i.e., the one fitting the complete phylogenies) could be inferred in most cases. Oversampling deep nodes, however, strongly biases inferences toward downturns, with simulations of structured and biased sampling suggesting that this occurs when sampling percentages drop below 80%. The magnitude of the effect and the sensitivity of diversification rate models is such that a useful rule of thumb may be not to infer rate downturns from real trees unless they have >80% species sampling.

A three-genome phylogeny of Momordica (Cucurbitaceae) suggests seven returns from dioecy to monoecy and recent long-distance dispersal to Asia.

The bitter gourd genus Momordica comprises 47 species in Africa and 12 in Asia and Australia. All have unisexual flowers, and of the African species, 24 are dioecious, 23 monoecious, while all Asian species are dioecious. Maximum likelihood analyses of 6257 aligned nucleotides of plastid, mitochondrial and nuclear DNA obtained for 122 accessions of Momordica and seven outgroups show that Momordica is monophyletic and consists of 11 well-supported clades. Monoecy evolved from dioecy seven times independently, always in Africa and mostly in savanna species with low population densities. Leaky dioecy, with occasional fruit-producing males, occurs in two African species and might be the first step in an evolutionary transition towards monoecy. Dated biogeographic analyses suggest that Momordica originated in tropical Africa and that the Asian species are the result of one long-distance dispersal event about 19million years ago. The pantropical vegetable Momordica charantia is of African, not Asian origin as had previously been suggested.

Inheritance of gynoecism in bitter gourd (Momordica charantia L.).

The inheritance of sex expression in cucumber (Cucumis sativus) and other cucurbits is well documented; however, the genetics of female sex (gynoecism) expression in bitter gourd (Momordica charantia) has not been described. Inheritance of gynoecism in bitter gourd was studied in a 100% gynoecious line (Gy263B). The F(2) and testcross segregation data revealed that gynoecism in Gy263B is under the control of a single, recessive gene. Following the gene nomenclature of cucurbits, it is proposed that the gene symbol, gy-1, be assigned for the expression of gynoecism in bitter gourd.

Barnase fusion as a tool to determine the crystal structure of the small disulfide-rich protein McoEeTI.

We present a fusion system suited to determine the crystal structure of small disulfide-rich proteins. McoEeTI, a hybrid inhibitor cystine knot microprotein, was produced as a soluble fusion to a catalytically inactive variant of the RNAse barnase in Escherichia coli. Functioning as a versatile tag, barnase facilitated purification, crystallization and high-resolution structure determination. Flexibility of the linker region allows for different relative orientations of barnase and the fusion partner in two crystallographically independent molecules and may thereby facilitate crystal packing. Nevertheless, the linker region is well ordered in both molecules. This system may prove more generally useful to determine the crystal structure of peptides and small proteins.