Biochemical and MALDI-TOF Mass Spectrometric Characterization of a Novel Native and Recombinant Cystine Knot Miniprotein from Solanum tuberosum subsp. andigenum cv. Churqueña.

Cystine-knot miniproteins (CKMPs) are an intriguing group of cysteine-rich molecules that combine the characteristics of proteins and peptides. Typically, CKMPs are fewer than 50 residues in length and share a characteristic knotted scaffold characterized by the presence of three intramolecular disulfide bonds that form the singular knotted structure. The knot scaffold confers on these proteins remarkable chemical, thermal, and proteolytic stability. Recently, CKMPs have emerged as a novel class of natural molecules with interesting pharmacological properties. In the present work, a novel cystine-knot metallocarboxypeptidase inhibitor (chuPCI) was isolated from tubers of Solanum tuberosum, subsp. andigenum cv. Churqueña. Our results demonstrated that chuPCI is a member of the A/B-type family of metallocarboxypeptidases inhibitors. chuPCI was expressed and characterized by a combination of biochemical and mass spectrometric techniques. Direct comparison of the MALDI-TOF mass spectra for the native and recombinant molecules allowed us to confirm the presence of four different forms of chuPCI in the tubers. The majority of such forms have a molecular weight of 4309 Da and contain a cyclized Gln in the N-terminus. The other three forms are derived from N-terminal and/or C-terminal proteolytic cleavages. Taken together, our results contribute to increase the current repertoire of natural CKMPs.

Plant cystine-knot peptides: pharmacological perspectives.

Cystine-knot miniproteins are a class of 30-50 amino acid long peptides widespread in eukaryotic organisms. Due to their very peculiar three-dimensional structure, they exhibit high resistance to heat and peptidase attack. The cystine-knot peptides are well represented in several plant species including medicinal herbs and crops. The pharmacological interest in plant cystine-knot peptides derives from their broad biological activities, mainly cytotoxic, antimicrobial and peptidase inhibitory and in the possibility to engineer them to incorporate pharmacophoric information for oral delivery or disease biomonitoring. The mechanisms of action of plant cystine-knot peptides are still largely unknown, although the capacity to interfere with plasma membranes seems a feature common to several cystine-knot peptides. In some cases, such as potato carboxypetidase inhibitor (PCI) and tomato cystine-knot miniproteins (TCMPs), the cystine-knot peptides target human growth factor receptors either by acting as growth factor antagonist or by altering their signal transduction pathway. The possibility to identify specific molecular targets of plant cystine-knot peptides in human cells opens novel possibilities for the pharmacological use of these peptides besides their use as scaffold to develop stable disease molecular markers and therapeutic agents.

Insecticidal activity of a recombinant knottin peptide from Loxosceles intermedia venom and recognition of these peptides as a conserved family in the genus.

Loxosceles intermedia venom comprises a complex mixture of proteins, glycoproteins and low molecular mass peptides that act synergistically to immobilize envenomed prey. Analysis of a venom-gland transcriptome from L. intermedia revealed that knottins, also known as inhibitor cystine knot peptides, are the most abundant class of toxins expressed in this species. Knottin peptides contain a particular arrangement of intramolecular disulphide bonds, and these peptides typically act upon ion channels or receptors in the insect nervous system, triggering paralysis or other lethal effects. Herein, we focused on a knottin peptide with 53 amino acid residues from L. intermedia venom. The recombinant peptide, named U2 -sicaritoxin-Li1b (Li1b), was obtained by expression in the periplasm of Escherichia coli. The recombinant peptide induced irreversible flaccid paralysis in sheep blowflies. We screened for knottin-encoding sequences in total RNA extracts from two other Loxosceles species, Loxosceles gaucho and Loxosceles laeta, which revealed that knottin peptides constitute a conserved family of toxins in the Loxosceles genus. The insecticidal activity of U2 -SCTX-Li1b, together with the large number of knottin peptides encoded in Loxosceles venom glands, suggests that studies of these venoms might facilitate future biotechnological applications of these toxins.

A cystine-knot miniprotein from tomato fruit inhibits endothelial cell migration and angiogenesis by affecting vascular endothelial growth factor receptor (VEGFR) activation and nitric oxide production.

SCOPE: Cystine-knot miniproteins are bioactive molecules with a broad range of potential therapeutic applications. Recently, it was demonstrated that two tomato cystine-knot miniproteins (TCMPs) exhibit in vitro antiangiogenic activity on human umbilical vein cells. The aim of the present study was to investigate the effects of a fruit-specific cystine-knot miniprotein of tomato on in vitro endothelial cell migration and in vivo angiogenesis using a zebrafish model. METHODS AND RESULTS: The cystine-knot protein purified from tomato fruits using gel filtration LC and RP-HPLC inhibited cell migration when tested at 200 nM using the wound healing assay, and reduced nitric oxide formation probed by 4-amino-5-methylamino-27-difluorofluoscescin diacetate. RT-PCR and Western blot analyses demonstrated that vascular endothelium growth factor A dependent signaling was the target of TCMP bioactivity. Angiogenesis was inhibited in vivo in zebrafish embryos treated with 500 nM TCMP. CONCLUSION: Our results demonstrate that cystine-knot miniproteins present in mature tomato fruits are endowed with antiangiogenic activity in vitro and in vivo. These molecules may confer beneficial effects to tomato dietary intake, along with lycopene and other antioxidants. Further investigation is warranted to explore the potential of these compounds as model scaffolds for the development of new drugs.

Allotides: Proline-Rich Cystine Knot α-Amylase Inhibitors from Allamanda cathartica.

Cystine knot α-amylase inhibitors belong to a knottin family of peptidyl inhibitors of 30-32 residues and contain two to four prolines. Thus far, only four members of the group of cystine knot α-amylase inhibitors have been characterized. Herein, the discovery and characterization of five cystine knot α-amylase inhibitors, allotides C1-C5 (Ac1-Ac5) (1-5), from the medicinal plant Allamanda cathartica are reported using both proteomic and genomic methods. Proteomic analysis showed that 1-5 are 30 amino acids in length with three or four proline residues. NMR determination of 4 revealed that it has two cis- and one trans-proline residues and adopts two equally populated conformations in solution. Determination of disulfide connectivity of 2 by differential S-reduction and S-alkylation provided clues of its unfolding process. Genomic analysis showed that allotide precursors contain a three-domain arrangement commonly found in plant cystine knot peptides with conserved residues flanking the processing sites of the mature allotide domain. This work expands the number of known cystine knot α-amylase inhibitors and furthers the understanding of both the structural and biological diversity of this type of knottin family.

Engineering knottins as novel binding agents.

Cystine-knot miniproteins, also known as knottins, contain a conserved core of three tightly woven disulfide bonds which impart extraordinary thermal and proteolytic stability. Interspersed between their conserved cysteine residues are constrained loops that possess high levels of sequence diversity among knottin family members. Together these attributes make knottins promising molecular scaffolds for protein engineering and translational applications. While naturally occurring knottins have shown potential as both diagnostic agents and therapeutics, protein engineering is playing an important and increasing role in creating designer molecules that bind to a myriad of biomedical targets. Toward this goal, rational and combinatorial approaches have been used to engineer knottins with novel molecular recognition properties. Here, methods are described for creating and screening knottin libraries using yeast surface display and fluorescence-activated cell sorting. Protocols are also provided for producing knottins by synthetic and recombinant methods, and for measuring the binding affinity of knottins to target proteins expressed on the cell surface.