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

Abstract

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.

Identification and characterization of a wolfberry carboxypeptidase inhibitor from Lycium barbarum

“…The Burke-Lineweaver plot showed that β-lybatide is a competitive inhibitor of bovine carboxypeptidase A with a Ki value of 6.62 nM, which is characteristic of the tight-binding inhibitors of carboxypeptidases. This result was consistent with previously reported inhibitors, in which the Ki values are 5.4, 4.8, 4.1 and 10.8 nM of PCI, tomato TCI (Hass & Hermodson, 1981), imaPCI (Lufrano et al, 2015) and chuPCI (Cotabarren et al, 2018b), respectively.…”

“…PCI inhibits fungal and insect carboxypeptidases to protect the plant from pathogen and insect attacks (Ryan, 1990). Since its discovery, only three additional plant CPIs have been characterized: tomato TCI (Hass & Hermodson, 1981), potato imaPCI (Lufrano et al, 2015) and chuPCI (Cotabarren et al, 2018b).…”

Molecular Insights into the Role of Cysteine-Rich Peptides in Induced Resistance to Fusarium oxysporum Infection in Tomato Based on Transcriptome Profiling

“…They display various biological activities including antifungal, insecticidal, and anticarcinogenic [ 36 ]. The knottins of Solanaceae plants act as carboxypeptidase inhibitors [ 38 ].…”