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

COTABARREN, JULIANA; TELLECHEA MARIANA; SEBASTIÁN TANCO; LORENZO JULIA; JAVIER GARCIA PARDO; FRANCESC X. AVILÉS; OBREGÓN, WALTER D.

INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES

MOLECULAR DIVERSITY PRESERVATION INTERNATIONAL-MDPI

Lugar: Basel; Año: 2018

1422-0067

Cystine-knot miniproteins (CKMPs) are members of cysteine-rich proteins families, comprising less than 50 aminoacids. The CKMPs have a characteristic knotted structure formed by three intramolecular disulfide bonds that confer remarkable stability to: pH, ionic strength, extreme temperature, chemical denaturation and proteolysis. In recent years, the spectrum of natural bioactivities of this class of miniproteins was further expanded by pharmaceutical and biomedical application. A novel cystine-knot inhibitor, named chuPCI, was isolated from tubers of Solanum tuberosum, subsp. andigenum cv. Churqueña. ChuPCI, member of the A/B-type metallocarboxypeptidase inhibitor family, was purified, cloned, expressed and characterized by biochemical and proteomic techniques. MALDI-TOF/MS analysis and comparison between native and recombinant spectra allowed us to prove the presence of two isoinhibitors of chuPCI (39 residues) that are naturally expressed; a first one with a modification of the N-terminus (Gln cyclized to pyroglutamic acid) and a second isoform that lacks the N-terminal residue. Native and recombinant chuPCI showed a molecular mass of 4,309 Da. This work contributes to the understanding of natural CKMPs, presenting a comparative analysis based on proteomic techniques and proves the identification of chuPCI both in natural extract as in recombinant fraction, revalidating the importance of proteomic analysis on present researches.