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; TANCO, SEBASTIÁN; LORENZO, JULIA; GARCIA-PARDO, JAVIER; AVILÉS, FRANCESC; OBREGÓN, WALTER

INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES

MOLECULAR DIVERSITY PRESERVATION INTERNATIONAL-MDPI

Año: 2018 vol. 19

1422-0067

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