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.

Keywords: Andean potatoes; Solanum tuberosum; carboxypeptidase inhibitor; cystine-knot miniproteins; plant inhibitor; protease.

Figure 1

Thermal stability characterization of the Churqueña tubers crude extract. (A) Carboxypeptidase A (CPA) inhibitory activity of the non-treated crude extract (CE) and of the samples of the crude extract after incubation at (T70) 70, (T85) 85 and (T100) 100 °C for 60 min. The inhibitory activity of these samples is shown as % compared to (CTRL) a positive control condition of CPA without inhibitor; (B) Tris-tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis (tris-tricine-SDS-PAGE) analysis of the crude extract (EC) and of the heat-treated samples after incubation at (T60) 60, (T70) 70, (T85) 85 and (T100) 100 °C for 60 min; (C) Isoelectrophocusing experiment of the crude extract (EC) and heat-treated samples incubated at (T60) 60, (T70) 70, (T85) 85 and (T100) 100 °C for 60 min. In panels (B,C) equal volumes (25 μL) of each sample were loaded in the gel, and the location of the potential S. tuberosum subsp. andigenum cv. Churqueña (native chuPCI) carboxypeptidase inhibitor is indicated with an arrow.

Figure 2

Purification and MALDI-TOF mass spectrometric analysis of a native carboxypeptidase inhibitor from Andean potatoes. (A) Schematic diagram of the one-step strategy followed for the purification of the native carboxypeptidase A inhibitor from potato tubers of S. tuberosum subsp. andigenum cv. Churqueña. Before purification, a crude extract of potato tubers was prepared and centrifuged. The clarified supernatant was incubated at 100 °C for 60 min. Once incubated, the sample was further centrifuged, and the resultant supernatant loaded onto an in-house prepared affinity resin for the specific purification of carboxypeptidase inhibitors; (B) Coomassie-stained tris-tricine-SDS-PAGE showing the purity of the final native protein. The location of the native chuPCI (nchuPCI) is indicated with an arrow; (C–E) MALDI-TOF mass spectra of (C) initial heat-treated crude extract, (D) unbound crude extract and (E) fraction eluted from the affinity chromatography. Numbers above the major peaks indicate the average mass of the MH+ ion (m/z). Note that the molecular mass is = mass of the MH+ −1 Da. MALDI-TOF spectra were recorded on a Ultraflextreme MALDI-TOF mass spectrometer (Bruker Daltonics) operating in in a linear positive mode. In panels D and E, dotted arrows indicate the mass of the MH+ ion (m/z) of the two faded signals (4198.415 and 4310.313 Da, corresponding to the peaks IIa and IIIa, respectively, in panel C).

Figure 3

Nucleotide and deduced amino acid sequences of the chuPCI precursor. (A) cDNA of chuPCI was isolated from the RNA of tuber buds from S. tuberosum subsp. andigenum cv. Churqueña, a variety of Andean potato. The organization of the primary structure is indicated: residues belonging to the signal peptide (sequences highlighted in yellow); N-terminal pro-segment of the inhibitor (sequences highlighted in green); mature chuPCI (sequences highlighted in blue and boxed); C-terminal extension (sequences highlighted in grey). Scissors indicates the specific post-translational cleavage sites required to obtain the mature chuPCI molecule; (B) Sequence alignment of the deduced amino acid sequences of mature chuPCI and the sequence of canonical PCI (S. tuberosum, subsp. tuberosum, GenPept: NP_001275048.1). Conserved amino acids are highlighted in blue, while non-conserved residues are shown unmarked.

Figure 4

Expression and purification of the recombinant inhibitor (rchuPCI). rchuPCI was produced in E. coli. After 20 h expression at 37 °C, the recombinant miniprotein from the culture supernatant subjected to a disulfide reshuffling procedure. Then, the rchuPCI was purified from the clarified supernatant through two purification steps: a mixed-mode chromatography with a cation exchange ligand (Streamline Direct HST) and a gel filtration chromatography (HiLoad 26/60 Superdex 30 prep grade column). (A) Mixed-mode chromatography elution profile. Protein elution was carried out with an increasing gradient of phosphate buffer at pH = 8.5 (left Y axis). The green dashed line indicates the percentage of buffer B (100 mM sodium phosphate buffer at pH 8.5); (B) Size-exclusion chromatography elution profile of chuPCI. The peak corresponding to the elution of rchuPCI is inidicated (rchuPCI); (C) Tris-tricine-SDS-PAGE electrophoresis of the purified rchuPCI. The arrow indicates the location of the purified miniprotein; (D) MALDI-TOF mass spectrum of purified rchuPCI. Numbers above the major peaks indicate the average masses of the MH+ ion (m/z). Note that the molecular mass is = mass of the MH+ −1 Da.

Figure 5

Peptide Mass Fingerprint profile of native chuPCI. MALDI-TOF mass spectra of the tryptic peptides resultant from the PMF analysis of the native chuPCI. Boxed peaks are those tryptic fragments that match the theoretical peptides generated by tryptic digestion of the rchuPCI sequence. For the analysis different post-translational modifications of rchuPCI were considered: (I) cyclization of the N-t Gln amino acid (conversion to pyroglutamic acid); (II) cleavage of the N-terminal Gln/cleavage of the N-terminal pyroglutamic acid; (III) cleavage of the C-terminal Gly. See Table S1 for more details. The inset shows the sequence coverage (red arrows) of each peptides from the PMF analysis. The final sequence coverage for the full-length inhibitor was 97.4% (100% for the C-terminally truncated inhibitor).