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.

Cold sweetening diversity in Andean potato germplasm from Argentina.

BACKGROUND: Cold-induced sweetening (CIS) is the accumulation of sucrose and reducing sugars in potato tubers at low temperatures. This process is central for the potato processing industry. During potato chip and French fry production, reducing sugars participate in the Maillard reaction to produce dark pigmented products not acceptable to consumers. Andean potatoes (Solanum tuberosum Group Andigena) constitute an enormous wealth of potato germplasm that can contribute to increase genetic diversity in breeding programs of many traits, including CIS. RESULTS: We analyzed reducing sugar content and chip quality in freshly harvested and cold-stored tubers from 48 native accessions. Andean accessions showed high variation in reducing sugar content and were classified in three types of CIS responses: type I, reducing sugar content before and after 4°C storage was lower than the value required by industry; type II, reducing sugar content before storage was acceptable, but after 4°C storage incremented up to non-acceptable levels; and type III, reducing sugar content was unacceptable before and after storage. CONCLUSION: Five Andean accessions presented acceptable reducing sugar content and good chip quality before and after 4°C storage in a consistent manner throughout several experiments. These features make them a useful source for improving the potato industry. © 2017 Society of Chemical Industry.

Purification and characterization of a novel trypsin inhibitor from Solanum tuberosum subsp. andigenum var. overa: Study of the expression levels and preliminary evaluation of its antimicrobial activity.

Protease inhibitors (PIs) have been traditionally recognized by their potential biomedical application in events with exacerbation of endogenous proteases activity. Plant PIs have gained interest as naturally occurring molecules, which usually show lower environmental impact residual toxicity than synthetic compounds. In this work, we isolated, cloned, expressed and purified a novel trypsin inhibitor from S. tuberosum subsp. andigenum var. overa, named oPTI. A significant over-expression of the oPTI coding gene after 48 h exposure of methyl jasmonate compared to the gene of reference. This inhibitor showed a molecular mass of 12 kDa and a Ki of 7.3 × 10-7 M. Finally, we evaluated the antimicrobial activity of oPTI against different pathogenic microorganisms. The oPTI demonstrated inhibitory effect on the growth of Acinetobacter baumannii S-1, Acinetobacter baumannii R, Acinetobacter calcoaceticus R, Acinetobacter calcoaceticus S, Bacillus stearothermophilus, Escherichia coli, Pseudomonas aeruginosa, Salmonella braenderup, Salmonella enteritidis, Salmonella typhimurium and Yersinia enterocolitica strains. This study represents the first report for the antimicrobial activity of a plant PI over a wide range of microorganisms. Our studies reinforce the importance of natural PIs as promising molecules for their potential application in the biomedical field and/or in the food industry as natural food preservatives.

Biodiversity of Andean potatoes: Morphological, nutritional and functional characterization.

Andean potatoes (Solanum tuberosum andigenum) are a staple food for Andean population; there is great biodiversity but only few varieties are cultivated nowadays. In order to contribute to biodiversity conservation of Andean potatoes, information about their morphological, nutritional and functional characteristics was generated. In gene bank (INTA-Balcarce), varieties collected from regional producers were preserved. Forty-four genotypes were multiplied and characterized. Morphological characteristics; proximate composition and functional compounds were analyzed. Cluster analysis separated them into 3 groups according to distinguishing characteristics, which define industrial or nutritional applications. Group 2 was characterized by higher content of macronutrients and Group 3 with the highest antioxidant activity, both would be advisable for direct consumption. Genotype CS 1418 had big size and oval form so it could be destined to potato chips industry. Knowledge on nutritional and functional properties of genotypes contributes to promoting the cultivation depending on properties and also to preserve biodiversity.

Improving the screening of potato breeding lines for specific nutritional traits using portable mid-infrared spectroscopy and multivariate analysis.

Efficient selection of potato varieties with enhanced nutritional quality requires simple, accurate and cost effective assays to obtain tuber chemical composition information. In this study, 75 Andean native potato samples from 7 Solanum species with different colors were characterized and quantified for their anthocyanin, phenolics and sugar content using traditional reference methods. IR (infrared) spectra of potato extracts were collected using a portable infrared system and partial least squares regression (PLSR) calibration models were developed. These models were validated using both full cross-validation and an independent sample set giving strong linear correlation coefficients of prediction (rPred)>0.91 and standard error of prediction (SEP) of 24mg/100g phenolics, 7mg/100g monomeric anthocyanins, 0.1g/100g reducing sugars and 0.12g/100g sucrose. Overall, portable infrared system with PLSR showed great potential to facilitate potato breeding and certain aspects of crop management, material selection for potato processing and related research by providing alternative prediction models.

Biochemical characterization of a novel carboxypeptidase inhibitor from a variety of Andean potatoes.

Natural protease inhibitors of metallocarboxypeptidases are rarely reported. In this work, the cloning, expression and characterization of a proteinaceous inhibitor of the A/B-type metallocarboxypeptidases, naturally occurring in tubers of Solanum tuberosum, subsp. andigenum cv. Imilla morada, are described. The obtained cDNA encoded a polypeptide of 80 residues, which displayed the features of metallocarboxypeptidase inhibitor precursors from the Potato Carboxypeptidase Inhibitor (PCI) family. The mature polypeptide (39 residues) was named imaPCI and in comparison with the prototype molecule of the family (PCI from S. tuberosum subsp. tuberosum), its sequence showed one difference at its N-terminus and another three located at the secondary binding site, a region described to contribute to the stabilization of the complex inhibitor-target enzyme. In order to gain insights into the relevance of the secondary binding site in nature, a recombinant form of imaPCI (rimaPCI) having only differences at the secondary binding site with respect to recombinant PCI (rPCI) was cloned and expressed in Escherichia coli. The rimaPCI exhibited a molecular mass of 4234.8Da by MALDI-TOF/MS. It displayed potent inhibitory activity towards A/B-type carboxypeptidases (with a Ki in the nanomolar range), albeit 2-4-fold lower inhibitory capacity compared to its counterpart rPCI. This result is in agreement with our bioinformatic analysis, which showed that the main interaction established between the secondary binding site of rPCI and the bovine carboxypeptidase A is likely lost in the case of rimaPCI. These observations reinforce the importance of the secondary binding site of PCI-family members on inhibitory effects towards A/B-type metallocarboxypeptidases. Furthermore, as a simple proof of concept of its applicability in biotechnology and biomedicine, the ability of rimaPCI to protect human epidermal growth factor from C-terminal cleavage and inactivation by carboxypeptidases A and B was demonstrated.