The swaposin-like domain of potato aspartic protease (StAsp-PSI) exerts antimicrobial activity on plant and human pathogens

Muñoz, Fernando Felipe; Mendieta, Julieta Renée; Pagano, Mariana; Paggi, Roberto A.; Daleo, Gustavo Raúl; Guevara, María Gabriela

doi:10.1016/j.peptides.2010.02.001

Cited by 49 publications

(52 citation statements)

References 76 publications

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“…Haemolysis assays were performed as described by Muñoz et al [27]. Fresh human red blood cells (hRBC) were rinsed in PBS and centrifuged for 10 min at 800 Â g three times and resuspended in PBS to a final erythrocyte concentration of 4% v/v.…”

Section: Haemolysis Assaymentioning

confidence: 99%

Fibrin(ogen)olytic and antiplatelet activities of a subtilisin-like protease from Solanum tuberosum (StSBTc-3)

et al. 2016

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a b s t r a c tPlant serine proteases have been widely used in food science and technology as well as in medicine. In this sense, several plant serine proteases have been proposed as potential anti-coagulants and antiplatelet agents. Previously, we have reported the purification and identification of a plant serine protease from Solanum tuberosum leaves. This potato enzyme, named as StSBTc-3, has a molecular weight of 72 kDa and it was characterized as a subtilisin like protease. In this work we determine and characterize the biochemical and medicinal properties of StSBTc-3. Results obtained show that, like the reported to other plant serine proteases, StSBTc-3 is able to degrade all chains of human fibrinogen and to produces fibrin clot lysis in a dose dependent manner. The enzyme efficiently hydrolyzes b subunit followed by partially hydrolyzed a and g subunits of human fibrinogen. Assays performed to determine StSBTc-3 substrate specificity using oxidized insulin b-chain as substrate, show seven cleavage sites: Asn3-Gln4; Cys7-Gly8; Glu13-Ala14; Leu15-Tyr16; Tyr16-Leu17; Arg22-Gly23 and Phe25-Tyr26, all of them were previously reported for other serine proteases with fibrinogenolytic activity. The maximum StSBTc-3 fibrinogenolytic activity was determined at pH 8.0 and at 37 C. Additionally, we demonstrate that StSBTc-3 is able to inhibit platelet aggregation and is unable to exert cytotoxic activity on human erythrocytes in vitro at all concentrations assayed. These results suggest that StSBTc-3 could be evaluated as a new agent to be used in the treatment of thromboembolic disorders such as strokes, pulmonary embolism and deep vein thrombosis.

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Section: Haemolysis Assaymentioning

confidence: 99%

Fibrin(ogen)olytic and antiplatelet activities of a subtilisin-like protease from Solanum tuberosum (StSBTc-3)

et al. 2016

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“…Moreover, PSI domains were shown to interact with lipids. Their anticipated role in vacuolar sorting and their antipathogenic effects are suggested to be dependent on protein-membrane interactions (2,(7)(8)(9). Egas and coworkers demonstrated that the recombinant PSI from cardosin A is able to interact with phospholipid vesicles and to induce leakage of their contents in a pH-and lipid-dependent manner (8).…”

mentioning

confidence: 99%

“…Egas and coworkers demonstrated that the recombinant PSI from cardosin A is able to interact with phospholipid vesicles and to induce leakage of their contents in a pH-and lipid-dependent manner (8). Moreover, it was reported that recombinant PSI of Solanum tuberosum aspartic protease (StAP) is able to kill bacterial pathogens and to inhibit sporulation of phytopathogenic fungi through interaction with microbial membranes (9). Recently, Bryksa and coworkers (10) determined the structure of the isolated recombinant StAP PSI domain and demonstrated its fusogenic activity via pH-dependent membrane interactions.…”

mentioning

confidence: 99%

“…Among the newly proposed antibiotics are antimicrobial peptides (AMPs), which might profit from high effectiveness, safety, and reduced levels of cross-resistance due to their mode of action (11)(12)(13). Structural similarities of PSI domains with SAPLIPs (2,10), in combination with their ability to interact with and permeabilize phospholipid membranes (8,10), resulting in antimicrobial activity (e.g., StAP PSI [9]), suggest a promising role for PSI domains as novel antibiotics.…”

mentioning

confidence: 99%

“…To date, PSI domains have only been produced using prokaryotic systems, such as Escherichia coli, giving only very low yields of nonglycosylated material (8,9). Increasing the production levels of such proteins has been a major challenge so far, with Bryksa and collaborators reporting their best results by expressing the StAP PSI fused to a solubility enhancement tag (10).…”

mentioning

confidence: 99%

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Establishing the Yeast Kluyveromyces lactis as an Expression Host for Production of the Saposin-Like Domain of the Aspartic Protease Cirsin

Curto¹,

Lufrano

Pinto

et al. 2014

Appl Environ Microbiol

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Typical plant aspartic protease zymogens comprise a characteristic and plant-specific insert (PSI). PSI domains can interact with membranes, and a role as a defensive weapon against pathogens has been proposed. However, the potential of PSIs as antimicrobial agents has not been fully investigated and explored yet due to problems in producing sufficient amounts of these domains in bacteria. Here, we report the development of an expression platform for the production of the PSI domain of cirsin in the generally regarded as safe (GRAS) yeast Kluyveromyces lactis. We successfully generated K. lactis transformants expressing and secreting significant amounts of correctly processed and glycosylated PSI, as well as its nonglycosylated mutant. A purification protocol with protein yields of ϳ4.0 mg/liter was established for both wild-type and nonglycosylated PSIs, which represents the highest reported yield for a nontagged PSI domain. Subsequent bioactivity assays targeting phytopathogenic fungi indicated that the PSI of cirsin is produced in a biologically active form in K. lactis and provided clear evidence for its antifungal activity. This yeast expression system thereby emerges as a promising production platform for further exploring the biotechnological potential of these plant saposin-like proteins.

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An Overview of Plant Proteolytic Enzymes

D‘Ippólito

Guevara

Tito

et al. 2018

Biotechnological Applications of Plant Proteolytic Enzymes

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The swaposin-like domain of potato aspartic protease (StAsp-PSI) exerts antimicrobial activity on plant and human pathogens

Cited by 49 publications

References 76 publications

Fibrin(ogen)olytic and antiplatelet activities of a subtilisin-like protease from Solanum tuberosum (StSBTc-3)

Fibrin(ogen)olytic and antiplatelet activities of a subtilisin-like protease from Solanum tuberosum (StSBTc-3)

Establishing the Yeast Kluyveromyces lactis as an Expression Host for Production of the Saposin-Like Domain of the Aspartic Protease Cirsin

An Overview of Plant Proteolytic Enzymes

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