Characterization of a novel cystatin type 2 from Rhipicephalus microplus midgut

Cardoso, Thyago Hermylly Santana; Lu, Stephen; Gonzalez, Boris R.G.; Torquato, Ricardo J.S.; Tanaka, Aparecida S.

doi:10.1016/j.biochi.2017.07.005

Cited by 10 publications

(8 citation statements)

References 30 publications

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“…The inhibition constant (K i ) in the order of nM (7.9 nM) is compatible with the one found in the literature for the dissociation constant of cathepsin L with human cystatins [1]. Similar K i values were obtained related to cystatins of hematophagous animals such as the bovine ectoparasite Rhipicephalus microplus, whose protein identified as Rmcystatin-4 was cloned, expressed, and purified, and has demonstrated inhibitory activity against cathepsin L with a K i of 11.1 nM [38]. The cystatin OmC2 from the tick genus Ornithodoros also presented similar K i values in the range of nM against lysosomal cathepsins S and C [17].…”

Section: Discussionsupporting

confidence: 86%

“…Furthermore, the use of the P. pastoris expression system, due to its limited production of endogenous secretory proteins, is known to favor an easy purification protein process [37]. In this sense, the isolation in two chromatography steps was sufficient to achieve a pure form of recombinant Cystatin-Hv, similar to the purification process performed by Cardoso [38], characterizing a tick cystatin that presented an inhibitory effect against the activity of a hemoglobin lytic enzyme.…”

Section: Discussionmentioning

confidence: 99%

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Novel Cysteine Protease Inhibitor Derived from the Haementeria vizottoi Leech: Recombinant Expression, Purification, and Characterization

Linhares

Faria

Kodama

et al. 2021

Toxins

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Cathepsin L (CatL) is a lysosomal cysteine protease primarily involved in the terminal degradation of intracellular and endocytosed proteins. More specifically, in humans, CatL has been implicated in cancer progression and metastasis, as well as coronary artery diseases and others. Given this, the search for potent CatL inhibitors is of great importance. In the search for new molecules to perform proteolytic activity regulation, salivary secretions from hematophagous animals have been an important source, as they present protease inhibitors that evolved to disable host proteases. Based on the transcriptome of the Haementeria vizzotoi leech, the cDNA of Cystatin-Hv was selected for this study. Cystatin-Hv was expressed in Pichia pastoris and purified by two chromatographic steps. The kinetic results using human CatL indicated that Cystatin-Hv, in its recombinant form, is a potent inhibitor of this protease, with a Ki value of 7.9 nM. Consequently, the present study describes, for the first time, the attainment and the biochemical characterization of a recombinant cystatin from leeches as a potent CatL inhibitor. While searching out for new molecules of therapeutic interest, this leech cystatin opens up possibilities for the future use of this molecule in studies involving cellular and in vivo models.

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Section: Discussionsupporting

confidence: 86%

Section: Discussionmentioning

confidence: 99%

Novel Cysteine Protease Inhibitor Derived from the Haementeria vizottoi Leech: Recombinant Expression, Purification, and Characterization

Linhares

Faria

Kodama

et al. 2021

Toxins

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“…Therefore, it has been proposed that aspartic and cysteine peptidase inhibitors can act as regulators of the digestion process. Not surprisingly, in R. microplus, cysteine and aspartic peptidases have been previously identified and implicated in blood digestion (Clara et al, 2011;Cruz et al, 2010) as well as cystatins (Cardoso et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

“…Cysteine peptidase inhibitors are classified into the cystatin superfamily based on their primary features (Barret et al, 1985;Rawlings et al, 2018). In the past years, several type 2 cystatins from ticks were identified and associated with different physiological processes, such as blood feeding (Karim et al, 2005;Yamaji et al, 2009), parasite-host relantionship (Kotsyfakis et al, 2006;Salat et al, 2010), blood digestion (Cardoso et al, 2017;Yamaji et al, 2010) and immune response (Lu et al, 2014;Zhou et al, 2006). On the other hand, only a handful of type 1 cystatins have been characterized.…”

Section: Introductionmentioning

confidence: 99%

A novel type 1 cystatin involved in the regulation of Rhipicephalus microplus midgut cysteine proteases

Rocha

Torquato

et al. 2020

Ticks and Tick-borne Diseases

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This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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“…In bloodsucking animals, such as ticks, cystatins are usually expressed in their salivary glands or the midgut, which suggests that cystatins might participate in the feeding process of bloodsuckers [11,12,13,14,15]; while in snakes, the cystatins usually exist in their venom glands, which have been reported to suppress the growth, invasion, and metastasis of B16F10 cells and MHCC97H cells, as well as to inhibit tumor angiogenesis [16,17,18,19]. At present, more cystatins have been identified from venomous insects, snakes, fishes, or mollusks through gene cloning, and transcriptomic and proteomic approaches [20,21,22,23,24].…”

Section: Introductionmentioning

confidence: 99%

The Anti-Angiogenic Activity of a Cystatin F Homologue from the Buccal Glands of Lampetra morii

Zhu

Wang

et al. 2018

Marine Drugs

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Cystatins are a family of cysteine protease inhibitors which are associated with a variety of physiological and pathological processes in vivo. In the present study, the cDNA sequence of a cystatin F homologue called Lm-cystatin F was cloned from the buccal glands of Lampetra morii. Although Lm-cystatin F shares a lower homology with cystatin superfamily members, it is also composed of a signal peptide and three highly conserved motifs, including the G in the N-terminal, QXVXG, as well as the PW in the C-terminal of the sequence. After sequence optimization and recombination, the recombinant protein was expressed as a soluble protein in Escherichia coli with a molecular weight of 19.85 kDa. Through affinity chromatography and mass spectrometry analysis, the purified protein was identified as a recombinant Lm-cystatin F (rLm-cystatin F). Additionally, rLm-cystatin F could inhibit the activity of papain. Based on MTT assay, rLm-cystatin F inhibited the proliferation of human umbilical vein endothelial cells (HUVECs) dose dependently with an IC50 of 5 μM. In vitro studies show that rLm-cystatin F suppressed the adhesion, migration, invasion, and tube formation of HUVECs, suggesting that rLm-cystatin F possesses anti-angiogenic activity, which provides information on the feeding mechanisms of Lampetra morii and insights into the application of rLm-cystatin F as a potential drug in the future.

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Characterization of a novel cystatin type 2 from Rhipicephalus microplus midgut

Cited by 10 publications

References 30 publications

Novel Cysteine Protease Inhibitor Derived from the Haementeria vizottoi Leech: Recombinant Expression, Purification, and Characterization

Novel Cysteine Protease Inhibitor Derived from the Haementeria vizottoi Leech: Recombinant Expression, Purification, and Characterization

A novel type 1 cystatin involved in the regulation of Rhipicephalus microplus midgut cysteine proteases

The Anti-Angiogenic Activity of a Cystatin F Homologue from the Buccal Glands of Lampetra morii

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