Abstract
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.
Highlights
Cystatins are a group of inhibitors, which could suppress the activity of C1 cysteine proteases and play important roles in a variety of physiological process to protect our tissues from inappropriate proteolysis [1,2]
Once the dynamic equilibrium between the levels of cystatins and their substrates is destroyed, people might suffer diseases, such as malignant tumor, neurodegenerative diseases, cardiovascular disease, and chronic kidney disease [1,7,8,9,10]. 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]
A Cystatin F Homologue was Identified from the Buccal Glands of L. morii
Summary
Cystatins are a group of inhibitors, which could suppress the activity of C1 cysteine proteases and play important roles in a variety of physiological process to protect our tissues from inappropriate proteolysis [1,2]. Once the dynamic equilibrium between the levels of cystatins and their substrates is destroyed, people might suffer diseases, such as malignant tumor, neurodegenerative diseases, cardiovascular disease, and chronic kidney disease [1,7,8,9,10] 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].
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