Molecular characterization, expression and immune functions of cystatin B in Japanese flounder (Paralichthys olivaceus).

Cystatin B is an intracellular inhibitor that regulates the activities of cysteine proteases. In this study, cystatin B in Japanese flounder (Paralichthys olivaceus) was characterized and its immune function was analyzed. This gene had a high similarity with the sequence of cystatin B in other fish species, and the derived peptide shared typical features of cystatin proteins including the QXVXG motif. The results of quantitative real-time PCR showed that cystatin B mRNA was constitutively expressed in all examined tissues, with the highest level in gill. The stimulations of lipopolysaccharide, peptidoglycan and polyinosinic-polycytidylic acid effectively increased the expression level of cystatin B mRNA. Functional analysis implied that the recombinant P. olivaceus cystatin B purified from Escherichia coli had cysteine protease inhibitory activity and could inhibit bacterial growth by binding to bacteria. Furthermore, we found that P. olivaceus cystatin B had no effects on the expression of inflammatory factors cytokines tumor necrosis factor α, interleukin 10, interleukin 1ß and interferon γ. These results indicate that cystatin B of P. olivaceus is potentially involved in immune responses against invading microbial pathogens, and provide a better understanding of the immune mechanisms of cystatins in teleosts.

Genetic and pharmacological evidence implicates cathepsins in Niemann-Pick C cerebellar degeneration.

Niemann-Pick C1 (NPC) disease, an autosomal recessive lipid trafficking disorder caused by loss-of-function mutations in the NPC1 gene, is characterized by progressive neurodegeneration resulting in cognitive impairment, ataxia and early death. Little is known about the cellular pathways leading to neuron loss. Here, we studied the effects of diminishing expression of cystatin B, an endogenous inhibitor of cathepsins B, H and L, on the development of NPC neuropathology. We show that decreased expression of cystatin B in patient fibroblasts enhances cathepsin activity. Deletion of the encoding Cstb gene in Npc1-deficient mice resulted in striking deleterious effects, particularly within the cerebellum where diffuse loss of Purkinje cells was observed in young mice. This severe pathology occurred through cell autonomous mechanisms that triggered Purkinje cell death. Moreover, our analyses demonstrated the mislocalization of lysosomal cathepsins within the cytosol of Npc1-deficient Purkinje cells. We provide evidence that this may be a consequence of damage to lysosomal membranes by reactive oxygen species (ROS), leading to the leakage of lysosomal contents that culminates in apoptotic cell death. Consistent with this notion, toxicity from ROS was attenuated in an NPC cell model by cystatin B over-expression or pharmacological inhibition of cathepsin B. The observation that Npc1 and Cstb deletion genetically interact to potently enhance the degenerative phenotype of the NPC cerebellum provides strong support for the notion that lysosomal membrane permeabilization contributes to cerebellar degeneration in NPC disease.

Identification of acid-resistant proteins in acquired enamel pellicle.

OBJECTIVES: This study characterized the proteome profile of the acquired pellicle formed in vivo on enamel. Changes in this proteome profile after exposure to lactic or citric acid were also evaluated. METHODS: Volunteers (n=8) were subjected to dental prophylaxis. After 2 h to allow the formation of the acquired pellicle, the teeth were isolated with cotton rolls and 1 mL of citric acid (1%, pH 2.5) or lactic acid (0.1 M pH 4.8) or deionized water was gently applied with a pipette on the anterior teeth (both maxillary and mandibular) for 10 s. In sequence, the pellicle was collected with an electrode filter paper soaked in 3% citric acid. This procedure was repeated for two additional days following a crossover protocol. Proteins were subjected to reverse phase liquid chromatography coupled to mass spectrometry (nLC-ESI-MS/MS). MS/MS data were processed and submitted to Proteome Discoverer software. Searches were done using SWISS-PROT and TrEMBL databases for human proteins. RESULTS: In total, seventy-two proteins were present in all groups and were submitted to quantitative analysis (SIEVE). Some of these proteins were increased more than two-fold after exposure to the acids. Among them, cystatin-B was increased 20- and 13-fold after exposure to citric and lactic acids, respectively. Additionally, some proteins were identified in only one of the groups (18, 5, and 11 proteins for deionized water, citric and lactic acids, respectively). CONCLUSIONS: Our results open new insights regarding potentially acid-resistant proteins that could be added to dental products to prevent acidic dissolution of the teeth.

Aspartic cathepsin D degrades the cytosolic cysteine cathepsin inhibitor stefin B in the cells.

Stefin B is the major general cytosolic protein inhibitor of cysteine cathepsins. Its main function is to protect the organism against the activity of endogenous potentially hazardous proteases accidentally released from lysosomes. In this study, we investigated the possible effect of endosomal/lysosomal aspartic cathepsins D and E on stefin B after membrane permeabilization. Loss of membrane integrity of lysosomes and endosomes was induced by a lysosomotropic agent L-Leucyl-L-leucine methyl ester (Leu-Leu-OMe). The rat thyroid cell line FRTL-5 was selected as a model cell line owing to its high levels of proteases, including cathepsin D and E. Permeabilization of acid vesicles from FRTL-5 cells induced degradation of stefin B. The process was inhibited by pepstatin A, a potent inhibitor of aspartic proteases. However, degradation of stefin B was prevented by siRNA-mediated silencing of cathepsin D expression. In contrast, cathepsin E silencing had no effect on stefin B degradation. These results showed that cathepsin D and not cathepsin E degrades stefin B. It can be concluded that the presence of cathepsin D in the cytosol affects the inhibitory potency of stefin B, thus preventing the regulation of cysteine cathepsin activities in various biological processes.

Olive flounder (Paralichthys olivaceus) cystatin B: cloning, tissue distribution, expression and inhibitory profile of piscine cystatin B.

Among the cystatin superfamily, cystatin B, also known as stefin B, is an intracellular inhibitor that regulates the activities of cysteine proteases, such as papain and cathepsins. In this study, the 536 bp cystatin B cDNA (referred to hereafter as PoCystatin B) was cloned from olive flounder (Paralichthys olivaceus) using a combination of the rapid amplification of cDNA ends (RACE) approach and olive flounder cDNA library screening. To determine the tissue distribution of PoCystatin B mRNA, the expression of PoCystatin B in normal and lipopolysaccharide (LPS)-stimulated flounder tissues were compared with that of the inflammatory cytokines interleukin (IL)-1ß, IL-6, and IL-8 by reverse transcription (RT)-polymerase chain reaction (PCR). The results of the RT-PCR analysis revealed ubiquitous PoCystatin B expression in normal and LPS-stimulated tissues. To characterize the enzymatic activity of PoCystatin B protein, recombinant PoCystatin B protein was overexpressed in Escherichia coli BL21(DE3) cells in the pCold™ TF DNA expression vector as a soluble fusion protein of 67-kDa. PoCystatin B inhibited papain cysteine protease, bovine cathepsin B, and fish cathepsins F and X to a greater extent, whereas fish cathepsins L, S, and K were inhibited to a lesser extent. These results indicate that the enzymatic characteristics of the olive flounder cystatin B are similar to those of mammalian cystatin B proteins, and provide a better understanding of the mechanisms of regulation of cathepsins and cystatins in marine organisms.

Cystatin B homolog from rock bream Oplegnathus fasciatus: genomic characterization, transcriptional profiling and protease-inhibitory activity of recombinant protein.

Cysteine proteases are present in all living organisms and, in animals, function in a vast array of physiological and pathological processes. Cysteine protease inhibitors act upon the cysteine proteases to regulate their activity. The cystatin superfamily of cysteine protease inhibitors has members represented in all living organisms studied to date. Here, we report the identification of a new member of the family 1 cystatin in Oplegnathus fasciatus rock bream (denoted as RbCyt B) and the characterization at the molecular level. The complete genomic sequence of RbCyt B consists of three exons and a promoter region. The open reading frame (ORF) encodes for a 100 amino acids length polypeptide with a single cystatin-like domain and a cysteine protease inhibitor signature motif. The conserved N-terminal glycine, glutamine-valine-glycine motif, QxVxG, and a variant of the proline-tryptophan, PW, motif were identified. RbCyt B showed closest phylogenetic distance to Dicentrarchus labrax cystatin B, and shared up to 73% amino acid identity and 90% amino acid similarity with known cystatin B genes. RbCyt B mRNA expression was detected in nine different tissues and was highly expressed in liver, spleen, gill, brain, intestine, kidney, head kidney, and blood, as compared with muscle. In vivo immune stimulation with Edwardsiella tarda bacteria caused significant up-regulation of RbCyt B mRNA in head kidney and spleen at 24h post-infection (P<0.05). Recombinant RbCyt B was expressed in Escherichia coli, and the purified protein demonstrated 82% papain inhibitory activity at 500 × 10(-3) µg µL(-1) in a concentration-dependent manner. These results suggest that RbCyt B is a member of family 1 cystatin with high homology to cystatin B, and is a biologically active protein possessing papain inhibitory activity and potentially involved in immune responses against invading Gram-negative bacteria in rock bream.

Scophthalmus maximus cystatin B enhances head kidney macrophage-mediated bacterial killing.

Cystatins form a large family of cysteine protease inhibitors found in a wide arrange of organisms. Studies have indicated that mammalian cystatins play important roles under both physiological and pathological conditions. However, much less is known about fish cystatins. In this report, we described the identification and analysis of a cystatin B homologue, SmCytB, from turbot Scophthalmus maximus. The open reading frame of SmCytB is 300bp, which encodes a 99-residue protein that shares high levels of sequence identities with the cystatin B of a number of fish species and contains the conserved cysteine protease inhibitor motif of cystatin B. Constitutive expression of SmCytB is high in muscle, brain, heart and liver, and low in spleen, blood, gill and kidney. Bacterial infection upregulates SmCytB expression in kidney, spleen, liver and brain but not in muscle or heart. Functional analysis showed that recombinant SmCytB purified from Escherichia coli exhibits apparent cysteine protease inhibitor activity. Transient overexpression of SmCytB in head kidney macrophages enhances macrophage bactericidal activity probably through a nitric oxide-independent mechanism. These results indicate that SmCytB is involved in the immune defense of turbot against bacterial infection.

VEGF-A induces angiogenesis by perturbing the cathepsin-cysteine protease inhibitor balance in venules, causing basement membrane degradation and mother vessel formation.

Tumors initiate angiogenesis primarily by secreting vascular endothelial growth factor (VEGF-A(164)). The first new vessels to form are greatly enlarged, pericyte-poor sinusoids, called mother vessels (MV), that originate from preexisting venules. We postulated that the venular enlargement necessary to form MV would require a selective degradation of their basement membranes, rigid structures that resist vascular expansion. To identify the specific proteases responsible for MV formation, we induced angiogenesis in mouse tissues with an adenoviral vector expressing VEGF-A(164) (Ad-VEGF-A(164)) or with VEGF-A-secreting TA3/St mammary tumors. We found that MV formation resulted from greatly increased activity of cathepsins (B>S>L) in venules transitioning into MV, as well as from a reciprocal decrease in the expression of several cysteine protease inhibitors (CPI), stefin A and cystatins B and C, by these same venules. Using a fluorescence probe that selectively binds cellular sites of cathepsin protease activity in vivo, we showed that increased cathepsin activity was localized exclusively to perivenular cells, not to venule endothelial cells. CPI strikingly inhibited angiogenesis in the Matrigel assay, and Ad-VEGF-A(164)-induced angiogenesis was reduced by approximately 50% in cathepsin B-null mice. Thus, VEGF-A, whether expressed by interstitial cells infected with an adenoviral vector or by tumor cells, upsets the normal cathepsin-CPI balance in nearby venules, leading to degradation of their basement membranes, an important first step in angiogenesis.