In vitro activation of neuraminidase in the β-galactosidase-neuraminidase-protective protein complex by cathepsin C

doi:10.1016/S0006-291X(88)80316-4

Biochemical and Biophysical Research Communications

Volume 157, Issue 2, 15 December 1988, Pages 770-775

https://doi.org/10.1016/S0006-291X(88)80316-4 Get rights and content

Neuraminidase can be activated by incubation of crude glycoprotein fractions at acidic pH for 90 minutes at physiological temperature. This activation is inhibited by leupeptin. Incubation of the purified neuraminidase-β-galactosidase-protective protein complex under the same conditions used for crude glycoprotein fractions did not lead to enhanced neuraminidase activity, but incubation in the presence of exogenous Cathepsin C at 4°C resulted in marked enhancement of neuraminidase activity. This activation was again inhibited by leupeptin. Cathepsin D treatment resulted in destruction of neuraminidase under the same conditions and this effect was again inhibited by leupeptin. β-galactosidase in crude glycoprotein fractions and in the complex was resistant to both Cathepsin C and D, while homogeneous β-galactosidase was inactivated by these enzymes. We suggest that in vitro activation of neuraminidase may mimic the in vivo intralysosomal conversion of the neuraminidase precursor into the mature form of the enzyme.

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Cathepsin C is a cysteine protease widely found in invertebrates and vertebrates, and has the important physiological role participating in proteolysis in vivo and activating various functional proteases in immune/inflammatory cells in the animals. In order to study the role of cathepsin C in the disease resistance of shrimp, we cloned cathepsin C gene (MjcathC) from Marsupenaeus japonicus, analyzed its expression patterns in various tissues, performed MjcathC-knockdown, and finally challenged experimental shrimps with Vibrio alginolyticus and WSSV. The results have shown the full length of MjcathC is 1782 bp, containing an open reading frame of 1350 bp encoding 449 amino acids. Homology analysis revealed that the predicted amino acid sequence of MjcathC shared respectively 88.42 %, 87.36 % and 87.58 % similarity with Penaeus monodon, Fenneropenaeus penicillatus and Litopenaeus vannamei. The expression levels of MjcathC in various tissues of healthy M. japonicus are the highest in the liver, followed by the gills and heart, and the lowest in the stomach. The expression levels of MjcathC were significantly up-regulated in all examined tissues of shrimp challenged with WSSV or V. alginolyticus. After knockdown-MjcathC using RNAi technology in M. japonicus, the expression levels of lectin and heat shock protein 70 in MjcathC-knockdown shrimp were significantly down-regulated, and the mortality of MjcathC-knockdown shrimp challenged by WSSV and V. alginolyticus significantly increased. Knockdown of the MjcathC reduced the resistance of M. japonicus to WSSV and V. alginolyticus. The above results have indicated that cathepsin C may play an important role in the antibacterial and antiviral innate immunity of M. japonicus.
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The effects of different stocking densities on Litopenaeus vannamei were investigated from the aspects of growth performance, immune response and transcriptome in this experiment. L. vannamei (initial body weight: 0.30 ± 0.02 g) were reared for 8 weeks at three stocking densities of 100 (LSD), 200 (MSD) and 300 (HSD) shrimp/m³, respectively. The results showed that the survival rate (SR), final body weight (FBW), weight gain rate (WGR), specific growth ratio (SGR) and protein efficiency ratio (PER) of L. vannamei significantly decreased, while the feed factor (FCR) significantly increased with the increase of stocking density. After Vibrio parahemolyticus infection, the SR of L. vannamei in the HSD group was significantly lower than that in the LSD and MSD groups. Increasing stocking density significantly increased the activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lysozyme (LYS) while significantly decreased the activities of catalase (CAT) and phenol oxidase (PO) in the serum of L. vannamei. Similar changes of the gene expression as the activities of immune enzymes were found in the hemocytes. Pairwise comparison between the LSD, MSD and HSD group in the transcriptome analysis identified that there were 304, 1376 and 2083 differentially expressed genes (DEGs) in LSD vs MSD, MSD vs HSD and LSD vs HSD, respectively. Among them, most of the immune-related DEGs were down-regulated and metabolism-related DEGs were up-regulated with the increasing stocking density. In addition, KEGG enrichment pathway analysis revealed that several immune and metabolic related pathways including PI3K-Akt signaling pathway and AMPK signaling pathway were significantly enriched. Of these, the PI3K-Akt signaling pathway had the most DEGs and was also the most significantly enriched pathway. Furthermore, 16 DEGs (such as FOXO, PCK2 and CTSC, etc.) and partial immune enzyme activity (such as AST, CAT and PO, etc.) changes were closely correlated with the increase of stocking density when partial immune-related DEGs and immune-related enzymes were analyzed jointly. All these results indicated that changes in stocking density had a significant effect on the growth performance, immunity and transcriptome of L. vannamei.
Development of the first internally-quenched fluorescent substrates of human cathepsin C: The application in the enzyme detection in biological samples
2016, Archives of Biochemistry and Biophysics
Citation Excerpt :
The mostly recognized physiological substrates of Cat C are granule-associated serine proteases: human neutrophil elastase (HNE), cathepsin G (Cat G), protainase 3 (PR3) and neutrophil serine proteinase 4 (NSP4) from neutrophils, mast cell chymase, as well as granzymes A and B from cytotoxic T lymphocytes and natural killer cells [10–13]. Cat C was also found to participate in neuraminidase activation [14], platelet factor XIII activation [15] and regulation of plasminogen-plasmin system [16]. It has been shown that mutations in human Cat C that lead to loss of its function result in a decrease in host defense and increased susceptibility to microbe infection being primarily responsible for Papillon-Lefevre [17] and Haim-Munk syndromes development [18].
Cathepsin C is a widely expressed cysteine exopeptidase that is mostly recognized for the activation of the granule-associated proinflammatory serine proteases in neutrophils, cytotoxic T lymphocytes and mast cells. It has been shown that the enzyme can be secreted extracellularly; however, its occurrence in human bodily fluids/physiological samples has not been thoroughly studied. In the course of this study, the first fluorescence resonance energy transfer peptides for the measurement of the activity of human cathepsin C were designed and synthesized. Two series of tetra- and pentapeptide substrates enabled the detailed S′ specificity study of cathepsin C, which has been examined for the first time. The extensive enzymatic studies of the obtained compounds resulted in the selection of the highly specific and selective substrate Thi-Ala(Mca)-Ser-Gly-Tyr(3-NO₂)-NH₂, which was successfully employed for the detection of cathepsin C activity in complex biological samples such as cell lysates, urine and bronchoalveolar lavage fluids. Molecular docking of the selected substrate was performed in order to better understand the binding mode of the substrates in the active site of cathepsin C.
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Involvement of Fenneropenaeus chinensis Cathepsin C in antiviral immunity
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Citation Excerpt :
The main functions of Cath C are protein degradation and activation of proenzymes. Cath C is involved in intracellular protein degradation [4], cell growth [8], neuraminidase activation [9], and platelet factor XIII activation [10]. Cath C and other cysteine proteases, such as cathepsins B, H, and L, were found to degrade intracellular proteins [4].
Cathepsin C (Cath C) is a lysosomal cysteine protease that belongs to the papain superfamily. Cath C is capable of activating many chymotrypsin-like serine proteases and is reported to be a central coordinator for the activation of many serine proteinases in immune and inflammatory cells. In this study, Cath C cDNA was cloned from Fenneropenaeus chinensis (Fc). The complete cDNA of Fc-Cath C in Chinese white shrimp was found to be 1445-base pairs (bp) long. It contained an open reading frame (ORF) 1356-bp long and encoded a 451-amino acid residue protein, including a 17-amino acid residue signal peptide. Real-time PCR analysis results indicated that Fc-Cath C was present in all the tissues detected and exhibited high level of transcription in the hepatopancreas. In hemocytes, hepatopancreas, gills and intestine, Fc-Cath C was upregulated after stimulation by the Vibrio anguillarum and the white spot syndrome viruses (WSSVs). Replication of the WSSV increased after the injection of Fc-Cath C antiserum or knockdown Cath C by RNA interference. These results implied that Cath C might play a crucial role in the antiviral immune response of shrimp.
Molecular cloning and mRNA expression of cathepsin C gene in black tiger shrimp (Penaeus monodon)
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Cathepsin C (dipeptidyl-peptidase I, DPPI) is a lysosomal cysteine proteinase belonging to the papain superfamily, which is capable of removing dipeptides sequentially from the amino terminus of peptide and protein substrates. In the present study, the cDNA of a cathepsin C was cloned from black tiger shrimp Penaeus monodon (designated PmcathepsinC) by homology cloning and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of PmcathepsinC consisted of 2051 nucleotides with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, and an open reading frame (ORF) of 1350 bp encoding a polypeptide of 449 amino acid residues with a predicted molecular weight of 50.0 kDa and theoretical isoelectric point of 5.65. The high identity of PmcathepsinC with Cathepsin C in other organisms indicated that PmcathepsinC should be a new member of the Cathepsin C family. By fluorescent quantitative real-time PCR, mRNA transcript of PmcathepsinC was detectable in all the examined tissues with higher level in ovary and heart. The temporal expression of PmcathepsinC mRNA in the hepatopancreas was up-regulated by lipopolysaccharide (LPS) stimulation and reached the maximum level at 4 h post-stimulation, and then dropped back to the original level gradually. These results indicated that PmcathepsinC was a constitutive and inducible acute-phase protein that perhaps involved in the immune defense of P. monodon.

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In vitro activation of neuraminidase in the β-galactosidase-neuraminidase-protective protein complex by cathepsin C

J. Biol. Chem.

Biochim. Biophys. Acta.

Biochim. Biophys. Acta.

Cell

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.