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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 30, 2002 as doi:10.1096/fj.01-0736fje. |
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Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon 305–701, Korea;
* Department of Anatomy, Yonsei University College of Medicine, Seoul 120–752, Korea; and
Biomass Team, Korea Institute of Energy Research, Taejon 305–343, Korea
2Correspondence: Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373–1 Kusong-dong Yusong-gu, Taejon 305–701, Korea. E-mail: sckim{at}mail.kaist.ac.kr
SPECIFIC AIMS
Parasin I, a 19-residue antimicrobial peptide abundant in the skin mucus of wounded, but not unwounded catfish, is produced from a larger precursor histone H2A. In the present study, we investigated the mechanism for the generation of parasin I from histone H2A in catfish skin mucosa by biochemical and immunohistochemical analysis. We also assessed the biological role of parasin I in the innate host defense of fish against invading microorganisms.
PRINCIPAL FINDINGS
1. Cathepsin D is responsible for the proteolytic cleavage of histone H2A to yield parasin I
To identify the protease responsible for the generation of parasin I from histone H2A, we first assessed the specific proteolytic activity that converts histone H2A to parasin I in the mucus extracts from normal (unwounded) and wounded catfish. Only the mucus extracts from wounded catfish had parasin I-producing activity, which was completely abolished by pepstatin A. These results indicate that an aspartic protease(s) is involved in the production of parasin I from histone H2A in the mucus of wounded catfish. We therefore sought to purify these aspartic protease(s) from the mucus of wounded catfish using a pepstatin A-agarose column. The fraction from the pepstatin A-agarose column that showed parasin I-producing activity was further separated into a single protein band of 
37 kDa by chromatography on an FPLC Mono Q column (Fig. 1
A, B). Amino acid sequence analysis of the purified enzyme identified 18 amino-terminal amino acid residues that closely matched the amino-terminal sequence of cathepsin D (Fig. 1C
). The proteolytic activity of cathepsin D was tracked throughout the purification by determination of the molecular size and antimicrobial activity of the processed parasin I-like peptide. Cathepsin D cleaved the Ser19-Arg20 bond of histone H2A to produce parasin I, as confirmed by amino acid sequence analysis of the processed parasin I-like peptide.
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2. Procathepsin D is activated to mature cathepsin D by a metalloprotease induced upon injury
Cathepsin D is constitutively expressed in almost all cells except some forms of cancer and in oocyte maturation of egg-laying animals. Therefore, the observation that parasin I production is induced by epidermal injury implies the presence of the responsible enzyme(s) in the mucus of wounded catfish subject to regulation. We hypothesized that cathepsin D might be secreted to the mucus as a latent proform and later activated to a mature form by an enzyme(s) induced in the mucus of wounded catfish. To address our hypothesis, we attempted to isolate procathepsin D from the mucus of normal catfish. The pepstatin A-bound fractions obtained from the mucus of normal catfish did not show parasin I-producing activity, but activity was generated when the pepstatin A flow-through fraction prepared from the mucus of wounded catfish was added. Fractions showing parasin I-producing activity upon addition of the pepstatin A flow-through fraction from wounded catfish were purified further by FPLC on a Mono Q column and a 40 kDa protein was purified. Amino acid sequence analysis and cDNA (GenBank accession no. AF396662) cloning identified the purified protein as procathepsin D. Western blot analysis showed that the 40 kDa procathepsin D was processed to 37 kDa mature cathepsin D by the pepstatin A flow-through fraction from the wounded catfish. This proceolytic activation was inhibited only by EDTA, which suggests that the cathepsin D-activating enzyme induced in the mucus of wounded catfish is a metalloprotease.
3. Parasin I is produced on the mucosal surface on epidermal injury
Immunohistochemical experiments were performed to determine the histological location of histone H2A, parasin I, and cathepsin D in cross sections of normal (Fig. 2
A–G) and wounded (Fig. 2H
) catfish skins. To distinguish the histone H2A present in cytoplasm from that in the nucleus, two specific antibodies—anti-acetylated (Ac-Lys5) histone H2A and anti-unacetylated histone H2A—were used. The anti-unacetylated histone H2A stained the cytoplasm of epithelial mucous cells (Fig. 2D
) and the anti-acetylated histone H2A stained only the nucleus (Fig. 2E
). The skin section subjected to periodic acid-Schiff (PAS) staining revealed that cells that were immunopositive for unacetylated histone H2A were also the PAS-positive cells responsible for the generation of mucus (Fig. 2B
). Incubation of the skin sections from normal (Fig. 2G
) and wounded (Fig. 2H
) catfish with a parasin I-specific antibody showed strong immunoreactivity only at the mucosal surface of the wounded skin. This indicates that parasin I was produced on the mucosal surface upon epidermal injury. An antibody against human cathepsin D localized the immunoreactivity to the cytoplasm of epithelial mucous cells, the same cells that contained unacetylated histone H2A (Fig. 2F
). Given that cathepsin D was isolated as a proenzyme from the mucus of normal catfish, our results indicate that the cathepsin D immunoreactivity in epithelial mucous cells of normal catfish resulted from procathepsin D, although the antibody cannot distinguish procathepsin D from cathepsin D.
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4. Parasin I is also present in the mucus of other fish species
Western blot analysis was performed to determine whether parasin I could be detected in other fish species. Results showed that the skin mucus of wounded catfish, eel, loach, and rainbow trout contained an immunoreactive peptide that corresponded to purified parasin I.
5. Parasin I shows good antimicrobial activity against fish-specific pathogens
Five fish-specific pathogens were used to test the bactericidal activity of synthetic parasin I: Aeromonas salmonicida, Cytophaga aquatilis, Yersinia ruckeri, Edwardsiella ictaluri, and Lactococcus garvieae. Results of the antimicrobial assays showed that parasin I is active against all pathogens tested (minimal inhibitory concentrations, 5–15 µg/ml).
CONCLUSIONS AND SIGNIFICANCE
In this study, we have demonstrated that the antimicrobial peptide parasin I is generated from unacetylated histone H2A in catfish skin mucosa by the action of cathepsin D, which is secreted to the mucosal surface as an inactive proenzyme (procathepsin D) and activated to the mature enzyme by a metalloprotease induced in response to epidermal injury (Fig. 3
). Cathepsin D is synthesized as an inactive proenzyme and activated most likely in the lysosome. It has been reported that 5–15% of procathepsin D escapes targeting to the lysosome and is secreted from cells, but little is known about the activation of secreted procathepsin D by extracellular proteases. Our protease inhibition assay revealed that the cathepsin D-activating enzyme induced in the mucus of wounded catfish is a metalloprotease. In vertebrates, metalloproteases, which are expressed or released in response to injury, disease, or inflammation, are typically associated with the immune response and tissue repair. Among them, matrilysin, whose expression is induced by exposure to bacteria, has been reported to function in the intestinal mucosal defense by regulating the activity of defensins. Cathepsin D is thought to function primarily in the normal degradation of cellular and phagocytosed proteins in the lysosome. It has also been postulated to play a role in antigen and prohormone processing, and its overexpression and increased secretion have been associated with the malignant behavior of human breast cancer. Our results suggest that the role of cathepsin D may be extended to the innate host defense system, specifically by processing histone H2A to yield the antimicrobial peptide parasin I.
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To elucidate the mechanism of parasin I generation in catfish skin mucosa, we performed immunohistochemical analyses. Immunohistochemical data indicate that unacetylated histone H2A, the precursor of parasin I, and procathepsin D are present in the epithelial mucous cells. This suggests they are secreted to the mucosal surface in a way similar to other mucous substances. It appears that a large amount of unacetylated histone H2A is available in the cytoplasm of epithelial mucous cells. This results in part from rapid regeneration of the skin mucosa and the active transcription in epithelial mucous cells reflected by the high expression of histone proteins. Of the total histone H2A synthesized in epithelial mucous cells, a limited amount moves into the nucleus and is acetylated; excess unacetylated histone H2A accumulates in the cytoplasm and eventually is secreted to the mucosal surface. The immunoreactivity of unacetylated histone H2A and procathepsin D did not change upon injury, whereas the immunoreactivity of parasin I at the mucosal surface of the skin was increased greatly in response to injury. This further confirms the existence of an inducible mechanism in parasin I production that is mediated by the activation of procathepsin D secreted to the mucosal surface.
The immunoreactivity of parasin I is extracellular, on the mucosal surface. This indicates that parasin I coats the mucosal surface, where it may contribute to the establishment of a local antimicrobial milieu. This notion is supported by our observation that parasin I showed good antimicrobial activity against fish-specific pathogens. Conservation of the amino acid sequence of histone H2A and cathepsin D in the fish phylum suggests that this parasin I sequence constitutes a biologically important part of the molecule that confers an advantage during selection and evolution in fish. Whether parasin I is ubiquitous in the skin mucosa of other fish is not clear. However, we confirmed by Western blot analysis the presence of parasin I in the mucus of eel, loach, and rainbow trout. Together, our results suggest that cathepsin D is responsible for the generation of parasin I from histone H2A upon epidermal injury and that parasin I represents an important innate antimicrobial defense against invading microorganisms in the skin mucosa of fish.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0736fje; to cite this article, use FASEB J. (January 30, 2002) 10.1096/fj.01-0736fje 
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