In Yup Park

Parasin I, an antimicrobial peptide derived from histone H2A in the catfish, Parasilurus asotus

In response to epidermal injury, Parasilurus asotus, a catfish, secreted a strong antimicrobial peptide into the epithelial mucosal layer. The molecular mass of the antimicrobial peptide, named parasin I, was 2000.4 Da, as determined by matrix‐associated laser desorption ionization mass spectrometry. The complete amino acid sequence of parasin I, which was determined by automated Edman degradation, was Lys‐Gly‐Arg‐Gly‐Lys‐Gln‐Gly‐Gly‐Lys‐Val‐Arg‐Ala‐Lys‐Ala‐Lys‐Thr‐Arg‐Ser‐Ser. Eighteen of the 19 residues in parasin I were identical to the N‐terminal of buforin I, a 39‐residue antimicrobial peptide derived from the N‐terminal of toad histone H2A [Kim et al. (1996) Biochem. Biophys. Res. Commun. 229, 381–387], which implies that parasin I was cleaved off from the N‐terminal of catfish histone H2A. Parasin I showed strong antimicrobial activity, about 12–100 times more potent than magainin 2, against a wide spectrum of microorganisms, without any hemolytic activity. Circular dichroism spectra of parasin I indicated a structural content of 11% α‐helix, 33% β‐sheet, and 56% random coils. The β‐sheet axial projection diagram of parasin I showed an amphipathic structure. Our results indicate that the catfish may produce parasin I from its histone H2A by a specific protease upon injury to protect against invasion by microorganisms.

A novel antimicrobial peptide from the loach, Misgurnus anguillicaudatus

A novel antimicrobial peptide, named misgurin, was isolated and characterized from the loach (mudfish), Misgurnus anguillicaudatus. The 21‐amino‐acid peptide with a molecular mass of 2502 Da was purified to homogeneity using a heparin‐affinity column and C18 reverse‐phase and gel‐permeation high‐performance liquid chromatography. The complete amino acid sequence of misgurin, which was determined by an automated amino acid sequencer, was Arg–Gln–Arg–Val–Glu–Glu–Leu–Ser–Lys–Phe–Ser–Lys–Lys–Gly–Ala–Ala–Ala–Arg–Arg–Arg–Lys. Misgurin is a strongly basic peptide which has 5 arginine and 4 lysine residues. Comparison of the amino acid sequence with those of other known antimicrobial peptides revealed that misgurin was a novel antimicrobial peptide. Misgurin showed a strong antimicrobial activity in vitro against a broad spectrum of microorganisms without significant hemolytic activity and was about 6 times more potent than magainin 2. Scanning electron microscopy confirmed that the peptide caused damage to the cell membrane by a pore‐forming mechanism similar to that of magainin 2. This damage occurred at the minimal inhibition concentration (MIC), but at higher concentration than MIC it lysed the cell.

Cathepsin D produces antimicrobial peptide parasin I from histone H2A in the skin mucosa of fish.

Parasin I is a potent 19‐residue antimicrobial peptide isolated from the skin mucus of wounded catfish (Parasilurus asotus). Here we describe the mechanism of parasin I production from histone H2A in catfish skin mucosa on epidermal injury. Cathepsin D is found to exist in the mucus as an inactive proenzyme (procathepsin D), and a metalloprotease, induced on injury, cleaves procathepsin D to generate active cathepsin D. This activated form of cathepsin D then cleaves the Ser19‐Arg20 bond of histone H2A to produce parasin I. Immunohistochemical analysis reveals that unacetylated histone H2A, a precursor of parasin I, and procathepsin D are present in the cytoplasm of epithelial mucous cells and that parasin I is produced on the mucosal surface on epidermal injury. Western blot analysis shows that parasin I is also present in the skin mucus of other fish species. Furthermore, parasin I shows good antimicrobial activity against fish‐specific bacterial pathogens. Taken together, these results indicate that cathepsin D and a metalloprotease participate in the production of parasin I from histone H2A and that parasin I contributes to the innate host defense of the fish against invading microorganisms.

Mechanism of anticancer activity of buforin IIb, a histone H2A-derived peptide

Buforin IIb is a novel cell-penetrating anticancer peptide derived from histone H2A. Here we analyzed the anticancer activity and cancer cell-killing mechanism of buforin IIb. Buforin IIb displayed selective cytotoxicity against 62 cancer cell lines by specifically targeting cancer cells through interaction with cell surface gangliosides. It traversed cancer cell membranes without damaging them and accumulated primarily in the nuclei. Once inside the cells, buforin IIb induced mitochondria-dependent apoptosis. In vivo analysis revealed that buforin IIb displayed significant tumor suppression activity in mice with tumor xenograft. Overall, these results suggest that buforin IIb constitutes a novel therapeutic agent for the treatment of cancers.

Structure-activity relations of parasin I, a histone H2A-derived antimicrobial peptide.

The structure-activity relations and mechanism of action of parasin I, a 19-amino acid histone H2A-derived antimicrobial peptide, were investigated. Parasin I formed an amphipathic alpha-helical structure (residues 9-17) flanked by two random coil regions (residues 1-8 and 18-19) in helix-promoting environments. Deletion of the lysine residue at the N-terminal [Pa(2-19)] resulted in loss of antimicrobial activity, but did not affect the alpha-helical content of the peptide. The antimicrobial activity was recovered when the lysine residue was substituted with another basic residue, arginine ([R(1)]Pa), but not with polar, neutral, or acidic residues. Progressive deletions from the C-terminal [Pa(1-17), Pa(1-15)] slightly increased the antimicrobial activity (1-4 microg/ml) without affecting the alpha-helical content of the peptide. However, further deletion [Pa(1-14)] resulted in nearly complete loss of antimicrobial activity and alpha-helical structure. Confocal microscopic analysis and membrane permeabilization assays showed that parasin I and its analogs with comparable antimicrobial activities localized to the cell membrane and subsequently permeabilized the outer and cytoplasmic membranes. Pa(1-14) also localized to the cell membrane, but lost membrane-permeabilizing activity, whereas Pa(2-19) showed poor membrane-binding and -permeabilizing activities. The results indicate that the basic residue at the N-terminal is essential for the membrane-binding activity of parasin I, and among the membrane-binding parasin I analogs, the alpha-helical structure is necessary for the membrane-permeabilizing activity.

Matrix metalloproteinase 2 is involved in the regulation of the antimicrobial peptide parasin I production in catfish skin mucosa

A 19‐residue antimicrobial peptide parasin I is generated from histone H2A in the skin mucus of catfish by the action of cathepsin D activated by a procathepsin D‐processing enzyme induced upon epidermal injury. Here we report the isolation and characterization of the procathepsin D‐processing enzyme in the mucus of wounded catfish. Sequence analysis of the cDNA identified the purified procathepsin D‐processing enzyme as matrix metalloproteinase 2 (MMP 2). By acting as a procathepsin D convertase upon epidermal injury, MMP 2 is involved in the regulation of parasin I production in catfish skin mucosa.

Molecular Cloning and Characterization of an Endoxylanase Gene of Bacillus sp. in Escherichia coli

A gene encoding an endoxylanase of Bacillus sp. was cloned and expressed in Escherichia coli. The entire nucleotide sequence of a 1,620 bp SmaI fragment containing the endoxylanase gene was determined. The endoxylanase gene was 639 bp long and encoded 213 amino acids which showed up to 96% amino acid homology with other endoxylanases. The endoxylanase produced by E. coli harboring pKJX4 was purified by ion-exchange chromatography (DE-52 and CM-52) and its N-terminal sequence was determined to be Ala-Gly-Thr-Asp-Tyr-Trp-Gln-Asn-Trp-Thr-Asp-Gly-Gly-Gly-Thr. The endoxylanase expressed in E. coli was identical to that of the original Bacillus sp. whose molecular weight was approximately 20,400. Most of the produced endoxylanase was localized in the periplasmic space of E. coli. When the endoxylanase was reacted with 2% oat spelts xylan (w/v) at 40 degrees C for 10 h, the major product was xylobiose which is known to be a selective growth stimulant to one of the healthy intestinal microflora, Bifidobacteria.

Effect of the replication mode of a plasmid on the stability of multimeric endoxylanase genes in Bacillus subtilis

Effect of the replication mode of a plasmid on the stability of tandemly multimerized endoxylanase genes and a gene dose-dependent expression of the endoxylanase were studied in Bacillus subtilis. The structural genes encoding an endoxylanase, carrying its original promoter and ribosomal binding sequence, were tandemly multimerized and cloned into the Escherichia coli-B. subtilis shuttle plasmid, pJH27 delta 88 or pMTL500e, which has a rolling circle-replicon or a theta (theta)-replicon in B. subtilis, respectively. The cloned dimers in pJH27 delta 88, which has a rolling circle-replicon, spontaneously rearranged to monomers in B. subtilis DB104, whereas those in pMTL500e, having a theta (theta)-replicon, were stably maintained. Expression level of the endoxylanase was proportional to the gene dosage in multimers. The endoxylanase activity in the supernatant increased from 80 U ml-1 with pMTL-1x containing a monomer of the gene to 165 U ml-1 with pMTL-4x containing a tetramer. These results indicate that high level expression of the endoxylanase gene can be obtained by tandemly multimerizing the genes in a plasmid with a theta (theta)-replicon.

Cathepsin D Produces the Potent Antimicrobial Peptide Parasin I from Histone H2A in Scaleless Fish Skin

Antimicrobial peptides have been known to act as the first line of mucosal host defense by exerting broad-spectrum microbicidal activity against invading pathogenic microbes [1,2]. Recently, a potent 19-residue linear antimicrobial peptide, named parasin I, was isolated from the skin mucus of wounded catfish, Parasilurus asotus [3]. The amino acid sequence of parasin I is KGRGKQGGKVRAKAKTRSS, which is identical to the N-terminal region of rainbow trout histone H2A in 16 of the 19 residues [4]. The very high homology between parasin I and the N-terminal region of histone H2A suggests that parasin I might be cleaved off from histone H2A by a specific protease cleavage similar to the case of buforin I, which is generated from the unacetylated histone H2A in the cytoplasm of gastric gland cells by the action of pepsin C isozymes in the toad stomach [5]. However, the protease that is responsible for the generation of parasin I from histone H2A has not been identified. Furthermore, the findings of Park et al. [3] that parasin I was found only in the skin mucus of wounded catfish and not in unwounded catfish suggests the involvement of an inducible mechanism in parasin I production. In this study, we describe the mechanism for the generation of parasin I from histone H2A in catfish skin mucosa upon epidermal injury. We also assess the biological role of parasin I in the innate host defense of the fish against invading microorganisms.