Moore KS et al. (1991), Antimicrobial peptides in the stomach of Xenopu...

XB-ART-24459

J Biol Chem 1991 Oct 15;26629:19851-7.

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Antimicrobial peptides in the stomach of Xenopus laevis.

Moore KS , Bevins CL , Brasseur MM , Tomassini N , Turner K , Eck H , Zasloff M .

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Antimicrobial peptides are widely distributed in nature and appear to play a role in the host defense of plants and animals. In this study we report the existence of antimicrobial peptides in the stomach of the vertebrate Xenopus laevis, an animal previously shown to store high concentrations of antimicrobial peptides in its skin. Antimicrobial activity was detected in extracts of X. laevis stomach tissue and nine antimicrobial peptides were then purified. A novel 24-amino acid peptide, designated PGQ, was isolated from these extracts, and has the following amino acid sequence: GVLSNVIGYLKKLGTGALNAVLKQ. PGQ is relatively basic and has the potential to form an amphipathic alpha-helix. The other peptides isolated are members of the magainin family of antimicrobial peptides, and include magainins I and II, PGLa, xenopsin precursor fragment, and four caerulein precursor fragments. None of these peptides had been previously identified in tissues other than the skin. The purification of the peptides from stomach extracts and subsequent protein sequence analysis reveals that the peptides have undergone the same processing as their dermal counterparts, and that they are stored in their processed forms. Northern blot analysis indicates that the magainin family of peptides are synthesized in the stomach, and immunohistochemical studies demonstrate that magainin is stored in a novel granular multinucleated cell in the gastric mucosa of Xenopus. This study demonstrates that the magainin family of antimicrobial peptides is found in the gastrointestinal system of X. laevis and offers an opportunity to further define the physiological role of these defense peptides.

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Species referenced: Xenopus laevis
Genes referenced: actl6a levi magainins pgla pgq xt6l

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	FIG. 1. Antimicrobial peptide isolation. P-30 gel filtration chromatogram with overlay of antimicrobial activity. A concentrated acetonitrile/trifluoroacetic acid extract of Xenopus stomach tissue was applied to a Bio-Gel P-30 column and eluted with 0.1% trifluoroacetic acid. An aliquot from each 3-ml fraction was assayed for antimicrobial activity against E. coli D31. Solid line, relative absorbance at 258 nm. Histogram, antimicrobial activity.
	FIG. 2. Antimicrobial peptide isolation. Reversed-phase HPLC chromatogram of fractions from P-30 column. Fractions 27- 36 were pooled and loaded onto a CI8 column and eluted with a linear gradient of acetonitrile in aqueous trifluoroacetic acid. Arrows indicate fractions which were purified and characterized. Solid line, absorbance; dashed line, % acetonitrile.
	FIG. 3. Antimicrobial activity of reversed-phase HPLC fractions. A 2-pl aliquot of each fraction was applied to a freshly poured lawn of E. coli D31. Clear areas indicate lysis of bacteria. 45- 80, HPLC fractions corresponding to those in Fig. 2. C, 100 ng of magainin I1 amide with glycine 18 omitted used as a positive control.
	FIG. 4. Determination of purity by capillary electrophoresis of reverse&phase HPLC fractions. samplweass injected onto a 72-cm capillary with 50 mM sodium phosphate, pH 2.5, at 45 "C, with a 25 kV current. A, fraction 57; B, fraction 61; C, fraction 73; D, fraction 75
	FIG. 5. Reversed-phase HPLC chromatograms of peptides subjected to protein sequence analysis. Sample was loaded onto a CI8 column and eluted with a linear gradient of acetonitrile in aqueous trifluoroacetic acid. Solid line, absorbance; dashed line, percentage acetonitrile. A, fraction 52 + 53, component 1; B, fraction 52 + 53, component 2; C, fraction 65; D, fraction 67; E, fraction 69.
	FIG. 6. Northern blot analysis of Xenopus tissues hybridized with magainin or PGLa probe. A, lane 1,20 pg of skin RNA; lane 2, 20 pg of stomach RNA; lane 3, 20 pg of liver RNA. The magainin cDNA clone is the probe for the top panel. B, lane 1,15 pg of skin RNA; lane 2, 20 pg of stomach RNA, lane 3, 20 pg of liver RNA. The PGLa oligonucleotide is the probe for the top panel. An actin cDNA is the probe for the bottom panel of both A and B. The positions of molecular weight markers (with units of kilobases) are shown on the side.
	FIG. 7. Xenopus stomach tissue which contains antimicrobial peptides. A, immunohistochemical stainingw ith magainin antisera (1:5000 dilution). Arrows indicate immunopositive cells; bur, 100 pm. B, control immunohistochemical staining with magainin immune- depleted sera (1:5000 dilution); bur, 100 pm. C, electron micrograph of granular cell in gastric mucosa. Arrows indicate granules which store the antimicrobial peptidesb;u r, 10 um.