Detection of Anionic Antimicrobial Peptides in Ovine Bronchoalveolar Lavage Fluid and Respiratory Epithelium
- Respiratory and Neurologic Disease Research Unit, National Animal Disease Center, Agricultural Research Service, U.S. Department of Agriculture, Ames, Iowa 500101 ;
- Department of Veterinary Pathology, Iowa State University, Ames, Iowa 500112 ; and
- Joint Program in Neonatology, Department of Medicine, Boston Children’s Hospital, Boston, Massachusetts 021153
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Fig. 1.The competitive ELISA was used to assess the specificity of monoclonal antibody 1G9-1C2. BSA-DDDDDDD-OH conjugate (50 ng of conjugate/well) was used as the adsorbed antigen, and varying graded concentrations (1.0 to 0.002 mM) of H-DDDDDDD-OH (peptide 1), H-GADDDDD-OH (peptide 2), H-TQDDGGK-OH (peptide 3), H-GGEEK-OH (peptide 4), H-VDDDDK-OH (peptide 5), and H-SGSGSGS-OH (peptide 6) were used as the competitive antigens in the presence of antibody 1G9-1C2 (1.0 μg/ml). Antibody 1G9-1C2 was specific and recognized H-GADDDDD-OH nearly as well as H-DDDDDDD-OH. However, the sequences with internal Asp or Glu residues (e.g., H-VDDDDK-OH, H-TQDDGGK-OH, and H-GGEEK-OH) were not recognized and did not cross-react. H-SGSGSGS-OH was a negative-control peptide.
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Fig. 2.The epitope binding sites of antibody 1G9-1C2 (25 μg/ml) were determined with 30 peptides (7 residues each) corresponding to a single-residue frameshift of the 7-residue sequence starting at residue 30 of the sequence identified in AP 87-8 of Fig. 3. For example, peptide 1 was H-DDGDDDG-OH, peptide 2 was H-DGDDDGA-OH, peptide 3 was H-GDDDGAD-OH, etc. Peptides were synthesized simultaneously on a derivatized cellulose sheet (SPOTs; Genosys). SPOT 32 contained H-DDDDDDD-OH and SPOT 33 contained H-EEEEEEE-OH as peptide controls. SPOTs were detected after the strip was incubated in casein-based blocking buffer, incubated in antibody, washed, incubated in secondary β-galactosidase-conjugated antibody, and incubated in Signal Development solution. Epitopes that contained more than 2 terminal Asp residues were recognized.
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Fig. 3.Nucleotide and predicted peptide sequences for candidate AP precursor genes, comprising exons from sheep genomic library clones. The predicted ORF for AP 87-8 extends in the 5′ direction. ORF sequences are capitalized, and potential AP coding sequences are in boldface.
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Fig. 4.RP-HPLC chromatograms of ovine BAL fluid (A) and ovine epithelial-cell extracts (B), showing peak fraction 5, eluted in 13 to 14% acetonitrile, with antimicrobial activity and evidence of AP by competitive ELISA with antibody 1G9-1C2. BSA-DDDDDDD-OH conjugate (50 ng of conjugate/well) was used as the adsorbed antigen, and RP-HPLC fractions were used as the competitive antigens in the presence of antibody 1G9-1C2 (1.0 μg/ml). To confirm the presence of AP, fractions were examined by MALDI mass spectrometry. H-DDDDDDD-OH [(M + H)+; 824.4 Da], H-GDDDDDD-OH [(M + H)+; 766.1 Da], and H-GADDDDD-OH [(M + H)+; 718.24 Da] were detected.
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Fig. 5.Western blot of ovine tracheal epithelium and lung homogenate. Lane 1, protein ladder (10 to 200 kDa) standard; lane 2, tracheal-cell homogenate stained with Coomassie blue; lane 3, tracheal-cell homogenate probed with antibody PAB96-1; lane 4, lung homogenate probed with antibody PAB96-1; lane 5, lung homogenate stained with Coomassie blue; lane 6, protein ladder standard. In tracheal epithelial cells, antibody PAB96-1 identified four bands with molecular masses of 53.7, 31.2, 28.0, and 25.7 kDa. In the lung, antibody PAB96-1 identified five bands with molecular masses of 53.5, 37.1, 31.2, 28.0, and 25.7 kDa. Identical results were seen with antibody 1G9-1C2 (data not shown). As controls, strips of membrane containing separated tracheal and lung proteins were incubated with preimmunized rabbit serum (1:200), preimmunized mouse serum (1:200), or peroxidase-labeled goat anti-rabbit IgG (0.5 μg/ml) or goat anti-mouse IgG (0.5 μg/ml). No reactions were seen (data not shown). In addition, preincubation of antibody PAB96-1 (5 μg/ml) or 1G9-1C2 (5 μg/ml) with 1.0 mM H-DDDDDDD-OH eliminated specific staining of all bands (data not shown). Blots were photographed (digital camera RD-175; Minolta), and the sizes of the reactive bands were determined (GelCompar, v. 4.0; Applied Maths) by extrapolation from a standard curve of the sizes of the stained bands on the protein ladder.
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Fig. 6.Tissue sections of ovine lung, incubated with antibody 1G9-1C2, show immunocytochemically stained regions of the alveoli (A) and bronchial epithelium (B) that were not seen in these regions when tissue sections were incubated in secondary antiserum alone (data not shown). Antibody 1G9-1C2 identified accumulated protein (arrows) in the cytoplasm of pulmonary endothelial cells and an occasional alveolar macrophage (A) and in the apical cytoplasm of the bronchial and bronchiolar epithelia (B). Goblet cells and epithelial cells of pulmonary alveoli were not stained.
- Copyright © 1998 American Society for Microbiology
