Germ-free and Colonized Mice Generate the Same Products from Enteric Prodefensins

• Published in: The Journal of biological chemistry Vol. 275; no. 51; pp. 40478 - 40482
• Main Authors: Pütsep, Katrin, Axelsson, Lars-Göran, Boman, Anita, Midtvedt, Tore, Normark, Staffan, Boman, Hans G., Andersson, Mats
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 22.12.2000; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Animals; Anti-Bacterial Agents - chemistry; Anti-Bacterial Agents - metabolism; Chromatography, High Pressure Liquid; Defensins - chemistry; Defensins - metabolism; Germ-Free Life; Intestine, Small - metabolism; Intestine, Small - microbiology; Medicin och hälsovetenskap; Mice; Peptide Fragments - chemistry; Peptide Fragments - metabolism; Protein Precursors - chemistry; Protein Precursors - metabolism; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M007816200

The use of germ-free mice offers the possibility to study antibacterial components in a gut uncolonized by bacteria. We have developed a method to extract and high pressure liquid chromatography-fractionate the antibacterial factors present in the small intestine of a single mouse. By mass spectrometry and sequence analyses of fractions exhibiting antimicrobial activity, we identified and characterized the defensin region in germ-free mice as well as in colonized mice. Defensins made up around 15% of the total antibacterial activity both in germ-free and colonized mice. The intestine of germ-free mice exhibited the same set of mature enteric defensins (defensins 1, 2, 3, 4, and 6) as mice colonized by a normal microflora. Mature defensins are generated through processing of larger precursors by enzymatic removal of a signal peptide and a propiece. We found that all prodefensins were cleaved at a Ser/Ala-Leu bond, giving 34-residue propiece peptides and only trace amounts of the predicted 39-residue peptide. This first step must be followed by the removal of a residual peptide to render the mature defensins, indicating that the processing is more complex than previously anticipated. The same propieces were found in both germ-free and colonized mice, suggesting that the same processing operates independent of bacterial presence in the intestine.