title: Impaired luminal processing of human defensin-5 in Crohn's disease: persistence in a complex with chymotrypsinogen and trypsin.
abstract: Human defensin (HD)-5 is an antimicrobial peptide expressed in small intestinal Paneth cells, and alterations in HD-5 expression may be important in Crohn's disease (CD) pathogenesis. Levels of HD-5 in Paneth cells and ileostomy fluid from control and CD patients were studied by quantitative immunodot analysis, immunohistochemistry, acid urea-polyacrylamide gel electrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis Western blotting, reverse phase-high performance liquid chromatography, N-terminal amino acid sequencing, and ES-QToF mass spectrometry. In both control and CD patients, HD-5 in Paneth cell extracts was present almost exclusively in the precursor form. HD-5 levels in ileostomy fluid were lower in CD patients (n = 51) than in controls (n = 20): median (range), 7.9 (5.5 to 35.0) microg/ml versus 10.5 (6.0 to 30.4) microg/ml; P = 0.05; this difference was most marked in CD patients with homozygous/compound heterozygous mutations in NOD2 (P = 0.03). In control ileostomy fluid, HD-5 was present in the mature form only. In contrast, CD patient ileostomy fluid contained both precursor and mature forms of HD-5, with the majority present in a complex with trypsin, chymotrypsinogen/chymotrypsin, and alpha1-anti-trypsin. Pro-HD-5 was not associated with trypsin or chymotrypsinogen in Paneth cell extracts. In conclusion, pro-HD-5 in the intestinal lumen is processed by trypsin in a complex in which chymotrypsinogen is also cleaved for activation. The persistence of this complex in CD may be attributable to increased luminal levels of proteinase inhibitors such as alpha1-anti-trypsin.
authors.affiliation: Institute of Infection, Immunity, and Inflammation, University of Nottingham, Nottingham, UK.
authors.forename: David
authors.initials: D
authors.lastname: Elphick
authors.forename: Susan
authors.initials: S
authors.lastname: Liddell
authors.forename: Yashwant R
authors.initials: YR
authors.lastname: Mahida
mesh_terms: D000328:Adult; D000595:Amino Acid Sequence; D001826:Body Fluids / Q000737:chemistry / Q000378:metabolism; D016022:Case-Control Studies; D002851:Chromatography, High Pressure Liquid; D002919:Chymotrypsinogen / Q000378:metabolism*; D003424:Crohn Disease / Q000378:metabolism*; D023082:Defensins / Q000378:metabolism* / Q000493:pharmacokinetics; D005260:Female; D006801:Humans; D007081:Ileostomy; D007413:Intestinal Mucosa / Q000378:metabolism*; D007421:Intestine, Small / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D008954:Models, Biological; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000378:metabolism; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D014020:Tissue Extracts / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism*; D000515:alpha 1-Antitrypsin / Q000378:metabolism; D023084:alpha-Defensins / Q000378:metabolism* / Q000493:pharmacokinetics
chemical_list: C500225:DEFA5 protein, human; D023082:Defensins; D046912:Multiprotein Complexes; D011498:Protein Precursors; D014020:Tissue Extracts; D000515:alpha 1-Antitrypsin; D023084:alpha-Defensins; D002919:Chymotrypsinogen; D014357:Trypsin