Activation of Paneth cell alpha-defensins in mouse small intestine.
Paneth cells in small intestine crypts secrete microbicidal alpha-defensins, termed cryptdins, as components of enteric innate immunity. The bactericidal activity of cryptdins requires proteolytic activation of precursors by matrix metalloproteinase-7 (MMP-7; matrilysin) (Wilson, C. L., Ouellette, A. J., Satchell, D. P., Ayabe, T., Lopez-Boado, Y. S., Stratman, J. L., Hultgren, S. J., Matrisian, L. M., and Parks, W. C. (1999) Science 286, 113-117). Here, we report on the intracellular processing of cryptdin proforms in mouse Paneth cells. Peptide sequencing of MMP-7 digests of purified natural procryptdins identified conserved cleavage sites in the proregion between Ser(43) and Val(44) as well as at the cryptdin peptide N terminus between Ser(58) and Leu(59). Immunostaining co-localized precursor prosegments and mature cryptdin peptides to Paneth cell granules, providing evidence of their secretion. Extensive MMP-7-dependent procryptdin processing occurs in Paneth cells, as shown by Western blot analyses of intestinal crypt proteins and proteins from granule-enriched subcellular fractions. The addition of soluble prosegments to in vitro antimicrobial peptide assays inhibited the bactericidal activities of cryptdin-3 and -4 in trans, suggesting possible cytoprotective effects by prosegments prior to secretion. Levels of activated cryptdins were normal in small bowel of germ-free mice and in sterile implants of fetal mouse small intestine grown subcutaneously. Thus, the initiation of procryptdin processing by MMP-7 does not require direct bacterial exposure, and the basal MMP-7 content of germ-free Paneth cells is sufficient to process and activate alpha-defensin precursors. MMP-7-dependent procryptdin activation in vivo provides mouse Paneth cells with functional peptides for apical secretion into the small intestine lumen.
Ayabe
Tokiyoshi
T
Department of Pathology, College of Medicine, University of California, Irvine, California 92697-4800, USA.
Satchell
Donald P
DP
Pesendorfer
Patrizia
P
Tanabe
Hiroki
H
Wilson
Carole L
CL
Hagen
Susan J
SJ
Ouellette
Andre J
AJ
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D023082:Defensins; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D007150:Immunohistochemistry; D007421:Intestine, Small / Q000378:metabolism*; D007930:Leucine / Q000737:chemistry; D020783:Matrix Metalloproteinase 7 / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D019879:Paneth Cells / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014633:Valine / Q000737:chemistry; D023084:alpha-Defensins / Q000378:metabolism*
D018076:DNA, Complementary; D023082:Defensins; D011506:Proteins; D023084:alpha-Defensins; C107642:cryptdin 3; C401847:cryptdin 4, mouse; D012694:Serine; D020783:Matrix Metalloproteinase 7; D007930:Leucine; D014633:Valine