Defensin related cryptdin

The modification of galactose with α1,2-fucose is involved in symbiosis with intestinal bacteria and elimination of pathogenic bacteria. It is postulated that α1,2-fucosylated mucin secreted from goblet cells is involved in defending an organism against infections, but the detailed molecular mechanisms are yet to be elucidated. It was previously reported that Paneth cells of the small intestine were positive for UEA-1 lectin staining. However, glycoproteins in Paneth cells carrying α1,2-fucose have not yet been identified. Glycoproteomic analysis of ileal lysates identified 3212 O-linked and 2962 N-linked glycopeptides. In particular, cryptdin-related sequence 1 (CRS1) expressed in Paneth cells was found to be α1,2-fucosylated. Unlike other antimicrobial α-defensin proteins, CRS1 contains unique Thr residues, which are modified with O-glycans, with 3HexNAc2Hex1Fuc1NeuAc being the main glycoform. Identification of α1,2-fucose on the O-glycans of CRS1 expressed in Paneth cells will pave the way for a mechanistic understanding of α1,2-fucose-dependent symbiosis with intestinal bacteria and elimination of pathogenic bacteria in the intestine.

The site of defensin-related CRS1C gene expression in mouse small bowel and the chromosomal location of the CRS1C locus, Defcr-rs1, have been determined. CRS1C (cryptdin-related sequence 1C) mRNA is an abundant small intestinal sequence that exhibits extensive similarity to the prepro-coding regions of defensin mRNAs yet does not encode a defensin (A. J. Ouellette and J. C. Lualdi, 1990, J. Biol. Chem. 265: 9831-9837). Using sequence-specific probes, CRS1C mRNA was detected in Paneth cells at the base of intestinal crypts by in situ hybridization. Southern blot analysis of genomic DNAs from inbred and recombinant inbred (RI) mouse strains, also conducted with probes specific for CRS1C, showed that the CRS1C locus maps to the proximal region of Chromosome 8. In 62 RI strains, no discordancies were found between Defcr-rs1 and Defcr, the cryptdin gene. Thus, both the Defcr-rs1 and the Defcr genes are expressed in Paneth cells and both are genetically inseparable within 1.58 cM on Chromosome 8. These studies identify a second defensin-related Paneth cell gene in mice.

Cryptdin mRNA codes for the apparent precursor to a corticostatin/defensin-related peptide that accumulates to high levels in mouse intestinal crypt epithelium during postnatal development. The primary structure, intestinal cell distribution, and developmental appearance of cryptdin mRNA have been determined. Cryptdin mRNA is 450-480 nucleotides long. Translation of the partial cryptdin cDNA sequence reveals a 70-amino acid open reading frame that includes 32 carboxy-terminal residues that align with those in the consensus sequence, C.CR...C....ER..G.C....CCR, which is a common feature of leukocyte defensins and lung corticostatins (Selsted, M. E., D. M. Brown, R. J. DeLange, S. S. L. Harwig, and R. I. Lehrer. 1985. J. Biol. Chem. 260:4579-4584; Zhu, Q., J. Hu, S. Mulay, F. Esch, S. Shimasaki, and S. Solomon. 1988. Proc. Natl. Acad. Sci. USA. 85:592-596). In situ hybridization of cryptdin cDNA to paraformaldehyde-fixed, frozen sections of adult jejunum and ileum showed intense and specific labeling of epithelial cells in the base of all crypts. Analysis of sections from suckling mice showed that cryptdin mRNA is detectable in 10-20% of crypts in 10-d-old mice, in approximately 80% of crypts in 16-d-old mice, and in all crypts of mice 20 d and older. During the fourth week, the sequence accumulates in crypts to the maximal adult level. Cryptdin mRNA content in adult small intestine is independent both of T cell involvement and luminal bacteria. The role of cryptdin in small bowel physiology remains to be determined: cryptdin may inhibit bacterial translocation, modulate intestinal hormone synthesis, influence hormonal sensitivity of the intestinal epithelium, or exhibit a multiplicity of related activities.