1. Positional specificity of defensin gene expression reveals Paneth cell heterogeneity in mouse small intestine
D Darmoul, A J Ouellette Am J Physiol. 1996 Jul;271(1 Pt 1):G68-74. doi: 10.1152/ajpgi.1996.271.1.G68.
Cryptdins are antimicrobial peptides of the defensin family that are expressed specifically by Paneth cells in small intestinal crypts (M.E. Selsted, S.I. Miller, A.H. Henschen, and A.J. Ouellette. J. Cell Biol. 118: 929-936, 1992), and at least 17 cryptdin isoforms have been reported in mouse small intestine (A.J. Ouellette, M.M. Hsieh, M.T. Nosek, D.F. Cano-Gauci, K.M. Huttner, R.N. Buick, and M.E. Selsted. Infect. Immun. 62: 5040-5047, 1994). Analysis of cryptdin gene expression in adult mouse small bowel revealed that the cryptdin-4 isoform is differentially expressed along the proximal-to-distal intestinal axis. By peptide-specific reverse transcriptase-polymerase chain reaction-based assays, cryptdin-4 mRNA was found to be absent from the proximal small bowel, increasing to maximal levels in the ileum. In contrast, intestinal content of cryptdin-1 and -5 mRNAs was equivalent in duodenum, jejunum, and ileum, and Northern blot hybridization experiments were consistent with both sets of data. Similarly, individual crypts isolated from duodenum contain cryptdin-1 mRNA but not cryptdin-4 mRNA. Taken together, the results show that Paneth cells are heterogeneous, depending on their position along the longitudinal axis of the small bowel. The positional specificity of defensin gene expression suggests that cryptdins may be useful markers for investigating the establishment and maintenance of this epithelial lineage in the mouse small intestine.
2. A novel mouse gene family coding for cationic, cysteine-rich peptides. Regulation in small intestine and cells of myeloid origin
A J Ouellette, J C Lualdi J Biol Chem. 1990 Jun 15;265(17):9831-7.
Cryptdin is a Paneth cell corticostatin/defensin in the mouse small bowel. To help define the intestinal role of cryptdin, cryptdin-related sequence (CRS) mRNAs have been characterized with respect to developmental regulation, sequence homology, putative coding function, and occurrence in myeloid cells. Cryptdin, CRS1C, and CRS4C mRNAs are transcribed from separate genes, occur at equivalent abundance in small intestine, and appear in the small bowel in concert during the 2nd and 3rd weeks postpartum. Cryptdin and CRS1C mRNAs are not detectable in adult mouse bone marrow, but probes specific for the 5'- or the 3'-untranslated regions of CRS4C mRNA hybridize to a moderately abundant 1.05-kilobase bone marrow mRNA in contrast to a highly abundant 0.75-kilobase mRNA in small intestine. Nucleotide sequences corresponding to the deduced prepro-coding regions of cryptdin, CRS1C, and CRS4C mRNAs contain a highly conserved 200-base pair region of 92% sequence similarity (CSE.2), but the mRNAs are not homologous otherwise. The deduced CRS1C and CRS4C polypeptides are apparent precursors of secreted, cationic, proline- and cysteine-rich peptides that contain Cys-Pro-X repeats. Unlike cryptdin, however, the proposed CRS1C and CRS4C mature peptide regions lack the structural motif characteristic of defensins. Attempts to find homologies between the putative CRS peptides and existing protein sequences have been unsuccessful, leading us to speculate that CRS1C and CRS4C represent a new family of nondefensin antimicrobial peptides in the mouse small bowel.
3. A family of defensin-like genes codes for diverse cysteine-rich peptides in mouse Paneth cells
K M Huttner, A J Ouellette Genomics. 1994 Nov 1;24(1):99-109. doi: 10.1006/geno.1994.1586.
Cryptdins constitute a diverse population of defensins in Paneth cells of intestinal crypts. In mice, certain intestinal mRNAs, termed "CRSIC" and "CRS4C," are considered to be cryptdin-related sequences, because their prepro-coding sequences are 94% identical to those of cryptdin-1 mRNA; however, their predicted products, which are cationic, cysteine-rich peptides are not defensins (A. J. Ouellette and J. C. Lualdi, J. Biol. Chem. 265: 9831-9837, 1990). Here we describe several mouse small intestinal mRNAs and genes that code for CRS4C prepropeptides. The 10-kDa deduced CRS4C proteins consist of a prepro sequence, potential monobasic or dibasic peptide cleavage sites, a predicted 3.7-kDa peptide that contains 7 [C]-[X]-[Y] repeats, and a C(N/K)CNPK carboxyl-terminal consensus sequence. In situ hybridization experiments showed that CRS4C mRNAs are found in Paneth cells of adult small bowel. The CRS4C genes closely resemble cryptdin genes, having a two-exon structure with highly conserved transcription start sites, intron-exon junctions, and a single intron of approximately 550 bp. Like the cryptdin genes, exon 1 of CRS4C genes consists of 5' untranslated sequences (UTS) and the prepro-coding region, and exon 2 codes for the predicted mature peptide and 3' UTS. Despite the similarity of first exons in CRS4C and cryptdin genes, their introns exhibit very little homology, and their second exons code for unrelated peptides. Analysis of introns suggests that the ancestral cryptdin and CRS4C genes may have diverged from a common ancestor in the distant past and expanded only recently. We speculate that the cryptdin/CRS genes evolved so that prepro regions encoded by exon 1 were conserved to allow the varied peptides coded by exon 2 to be directed into Paneth cell secretory granules.
