INTRODUCTION

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Located in the crypts of Lieberkühn, Paneth cells synthesize and release proteinaceous granules into the lumen of the small bowel. These secretory granules are rich in antimicrobial peptides and proteins, supporting the hypothesis that these cells contribute to innate immunity of the intestinal mucosa (20). Paneth cell -defensins, also termed cryptdins, are gene-encoded antibiotic peptides expressed by this epithelial lineage, and mouse and human Paneth cells contain high levels of these peptides and their corresponding mRNAs (15, 16, 18). Six cryptdin peptide variants have been purified from mouse small intestine (7, 26), and five of those isoforms, cryptdins 1 to 3, 5, and 6, are coded by separate genes clustered in the proximal region of chromosome 8 (18). Transcripts of cryptdin genes are approximately 1 kb in length and coded by two-exon genes in which the first exon codes for the preprosegment of the precursor and the second exon codes for the mature -defensin peptide (14). Human Paneth cells express two -defensin genes, HD-5 and HD-6 (2, 15, 16), but the mouse -defensin gene family is larger, coding for at least 19 different cryptdin isoforms (18).

At the level of both the peptide and the gene, cryptdin 4 has several features that distinguish it from the known Paneth cell -defensins. For example, the peptide has the highest antimicrobial activity of the known mouse enteric -defensins in in vitro assays (18), and the cryptdin 4 gene has a unique pattern of expression along the longitudinal axis of the small intestine (5). In contrast to cryptdins 1 and 5, whose mRNAs occur at approximately equivalent levels along the length of the mouse small bowel, cryptdin 4 mRNA levels increase along the proximal-to-distal axis to reach maximal concentration in the distal ileum. To test for potential structural differences correlating with differential cryptdin 4 gene activity, DNA sequences up-stream of mouse Paneth cell -defensin gene transcription initiation sites have been analyzed and compared.

Here, we demonstrate that the cryptdin 4 peptide is a Paneth cell granule constituent, and thus is secreted into the lumen, and that peptide distribution corresponds to the increasing cryptdin 4 mRNA concentration along the length of the gut. Also we report on the structure of the 129/SVJ mouse cryptdin 4 gene and show that its two exons are identical in sequence to cryptdin 4 cDNAs from intestinal crypts of C3H/HeJ and outbred Swiss mice. The region just upstream of the cryptdin 4 gene transcriptional initiation site contains a 130-bp repeated element that does not occur in other known mouse -defensin genes.

(This work was presented in preliminary form at the Annual Meeting of the American Gastroenterological Association, Washington, D.C., May 1997.)

	MATERIALS AND METHODS

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Solid-phase synthesis of cryptdin 4. The cryptdin 4 peptide chain was assembled on a Millipore 9050 automated continuous flow peptide synthesizer by sodium 9-fluorenylmethoxycarbonyl methodology (3). Amino acids were activated in situ with N,N-diisopropylcarboxydiimide-N,N-diisopropylethylamine-1-hydroxybenzotriazole chemistry (27), and the coupling yield was monitored on line with quinoline yellow (29). The synthetic peptide was released from the polyethylene glycol-polystyrene resin support by treatment with trifluoroacetic acid-water-phenol-triisopropylsilane (88:5:5:2, reagent B [27]) for 6 h at 20°C and filtered. The peptide solution was adjusted with acetic acid (HOAc) to a final concentration of 30%, extracted three times with equal volumes of dichloromethane, diluted fivefold with H₂O, and lyophilized. The lyophilized peptide was dissolved in 10% HOAc and lyophilized again prior to biochemical analysis.

Preparation of anti-cryptdin 4 antibody. A peptide-specific polyclonal rabbit antibody to the synthetic, folded cryptdin 4 was prepared. A sample (4 mg) of cryptdin 4 conjugated to ovalbumin was used to immunize two New Zealand White rabbits (Zymed Laboratories, Inc., San Francisco, Calif.). Serum samples were collected for 8 to 12 weeks, until the anti-cryptdin 4 titer, determined by enzyme-linked immunosorbent assay, reached 1:10,000 for each rabbit. Immunoglobulin G (IgG) was isolated from antiserum by DEAE Econo-Pac chromatography (Bio-Rad, Richmond, Calif.) as described by the manufacturer. Peptide specificity of the antibody was determined by assessing immunoreactivity against cryptdins 1 to 6 immobilized on polyvinylidene difluoride and nitrocellulose membranes, with the antibody demonstrating reactivity only with cryptdin 4.

Immunohistochemical detection of cryptdin 4. Paraffin sections of formalin-fixed mouse small bowel were deparaffinized with xylenes, treated for 30 min with 0.3% H₂O₂, and washed extensively with water and then with phosphate-buffered saline (PBS). Slides were incubated three times for 5 min each in the microwave oven with antigen unmasking solution (Vector Laboratories, Inc., Burlingame, Calif.) and then cooled in unmasking solution (Vector Laboratories, Inc.) for 30 min at room temperature. After being rinsed with PBS, the sections were blocked by incubation with normal goat serum for 30 min and with Avidin D blocking solution for 15 min, rinsed briefly with PBS, and then incubated with biotin blocking solution (Vector Laboratories, Inc.) for 15 min. Slides were incubated with a 1:50 dilution of rabbit anti-cryptdin 4 IgG, a 1:10 dilution of anti-cryptdin 4 IgG in 1× PBS preabsorbed at 4°C overnight with 80 µg of synthetic, folded cryptdin 4, or with IgG prepared from serum of rabbits prior to immunization. After 30 min, slides were washed three times with PBS, incubated for 30 min with a 1:200 dilution of biotinylated goat anti-rabbit IgG, and washed as before. After 60 min of incubation with Vectastain ABC peroxidase reagent (Vector Laboratories, Inc.), slides were washed and flooded for 3 min with diaminobenzidine as a peroxidase substrate, washed, counterstained with hematoxylin, cleaned, and mounted.

Cryptdin 4 gene isolation. Bacteriophages containing cryptdin 4 gene sequences were identified in a 129/SVJ mouse genomic library in  DASH II (Stratagene Cloning Systems, La Jolla, Calif.). Phage plates (1.1 × 10⁵ PFU/dish) were screened in duplicate by hybridization of filter lifts with oligonucleotide cryp4 (5'-CGGCG GGGGC AGCAG TA-3'), the reverse complement of nucleotides 259 to 275 in cryptdin 4 cDNA, labeled with [-³²P]ATP by using polynucleotide kinase (Promega Corporation, Madison, Wis.). Conditions for hybridization and washing of filters have been described previously (5). Subsequently, a mixed probe for putative cryptdin gene promoter sequences was prepared by amplification of mouse genomic DNA (30 cycles of 94°C for 40 s, 60°C for 40 s, and 72°C for 40 s) with oligonucleotide Defcr_m150 (5'-GCTGC TCCTC AGTAT TAGTC TCTT-3') paired with sense strand primer CIA7 (5'-TAAAT (A/G)(C/T)TCA AGCAG ATGG-3'). Defcr_m150 primes on the antisense strand of all known cryptdin cDNAs near the midpoint of exon 1, and the CIA7 priming site is conserved in several mouse Paneth cell -defensin genes between residues 216 and 234 relative to the transcription start site (13a). This 384-bp PCR product was used to screen Southern blots of genomic clones to identify isolates containing sequences upstream of the transcription start site of this gene.

DNA sequencing and analysis. Denatured plasmid DNAs from subcloned fragments of genomic clones were sequenced by dideoxy nucleotide termination (23) with modified Sequenase II (United States Biochemical Corp., Cleveland, Ohio). Computations for similarity searches of DNA sequences in nonredundant nucleic acid and protein sequence databases were performed at the National Center for Biotechnology Information by using the BLAST network service (1, 12). DNA sequences were analyzed by using programs in the University of Wisconsin Genetics Computer Group suite (6) as well as routines in the MacVector version 4.5.3 program (Eastman Kodak Company, Rochester, N.Y.).

Nucleotide sequence accession numbers. The GenBank accession number for the cryptdin 4 gene sequence (Fig. 4A) is AF178040. Those for the 5' UTRs of cyrptdin 1, cryptdin 5, cryptdin 6, and CRS4C-2 (Fig. 4B) are AF178043, AF178042, AF178041, and AF178044, respectively.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

Analysis of synthetic cryptdin 4. The cryptdin 4 peptide (GLLCY CRKGH CKRGE RVRGT CGIRF LYCCP RR), isolated previously from full-length, full-thickness mouse small intestine (26), is the most potent of the known mouse Paneth cell -defensins (18). Because the overall recovery of cryptdin 4 from intestinal extracts is low relative to that of cryptdins 1, 2, 5, and 6, which are expressed along the entire length of the small bowel, the peptide was synthesized to obtain sufficient quantities of peptide for biochemical analysis and antibody production (see Materials and Methods). The overall peptide yield was estimated at 4.1% with a recovery of 35 mg of RP-HPLC-purified, folded cryptdin 4 recovered from 860 mg of crude peptide. The purified synthetic cryptdin 4 peptide was characterized by mass spectroscopy, amino acid analysis, and analytical RP-HPLC and shown to be identical to the natural cryptdin 4 reference peptide in all respects (data not shown). In acid-urea polyacrylamide gel electrophoresis (AU-PAGE), a system that provides high resolution of the protonated forms of defensins and small basic peptides (21, 25), the synthetic and natural cryptdin 4 peptides had identical mobilities (Fig. 1A). As may be predicted from their identical biochemical properties, synthetic and natural cryptdin 4 were functionally equivalent in assays of antimicrobial activity against Escherichia coli ML35 and Staphylococcus aureus (Fig. 1B). Therefore, this synthetic cryptdin 4 peptide was used for preparation of antiserum to analyze the tissue distribution of cryptdin 4 peptide in Paneth cells along the length of the mouse small intestine.

View larger version (17K): [in this window] [in a new window]   FIG. 1.   Characterization of synthetic cryptdin 4. Panel A, AU-PAGE of natural (lane 1) and synthetic (lane 2) cryptdin 4. Samples (1 µg) of peptide dissolved in 2% acetic acid were electrophoresed for 6 h at 200 V in 12.5% polyacrylamide gels as described previously (25) with migration toward the cathode proceeding from top to bottom. The gel was stained with Coomassie blue as described previously (24). Panel B, antimicrobial activities of natural and synthetic cryptdin 4. The combined bactericidal and bacteriostatic activities of the peptides were evaluated by a radial diffusion assay (see Materials and Methods [18]). The similarity of zones of bacterial growth inhibition obtained with the corresponding natural or synthetic peptides shows that they are functionally equivalent. Symbols: , E. coli exposed to natural cryptdin 4; , E. coli exposed to synthetic cryptdin 4; , S. aureus exposed to natural cryptdin 4; , S. aureus exposed to synthetic cryptdin 4.

Localization of cryptdin 4 in mouse small intestine. In the small bowel, cryptdin 4 gene expression is restricted to Paneth cells, and the highest levels of peptide occur in the distal regions of the organ. Immunoperoxidase staining of full-thickness sections of mouse small intestine with anti-cryptdin 4 IgG failed to identify the peptide in the duodenum (Fig. 2A), showed intermediate levels along the jejunum (Fig. 2B to D), and demonstrated maximal levels in terminal ileum (Fig. 2G). In contrast to these results, Paneth cells in duodenum are strongly immunoreactive with the anti-cryptdin 1 antibody (26). Cryptdin 4 is present in Paneth cells at the base of every ileal crypt (Fig. 2E to G). When sections of ileum stained with the antibody were viewed at higher magnification, the cryptdin 4 antigen was found to be concentrated within Paneth cell secretory granules (Fig. 3A and B). Thus, as with the known enteric -defensins, cryptdin 4 is a constituent of secretory granules and is therefore released into the crypt lumen.

View larger version (122K): [in this window] [in a new window]   FIG. 2.   Immunohistochemical localization of cryptdin 4 peptide in Paneth cells of distal mouse small intestine. Sections of adult mouse duodenum (A), jejunum (B through D), and ileum (E through G) were analyzed for the presence of the cryptdin 4 peptide, by using purified IgG from a polyclonal rabbit cryptdin 4-specific antibody (see Materials and Methods). In each panel, arrows indicate immunoperoxidase localization in Paneth cells of the crypts. Magnification, ×40. IgG prepared from preimmune serum is negative (H). Note that duodenal crypts are negative, jejunal crypts are modestly positive, and ileal crypts are strongly immunoreactive.

View larger version (133K): [in this window] [in a new window]   FIG. 3.   Cryptdin 4 in Paneth cell secretory granules. As described for Fig. 2, sections of adult mouse ileum were incubated with IgG prepared from polyclonal rabbit cryptdin 4-specific antiserum (A and B), with preimmune serum (C), or with immune IgG preadsorbed with 80 µg of synthetic cryptdin 4 (D). Magnification, ×100. In panels A and B, the arrows indicate secretory granules in Paneth cells that are strongly immunoreactive; in panels C and D, arrows indicate eosinophilic granules that are immunoperoxidase negative after exposure to preimmune and preadsorbed IgG controls, respectively.

Cryptdin 4 gene structure. The structural features of the cryptdin 4 gene are conserved relative to those of known mouse and human Paneth cell -defensin genes (Fig. 4 [14-16]). Bacteriophage clones in a 129/SVJ mouse genomic library were identified by hybridization with the cryp4 oligonucleotide (4, 5), and several isolates contained a positive, 2-kb EcoRI fragment that contained the complete coding sequence for the cryptdin 4 precursor (see Materials and Methods). DNA sequencing of those clones showed that the cryptdin 4 gene primary transcript is 987 nucleotides long and contains two exons (Fig. 4A). Exon 1 contains a 45-bp 5' untranslated region and sequence coding for the preprosegment of the precursor; exon 2 codes for the mature cryptdin 4 peptide and contains approximately 100 bp of 3' untranslated sequence (Fig. 4A). The two cryptdin 4 exons of the 129/SVJ mouse are identical to cryptdin 4 cDNA from C3H/HeJ and outbred Swiss mice (18). The sequences upstream of transcriptional start sites of mouse cryptdin genes are conserved (Fig. 4B). A multiple sequence alignment produced by the program Pileup (6) illustrates the extensive sequence similarity between the 5' upstream regions of the cryptdin 1, 4, 5, and 6 genes and also with the defensin-related mouse CRS4C-2 (13) gene (Fig. 4B). Also, cryptdin 4-coding genomic sequences from 129/SVJ (Fig. 4) and C3H/HeJ, BALB/cJ, and outbred Swiss mice are identical (data not shown), as are cryptdin 4 cDNAs from outbred Swiss and several inbred strains of mice (20a).

View larger version (76K): [in this window] [in a new window]   FIG. 4.   Nucleotide sequence and structure of the mouse cryptdin 4 gene. (A) Overlapping fragments of cryptdin 4 genomic clones were sequenced as described above (see Materials and Methods). The transcribed sequence is depicted in uppercase characters; the sequence coding for the deduced cryptdin 4 precursor is underlined; the intron, determined by sequence alignment with cryptdin 4 cDNA (18), is shown in uppercase italics; and untranscribed sequences in the 5' and 3' flanking regions of the gene are shown in lowercase. The deduced amino acid sequence of the cryptdin 4 precursor is shown in uppercase, single-letter notation above the coding regions, with an asterisk denoting the termination codon. The deduced polyadenylation start site is shown as double underlined characters, beginning 21 nucleotides from the transcription termination site. Nucleotide residue positions are numbered with the first nucleotide of the transcription start site assigned residue position +1. (B) Alignment of 5' upstream sequences from mouse -defensin family genes. Sequences corresponding to nucleotide positions 630 to +5 in four mouse cryptdin genes (14) and in the related CRS4C-2 gene (13) were aligned with the UWGCG program Pileup (6) by using a gap weight of 5.00 and a gap length weight of 0.30. Dot characters (.) in lines of sequence were introduced by the program to maximize the alignment score. The cryptdin 4 nucleotide sequence is shown in uppercase type with a tandemly repeated element denoted as shaded text. Residue positions are numbered in relation to the transcriptional start site, designated to be position +1.

Detection of new cryptdin 4 isoform. The mouse cryptdin 1-like -defensin subfamily has undergone gene duplication events to generate many peptide isoforms in Mus musculus (14, 18). However, evidence of cryptdin 4 isoforms has not been found in outbred Swiss or in inbred strains of mice. Studies of cryptdin 4-coding sequences amplified from genomic DNA of wild-derived strains revealed that the Mus czech II/Ei genome codes for the following deduced cryptdin 4 peptide variant: DLTCYCRKGHCKRGGRVRGTCGIRFLYCCPRR (replaced amino acids are indicated by italics), compared to GLLCYCRKGHCKRGERVRGTCGIRFLYCCPRR, the primary structure of cryptdin 4 isolated from M. musculus domesticus. To our knowledge, the M. czech II/Ei cryptdin 4 variant, with D1G, T3L, and G15E amino acid replacements, represents the first evidence of evolutionary diversity of this particular -defensin gene in mice.

Tandemly repeated element specific to the cryptdin 4 gene. Dot matrix alignments of sequences corresponding to 600 bp 5' of the transcriptional start sites of the cryptdin 1 gene and the cryptdin 5 and 6 genes produce continuous diagonal lines, indicative of their 95% nucleotide sequence identities (Fig. 5Aa and Ab). However, a similar comparison of the cryptdin 1 and cryptdin 5 genes with the cryptdin 4 gene sequence showed that the 5' flanking DNA of the cryptdin 4 gene contains a distinctive, tandemly repeated element (Fig. 5Ac and 5d). The alignment of the cryptdin 1 and cryptdin 4 upstream regions becomes discontinuous near nucleotide position 300 (Fig. 5Ac), indicative of a direct repeat in the region upstream of the cryptdin 4 gene transcriptional start site. We infer that the second repeat and the 70 bp adjacent to its 3' end, i.e., nucleotides 270 to 70, have replaced DNA present in other mouse Paneth cell -defensin genes, because the corresponding regions (nucleotides 270 to 70) of the cryptdin 1, 5, and 6 genes lack similarity with the cryptdin 4 repeated element (data not shown). The tandem repeats, located from nucleotides 495 to 365 and 270 to 140 (Fig. 5B) are nearly identical in sequence, except that the element distal to the initiation site has a 3-bp gap (data not shown). The role of this duplicated element and the doubling of certain putative transcription factor binding sites in the regulation of cryptdin 4 gene activity remains to be determined, but to our knowledge, no known -defensin genes contain such a structural feature.

View larger version (42K): [in this window] [in a new window]   FIG. 5.   A repeated sequence element upstream of the cryptdin 4 gene transcriptional start site. (A) DNA sequences in the region from positions +1 to 700 upstream of the cryptdin genes shown were compared by the Pustell dot matrix alignment routine in the MacVector suite (see Materials and Methods). In contrast to comparisons of cryptdins 1 and 6 (a) as well as of cryptdins 1 and 5 (b), which were colinear and nearly identical, alignment comparisons involving the cryptdin 4 gene revealed a discontinuity in the sequence alignment that is indicative of a tandem repeat (c and d). (B) Potential transcription factor recognition sites (10, 11, 28) in the region upstream of the cryptdin 4 transcriptional start site are indicated in uppercase type and identified above lines of nucleotide sequence. The duplicated element described for panel A, from nucleotides 494 to 367 and 269 to 140, is indicated by shaded text. (C) The only region of similarity between the 1.2 kb of nucleotide sequence 5' of the mouse cryptdin 4 and human HD-5 Paneth cell -defensin gene transcription start sites is shown. Nucleotide sequences from the 5' untranscribed regions of cryptdin 4 and HD-5 genes were aligned using the program Bestfit. Pluses between lines of DNA sequence denote identical nucleotide positions. Conserved potential DNA recognition sequences for AP-1 (italics), Oct 1.4 (double underlining), and GCRE/GCN4-HIS (single underlining) transcription factors are indicated.