Alpha-defensins in enteric innate immunity: functional Paneth cell alpha-defensins in mouse colonic lumen

Abstract

Paneth cells are a secretory epithelial lineage that release dense core granules rich in host defense peptides and proteins from the base of small intestinal crypts. Enteric alpha-defensins, termed cryptdins (Crps) in mice, are highly abundant in Paneth cell secretions and inherently resistant to proteolysis. Accordingly, we tested the hypothesis that enteric alpha-defensins of Paneth cell origin persist in a functional state in the mouse large bowel lumen. To test this idea, putative Crps purified from mouse distal colonic lumen were characterized biochemically and assayed in vitro for bactericidal peptide activities. The peptides comigrated with cryptdin control peptides in acid-urea-PAGE and SDS-PAGE, providing identification as putative Crps. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry experiments showed that the molecular masses of the putative alpha-defensins matched those of the six most abundant known Crps, as well as N-terminally truncated forms of each, and that the peptides contain six Cys residues, consistent with identities as alpha-defensins. N-terminal sequencing definitively revealed peptides with N termini corresponding to full-length, (des-Leu)-truncated, and (des-Leu-Arg)-truncated N termini of Crps 1-4 and 6. Crps from mouse large bowel lumen were bactericidal in the low micromolar range. Thus, Paneth cell alpha-defensins secreted into the small intestinal lumen persist as intact and functional forms throughout the intestinal tract, suggesting that the peptides may mediate enteric innate immunity in the colonic lumen, far from their upstream point of secretion in small intestinal crypts.

FIGURE 1.

Isolation of putative α-defensins from mouse colon. Proteins extracted from complete ileum (organ plus luminal contents) of 12 mice (A), complete colon (organ plus luminal contents) of 12 mice (B), colonic tissue of 9 mice (C), and colonic luminal contents of 9 mice (D) were separated by P-60 gel-permeation chromatography (“Experimental Procedures”). Beginning 10 fractions after elution of the void volume, every third fraction was analyzed in AU-PAGE, and gels were stained with Coomassie Blue. The arrows indicate increasing elution time from the P-60 columns. The boxed region denotes either α-defensins (A) or the anticipated position of α-defensin elution from P-60 gel-permeation chromatography columns and rapid migration in AU-PAGE (B–D). Note that no apparent α-defensin peptides were evident in the colonic tissue extract (C). Recombinant Crp4 was loaded in the far left lane in panels A–C.

FIGURE 2.

Colonic α-defensins are structurally intact. α-Defensin preparations from ileum tissue, complete ileum, and complete colon were analyzed by SDS-PAGE under reducing conditions (“Experimental Procedures”) along with pro-Crp4, Crp4, and Crp3 control peptides. Pro-Crp4 has a molecular mass of 8221 Da, and mature, processed α-defensins have molecular masses of ∼3700–4500 Da. 5% of the total defensin preparation generated from protein extracts from four mice separated by gel-permeation chromatography and CM-Sepharose was electrophoresed in the lanes as described. Lanes: M, molecular weight markers; 1, proCrp4; 2, Crp4; 3, Crp3; 4–6, α-defensin preparations from ileal tissue, complete ileum, and complete colon, respectively. Because of their similar sizes, reduced α-defensins migrate with indistinguishable mobilities in SDS-PAGE, as observed. The combined colonic α-defensins migrate as a single band with the same apparent molecular weights as control α-defensins, indicating that intact α-defensins were isolated from the colon.

FIGURE 3.

Primary structures of colonic α-defensins deduced by MALDI-TOF MS. Masses of colonic α-defensins were determined by MALDI-TOF MS (“Experimental Procedures”). Experimental masses were compared with the theoretical masses deduced from previously characterized mouse Paneth cell α-defensin peptide and mRNA sequences and used to deduce peptide primary structures and identities. All masses are given in atomic mass units (A.M.U.). Dash characters were introduced into the alignment of certain colonic α-defensin primary structures to maintain cysteine spacing. The canonical α-defensin disulfide array is depicted for Crp1 above the aligned colonic α-defensins. Residues proximal to the first cysteine are boxed to highlight the variable N termini of colonic α-defensins. A form of each Crp was identified in the colon.

FIGURE 4.

N-terminal sequencing confirms the identities of colonic α-defensins. A, contains the following samples electrophoresed in a non-reducing AU-PAGE gel: Crp3 (lane 1), Crp4 (lane 2), a preparation of α-defensins from complete ileum (lane 3), a preparation of α-defensins from complete colon (lane 4). Individual bands from the complete colon α-defensin preparation in lane 4 are labeled with lowercase letters a–e. Peptide bands were transferred to polyvinylidene difluoride membrane as preparation for 11 cycles of N-terminal sequencing (“Experimental Procedures”) with the resulting deduced peptide identities and primary structures shown in B. Underlined sequence represents amino acid residues determined by sequencing. Because residue position 10 is the first variable amino acid among full-length Crps 1–3 and 6, it is boxed to highlight the identities of individual peptides.

FIGURE 5.

α-Defensins isolated from the complete colon and colonic lumen retain bactericidal activity. Bactericidal peptide assays were performed against L. monocytogenes (A and B), E. coli ML35 (C and D) and S. enterica sv. typhimurium ΔphoP (E and F), surviving bacteria were counted, and data were expressed as percent of no peptide control. A, C, and E symbols: ♦, Crp4; ♢, Crp Mix; ●, α-defensins from complete ileum; ○, α-defensins from complete colon. B, D, and F symbols: ■, Crp3; ●, α-defensins from complete ileum; ○, α-defensins from complete colon; ▼, α-defensins from ileal tissue; ▽, α-defensins from colonic lumen. α-Defensin preparations from the complete colon and colonic luminal contents were bactericidal. Data are representative of multiple experiments.

FIGURE 6.

Differential susceptibility of α-defensins and β-defensins to proteolysis by trypsin and chymotrypsin. Representative mouse α-defensins, including Crp2, Crp3, and Crp4, and β-defensins, hBD-1, hBD-3, and rhBD-4, were incubated without enzyme (−), with trypsin (T), or with chymotrypsin (C). (6 C/A)-Crp4, a peptide lacking the disulfide bonds common to all α-defensins, was included as a susceptible control peptide. Samples representing 85% of each digest were analyzed by AU-PAGE (A), and the remainder of each digest was subjected to MALDI-TOF MS to determine the masses of enzymatic cleavage products larger than 2 kDa. In B, the primary structures of the peptides are aligned with the canonical cysteine positions boxed in gray. Trypsin cleavage sites deduced from the MALDI-TOF MS analysis are shown with filled arrows, and chymotrypsin cleavage sites are shown with open arrows.