Paneth cell alpha-defensins from rhesus macaque small intestine

Abstract

Antimicrobial peptides are secreted by small intestinal Paneth cells as components of innate immunity. To investigate the role of alpha-defensins in enteric host defenses in nonhuman primates, alpha-defensin cDNAs were isolated, alpha-defensin peptides were purified from rhesus macaque small bowel, and the bactericidal activities of the peptides were measured. Six rhesus enteric alpha-defensin (RED) cDNAs, RED-1 to RED-6, were identified in a jejunum cDNA library; the deduced RED peptides exhibited extensive diversity relative to the primary structures of rhesus myeloid alpha-defensins. RED-4 was purified from monkey jejunum, and N-terminal peptide sequencing of putative RED-4 peptides identified two N termini, RTCYCRTGR. and TCYCRTGRC.; these corresponded to alternative N termini for the RED-4 molecules, as deduced from their molecular masses and RED cDNAs. In situ hybridization experiments localized RED mRNAs exclusively to small intestinal Paneth cells. Recombinant RED-1 to RED-4 were purified to homogeneity and shown to be microbicidal in the low micromolar range (</=10 micro g/ml) against gram-positive and gram-negative bacteria, with individual peptides exhibiting variable target cell specificities. Thus, compared to myeloid alpha-defensins from rhesus macaques, enteric alpha-defensin peptides are highly variable in both primary structure and activity. These studies should facilitate further analyses of the role of alpha-defensins in primate enteric immunity.

FIG. 1.

Alignment of RED cDNA sequences. α-Defensin clones from a rhesus macaque small bowel cDNA library were identified by hybridization with an amplified HD5 cDNA probe and sequenced (see Materials and Methods). Six obtained sequences were aligned with CLUSTALW. Dots in aligned sequences denote identities with RED-6; dashes denote spaces introduced to optimize the alignment.

FIG. 2.

Alignment of deduced RED-1 to RED-6 precursor proteins. RED precursor sequences were predicted from corresponding cDNAs (Fig. 1). N termini of RED-1 to RED-6 were deduced from N termini identified for the natural RED-4 peptide (see Fig. 3), which is conserved in the six deduced pro-RED sequences. Shaded residues denote conserved arginines that may serve as canonical trypsin-processing sites (8). Signal peptides and prosegments are highly conserved, and mature RED peptides show extensive variations between sequences.

FIG. 3.

Alignment of myeloid and enteric α-defensins from rhesus macaques. The primary structures of RMADs and REDs are shown aligned with human myeloid α-defensin HNP-1 and human Paneth cell α-defensin HD5. RMAD-1 to RMAD-3 and RMAD-8 residue positions that differ from those of HNP-1 are shaded. Similarly, RMAD-4 to RMAD-7 and RED residue positions that differ from those of HD5 also are shaded. N-terminal residues within parentheses represent alternative N termini determined here and by Selsted et al. (29). Note that RED peptides are markedly more varied than RMAD peptides.

FIG. 4.

Localization of RED mRNAs in Paneth cells of rhesus macaque jejunum. Sections of monkey jejunum were hybridized to a RED-4 prosegment cDNA probe as described in Materials and Methods. (A and B) Hybridization with anti-sense-strand and sense-strand riboprobes, respectively, shown at a magnification of ×95. Arrows indicate the base of jejunum crypts occupied by Paneth cells. Note the absence of hybridization with the negative control sense-strand probe in panel B. (C) Like panel A but shown at a magnification of ×380. Arrows indicate strongly positive individual Paneth cells.

FIG. 5.

Recombinant RED peptides. (A) Samples (1 μg) of purified recombinant RED-1 to RED-4 were separated by acid urea-PAGE and stained with Coomassie blue. Lanes: 1, RED-1; 2, RED-2; 3, RED-3; 4, RED-4; 5, Crp4. (B) Peptide samples (4 μg) were analyzed separately on an analytical C₁₈ RP HPLC column and eluted with a gradient of 0 to 40% acetonitrile for 60 min. Numerals 1 to 4 above the peaks of the tracings denote the relative elution positions for RED-1 to RED-4, respectively.

FIG. 6.

Biochemical features of recombinant RED and RMAD peptides. Recombinant RED and RMAD amino acid sequences and their theoretical (theor.) molecular weights (M.W.) are shown along with their experimental M.W. observed by MALDI-TOF MS. Net charges were calculated from the following values: Arg, +1; Lys, +1; His, +0.5; Asp, −1; and Glu, −1.

FIG. 7.

Bactericidal peptide activities of recombinant RED peptides. Individual peptides at the concentrations shown were incubated with 10⁶ CFU of log-phase bacterial cells/ml in 50 μl for 1 h and then plated on semisolid media (see Materials and Methods). Surviving bacterial cells were determined as bacterial CFU by counting after overnight growth. The results shown are representative of duplicate or triplicate experiments performed on separate days, and individual data points denote single determinations. (A) V. cholerae. (B) E. coli. (C) L. monocytogenes. (D) S. aureus. Symbols: •, RED-1; ○, RED-2; ▾, RED-3; ▿, RED-4; ▪, Crp4.

FIG. 8.

Bactericidal peptide activities of recombinant RMAD peptides. Peptides were assayed for bactericidal activities as described in the legend to Fig. 7. Again, the results shown are representative of duplicate or triplicate experiments performed on separate days, and individual data points denote single determinations. (A) V. cholerae. (B) E. coli. (C) L. monocytogenes. (D) S. aureus. Symbols: •, RMAD-3; ○, RMAD-4; ▾, RED-4.