Host defense functions of proteolytically processed and parent (unprocessed) cathelicidins of rabbit granulocytes

Abstract

Members of the cathelicidin family are present in all mammals studied. Generally, these proteins contain a conserved N-terminal domain and a structurally and functionally divergent C-terminal region that expresses antibacterial or other activities when proteolytically released. Rabbit granulocytes produce CAP18, a cathelicidin that conforms to this structural and functional organization, and also 15-kDa protein isoforms (p15s) that share several key structural features with other cathelicidins but apparently do not undergo processing with release of an active peptide. To further define the importance of proteolysis in the antibacterial activities of these proteins, we have purified from granulocytes proCAP18, its C-terminal peptide (CAP18p), and two p15 isoforms to apparent homogeneity. Of these four polypeptides, only CAP18p was independently cytotoxic to encapsulated Escherichia coli (90% inhibitory concentration, approximately 600 nM) but it was approximately 50-fold less potent on a molar basis than the bactericidal/permeability-increasing protein (BPI). However, all four cathelicidin species, notably including proCAP18, exhibited antibacterial synergy with BPI, and the p15s also displayed synergy with CAP18p in the absence of BPI. Subnanomolar concentrations of proCAP18 blocked lipopolysaccharide-induced chemiluminescence of human leukocytes, showing a molar potency more than 100-fold greater than that of CAP18p ( approximately 20 nM) or BPI ( approximately 50 nM). Thus, while independent bactericidal activity of cathelicidins requires processing, other host-defense functions do not and are more potently expressed by the unprocessed protein than by the C-terminal peptide.

FIG. 1.

Purification of rabbit cathelicidins. (A) FPLC separation of cathelicidins by employing an SP-Sepharose column and a complex gradient of NaCl (solid line). (B) FPLC fractions were trichloroacetic acid (TCA) precipitated, resolved on gradient SDS-PAGE PhastGEL, and stained with Coomassie blue. Molecular mass standards (in kDa) are indicated on the left edge of the gels. Immunoblots of TCA-precipitated FPLC fractions were probed for BPI (C), CAP18 forms (D), and p15s (E). (F) Further resolution of proteins from FPLC fractions was accomplished by RP-HPLC with a C₄ column and a complex acetonitrile gradient, as shown by the solid line. The figure is a composite of individual chromatograms of each purified protein run separately and detected at 214 nm (with background subtracted). The most hydrophilic protein, p15A, is the first to elute (heavy solid line), followed by p15B (gray solid line), CAP18p (dotted line), and proCAP18 (light solid line). (G) Immunoblots of proCAP18 and p15 isoforms (1 μg each) probed with antiserum against CAP18p.

FIG. 2.

Antimicrobial activities of purified cathelicidins against E. coli K1/r in isotonic media. Independent antimicrobial activity of rabbit cathelicidins and BPI against E. coli K1/r(pCGLS1) at a concentration of 1 × 10⁶/ml was determined as described in Materials and Methods. Points represent the means of 3 to 20 independent determinations. Bioluminescence of bacterial ATP-dependent luciferase was measured as described in the text after 2 h of incubation. The luminescence of untreated growing control bacteria is 100%, and the antimicrobial effect is the percentage of control light counts remaining after 2 h of incubation.

FIG. 3.

Cathelicidins synergize with BPI. E. coli K1/r(pCGLS1) was incubated alone (100%) or with dilutions of BPI in the presence of different doses of p15A (A), p15B (B), proCAP18 (C), and CAP18p (D). Data represent the means of 3 to 6 independent experiments.

FIG. 4.

Synergy between CAP18p and p15s. E. coli K1/r(pCGLS1) was incubated alone (100%) or with dilutions of CAP18p in the presence of increasing doses of p15A (A) and p15B (B). Data represent the means of three to six independent experiments.

FIG. 5.

Inhibition of LPS by rabbit cathelicidins and BPI. (A) LPS stimulates chemiluminescence in purified primary human leukocytes in a dose-dependent fashion in the presence of 10 nM LBP and lucigenin. (B) To determine LPS-neutralizing activity, BPI (closed squares), p15A (open triangles), p15B (open circles), CAP18p (open diamonds), or proCAP18 (closed diamonds) was added to leukocyte suspensions before the addition of 0.1 ng of E. coli J5 LPS/ml. Data presented represent the means ± standard errors of the means for 10 to 20 determinations (A) or the means of 4 to 8 determinations (B).