Engineering disulfide bridges to dissect antimicrobial and chemotactic activities of human beta-defensin 3 - PubMed (original) (raw)

Engineering disulfide bridges to dissect antimicrobial and chemotactic activities of human beta-defensin 3

Zhibin Wu et al. Proc Natl Acad Sci U S A. 2003.

Abstract

Human defensins form a family of small, cationic, and Cys-rich antimicrobial proteins that play important roles in innate immunity against invading microbes. They also function as effective immune modulators in adaptive immunity by selectively chemoattracting T lymphocytes and immature dendritic cells. On the basis of sequence homology and the connectivity of six conserved Cys residues, human defensins are classified into alpha and beta families. Structures of several beta-defensins have recently been characterized, confirming the disulfide connectivity conserved within the family, i.e., Cys1-Cys5, Cys2-Cys4, and Cys3-Cys6. We found that human beta-defensin 3 (hBD3), a recently described member of the growing beta family, did not fold preferentially into a native conformation in vitro under various oxidative conditions. Using the orthogonal protection of Cys1-Cys5 and of Cys1-Cys6, we chemically synthesized six topological analogs of hBD3 with predefined disulfide connectivities, including the (presumably) native beta pairing. Unexpectedly, all differently folded hBD3 species exhibited similar antimicrobial activity against Escherichia coli, whereas a wide range of chemotactic activities was observed with these analogs for monocytes and cells transfected by the chemokine receptor CCR6. Furthermore, whereas substitution of all Cys residues by alpha-aminobutyric acid completely abolished the chemotactic activity of hBD3, the bactericidal activity remained unaffected in the absence of any disulfide bridge. Our findings demonstrate that disulfide bonding in hBD3, although required for binding and activation of receptors for chemotaxis, is fully dispensable for its antimicrobial function, thus shedding light on the mechanisms of action for human beta-defensins and the design of novel peptide antibiotics.

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Figures

Figure 1

Figure 1

Fig. 1.

Fig. 1.

(A) Folding of hBD1 and hBD2 aided by cysteine/cystine. Fully reduced, purified peptides were analyzed by C18 RP-HPLC (thin lines) with a linear gradient of 5–65% acetonitrile over 30 min. Peptides folded after 24 h (thick lines) were analyzed under the same chromatographic conditions. (B) Folding of hBD3 under various oxidative conditions: DMSO, air, and cysteine/cystine. Analyses by C18 RP-HPLC were carried out using a linear gradient of 20–35% acetonitrile over 30 min.

Fig. 2.

Fig. 2.

(A) Strategy for preparation of six topological analogs of hBD3. Fully reduced hBD3 peptides with Cys1/Cys5 or Cys1/Cys6 selectively protected by Acm were subjected to oxidative folding aided by cysteine/cystine, resulting in six intermediate products (a–f), each containing two disulfide bridges. After determination of the disulfide connectivity in each species, deprotection and spontaneous oxidation of Cys1/Cys5 or Cys1/Cys6 were achieved by treatment with I2 at pH 4. (B) Formation of two disulfide bridges in Cys1/Cys5- or Cys1/Cys6-protected hBD3. C18 RP-HPLC analyses were performed using a linear gradient of 5–65% acetonitrile over 30 min. All six resultant oxidation products of identical masses, a–f, were pooled for identification of the disulfide connectivity. The minor peak between species a and_b_ was a partially oxidized product with only one disulfide bridge formed as indicated by ESI-MS. (C) Analysis of the six purified topological analogs of hBD3 with defined disulfide connectivities on C18 RP-HPLC running a linear gradient of 5–65% acetonitrile over 30 min. Note that the order of elution changes upon formation of the third disulfide bridge.

Fig. 3.

Fig. 3.

Salt dependence of antimicrobial activity of linear and disulfide bridged hBD3 against E. coli. Oxidized hBD3 and [Abu]-hBD3 were assayed against E. coli ATCC 25922 at a fixed concentration of 20 μg/ml, as described in Materials and Methods, except that NaCl at concentrations noted in the figure were included in the assay buffer (10 mM potassium phosphate, pH 7.4).

Fig. 4.

Fig. 4.

Comparative analysis of synthetic hBD3 with the β topology and commercial hBD3 (1 μg each) on a Vydac C18 RP column (4.6 × 150 mm). The chromatographic data were collected using a linear gradient of 20–35% acetonitrile over 30 min.

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