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

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 ␣ and ␤ families. Structures of several ␤-defensins have recently been characterized, confirming the disulfide connectivity conserved within the family, i.e., Cys 1 -Cys 5 , Cys 2 -Cys 4 , and Cys 3 -Cys 6 . We found that human ␤-defensin 3 (hBD3), a recently described member of the growing ␤ family, did not fold preferentially into a native conformation in vitro under various oxidative conditions. Using the orthogonal protection of Cys 1 -Cys 5 and of Cys 1 -Cys 6 , we chemically synthesized six topological analogs of hBD3 with predefined disulfide connectivities, including the (presumably) native ␤ 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 ␣-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 ␤-defensins and the design of novel peptide antibiotics.

β‐Defensins chemoattract macrophages and mast cells but not lymphocytes and dendritic cells: CCR6 is not involved

“…This could imply that disulfide bonding is essential to the functional structure of the molecules. However, in the case of HBD-3, disulfide bonding was relevant only for its chemotactic but not its antimicrobial activities [17,33]. In keeping with these studies, we observed that a linear form of HBD-3 had lost its ability to chemoattract macrophages.…”

“…The differential migration of DC and macrophages in response to CCL20 and b-defensins argued against CCR6 being a defensin receptor which is in contrast to previously published data [12, [16][17][18]. Therefore, we investigated two cell lines stably expressing human CCR6 which were derived from RBL-2H3 (Fig.…”

“…They modulate the cytokine response of mononuclear blood cells [11], and promote systemic antigen-or tumor-specific immune responses [12][13][14][15]. In keeping with these findings, human b-defensin (HBD)-1 to -3 as well as murine b-defensin (mBD)-2, -3 and -29 were shown to selectively chemoattract memory T lymphocytes and/or immature DC via CCR6 [12, [16][17][18]. As some b-defensins additionally induced migration of monocytes and mast cells [19][20][21][22] that do not express CCR6, further receptors for b-defensins were postulated [23].…”

In vitro antibacterial and hemolytic activities of crotamine, a small basic myotoxin from rattlesnake Crotalus durissus

“…Recent studies have revealed that the three intramolecular disulfide bonds of b-defensins are dispensable for antibacterial activity, but required for chemotactic activity and for their resistance to protease degradation. 10,11 In light of these surprising findings, the significance of the three intramolecular disulfide bonds in the antibacterial activity and hemolysis of crotamine was tested. We clearly revealed that besides structural similarity, the crotamine resembles b-defensins in its mode of action, but with a narrow antibacterial spectrum.…”

Topology of the disulfide bonds in the antiviral lectin scytovirin

“…Upon tryptic digestion at pH 6, rSD1 yielded a disulfide pattern identical to the one found for native SVN and SD1B. The m/z ¼ 2529.0 species, as well as its thermolytic fragment, residues [25][26][27][28][29][30]-[37-43] (m/z ¼ 1444.6), were sequenced. Under these conditions, the heterodimer of m/z ¼ 2511.0 was not found in the digest.…”