Engineering disulfide bonds of the novel human β‐defensins hBD‐27 and hBD‐28: Differences in disulfide formation and biological activity among human β‐defensins

Abstract

Human beta-defensins comprise a large number of peptides that play a functional role in the innate and adaptive immune system. Recently, clusters of new beta-defensin genes with predominant expression in testicular tissue have been discovered on different chromosomes by bioinformatics. beta-Defensins share a common pattern of three disulfides that are essential for their biological effects. Here we report for the first time the chemical synthesis of the new fully disulfide-bonded beta-defensins hBD-27 and hBD-28, and compare the results with synthetic procedures to obtain the known hBD-2 and hBD-3. While hBD-27 was readily converted into a product with the desired disulfide pattern by oxidative folding, hBD-28 required a selective protective group strategy to introduce the three disulfide bonds. The established synthetic processes were applied to the synthesis of hBD-2, which, like hBD-27, was accessible by oxidative folding, whereas hBD-3 required a selective strategy comparable to hBD-28. Experimental work demonstrated that trityl, acetamidomethyl, and t-butyl are superior to other protection strategies. However, the suitable pairwise arrangement of the protective groups can be different, as shown here for hBD-3 and hBD-28. Determination of the minimum inhibitory concentration against different bacteria revealed that hBD-27, in contrast to other beta-defensins tested, has virtually no antimicrobial activity. Compared to the other peptides tested, hBD-27 showed almost no cytotoxic activity, measured by hemoglobin release of erythrocytes. This might be due to the low positive net charge, which is significantly higher for hBD-2, hBD-3, and hBD-28.

Codon optimization enhances the expression of porcine β-defensin-2 in Escherichia coli

“…The mixtures were centrifuged after cultivation at 37°C for 1 h. Then, 100-μL aliquots of the supernatant were investigated at 450 nm (reference 630 nm) using a plate reader. The percentage of hemolysis was counted by comparison with the controls without peptide and with 1% Tween-20 (Schulz et al, 2005;Bai et al, 2009). All assays were implemented three times.…”

A new bioproduction route for a novel antimicrobial peptide

“…Unlike the well characterized hBDs1, 2, and 3 which are commonly found in skin or airways, hBD28 is expressed only at the epithelial cell layer of the testis and epididymis (Pazgiera et al, 2006), suggesting that hBD28 could have a specific role at its site of expression (Rodríguez-Jiménez et al, 2003). A very attractive antimicrobial characteristic of hBD28 is its broad spectrum activity at low minimum inhibitory concentration (MIC) values against clinically relevant pathogens (Schulz et al, 2005). To date, limited knowledge is available regarding the structural motif and mechanistic activity of hBD28.…”

“…An in vitro production platform that can efficiently and rapidly bulk manufacture hBD28 is therefore important. The in vitro production of hBD28 by chemical synthesis has been recently reported (Schulz et al, 2005). That chemical synthesis method, however, is inadequate as a sustainable production route for hBD28 because the need for a subsequent in vitro cysteine oxidation step was found to significantly decrease product yield, where correct disulfide pairing formed only the minor products.…”

Studies of the Biological Properties of Human β-Defensin 1

“…Three human ␤-defensins, hBD1-3, 4 have been isolated from natural sources and characterized in detail (8 -11). Additionally, the recombinant or synthetic preparations of hBD4, hBD27, and hBD28 have also been described and characterized functionally (12,13). Analysis of the human genome indicates the existence of over 40 potential coding regions for these peptides (14,15).…”