Translocation of a Thioether-Bridged Azurin Peptide Fragment via the Sec Pathway in Lactococcus lactis

Abstract

This study demonstrates for the first time that a thioether-containing peptide, an azurin fragment, can be translocated via the Sec pathway. This methyl-lanthionine was introduced by the nisin modification enzymes. The Sec pathway can therefore be a successful alternative for those cyclized peptides that are inefficiently transported via NisT.Azurin, a cupredoxin produced by Pseudomonas aeruginosa, can selectively enter human cancer cells and induce apoptosis (24) via binding to the tumor suppressor protein p53 (1). The azurin peptide fragment p28, containing amino acids 50 to 77 (LSTAADMQGVVTDGMASGLDKDYLKPDD), still enters human cancer cells and inhibits tumor proliferation (20). Importantly, novel cancer treatments can be based on azurin peptide fragments and derivatives thereof (T. Das Gupta and A. Chakrabarty, 20 March 2008, patent application WO2008033820). Although the pharmacokinetic property of therapeutic peptides is promising, lack of biostability is the major hurdle for their successful application. Consequently, it is very relevant to explore the possibilities for enhancing biostability of these peptides.In our group, we developed a technology to improve the stability of therapeutic peptides by exploiting the nisin synthetase enzymes NisB and NisC for the introduction of thioether bridges. We applied a two-plasmid expression system (7,8,12), in which the NisBTC-encoding plasmid is compatible with the substrate-peptide-encoding plasmid. Lactococcus lactis containing this expression system can secrete nonlantibiotic peptides which are dehydrated or stabilized by a thioether ring (8, 16). NisB dehydrates serines and threonines in substrate peptides, NisC couples dehydrated residues stereo-and regioselectively to cysteines, and NisT, the ABC transporter, translocates the modified peptides out of the cell (10,11,13,15). The leader peptide is essential for targeting and modification of the propeptides (23).When transport via NisT is impaired or is less efficient, the Sec pathway of L. lactis is a successful alternative in translocation of dehydrated peptides. When the nisin leader is preceded by a Sec signal peptide or a Tat signal peptide 27 or 44 amino acids long, respectively, modification by NisB and NisC still occurs (12; G. N. Moll, A. Kuipers, R. Rink, A. J. M. Driessen, and O. P. Kuipers, 15 June 2006, patent application WO 2006062398). However, NisC-cyclized prenisin was not translocated via the Sec system (12). This is likely due to the dimensions of fully modified nisin (3), which is too large to fit in the SecY pore (12, 21). Here, we report for the first time that the Sec pathway of L. lactis can translocate a p28 azurin fragment analog with a thioether ring.

Bioengineering of a Nisin A‐producing Lactococcus lactis to create isogenic strains producing the natural variants Nisin F, Q and Z

“…Thus the basis for the non‐production of these peptides is not apparent. We anticipate that the use of alternate expression systems, which differ more dramatically from that in a wild‐type cell (such as those used to introduce lanthionine residues into non‐nisin peptides (Rink et al ., 2005; Kuipers et al ., 2006; Rink et al ., 2007; Kluskens et al ., 2009; Kuipers et al ., 2009; Majchrzykiewicz et al ., 2010)), could be employed to generate Nisin U and U2. However, the inability of the Nisin A machinery in its natural form (i.e.…”

“…In recent years it has been shown that Nisin biosynthetic proteins can be harnessed, both in vivo and in vitro . This technology has facilitated the incorporation of lantibiotic‐associated post‐translational modifications into a range of unrelated peptides (Kuipers et al ., 2006; Kluskens et al ., 2009; Kuipers et al ., 2009; Majchrzykiewicz et al ., 2010; for review see Moll et al ., 2010). In addition to the long established use of Nisin as a food preservative, the high potency and multiple mechanisms of action have also been the focus of studies designed around using Nisin in clinical or veterinary applications against drug‐resistant pathogens.…”

Methodologies and Strategies for the Bioengineering of Lantibiotics

“…For example, a lanthionine bridge was introduced in the azurin fragment, novel agent for cancer treatment, to enhance its biostability. NisTmediated transport could not secrete the lanthioninecontaining azurin fragment, but Sec-mediated transport was successful [45]. In another example, a lanthionine bridge was introduced into an angiotensin-(1-7) heptapeptide that plays a protective role in hepatic disease; this modified peptide was secreted using NisT-mediated transport system [46].…”

Biosynthesis of lanthionine-constrained agonists of G protein-coupled receptors

“…In the C-terminal part of the nisin leader peptide, general cleavage sites could be engineered to allow convenient clean removal of the leader peptide from the produced peptide of interest [29]. By engineering a signal sequence at the N-terminus of the leader peptide constructs peptides could also be exported out of L. lactis via the SEC secretion system provided they had limited bulkiness [36,37]. Excellent in vitro studies by Wilfred van der Donk and co-workers furthermore demonstrated that a bifunctional lanthionine-introducing LanM enzyme, which performs both the dehydration as well as the cyclization step, also has a broad substrate specificity [38].…”