Transglutaminase-mediated crosslinking of a host defence peptide derived from human apolipoprotein B and its effect on the peptide antimicrobial activity
Introduction
Host defence peptides (HDPs) are normal components of the innate immune system. Natural HDPs have a size ranging from 12 to 50 amino acids, are mostly cationic owing to the presence of a high content in Lys and Arg residues and contain over 50% of hydrophobic amino acids [1,2]. These features are responsible for the high selectivity of HDPs for the negatively charged bacterial membranes with respect to the zwitterionic ones of eukaryotic cells [2]. Although HDPs were initially studied as antimicrobial agents in the treatment of chronic infections, their applicability in wider fields of human health has attracted growing interest over the years. Numerous researches proved that HDPs are able to counteract microbial contamination and food spoilage. Indeed, they are active towards foodborne pathogens directly as antimicrobials or indirectly by exploiting different activities, such as anti-biofilms and immunomodulatory agents. Besides their interesting mechanism of action and broad-spectrum activity, it has been shown that HDPs are able to preserve food samples without altering their qualities and organoleptic properties. The first peptide recognized as food preservative approved from the Food and Drug Administration was nisin, a bacteriocin isolated from the lactic acid bacteria. Nisin is heat-stable, harmless and effective on Gram-positive bacteria, such as Listeria monocytogenes [3] and it has been used to functionalize different kinds of active packaging materials [4,5]. On the other hand, beyond to be ineffective towards Gram-negative bacteria, its antimicrobial activity is strongly dependent on pH. In fact, at neutral pH, nisin antibacterial activity is strongly compromised. Several further peptides have been used to functionalize packaging materials. Among these, sonoresin, a bacteriocin derived from Bacillus sonorensis, has been employed to functionalize low density polyethylene (LDPE) film [6], conferring to it the ability to prevent Staphylococcus aureus growth, as well as chicken meat and fresh tomatoes spoilage [6]. Moreover, other bacteriocins, such as the enterocins, lactocins, natamycin and pediocins have been used to render various polymeric coatings endowed with antibacterial and antifungal activity [[7], [8], [9], [10]].
Being HDPs produced in all living organisms, further peptides derived from different sources have been investigated as novel bio-preservatives beyond the bacteriocins. Among these, pleurocidins are cationic mucus derived HDPs isolated from the Atlantic flounders [11]. They have been found to be endowed with significant antimicrobial activity towards main foodborne pathogens, such as Vibrio parahemolyticus, L. monocytogenes, E. coli O157:H7, Saccharomyces cerevisiae, and Penicillium expansum [12]. Defensins from vertebrates, produced by mammalian phagocytes and chicken epithelial cells, have been shown to possess antimicrobial activity against several bacteria, fungi, and viruses. In particular, gallinacin-6, a defensin produced in chicken digestive tract, showed antimicrobial activity towards many foodborne pathogens, such as E. coli, Salmonella, Staphylococcus aureus, Saccharomyces cerevisiae and Candida albicans [13]. Furthermore, milk is one of the main sources of antimicrobial agents. Proteins like casocidin, caseins A and B, as well as lactoferrin, are able to inhibit the growth of foodborne pathogens [14]. Lactoferrin is present in different meat products, where it exerts antimicrobial activity towards Carnobacterium, L. monocytogenes, E. coli, and Klebsiella [15].
Recently, two novel antimicrobial peptides of different length were identified in silico in human apolipoprotein B sequence, and then recombinantly produced and characterized. They have been named r(P)ApoBL and r(P)ApoBS because of the presence of a Pro residue becoming the N-terminus of the peptides released by the acidic cleavage of an Asp-Pro bond; L and S subscripts, instead, refer to a longer or a shorter version of the identified peptide sequence, respectively [[16], [17], [18], [19]]. Both HDPs were found to possess a broad-spectrum antimicrobial activity, whereas they were found to be neither toxic nor haemolytic when tested towards eukaryotic cells. In addition, they were found to exert significant anti-biofilm activity and to be able to act in synergism with commonly used antibiotics and with EDTA.
In this paper, the ability of r(P)ApoBL to act as substrate of transglutaminase was investigated, since six Lys residues are present in its amino acid sequence (PHVALKPGKLKFIIPSPKRPVKLLSGGNTLHLVSTTKT). Transglutaminases (EC 2.3.2.13), enzymes widely distributed in nature, catalyse the acyl transfer reaction of γ-glutamyl residues, present in protein and peptide substrates (Gln acyl donors), to amino donor substrates (Lys inside peptides and proteins, or small molecular weight free amines, acyl acceptors). A variety of different reaction products, depending on the involved substrate molecules, can be obtained [20,21]. The transamidation reaction gives rise to ε-(γ- glutamyl)lysine intra- and/or inter-crosslinks, generating both homo- and hetero-polymers, as well as to protein–amine conjugates. The most studied microbial transglutaminase (mTG) has been purified from the culture medium of Streptoverticillium sp. S-8112 [22], which has been identified as a variant of Streptoverticillium mobaraense and also known as Streptomyces mobaraensis [23]. In contrast to the other enzymes of the same family, mTG is Ca2+ independent and is remarkably stable over a wide range of temperatures and pHs. These characteristics, including the higher reaction rate, the broad substrate specificity and the low-cost mass production by traditional fermentation technology, make mTG particularly useful for industrial and biotechnological applications as a food-grade additive capable of improving many important features of different protein-based foods [24,25]. In addition, mTG was applied in the bioplastic sector, in order to enhance the technological performance of the protein-based materials [26,27].
In the present study it was demonstrated that r(P)ApoBL effectively acts as mTG acyl acceptor by using different peptide and protein acyl donor substrates. Furthermore, mass spectrometric analyses were carried out to identify the specific lysine residue(s) involved in the reaction, and the antimicrobial activity of a protein-based film was finally investigated after r(P)ApoBL incorporation in the absence or presence of mTG.
Section snippets
Materials
Expression and isolation of recombinant peptide r(P)ApoBL was carried out as previously described [16]. Monodansylcadaverine (MDC), N,N-dimethylcasein (DMC), N-α-acetyl- lysine, and Substance P (SP) were purchased from Sigma Chemical Company (St. Louis, MO,USA). Bitter vetch (BV) seeds were obtained from a local market (Gallicchio, PZ, Italy). mTG (Activa WM), derived from the culture of Streptoverticillium spp., was supplied by Ajinomoto Co. (Japan). The enzyme was prepared by dissolving the
r(P)ApoBL as acyl acceptor substrate of mTG
r(P)ApoBL is a cationic peptide containing six Lys residues, potentially able to act as acyl acceptors for mTG, without Gln residues as possible acyl donors. Hence, to demonstrate whether one or more Lys residue(s) could be reactive in mTG-catalyzed reaction, it was carried out a fluorimetric enzymatic assay [28] based on the covalent incorporation of MDC (a fluorescent acyl acceptor) into DMC (a protein acyl donor) and on verifying a possible inhibiting or alternative substrate activity by r(P
Conclusions
The results of this study highlight the potential of mTG as a biotechnological tool to crosslink r(P)ApoBL to various acyl donor substrates and identify Lys-18 of the peptide as an effective acyl acceptor site for the enzyme. Furthermore, an edible film with enhanced antimicrobial properties was obtained by grafting BV seed proteins with r(P)ApoBL. Notably, the biological activity of the BVPC/r(P)ApoBL film was lost when mTG was added into the film forming solution. All together these findings
Declaration of Competing Interest
The authors declare that they no conflict of interest.
Acknowledgments
This work was supported by University of Naples ‘Federico II’ and University of Campania “Luigi Vanvitelli”.
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Both authors contributed equally to this work.