aphy & S r ep og a t r a a t m i o o r n Lilla et al., J Chromatograph Separat Techniq 2012, 3:2 h T e C c f Journal of Chromatography h DOI: 10.4172/2157-7064.1000122 o n l i a q ISSN:n 2157-7064 u r e u s o J Separation Techniques Research Article OpenOpen Access Access Structural Characterization of Transglutaminase-Catalyzed Casein Cross- Linking Sergio Lilla1,2, Gianfranco Mamone2, Maria Adalgisa Nicolai1, Lina Chianese1, Gianluca Picariello2, Simonetta Caira2 and Francesco Addeo1,2* 1Dipartimento di Scienza degli Alimenti, University of Naples “Federico II”, Parco Gussone, Portici 80055, Italy 2Istituto di Scienze dell’Alimentazione (ISA) – CNR, Via Roma 64, 83100 Avellino, Italy Abstract Microbial transglutaminase is used in the food industry to improve texture by catalyzing protein cross-linking. Casein is a well-known transglutaminase substrate, but the complete role of glutamine (Q) and lysine (K) residues in its cross-linking is not fully understood. In this study, we describe the characterization of microbial Transglutaminase -modified casein using a combination of immunological and proteomic techniques. Using 5-(biotinamido)pentylamine as an acyl acceptor probe, three Q residues of β-casein and one of αs1-casein were found to participate as acyl donors. However, no Q-residues were involved in network formation with κ-casein or αs2-casein. Q and K residues in the ε-(γ-glutamyl)lysine-isopeptide bonds β-casein were identified by nanoelectrospray tandem mass spectrometry of the proteolytic digests. This work reports our progress toward a better understanding of the function and mechanism of action of microbial transglutaminase-mediated proteins. The results suggest a possible role for transglutaminase in the formation of casein micelles. Keywords: Microbial transglutaminase; Casein; Milk; ε-( γ After ingestion, ε-(γ-glutamyl) lysine isopeptide is somewhat resistant -glutamyl)-lysine isodipeptide bond; 5-(biotinamido) pentylamine; to digestive enzymes [21]. The ε-(γ-glutamyl) lysine isopeptide bond Tandem mass spectrometry is cleaved by γ-glutamylcyclotransferase which releases L-Lys and 5-oxoproline. The 5-oxoproline is then further metabolized to glutamic Introduction acid by 5-oxoprolinase (5-OP) [1]. Foods treated with mTG have Transglutaminase (TG) is one of a family of enzymes that catalyzes been approved for human consumption by the US Food and Drug an acyl transfer reaction in the presence of Ca2+. This reaction forms Administration, and its application has expanded to all foods. amide bonds between the ε--carboxamide groups of peptide-bound Many food proteins such as casein (CN) are excellent substrates for glutamine (Q) residues and a variety of primary amines [1]. In TG [22-24], primarily due to their highly accessible and flexible open the reaction catalyzed by TG, Q residues serve as the acyl donors chain structure. Milk with a high degree of cross-linking is unable to and ε-amino groups of lysine (K) as the acyl acceptors. TG shows coagulate after the addition of rennet, probably due to failure during substrate selectivity for Q residues as the acyl donors. However, it the primary phase of milk clotting. This hinders the nonenzymatic is less selective with regards to the acyl acceptor (K-residue) [2,3]. secondary phase that is initiated when a sufficient amount of k-CN is The K-residue can be replaced by low molecular weight amines, hydrolyzed [25,26]. such as monodansylcadaverine, fluoresceincadaverine [4,5] and 5-biotinamidopentylamine [6], and also by dansylated or biotinylated CN is a better substrate for TG than whey proteins. However, the glutamine-containing peptides [7]. In the absence of a K-residue or denaturation of whey proteins makes their amino groups accessible to other amines, water can act as the nucleophile, which results in the TG, which allows cross-linking to occur [27-29]. CN is an amphiphilic deamidation of Q-residues to glutamic acid [4,8,9]. The ability of protein which can self-assemble into micelles stabilized by both TG to catalyze cross-linking, incorporate of amines, and deamidate hydrophobic interactions and calcium phosphate bridges. The amino 2+ modifies the functional properties of proteins [10-13]. Ca -dependent acid sequences of αs1-, αs2-, β-, and κ-CN, their relative proportions and mammalian TG (i.e., blood coagulation factor FaXIII and tissue TG) total CN vary in whole milk depending on the species. was the first TG to be investigated for application in the food industry TG-mediated modification of CN and its exact mechanisms have [11,14,15], but the high cost of its extraction and purification limited its use. Hence, the major source of the enzyme has become Ca2+- independent microbial TG (mTG), which is largely produced through *Corresponding author: Francesco Addeo, Dipartimento di Scienza degli fermentation technology utilizing Streptoverticullium species [16 ]. Alimenti, University of Naples “Federico II”, Parco Gussone, Portici 80055, Italy, Ca2+-independent mTG is useful for the modification of the functional E-mail: [email protected] properties of food proteins (i.e., milk proteins, soybean globulins, Received March 14, 2012; Accepted April 06, 2012; Published April 08, 2012 gluten, actin, myosin, and egg proteins) [17]. The amino acid sequence Citation: Lilla S, Mamone G, Nicolai MA, Chianese L, Picariello G, et al. (2012) of mammalian TG is slightly different from that of mTG, although they Structural Characterization of Transglutaminase-Catalyzed Casein Cross-Linking. both have Cys in the active site. mTG is 331 amino acids long with a J Chromatograph Separat Techniq 3:122. doi:10.4172/2157-7064.1000122 molecular mass of 37,863 Da, and its optimum pH is in the range from Copyright: © 2012 Lilla S, et al. This is an open-access article distributed under 5-8 [18]. Digestibility of products containing ε-(γ-glutamyl) lysine the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and isopeptide has been investigated in different circumstances [19,20]. source are credited. J Chromatograph Separat Techniq ISSN:2157-7064 JCGST, an open access journal Volume 3 • Issue 2 • 1000122 Citation: Lilla S, Mamone G, Nicolai MA, Chianese L, Picariello G, et al. (2012) Structural Characterization of Transglutaminase-Catalyzed Casein Cross-Linking. J Chromatograph Separat Techniq 3:122. doi:10.4172/2157-7064.1000122 Page 2 of 9 not been fully elucidated. The interaction of TG with its substrate in PBS/0.1%SDS, and the beads were washed as described in “mTG depends not only on the primary amino acid sequence, but also the reaction” section. The enriched biotinylated peptides were eluted by secondary and/or tertiary sequence. It also appears necessary that Q be adding 25 ml of 70% acetonitrile/2% formic acid/0.2 mM biotin and exposed on the surface of CN fractions so that it can react with TG. It is incubating for 30 min at 37˚C. The assay was performed in parallel on possible for Q residues to be partially exposed on the outer CN fractions a sample incubated with no mTG and 5BP. of micelles. However, other Q residues may be partially blocked by RP- HPLC and LC-ES-MS analysis steric hindrance, which will make the protein substrate less accessible to TG. In any case, it seems that only a small fraction of Q residues Liquid chromatography was performed using a 2.1 mm i.d. x 250 is transamidated at any given time [30]. Because CN preparations mm, C18, 5 µm reverse-phase column (Vydac, Hesperia, CA, USA) incubated for different lengths of time present different rheological with a flow rate of 0.2 ml/min on an Agilent 1100 modular system (Palo properties, the cross-linking of CN from four species (cow, sheep, goat, Alto, CA, USA). Solvent A was 0.1% trifluoroacetic acid (TFA) (v/v) in and buffalo) has not been investigated. Ovine milk in particular has water, and solvent B was 0.1% TFA in acetonitrile. The proteins were received little attention, even though it possesses the highest level of separated using a gradient from 35% to 50% B over 60 min. Peptide CN. The present work aims to identify the in vitro susceptibility of CN separation was instead performed using a gradient from 5% to 70% fractions to mTG-mediated modification. Some Q-donor sites have B over 90 min. Peaks were monitored by UV detection at 220 nm. been examined for their role in the assembly and structure of the CN Mass spectrometry (MS) was performed using a Platform (Micromass, micelle. This study reports the identification of several amino acid Manchester, UK) equipped with standard electrospray. The eluent sequences of ovine CN that act as glutamine-donor substrates of mTG. from the Vydac column chromatography was injected into the mass Materials and Methods spectrometer online with the UV detector via a 75 µm i.d. fused silica capillary. The mass spectra were scanned from 2000 to 400 atomic mass Trypsin, endoproteinase Glu-C, dithiothreitol (DTT), units at a scan cycle of 5 s/scan. The source temperature was held at 5-(biotinamido) pentylamine (5-BP), α-cyano-4-hydroxycinnamic 200˚C and the cone voltage at 40 V. Mass scale calibration was obtained acid, and sinapinic acid, renin substrate tetradecapeptide porcine using myoglobin as the reference compound. were purchased from Sigma (St. Louis, Missouri, USA). mTG from MALDI -TOF MS analyses Streptoverticillium mobaraense was obtained from Ajinomoto (Tokyo, Japan). MALDI-MS TOF experiments were carried out on a PerSeptive Biosystems (Framingham, MA, USA) Voyager DE-PRO instrument Ovine CN from single milk was skimmed by centrifugation at 3000 equipped with a N2 laser (337 nm, 3 ns pulse width). Each spectrum rpm, and the isoelectric CN was prepared according to the procedure of was taken with the following procedure: a 0.5 µL-aliquot of sample was Aschaffenburg [31].