Abstract

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Matrix metalloproteinases (MMPs), the class of enzymes involved in the degradation of extra cellular matrix and surrounding cells are known to be expressed during cancer cell invasion, arthritis and metastasis and MMP-9, in particular seems to be a key protease associated with tumor progression. Therefore development of inhibitors of MMP-9 is a challenging task which can have therapeutic benefit for patients suffering from various cancers.β-lactoglobulin (β-LG), a core member of lipocalin (Lcn)family is an abundant whey protein in milk of ruminants and has attracted considerable attention as a rich source of bioactive peptides involved in diverse biological functions. Many of the lipocalins are shown to possess protease inhibitory activity including Lcn-2 and β-LG being a major lipocalin, its tryptic peptides were verified for the inhibitory effect on MMP-9.Hence, to investigate the effect of β-LG peptides on the protease activity, we have used β-LG purified from buffalo colostrum.The tryptic peptides of β-LG were separated and analyzed by LC-MS/MS. Based on in silico analysis the sequence information for singly charged peptide ions, m/z-573 IIAEK (P1), m/z-673 GLDIQK (P2), m/z-916 IDALNENK(P3) and m/z-933 IIVTQTMK (P4) were deduced and the interaction of these peptides with MMP-9 was verified by validated target of Matrix metalloproteinase-9 (PDB: 1GKC) using MolDock. A good correlation was observed in binding affinity of hypocholesterolemic peptides (IIAEK & GLDIQK) along with other two peptides (IDALNENK & IIVTQTMK) of β-LG from the buffalo colostrum implicating its utilization in functional foods.
Keywords: β-lactoglobulin, LC-MS/MS, Collision induced dissociation, In silico analysis, Matrix metalloproteinases, Molecular docking.

Introduction

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Milk contains a large number of bioactive peptides with various biological attributes. These peptides either exist naturally in milk or released after milk protein proteolysis. The natural bioactive peptides found in milk include epidermal growth factor (EGF), transforming growth factor (TGF), nerve growth factor (NGF), insulin, and insulin like growth factors I and II (IGF-I and IGF-II) and the concentrations of these peptides are generally high in colostrum, often higher than those found in blood circulation. Due to a limited protease activity in the gastrointestinal tract of neonates and the existence of protease inhibitors in the milk, these milk borne bioactive peptides are likely to survive the gastrointestinal digestion in suckling neonates. It is speculated that colostrum-borne bioactive peptides may play a role in regulating postnatal gut development in suckling neonates. Research in the field of bioactive peptides has intensified during the past two decades and has been extensively reviewed and an increasing number of in vitro and in vivo studies revealed that peptides released after enzymatic digestion can play very important role in human health [1-4] and modulate several regulatory processes [5]. Thus the peptides derived from milk proteins have been shown to improve immunity in the newborn with varied bioactivities like antihypertensive, antimicrobial, antioxidative, antithrombotic, immunomodulatory, mineral-carrying and opioid. Hence there is increasing commercial interest in the production of bioactive peptides with the purpose of using them as active ingredients in functional foods. Presently peptides derived from natural sources are of great demand with multifunctional abilities.
β-lactoglobulin (β-LG), a small soluble secretary protein is the major whey protein in ruminants belong to lipocalin family of proteins. Lipocalins are small secretary proteins consisting of a highly conserved eight stranded anti-parallel hydrophobic barrel bordered on one side by β-helix. More than 20 lipocalins have been identified so far involved in diverse biological functions varying from transport of small hydrophobic molecules to tumor progression and metastasis. The interest concerning lipocalins in cancer has so far been focused to the variations in concentration and the modification of lipocalin expression in distinct cancer forms [6].In addition, lipocalins have been assigned a role in cell regulation although influence of extracellular lipocalins on intracellular cell regulation events however is not fully understood. Some of the lipocalins also have protease inhibitory properties and possess the ability to interact with tumor specific proteases, revealing another pathway for lipocalins to interact with cancer cells. Lcn-1/ Tear lipocalin [7], Von Ebner’s gland protein [8], Mouse urinary protein [9], Triabin[10] are reported to have cysteine and papain, trypsin, serine protease inhibitory properties while, Lcn-2 is a ligand for matrix metallo protenase-9 (MMP-9) upregulated in many of the solid tumors [11]. MMP-9, a Zn²± dependent endopeptidase is markedly increased during metastasis and after myocardial infarction [12]. Therefore inhibition of MMP-9 is considered to be a novel approach controlling cancer and hypertension.
Development of low molecular weight protease inhibitors with minimal cytotoxicity may have effective therapeutic potential as a new class of anticancer drugs [13]. Hence histidine containing tetrapeptide sublibraries from different natural and non natural amino acids, from which new inhibitors of gelatinases (MMP-9 and MMP-2) have been generated as potential anticancer drugs [14]. MMPs catalytic domain containing N terminal HExxHxxGxxH Zn²+ binding consensus sequence and C terminal hemopexin-like domain are known to play key role in determining the substrate specificities of the various MMPs [15]. A conserved cysteine residue in the prodomain ligates the Zn²+ in the active site of the proteases, causing inactivation of the catalytic domain. Hence cysteine containing peptides mimik this to inactivate the enzyme activity and also the presence of free sulfhydryl or hydroxamate groups were found capable of inhibiting the activity by chelating Zn²+ in the active site of MMPs [16].
The bioactivities reported for milk β-LG derived peptides exhibits antihypertensive, antimicrobial, antioxidant, opioid and immunostimulatory effects [17-19]. The peptides of β-LG released after the proteolysis with trypsin, chymotrypsin, thermolysin and protenase K were shown to possess angiotensin I converting enzyme (ACE) inhibitory property, a key enzyme that controls hypertension [20,21]. Further, the tryptic peptides of β-LG inhibited cholesterol absorption as evaluated by in vivo studies and was shown to be greater than β-sitosterol [12]. Hence, β-LG is incorporated as active ingredient in Biozate, a pharmaceutical preparation [1]. Although structure function relationship is well ascribed for the peptides having ACE inhibitory activity, no such correlation exists for vast majority of functional peptides of β-LG. Incidentally; several lipocalins are reported to have protease inhibitory activity and are known to interact with tumor specific proteases [7-10]. Hence, in the present study we have made an attempt to deduce the sequence information of some of the tryptic peptides of β-LG purified from buffalo colostrum by in-silico analysis and assign their functional attributes as inhibitors of MMP-9 by docking analysis.

Methods

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The β-LG isolated and purified from buffalo colostrum [22], was used for the analysis.
Isolation and purification of BLG-col
BLG-col from buffalo (B. bubalis) colostrum was isolated by ammonium sulfate fractionation and purified by gel permeation chromatography on a Sephadex G-100 as described earlier [22].
Trypsin digestion of BLG-col
The enzyme digestion of BLG-col was performed using trypsin in 50mM NH4HCO3 (pH 8). Enzyme to BLG-col ratio was maintained at 1:50 and the reaction mixture was incubated at 37°C for 12h [21]. The digested sample was fractionated on C18 column (Vydac, 4.6×250mm, 5µm) using a linear gradient of acetonitrile (2%/min) containing 0.1% trifluroacetic acid. Fractions were collected and concentrated in a Speed Vac system and reconstituted in 5µL of 0.2% v/v TFA solution.
LC-MS/MS analysis
The LC-MS/MS analysis of the tryptic peptides of BLG-col were obtained on a HCT ULTRA ETD II mass spectrometer (Bruker Daltonics, Bremen, Germany), equipped with two Octapole followed by an ion trap and a separate hexapole next to negative chemical ionization chamber (nCI). Fluoranthene was used as the electron transfer reagent and methane was used for the chemical ionization. The reaction time of electron transfer was typically set at 100ms. Helium was used as the collision gas for collision induced dissociation (CID) experiments.
Database search
The raw data files of MS/MS in .mgf file format were extracted using data analysis 4.0 software and further searched in Mascot MS/MS ion search database http://www.matrixscience.com/cgi/search_form.pl?FORMVER=2&SEARCH=MIS using MSDB (MSDB 20060831; 3239079 sequences; 1079594700 residues), non-redundant NCBI (NCBInr 20090820; 9511482 sequences; 3251602805 residues) and SWISS-PROT (SwissProt 57.6; 495880 sequences; 174780353 residues) databases.
Molecular Docking analysis
The model for MMP-9 used in this study was derived from the co-ordinates of the structure labeled 1GKC in the protein data bank, which represents the human MMP-9 bound to NFH at 2A° resolution [23]. The structure of the peptide was constructed using automated ArgusLab 4.0.1 web server (http://www.arguslab.com). The possibility of binding, precise location of binding site and the mode of binding of the ligand was carried out using an automated docking software Molegro Virtual Docker 2008, version 3.2.1. The possible interacting conformations and orientations were analyzed by clustering methods embedded in MolDock [24].
All docking studies were carried out using the construct MMP-9 molecular model complexed with inhibitor NFH (N-formylhydroxylamine). The molecular structure of MMP-9 active domain was prepared from the coordinate sets of 1GKC chain A as the template [23]. All water molecules were excluded where as, zinc and chloride atoms were retained in the active site fixed to their crystal position through out the docking process. The binding site of the enzyme was computed within a cavity volume 77.312 A° and surface 271.36 A° such that the binding site of MMP-9 was well sampled with grid resolution of 0.3A°. The value of population size and maximum interactions 100 and 10,000 respectively were used for each run and 5 best poses were retained for ligand NFH docked into the template structure as a positive control and the RMSD was obtained.

Results and Discussions

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The LC-MS spectra of the tryptic peptides of colostrum β-LG clearly indicated the presence of tryptic peptides of masses m/z-573 [M+H]+, m/z-673 [M+H]+,m/z-916  [M+H]+ and m/z- 933 [M+H]+ in analogy to the PMF data reported earlier for BLG-col [22] These ions were numbered accordingly as P1-P4 respectively and analyzed for sequence information.
LC-ESI-MS/MS analysis using ion trap mass spectrometer with CID capability facilitated in fragmentation analysis of singly charged ion pairs m/z-573 (P1, Illustration 1
A), 673 m/z-( P2, Illustration 1B), m/z-916 (P3, Illustration 1C) and m/z-933 (P4, Illustration 1D). The direct submission of .mgf file for  MS/MS of m/z- 573, 673, 916 and 933 to Mascot MS/MS ion search yielded peptide sequences IIAEK (P1), GLDIQK (P2), IDALNENK (P3) and IIVTQTMK (P4) respectively (Supplimentary file 1-4). Notably, the sequences of P1 and P2 completely matched to hypocholesterolaemic peptides of bovine milk β-LG and these sequences were deposited in the peptidome database with an accession number PSE 141. Similarly IIVTQTMK corresponded to N- terminal sequence of buffalo colostrum [22] and milk β-LG [25] while, IDALNENK matched to the milk β-LG. The in-silico analysis of CID-MS/MS based fragmented peptides thus revealed complimentary data to PMF data of colostrum β-LG, besides the sequence tags were in compliance to f1-8 (P4), f9-14 (P2), f71-75 (P1) and f84-91(P3) of the β-LG of buffalo milk and cDNA sequences deposited in the database [26].
In the present study, peptide sequences IIAEK (m/z-573), GLDIQK (m/z-673), IDALNENK (m/z-916) and IIVTQTMK (m/z-933) from BLG-col were confirmed after in silico analysis. In order to understand the interaction of these peptides with MMP-9, we performed docking calculations. The catalytic centre of MMP-9 is composed of the active site Zn²+, co-ordinated by histidine (401, 405 and 411) and glutamic acid (402) residues. The cavity information of the enzyme was obtained as cavity volume 77.312 A° and surface 271.36 A°. We observed that hydrogen bond, electrostatic interaction and hydrophobic interaction were the main stabilizing forces in the enzyme-ligand interaction. The known MMP-9 inhibitor NFH interacted with His411, His405, Glu402, Leu188, Pro421, Gly186, Leu187, Leu188 and Tyr423 while, the interactions of the peptides IIAEK (P1), GLDIQK (P2), IDALNENK (P3) and IIVTQTMK (P4) to the  active pocket amino acids of MMP-9 were as shown in (Illustration 2,3,4 &5). Analogous to the well known inhibitor, all the peptides were found to interact with the active site amino acid residues based on hydrogen bond parameters. To evaluate the effectiveness of docked tryptic peptides in the Molegro docking programme, we first docked NFH and compared the resulted geometry with the corresponding crystal structure of MMP-9 complex (PDB: 1GKC). The best return poses in presence of Zn(II) with the docking score showed same interactions to be considered for high potency of the peptide and interestingly binding energy of P3 was found much higher (-74.69 kJ/mol) than NFH (-120.745 kJ/mol). Similarly, the binding energies of P1(-137.536 kJ/mol), P2(-130.475 kJ/mol) and P4 (-123.404 kJ/mol) were found in close match to the effect seen with respect with the inhibitor. These validated results of the docking analysis demonstrate the inhibitory effect of BLG-col peptides on MMP-9, hence a newer role can be assigned for BLG-col.

Conclusion

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Currently whey proteins have attracted attention as biologically active ingredients with important nutritional and functional properties. The peptides of β-LG are known to exhibit well-defined biological functions including ACE inhibitory activity. Further, as ACE inhibitory peptides also known to possess MMP-9 inhibition property, the present study would be instrumental to verify similar such effects for ACE inhibitory peptides of BLG-col. Since naturally derived peptides would be expected not to have side effects otherwise associated with synthetically produced drugs, our study explores structural specificity of the milk derived peptides and provides valuable insights in designing highly specific inhibitors of MMP-9 from food origin.

Acknowledgements

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We thank University Grant Commission, New Delhi, India for sanction of grant (F31-294/2005-06) to undertake this investigation and Rohit A. Chougule thank ICMR for award of SRF.

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Source(s) of Funding

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None

Competing Interests

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No competing interest

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