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Peptide Profiling of Igg and Their Interaction with Receptor Outer Membrane Porin of Klebsiella Pneumoniae

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Peptide Profiling of Igg and Their Interaction with Receptor Outer Membrane Porin of Klebsiella Pneumoniae Central JSM Biochemistry & Molecular Biology Bringing Excellence in Open Access Research Article *Corresponding author Dr. Aparna HS, Department of Studies in Biotechnology, University of Mysore, Mysore-570006, Karnataka, India, Peptide Profiling of IgG and Tel: 91-821-2419482(O); Fax: 91-821-2419363(O); Email: their Interaction with Receptor Submitted: 03 April 2017 Accepted: 02 June 2017 Published: 05 June 2017 Outer Membrane Porin of ISSN: 2333-7109 Copyright Klebsiella pneumoniae © 2017 Mamatha et al. Mamatha Bhanu LS and Aparna HS* OPEN ACCESS Department of Biotechnology, University of Mysore, India Keywords • Buffalo colostrum Abstract • Immunoglobulin G • nanoLC-MS/MS Immunoglobulins make up the largest portion of the humoral immunity, of which Immunoglobulin • Peptides G (IgG) is the main class of antibodies present in ruminant colostrum. Proteolytic enzymes have • Porin been successfully used to generate peptides for reagent and therapeutic use. IgG purified from buffalo (Bubalus bubalis) colostrum was proteolytically digested by pepsin and pancreatin to generate peptide fragments and analyzed by nanoLC-MS/MS (Liquid chromatography - mass spectrometry). We identified 25 peptide sequence matches to heavy and light chains in the complimentary determining region of IgG. The small peptides generated were used to examine their interaction on Klebsiella pneumoniae outer membrane protein (OMP), a drug target site. IgG peptides were found to interact with porin emaluating standard antibiotic ampicillin suggesting their probable interaction to the drug target. A more detailed investigation would be interesting to establish newer functionalities to peptides of IgG which can have profound implications in developing therapeutic formulations. ABBREVIATIONS binding sites in the variable domains of the antigen binding FC: Fragment Crystallizable Region; FAB: Antigen Binding fragment (Fab) and alert immune cells to putative threats Fragment; F(ab’)2: Two Antigen Binding Fragment; Fd: Heavy through interaction sites in the constant domain of the tail Chain; IgG: Immunoglobulin G; VH: Heavy Chain; VL: Light Chain; ESBL: Extended-Spectrum Beta-Lactamases; CDRs: Complemen- and solvent-exposed, Fc (fragment crystallizable), Fab (antigen region [7]. Because of the hinge region IgG molecules is flexible tarity-Determining Regions, HR-LC-MS: High Resolution-Liquid binding fragment), and F(ab’)2 (two antigen binding fragment) Chromatography Mass Spectrometry; OMP: Outer Membrane fragments can be readily generated by enzymatic cleavage under Porin; Gscore: Glide Score native condition [8]. IgG molecules composed of only antigen binding portions can be advantageous and have potential for INTRODUCTION immunotherapeutic applications in cancer treatment, infection Immunoglobulins (Igs) are glycoproteins that form an clearance, and targeted drug delivery [9]. The complementarity- important part of the immune system. Ruminant colostrum is determining regions (CDRs) that reside in the variable regions characterized by its very high level of immunoglobulin G (IgG) of light chains and heavy chain (Fd) are responsible for the [1]. In colostrum, Igs are the principal agents that protect the gut mucosa against pathogenic microorganisms, and confer on its potency [10]. The smaller size enables in better tissue antigen binding specificity and consequently, have an effect passive immunity to the ruminant neonate until its own immune penetration thus facilitating better antigen recognition in system is developed [2]. Oral administration of immunoglobulin immunochemistry and less steric hindrance leading to more preparations from human serum as well as bovine colostrum sensitive antigen detection. The enzyme pepsin cleaves the Fc and serum have been evidenced to be safe as well as effective in portion of an immunoglobulin into small sub fragments leaving human clinical trials in treating gastrointestinal tract infections a F(ab’)2 fragment with two antigen binding sites connected by [3] and other microbial infections [4]. Hence, colostrum and milk are categorized as medicinal nutrition [5]. IgGs are large proteins survive gasterointestinal digestion was performed by pepsin and disulfide bonds [11]. Colostrum IgG, a neonatal food component, of approximately 150 kDa, made of two identical heavy chains pancreatin, the resulted peptide fractions analysed by HR-LC/MS (50 kDa) and two identical light chains (25 kDa) [6]. Antibodies (High performance liquid chromatography mass spectrometry). recognize foreign molecules (antigens) through epitope- The outer membrane of gram-negative bacteria is the first Cite this article: Aparna HS, Mamatha Bhanu LS (2017) Peptide Profiling of IgG and their Interaction with Receptor Outer Membrane Porin of Klebsiella pneumoniae. JSM Biochem Mol Biol 4(2): 1026. Mamatha et al. (2017) Email: Central Bringing Excellence in Open Access line of defense against toxic compounds and provides a variety 98% buffer B over 20 min. Peptides were introduced to the mass of functions including passive and active transport, host- analyzer from the LC through Agilent 1260 Chip-cube operating pathogen recognition, signal transduction, and catalysis. Due to in ESI-positive (Jet Stream Ionization) ion mode; precursor mass their exposed epitopes on the cell surface and highly conserved within a range of 300-3200 m/z were selected for MS/MS with a immunogenicity [12,13], Omps could be used as candidates to threshold absorbance count of above 2000 and ramped collision develop vaccines for combating bacterial infections [14,15]. energy was limited to charge 2, 3 and more than 3 . The MS scan As a most important member of Omps, the outer membrane rate (acquisition rate) was 6 spectra/sec and acquisition time of protein A (OmpA) is a class of highly conserved proteins among 166.6 ms. MS/MS spectra were collected with an isolation width the enterobacteriaceae family at medium (~4 amu) resolution and ramped collision energy multifunctional protein that plays an important role in bacterial for different charge states. MS/MS spectra were scanned from physiology and pathogenesis. [12].It can OmpA function is confirmedas an adhesin as a 0-3200 m/z with acquisition rate of 3 spectra/sec and acquisition time of 333.3 ms. Precursor/parent ions were excluded for 0.15 immune target and evasin, and serve as a receptor for several min following selection for MS/MS. and invasin, participate in biofilm formation, act as both an bacteriophages [13]. OmpA (outer membrane protein A) is one Molecular docking of the best characterized OM proteins and highly conserved among the Enterobacteriaceae family. K. pneumoniae produces The peptides obtained from IgG after in vitro gastrointestinal three major proteins, OmpK35, OmpK36, and OmpA, in its outer simulation digestion were used for docking analysis targeting membrane [14]. Based on the sequence similarities of ompK35 outer membrane porin of K. pneumoniae (www.rcsb.org; PDB ID: and ompK36 genes, OmpK35 is a homolog of OmpF [15], and 1OSM) using Schrodinger software. The protein was prepared for OmpK36 [16] is a homolog of OmpC [17]. Clinically, most of molecular docking by protein preparation wizard for the addition the (Extended-spectrum beta-lactamases) ESBL-producing K. of missing atoms, hydrogens, assigning bond orders, setting pneumoniae strains express only OmpK36. This barrier comprises proper ionization states of residues and capping the termini. a lipid bilayer that is impermeable to hydrophobic antibiotics and other compounds [18]. Thus by evaluating interactions of the orientations, at neutral pH) and energy was minimized with OPLS The receptors were then refined with H-bond assignment (water ligand/s with the receptor with energy-based score or scoring function for each pose, provide clues of the potential bioactive to the residues (Phe 189, Thr 229, Tyr 231, Phe 257; PDBSUM 2005 force field. The receptor grid specified was with reference peptide ligands of IgG peptides across OMP. In this context, IgG software). The receptor grid scaling of Vander Waals radii was characterized from buffalo colostrum [19] possessing growth set at 0.8 and partial charge cut off value of 0.15. The ligands inhibitory potential on K. pneumonia [20] was digested with the (FIFPP, IKSFRI, IVKPGASV, FDVWGTGT) along with standard enzymes pepsin and pancreatin and the peptides were analysed antibiotic ampicillin (PDB ID: 4GCP) were prepared in ligprep by nanoLC-MS/MS. Based on the in vitro results, the probable interaction of IgG peptides was studied in silico by targeting outer at target pH: 7.0 ± 2.0, tautomers and stereo isomers (generate with the following parameters, force field: OPLS2005, ionization membrane porin (OMP) of K. pneumoniae. from 3D structure) with at most 6 ligands to be generated. MATERIALS AND METHODS Finally,all combinations the ligands of werespecified docked chiralities by XP (Extraand determine Precision) chiralities method In vitro gastrointestinal digestion of IgG results were exported with XP descriptors information by adding In vitro gastrointestinal digestion was carried out in triplicates Epikwith flexibilityState penalties (nitrogen to docking inversions score. and ring conformations). The as described earlier [21]. A 3.5 % (w/v) IgG sample in 0.1 M KCl- RESULTS AND DISCUSSION HCl (pH 2) buffer was incubated with pepsin (4% w/w) for 4 h at 37°C and the reaction was terminated by keeping in boiling HR-LC-MS analysis water for 10 min. The suspension was neutralized with addition - of 2 N (Normality) NaOH
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