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 Submitted: 03 April 2017 Accepted: 02 June 2017 Published: 05 June 2017 Outer Membrane 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 inthe complimentary determining region of IgG. The small peptides generated were used to examine their interaction on Klebsiella pneumoniae outer membrane (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 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 confirmed as 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 to pH 7 and digested with 4% (w/v) nation of proteases, pepsin and prancreatin. The resultant pep- pancreatin at 37°C for 4 h. The enzyme was inactivated by boiling tidesThe obtained purified after buffalo HR-LC/MS colostrum analysis IgG was searcheddigested byagainst a combi non for 10 min and centrifuged (10,000xg, 30 min) and desalted. redundant NCBI database. Search results revealed the identity of HR-LC/MS analysis 25 peptide sequences matching to IgG heavy and light chains of - The desalinized peptide mixture was fractionated on Agilent lecular ions matching to IgG light chain were m/z - 1165.5583+, 6550 iFunnel QTOF mass spectrometer (Agilent Technologies) mouse,m/z-1576.8006 human and+, m/z-1818.8915 bovine species.+ The, m/z-2345.0489 identified protonated+ and m/z- mo 3454.6617+ corresponded to LSVSSVTAEDSA, LIYSTNQWP- SPAVT, LSPGERATLSCRASHSF, LPGTAAISELQSEDEGDYYCAL coupled to an Agilent G1376A Infinity Capillary Pump and G2226A permeate was loaded on to a Protein ID-Chip (G4240-62030 and LSVATGEKVTIRCITNTDIDDDMNWYQQKP sequences (Table HighInfinity Performance Nanoflow Chip, Pump 360 LC nl system enrichment (Agilent). column, The 150 desalted mm x

Agilent Technologies) in buffer A (0.1% formic acid in MilliQ antibody.1). In mammals Among there these are peptides,two types LSVSSVTAEDSA of Ig light chains, and lambda LSVAT (λ)- 75Âμm) via an Infinity Auto sampler (Low Flow Hip Sampler, anda kapp (κ) but only one type of light chain is present in any of nano Pump. Peptides were eluted into the mass spectrometer mouse, LIYSTNQWPSPAVT and LPGTAAISELQSEDEGDYYCAL throughwater) at aa flow linear rate gradient of 1 µl/min with of initial Capillary conditions Pump and of 0.3 3% µl/min buffer GEKVTIRCITNTDIDDDMNWYQQKP matched to κ chain from B (90% Acetonitrile, water, 0.1% formic acid) increasing up to n from variable domain. matched to λ chain from human while, LSPGERATLSCRASHSF was found similar to bovine κ light chai JSM Biochem Mol Biol 4(2): 1026 (2017) 2/8 Mamatha et al. (2017) Email:

Central Bringing Excellence in Open Access Each Ig heavy chain has an N-terminal variable (V) region unwanted binding events, leaving a bivalent Fab’2 fragment containing the antigen-binding site and a C-terminal constant (C) and Fab’ is important for better tissue penetration (derived region, encoded by an individual C region gene, that determines from Fab’2) or Fab may be preferred. Antibody fragments are the isotype of the antibody and provides effectors or signaling commonly used as the starting point for drug molecules as a functions [21]. result of having lower immunogenicity than intact antibodies. The molecular ions m/z - 882.39991+, m/z - 935.495211+, and Therefore sequential digestion by pepsin and pancreatin was m/z - 1625.88051+ were matched to sequences – FDVWGTGT, FI- found to enhance the effectiveness and economic feasibility of

ISRDNA and WVRGVPEKGLEWVA homologues to V-D-J-region. passive oral immunoprophylaxis with bovine milk Igs [24]. VDJ gene segments encode the variable region of the heavy chain. Molecular docking - tion of the human antibody repertoire which allow rapid humoral The small peptides (5-8 residues) obtained after in vitro immuneV (D) J recombination responses to a is wide the rangeprimary of pathogenicmechanism challenges for diversifica [24]. gastrointestinal digestion of IgG were used to examine their effect The peptide sequences FIFPP (m/z - 619.33696+), IKSRFI (m/z - on osmoporin of K. pneumoniae by molecular docking to further 763.48319+), IVKPGASV (m/z - 769.46976+), LLKPSETLSL (m/z validate the results of in vitro antibacterial property of buffalo - 1100.65685 2+), LKSRLSIMKD (m/z - 1190.69325+), WIRQAPG- colostrum IgG [20]. Osmoporin is a transmembrane pore in the KGL (m/z - 1124.64543+), IVQPGGMKLSCA (m/z - 1202.61511+), outer membrane of Gram-negative bacteria. It exists as a trimer WVRQVPEKGL (m/z - 1210.68221+), IRQPPGKGLEWIM consisting of chains A, B and C. Chain A of OSM (length 191.8Å) (m/z - 1523.82821+), LSLTCTTSGFSLS (m/z - 1316.64095+), was used for computational analysis (Figure 1). The docked LVKQSQTLSLTCT (m/z - 1421.76755+), LESLTCSVSGGSIS (m/z complexes of FDVWGTGT, IVKPGASV, IKSRFI and FIFPP showed - 1339.64167+), LRDTSVTAAL (m/z - 1046.58475+), VKQRPG- strong interaction with the residues Phe 189, Thr 229, Tyr 231, GGLGWIF (m/z - 1414.79606+), ISDYQMNWIRQTGKGLEWLYM Phe 257. These were imported in XP visualiser and corresponding (m/z - 2632.26932+), IRQPPGKGLEWIGCVSDSGSTSYYPT (m/z - docking poses were viewed in workspace and compared with the 2697.2984+), and FTFSDYYMAWVRQVPEKGLEWVANINYDGSST standard drug ampicillin (PDB ID: 4GCP). The overall dock scores (m/z - 3859.6617+) matched to the Ig heavy chain variable re- (GScore) for the 5 ligands 4GCP, FDVWGTGT, IVKPGASV, IKSRFI gion. Most of the sequence identity was from human Ig and a and FIFPP were -4.9, -4.8, -4.7, -4.6 and -4.3 respectively (Table very few matches were from mouse and bovine species (Table 2). 3). Hydrogen bond interactions in ampicillin were commonly However no data is available on the buffalo IgG either from serum found with Gly 216, Gly 217, Thr 229 and hydrophobic interaction or milk in the existing database. Hence these sequences can be with Ala 187, Phe 189, Leu 218, Tyr 231, Phe 257 (Figure 2). The uploaded to the protein database. This method can be exploited IgG peptide, FDVWGTGT hydrogen bonded with Tyr 214 and for generating antibody fragments with potential use as drugs, Gln 233 while hydrophobic bonding with Ala 187, Phe 189, Tyr so-called single-domain antibodies [22]. In analogy to our ap- 214, Leu 218, Tyr 231, Tyr 235 (Figure 3) was evident. Similarly, IVKPGASV, hydrogen bond interaction showed with Gln 233, monoclonal IgG2a F(ab’)2 fragment was developed by Stowers Gln 255 and hydrophobic interaction with Ala 187, Phe 189, Leu proach, a more economical and efficient production of mouse [9]for immunotherapeutic applications. 218, Tyr 231, Tyr 214, Phe 257 (Figure 4) and IKSRFI, hydrogen The use of antibodies in immunoassays such as ELISA bond interacted with Gly 217, Tyr 231 and hydrophobic with or western blotting is critical for antigen detection. However, Ala 187, Leu 218, Tyr 220, Leu 227, Ala 228, Tyr 231, Phe 257 (Figure 5). In FIFPP, Gln 233 involved in hydrogen interaction of the whole antibody itself. By selectively cleaving an antibody and hydrophobic interaction was found with Val 178, Phe 189, withsometimes proteases it is and beneficial using reducing to use a portionagents, itof is the possible molecule to engineer instead Tyr 214, Tyr 231 and Tyr 235 [Figure 6]. Some of the interactive fragments with discrete characteristics. Following reduction amino acid residues were found similar between ampicillin and selected peptides. The Gscore of IgG peptides were found closer were characterized by LC-MS, capillary isoelectric focusing and to the score of standard antibiotic ampicillin suggesting their glycanof disulfide mapping bonds, [23]. three It is also antibody possible domains with genetic (LC, Fd, engineering and Fc/2) probable interaction to the drug target. Further the lipophilic, techniques to express small fragments directly. In some cases it electrostatic interactions and binding energy among the ligand may be desirable to remove the Fc domain, which may mediate and the receptor residues were also shown in Table (3).

Table 1: Light chain matched peptide sequences of colostrum IgG treated with pepsin and pancreatin. Sl. Accession M/Z Sequence Protein Organism No. number Immunoglobulin kappa chain variable 1 1165.5583 LSVSSVTAEDSA CCM09771.1 Mouse region 2 1576.8006 LIYSTNQWPSPAVT BAN63134.1 IgG L chain Human 3 1818.8915 LSPGERATLSCRASHSF S16832 Ig kappa chain V region Bovine Immunoglobulin lambda chain variable 4 2345.0489 LPGTAAISELQSEDEGDYYCAL CAC94285.1 Human region Immunoglobulin kappa light chain 5 3454.6617 LSVATGEKVTIRCITNTDIDDDMNWYQQKP AAD39779.1 Mouse variable region

JSM Biochem Mol Biol 4(2): 1026 (2017) 3/8 Mamatha et al. (2017) Email:

Central Bringing Excellence in Open Access

Table 2: Heavy Chain matched peptide sequences of colostrum IgG treated with pepsin and pancreatin. Sl. Accession M/Z Sequence Protein Organism No. number 1 619.336962 FIFPP AAP205491 Immunoglobulin G heavy chain Human 2 763.48319 IKSRFI AAD24790.1 Immunoglobulin heavy chain variable region Mouse 3 769.46976 IVKPGASV CAC29289.1 Immunoglobulin heavy chain Human 4 882.39991 FDVWGTGT AEC22872.1 Immunoglobulin heavy chain V(D)J region Human 5 935.49521 FIISRDNA AAA37969.1 Immunoglobulin heavy chain V-D-region Mouse 6 1046.58475 LRDTSVTAAL AAR32583.1 Immunoglobulin heavy chain Human

7 1100.65685 LLKPSETLSL JH0428 Ig gamma chain V region (VH4-T14) Human Immunoglobulin G heavy chain variable 8 1124.64543 WIRQAPGKGL AIT38804.1 Human region 9 1190.69322 LKSRLSIMKD AAA98646.1 Immunoglobulin heavy chain variable region Bovine 10 1202.61511 IVQPGGMKLSCA AGA92777.1 Immunoglobulin heavy chain Mouse 11 1210.68221 WVRQVPEKGL AAK19265.1 Immunoglobulin heavy chain variable region Mouse 12 1316.64095 LSLTCTTSGFSLS ABF48111.1 Immunoglobulin gamma heavy chain Bovine 13 1339.64167 LESLTCSVSGGSIS CEF91268.1 Immunoglobulin heavy chain Bovine 14 1414.79606 VKQRPGGGLGWIF AAG39133.1 Immunoglobulin heavy chain Mouse 15 1421.76755 LVKQSQTLSLTCT AAC48521.1 Immunoglobulin heavy chain variable region Bovine Immunoglobulin G heavy chain variable 16 1523.82821 IRQPPGKGLEWIM AET35388.1 Human region 17 1625.88051 WVRGVPEKGLEWVA AAB48326.1 Ig heavy chain V-D-J-region Mouse 18 2632.26936 ISDYQMNWIRQTGKGLEWLYM AAQ05424.1 Ig heavy chain variable region, VH3 family Human 19 2697.2984 IRQPPGKGLEWIGCVSDSGSTSYYPT CAD82966.1 Ig heavy chain Human 20 3859.6617 FTFSDYYMAWVRQVPEKGLEWVANINYDGSST ADM44572.1 Immunoglobulin G1 heavy chain V- D-region Human

Table 3: Docking of 1OSM with IgG peptides from XP method. Binding energy Ligandsa G Score Lipophilic H-bond Electrostatic (kJ mol-1 ) 4GCP_chainA -4.9 -3.0 -1.4 0.1 -6.12 FDVWGTGT -4.8 -4.6 -0.8 -0.3 -5.89 IVKPGASV -4.7 -5.2 -0.7 -0.3 -5.56 IKSRFI -4.6 -4.5 -0.7 -0.1 -5.24 FIFPP -4.3 -4.3 -0.9 -0.1 -4.26

Molecular modeling has proven increasingly important in interaction with Omp may be new promising therapeutic complementing in vitro experimental results. Osmoporins are molecules to treat infections caused by Klebsiella. the transmembrane channel which allows diffusion of antibiotics and many other substances and is considered as an ideal target CONCLUSIONS for developing a drug against several bacterial infections Immunoglobulins are becoming major target oriented [25]. Typically, diffusion of IgG peptides depends on multiple biotherapeutics to treat an array of human diseases. IgG are the electrostatic interactions, hydrogen bonds and hydrophobic fastest growing class of therapeutic drugs, because of their high interaction that engage chemical groups on the antibiotic and amino acids of the constriction zone of the porin. The preliminary immunoglobulin has only recently become feasible with the study carried out using ovine β- casein hydrolyzate after pepsin, abilityspecificities to generate to target peptidecells. Proteomic motifs analysis of heavy of andbuffalo light colostrum chains. trypsin and chymotrypsin digestion was used as a biosensor for quorum sensing molecules and it indicated that the antimicrobial interpretation due to the frequency of interspersed variable and peptides from ovine β-casein may interfere with cell-signaling conservedSequences aminostill present acid sequences significant within challenges the same for masspeptides. spectral The in bacterial populations [26]. Earlier studies indicate OmpK35 observation of similar sequence properties in bovine, mouse and OmpK36 provide a channel that allows a wide range of and human data sets indicates that these are intrinsic features of antibiotics to penetrate the K. pneumoniae . These studies immunoglobulin primary structure which should be accounted for in any proteomic analysis of antibody repertoire, regardless of cephalosporin than OmpK36 does. Similarly, the IgG peptide of species. Moreover, antibodies can act as guides in the quest have shown that OmpK35 allows for more efficient penetration

JSM Biochem Mol Biol 4(2): 1026 (2017) 4/8 Mamatha et al. (2017) Email:

Central Bringing Excellence in Open Access

Figure 1 X-ray crystallographic structure of Outer Membrane Porin (OMP) of K. pneumoniae (PDB ID: 1OSM).

Figure 2 Receptor ligand interaction of ampicillin with OMP of K. pneumoniae showing interactive amino acid residues in the grid.

JSM Biochem Mol Biol 4(2): 1026 (2017) 5/8 Mamatha et al. (2017) Email:

Central Bringing Excellence in Open Access

Figure 3 Receptor ligand interaction of FDVWGTGT with OMP of K. pneumoniae showing interactive amino acid residues in the grid.

Figure 4 Receptor ligand interaction of IVKPGASV with OMP of K. pneumoniae showing interactive amino acid residues in the grid.

JSM Biochem Mol Biol 4(2): 1026 (2017) 6/8 Mamatha et al. (2017) Email:

Central Bringing Excellence in Open Access

Figure 5 Receptor ligand interaction of IKSFRI with OMP of K. pneumoniae showing interactive amino acid residues in the grid.

Figure 6 Receptor ligand interaction of FIFPP with OMP of K. pneumoniae showing interactive amino acid residues in the grid.

JSM Biochem Mol Biol 4(2): 1026 (2017) 7/8 Mamatha et al. (2017) Email:

Central Bringing Excellence in Open Access for small molecules (peptides) that have the ability to modulate 13. Smith SG, Mahon V, Lambert MA and Fagan RP. A molecular Swiss protein–protein interactions, which have traditionally only been army knife: OmpA structure, function and expression. FEMS Microbiol considered to be tractable targets for biological drugs. Hence, Lett. 2007; 273: 1-11. future studies are necessary to determine a safe and effective 14. Sugawara E, Kojima S, Nikaido H. Klebsiella pneumoniae major porins therapeutics or vaccine/s. their homologs OmpF and OmpC. J Bacteriol. 2016; ACKNOWLEDGEMENTS 198:OmpK35 3200-3208. and OmpK36 allow more efficient diffusion of β-lactams than Authors thank SAIF, IIT, Bombay for nanoLC-MS/MS facility, 15. Doménech-Sánchez A, Martínez-Martínez L, Hernández-Allés S, Schrodinger software, IOE instrumentation facility to carry out del Carmen Conejo M, Pascual Á, Tomás JM, et al., Role of Klebsiella this study. Mamatha Bhanu thank University Post-graduate cell pneumoniae OmpK35 porin in antimicrobial resistance. Antimicrob Agents Chemother. 2003; 47: 3332-3335. for the award of fellowship. 16. REFERENCES term low-frequency hospital outbreak of KPC-producing Klebsiella Tofteland S, Naseerinvolving U, Lislevand Intergenus JH, plasmid Sundsfjord diffusion A, Samuelsen and a persisting O. A long- 1. Abdel-Salam Z, Abdel Ghany Sh, Harith MA. Evaluation of pneumoniae environmental reservoir. PLoS One. 2013; 8: e59015. immunoglobulin’s in bovine colostrum using laser induced 17. Albertí S, Rodríquez-Quiñones, Schirmer T, Rummel G, Tomás JM, Rosenbusch JP, Benedí VJ. A porin from : 2. Jiang X, Hu J, Thirumalai D, Zhang X. Immunoglobulin transporting Klebsiella pneumoniae fluorescence. Talanta. 2014; 129: 15-19. sequence homology, three-dimensional model, and complement receptors are potential targets for the immunity enhancement and binding. Infect Immun. 1995; 63: 903-910. generation of mammary gland bioreactor. Front Immunol. 2016; 7: 214. 18. Kawai K, Liu Y, Ohnishi K, Oshima S. A conserved 37 kDa outer membrane protein of is an effective vaccine 3. Hurley WL, Peter KT. Perspectives on immunoglobulins in colostrum Edwardsiella tarda candidate. Vaccine. 2004; 22: 3411-3418. and milk. Nutrients. 2011; 3: 442-474. 19. Mamatha Bhanu LS, Nishimura SI, Aparna HS. Glycome characterization 4. El-Loly MM. Bovine milk immunoglobulins in relation to human of immunoglobulin G from buffalo ( ) colostrums. health. Int J. Dairy Sci. 2007; 2: 183-195. Bubalus bubalis Glycoconj J. 2015; 32: 625-634. 5. Jasion VS, Burnett BP. Survival and digestibility of orally-administered 20. Mamatha Bhanu LS, Nishimura SI, Aparna HS. Inhibitory potential of immunoglobulin preparations containing IgG through the Buffalo ( ) colostrum immunoglobulin G on gastrointestinal tract in humans. Nutr j. 2015; 14: 22. Bubalus bubalis Klebsiella pneumoniae. Int J Biol Macromol. 2016; 88: 138-145. 6. Mix E, Robert G, Uwe KZ. Immunoglobulins-basic considerations. J 21. Harmsen MM, Haard HJ. Properties, production, and applications Neurol. 2006; 255: 9-17. of camelid single-domain antibody fragments. Appl Microbiol 7. Boutz DR, Horton AP, Wine Y, Lavinder JJ, Georgiou G, Marcotte EM. Biotechnol. 2007; 77: 13-22. 22. An Y, Zhang Y, Mueller HM, Shameem M, Chen X. A new tool for Chem. 2014; 10: 4758-4766. Proteomic identification of monoclonal antibodies from serum. Anal monoclonal antibody analysis: application of IdeS proteolysis in IgG 8. Carter Paul J. Potent antibody therapeutics by design. Nat Rev Immunol. 2006; 6: 343-357. 23. domain-specificPetschow BW, Talbott characterization. RD. Reduction MAbs. in 2014;virus-neutralizing 6: 879-893. activity of

9. Stowers RS, Callihan JA, Bryers JD. Optimal Conditions for F (ab’)2 a bovine colostrum immunoglobulin concentrate by gastric acid and Antibody Fragment Production from Mouse IgG2a. Journal of digestive enzymes. J Pediatr Gastroenterol Nutr. 1994; 19: 228-235. undergraduate research in bioengineering. 2010. 24. Fernández, Lucía, Robert EW Hancock. Adaptive and mutational 10. Boushaba R, Peter K, Nigel Slater KH. Kinetics of whole serum Microbiol Rev. 2012; 25: 661-681. Biotechnol prog. 2003; 4: 1176-1182. resistance: role of porins and efflux pumps in drug resistance. Clin and prepurified IgG digestion by pepsin for F(ab’) 2 manufacture. 25. Lou H, Chen M, Black SS, Bushell SR, Ceccarelli M, Mach T, Bayley H, et 11. Jiang XR, Song A, Bergelson S, Arroll T, Parekh B, May K, et al. Advances al. Altered antibiotic transport in OmpC mutants isolated from a series in the assessment and control of the effector functions of therapeutic of clinical strains of multi-drug resistant E. coli. PLoS One. 2011; 6: antibodies. Nat Rev Drug Discov. 2011; 10: 101-111. e25825. 12. Jeannin P, Magistrelli G, Goetsch L, Haeuw JF, Thieblemont N, Bonnefoy 26. Expósito IL, Recio I, Antibacterial activity of peptides and folding JY, et al. Outer membrane protein A (OmpA): a new pathogen- variants from milk proteins. j idairyj. 2006; 16: 1294-1305. associated molecular pattern that interacts with antigen presenting cells-impact on vaccine strategies. Vaccine. 2002; 20: A23-A27.

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.

JSM Biochem Mol Biol 4(2): 1026 (2017) 8/8