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• W2108475283 abstract "Paraoxonases (PONs) are a family of lactonases with promiscuous enzyme activity that has been implicated in multiple diseases. PON2 is intracellularly located, is the most ubiquitously expressed PON, and has the highest lactonase activity of the PON family members. Whereas some single-nucleotide polymorphisms (SNPs) in PON1 have resulted in altered enzymatic activity in serum, to date the functional consequences of SNPs on PON2 function remain unknown. We hypothesized that a common PON2 SNP would result in impaired lactonase activity. Substitution of cysteine for serine at codon 311 in recombinant PON2 resulted in normal protein production and localization but altered glycosylation and decreased lactonase activity. Moreover, we screened 200 human lung samples for the PON2 Cys311 variant and found that in vivo this mutation impaired lactonase activity. These data suggest that impaired lactonase activity may play a role in innate immunity, atherosclerosis, and other diseases associated with the PON2 311 SNP. Paraoxonases (PONs) are a family of lactonases with promiscuous enzyme activity that has been implicated in multiple diseases. PON2 is intracellularly located, is the most ubiquitously expressed PON, and has the highest lactonase activity of the PON family members. Whereas some single-nucleotide polymorphisms (SNPs) in PON1 have resulted in altered enzymatic activity in serum, to date the functional consequences of SNPs on PON2 function remain unknown. We hypothesized that a common PON2 SNP would result in impaired lactonase activity. Substitution of cysteine for serine at codon 311 in recombinant PON2 resulted in normal protein production and localization but altered glycosylation and decreased lactonase activity. Moreover, we screened 200 human lung samples for the PON2 Cys311 variant and found that in vivo this mutation impaired lactonase activity. These data suggest that impaired lactonase activity may play a role in innate immunity, atherosclerosis, and other diseases associated with the PON2 311 SNP. The paraoxonase (PON) 3The abbreviations used are: PONparaoxonaseSNPsingle-nucleotide polymorphism3OC12-HSLN-3-oxododecanoyl homoserine lactonePNGase Fpeptide-N-glycosidase FEndo Hendoglycosidase HALSamyotrophic lateral sclerosisCHOChinese hamster ovaryERendoplasmic reticulum. family consists of three members: PON1, PON2, and PON3 (1.Primo-Parmo S.L. Sorenson R.C. Teiber J. La Du B.N. Genomics. 1996; 33: 498-507Crossref PubMed Scopus (586) Google Scholar, 2.Mochizuki H. Scherer S.W. Xi T. Nickle D.C. Majer M. Huizenga J.J. Tsui L.C. Prochazka M. Gene. 1998; 213: 149-157Crossref PubMed Scopus (151) Google Scholar, 3.Camps J. Marsillach J. Joven J. Crit. Rev. Clin. Lab. Sci. 2009; 46: 83-106Crossref PubMed Scopus (210) Google Scholar). PON1 was the first family member described and was named for its ability to degrade the organophosphate paraoxon. PON1 was subsequently found to inhibit low density lipoprotein oxidation and be important for cardiovascular disease (4.Durrington P.N. Mackness B. Mackness M.I. Arterioscler. Thromb. Vasc. Biol. 2001; 21: 473-480Crossref PubMed Scopus (726) Google Scholar). In addition, analysis of PON1 single-nucleotide polymorphisms (SNPs) has linked PON1 to the pathogenesis of numerous other human disorders including atherosclerosis, diabetes, cerebrovascular disease, Alzheimer disease, amyotrophic lateral sclerosis, organophosphate susceptibility, and Parkinson disease (5.Furlong C.E. J. Biochem. Mol. Toxicol. 2007; 21: 197-205Crossref PubMed Scopus (60) Google Scholar, 6.Bhattacharyya T. Nicholls S.J. Topol E.J. Zhang R. Yang X. Schmitt D. Fu X. Shao M. Brennan D.M. Ellis S.G. Brennan M.L. Allayee H. Lusis A.J. Hazen S.L. JAMA. 2008; 299: 1265-1276Crossref PubMed Scopus (454) Google Scholar, 7.Erlich P.M. Lunetta K.L. Cupples L.A. Huyck M. Green R.C. Baldwin C.T. Farrer L.A. Hum. Mol. Genet. 2006; 15: 77-85Crossref PubMed Scopus (83) Google Scholar, 8.Humbert R. Adler D.A. Disteche C.M. Hassett C. Omiecinski C.J. Furlong C.E. Nat. Genet. 1993; 3: 73-76Crossref PubMed Scopus (770) Google Scholar, 9.Zintzaras E. Hadjigeorgiou G.M. J. Hum. Genet. 2004; 49: 474-481Crossref PubMed Scopus (95) Google Scholar, 10.Furlong C.E. Cole T.B. Jarvik G.P. Pettan-Brewer C. Geiss G.K. Richter R.J. Shih D.M. Tward A.D. Lusis A.J. Costa L.G. Neurotoxicology. 2005; 26: 651-659Crossref PubMed Scopus (72) Google Scholar, 11.Draganov D.I. La Du B.N. Naunyn. Schmiedebergs Arch. Pharmacol. 2004; 369: 78-88Crossref PubMed Scopus (366) Google Scholar). Despite these numerous association studies, the mechanism(s) underlying the role of PON in disease pathogenesis remains to be fully determined, in part because of uncertainty regarding the endogenous or natural substrate of PON. paraoxonase single-nucleotide polymorphism N-3-oxododecanoyl homoserine lactone peptide-N-glycosidase F endoglycosidase H amyotrophic lateral sclerosis Chinese hamster ovary endoplasmic reticulum. However, the native enzyme activity of PON was recently found to be as a lactonase (12.Khersonsky O. Tawfik D.S. Biochemistry. 2005; 44: 6371-6382Crossref PubMed Scopus (372) Google Scholar), suggesting that despite its known enzymatic promiscuity for other substrates including esters and phosphotriesters, its endogenous substrates are lactones. Importantly, all three PON family members have conserved their lactonase activity, but individual PON family members have widely disparate enzymatic activities toward other substrates. One specific lactone-containing molecule that PONs can degrade is the bacterial quorum-sensing molecule, N-3-oxododecanoyl homoserine lactone (3OC12-HSL) (13.Ozer E.A. Pezzulo A. Shih D.M. Chun C. Furlong C. Lusis A.J. Greenberg E.P. Zabner J. FEMS Microbiol. Lett. 2005; 253: 29-37Crossref PubMed Scopus (176) Google Scholar, 14.Teiber J.F. Horke S. Haines D.C. Chowdhary P.K. Xiao J. Kramer G.L. Haley R.W. Draganov D.I. Infect. Immun. 2008; 76: 2512-2519Crossref PubMed Scopus (129) Google Scholar, 15.Draganov D.I. Teiber J.F. Speelman A. Osawa Y. Sunahara R. La Du B.N. J. Lipid Res. 2005; 46: 1239-1247Abstract Full Text Full Text PDF PubMed Scopus (564) Google Scholar, 16.Teiber J.F. Draganov D.I. La Du B.N. Biochem. Pharmacol. 2003; 66: 887-896Crossref PubMed Scopus (132) Google Scholar). 3OC12-HSL is an acyl-homoserine lactone that Pseudomonas aeruginosa, a common cause of hospital-acquired infections and an important cause of pulmonary morbidity and mortality in cystic fibrosis, uses to control biofilm formation and virulence factor production (17.Bassler B.L. Losick R. Cell. 2006; 125: 237-246Abstract Full Text Full Text PDF PubMed Scopus (836) Google Scholar, 18.Parsek M.R. Greenberg E.P. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 8789-8793Crossref PubMed Scopus (487) Google Scholar). Of the three PON family members, PON2 has the greatest lactonase activity toward 3OC12-HSL (13.Ozer E.A. Pezzulo A. Shih D.M. Chun C. Furlong C. Lusis A.J. Greenberg E.P. Zabner J. FEMS Microbiol. Lett. 2005; 253: 29-37Crossref PubMed Scopus (176) Google Scholar, 19.Stoltz D.A. Ozer E.A. Ng C.J. Yu J.M. Reddy S.T. Lusis A.J. Bourquard N. Parsek M.R. Zabner J. Shih D.M. Am. J. Physiol. Lung Cell. Mol. Physiol. 2007; 292: L852-L860Crossref PubMed Scopus (108) Google Scholar). In contrast to PON1 and PON3, PON2 is not present in serum, and it has minimal arylesterase and paraoxonase activity. Human airway epithelial cells degrade 3OC12-HSL (19.Stoltz D.A. Ozer E.A. Ng C.J. Yu J.M. Reddy S.T. Lusis A.J. Bourquard N. Parsek M.R. Zabner J. Shih D.M. Am. J. Physiol. Lung Cell. Mol. Physiol. 2007; 292: L852-L860Crossref PubMed Scopus (108) Google Scholar, 20.Chun C.K. Ozer E.A. Welsh M.J. Zabner J. Greenberg E.P. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 3587-3590Crossref PubMed Scopus (236) Google Scholar), and murine PON2-deficient airway epithelial cells have an impaired ability to inactivate 3OC12-HSL (19.Stoltz D.A. Ozer E.A. Ng C.J. Yu J.M. Reddy S.T. Lusis A.J. Bourquard N. Parsek M.R. Zabner J. Shih D.M. Am. J. Physiol. Lung Cell. Mol. Physiol. 2007; 292: L852-L860Crossref PubMed Scopus (108) Google Scholar). In addition, we have recently found that PON1-transgenic Drosophila melanogaster are protected from organophosphate poisoning and P. aeruginosa lethality (21.Stoltz D.A. Ozer E.A. Taft P.J. Barry M. Liu L. Kiss P.J. Moninger T.O. Parsek M.R. Zabner J. J. Clin. Invest. 2008; 118: 3123-3131Crossref PubMed Scopus (75) Google Scholar). PON2 activity in airway epithelial cells may represent a novel antibacterial defense mechanism against invading pathogens that utilize acyl-homoserine lactones for quorum sensing and virulence regulation. Genetic variation in human genes can have a substantial impact on host responses, and SNP analysis may be used in anticipating responses and outcomes or in adjusting drug dosing. In this study we asked whether a common PON2 SNP, a serine to cysteine amino acid change at codon 311 in PON2 (2.Mochizuki H. Scherer S.W. Xi T. Nickle D.C. Majer M. Huizenga J.J. Tsui L.C. Prochazka M. Gene. 1998; 213: 149-157Crossref PubMed Scopus (151) Google Scholar), alters PON2 lactonase activity for 3OC12-HSL. This SNP is fairly common in the general population and is strongly conserved evolutionarily. We chose this PON2 SNP because PON2 has the greatest lactonase activity of PON family members, phylogenetic analysis suggests that PON2 is the oldest PON family member (11.Draganov D.I. La Du B.N. Naunyn. Schmiedebergs Arch. Pharmacol. 2004; 369: 78-88Crossref PubMed Scopus (366) Google Scholar), and several PON1 SNPs are in linkage disequilibrium with PON2 SNPs. We found that recombinant PON2 Cys311 exhibits an impaired ability to inactivate 3OC12-HSL and that airway epithelial cells from humans homozygous for Cys/Cys at PON2 amino acid position 311 also have an impaired ability to degrade 3OC12-HSL. Chinese hamster ovary (CHO) cells were used for transfection studies because these cells have no endogenous PON or lactonase activity (13.Ozer E.A. Pezzulo A. Shih D.M. Chun C. Furlong C. Lusis A.J. Greenberg E.P. Zabner J. FEMS Microbiol. Lett. 2005; 253: 29-37Crossref PubMed Scopus (176) Google Scholar). CHO cells were cultured as monolayers in plastic dishes and transfected with plasmids expressing hPON2 Cys311 or Ser311 using Lipofectamine 2000 (Invitrogen), according to the manufacturer's protocol, and allowed to express for 24–48 h. For epithelial cell lysate experiments, epithelia were first washed with cold phosphate-buffered saline (containing calcium and magnesium), and then 50 μl of lysis buffer (50 mm Tris·HCl, pH 6.9, 150 mm NaCl, 10 μm leupeptin, 10 μm aprotinin, 1 μm pepstatin A, 1 mm phenylmethylsulfonyl fluoride, 0.1 mg/ml benzamidine) was added to the apical surface. After 20 min of rocking in lysis buffer at 4 °C, the cells were scraped free from the membrane with a pipette tip and lysed by sonication (10 pulses, pulse duration of ∼1 s) (Branson Sonifier 250, Danbury, CT). The cellular debris was cleared from the lysate by centrifugation (4500 × g for 30 s at 4 °C). The relative protein concentrations were determined by the Bio-Rad protein assay (500-0006). Lysate preparations (10–20%) were diluted in phosphate-buffered saline (containing calcium and magnesium) and incubated in the presence of 3OC12-HSL (RTI International, Research Triangle Park, NC) at 37 °C. 3OC12-HSL in acidified ethyl acetate was dried under a nitrogen gas stream and then dissolved in phosphate-buffered saline (containing calcium and magnesium) to achieve a final concentration of 10 μm 3OC12-HSL. At various time points, 6-μl aliquots were collected, added to 100 μl of ethyl acetate, and stored in airtight glass vials at −20 °C. 3OC12-HSL was obtained from RTI International. 3OC12-HSL was measured in a quantitative bioassay as previously described using Escherichia coli MG4 (pKDT17) (22.Pearson J.P. Gray K.M. Passador L. Tucker K.D. Eberhard A. Iglewski B.H. Greenberg E.P. Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 197-201Crossref PubMed Scopus (809) Google Scholar). PON2 chemical deglycosylation was performed by first transfecting CHO cells with a plasmid expressing PON2 Cys311 or Ser311. 30 min later, tunicamycin was added to the cell culture medium at a final concentration of 0.5 μg/ml. 36–48 h later cell lysates were harvested, and SDS-PAGE was performed for PON2. Peptide-N-glycosidase F (PNGase F) (Sigma) digestion was performed according to the manufacturer's directions. Briefly, cell lysates were collected in lysis buffer (50 mm Tris·HCl, pH 7.5, 138 mm NaCl, 5 mm EDTA, 1 mm EGTA, 1 mm NaF, phenylmethylsulfonyl fluoride, leupeptin, pepstatin, and aprotinin) and incubated with PNGase F (10 units) for 4 h at 37 °C. The reaction was stopped by incubating at 100 °C for 5 min. Deglycosylation was then assessed with SDS-PAGE. Endoglycosidase H (Endo H) (Sigma) digestion was performed according to the manufacturer's directions. The cell lysates were incubated with reaction buffer and denaturation solution and heated for 5 min at 100 °C. Following cooling of the sample, Endo H was added to the samples and incubated for 4 h at 37 °C. Digestion was then determined using SDS-PAGE. CHO cell lysates were made by incubation in lysis buffer (50 mm Tris·HCl, pH 7.5, 138 mm NaCl, 5 mm EDTA, 1 mm EGTA, 1 mm NaF, phenylmethylsulfonyl fluoride, leupeptin, pepstatin, and aprotinin) with 1% Triton X-100 for 20 min at 4 °C on a rocker. The cells were scraped from the tissue culture plates and transferred to a microcentrifuge tube. Sonication was performed for 10 s, and cell debris was cleared by centrifugation at 4,500 × g for 30 s at 4 °C. The lysates were then combined with loading buffer and separated by SDS-PAGE. Protein was then transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore, Billerica, MA). Polyvinylidene difluoride membranes were blocked either overnight at 4 °C or for 2 h at room temperature in 5% bovine serum albumin in phosphate-buffered saline. The membranes were then incubated with the PON2 antibody (1:1000; rabbit, polyclonal anti-human PON2; Orbigen, San Diego, CA) for 2 h at room temperature and then washed three times in 1× TTBS (137 mm NaCl, 2.7 mm KCl, 2.5 mm Tris, 0.05% Tween 20). Secondary antibodies were conjugated to horseradish peroxidase (Amersham Biosciences) and used at 1:10,000 for 1 h. Following three washes with 1× TTBS, immunoreactive bands were detected with SuperSignal solution (Pierce) and exposed to film. Site-directed mutagenesis was performed with the Stratagene QuikChange™ kit according to the manufacturer's standard protocol (Stratagene, Cedar Creek, TX). Double-stranded DNA template was prepared by a standard megaprep protocol (Qiagen). Mutant strand synthesis was performed with the following primers for N226Q (5′-GATTCAGCAAATGGGATCCAGATTTCACCTGATGATAAG-3′), for N254Q (5′-GTTTTGGAAAAACACACTAATATGCAGTTAACTCAGTTGAAGG-3′), for N269Q (5′-TGGATACACTGGTGGATCAGTTATCTATTGATCCTTCC-3′), for N323Q (5′-ACTACAGTTTATGCCAACCAGGGGTCTGTTCTCCAAGG-3′), for N254A (5′-GTTTTGGAAAAACACACTAATATGGCTTTAACTCAGTTGAAGG-3′), and for N323A (5′-ACTACAGTTTATGCCAACGCTGGGTCTGTTCTCCAAGG-3′). The reaction products were then treated with the restriction endonuclease DpnI to enrich for multiply mutated single-stranded DNA. This reaction mixture was then transformed into XL10-Gold® Ultracompetent cells and spread on Luria-Bertani ampicillin agar plates (containing 80 ng/ml 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal) and 20 mm isopropyl β-d-thiogalactopyranoside). The colonies were picked, and site-specific mutations were confirmed by DNA sequencing (DNA Facility, University of Iowa). CHO cells were plated on collagen-coated four-well chamber slides and transfected with a plasmid encoding either human PON2 Cys311 or Ser311. At 36 h following transfection, the cells were fixed with 1% paraformaldehyde in methanol, blocked in 2% bovine serum albumin in SuperBlock (Pierce), and stained with anti-human PON2 antibody (rabbit polyclonal, Orbigen) and a monoclonal antibody specific for protein-disulfide isomerase to localize the endoplasmic reticulum (Assay Designs, Ann Arbor, MI). Confocal images were acquired with a Bio-Rad MRC-1024 laser scanning confocal system mounted on a Nikon E600 microscope. Primary human airway epithelial cells were isolated from the trachea and bronchi of human donor lungs. The isolated cells were seeded onto collagen-coated, semi-permeable membranes (0.6-cm2 Millicell-HA; Millipore, Bedford, MA) and grown at the air-liquid interface as previously described (23.Karp P.H. Moninger T. Weber S.P. Nesselhauf T.S. Launspach J. Zabner J. Welsh M.J. Epithelial Cell Culture Protocols.in: Wise C. Humana Press, Inc., Totowa, NJ2002: 115-137Google Scholar). After genotypic identification of PON2 SNPs in donor fibroblasts, primary or passage 1 cryopreserved airway epithelial stocks were thawed and expanded for the described studies. All of the experiments were performed on well differentiated human airway epithelia (more than 14 days after seeding of cells). The samples were collected with the approval of the University of Iowa Institutional Review Board. DNA samples were genotyped for the presence of several common coding and noncoding polymorphisms, including PON2 Cys311 → Ser. PCR primers for amplifying the sequences containing the SNP were designed using Primer 3. The PCR primers are listed in the supplemental materials. Sense and antisense probes used for fluorescence polarization-single base extension detection of SNPs were designed using Primer Premier. The sense and antisense probes for genotyping the PON2 Cys311 → Ser SNP are: 5′-CTG TAG TCA CTG TAG GCT TCT CA-3′ and 5′-CCG CAT CCA GAA CAT TCT AT-3′, respectively. Each genotyping reaction consisted of a polymerase chain reaction followed by a single base pair extension reaction using either the sense or antisense probe and dideoxynucleotides labeled with carboxytetramethylrhodamine (TAMRA) or R110 for the alternative alleles. The fluorescence signal was detected by fluorescence polarization using the analyst HT (Molecular Devices, Sunnyvale, CA), and genotypes were determined based on the plot of TAMRA versus R110 signal values. All of the experiments were performed in at least triplicate, and the data are presented as the means ± S.E. of the mean (S.E.). Comparisons between two groups were made with Student's t test. We cloned PON2 and asked whether mutating the 311 amino acid, from a serine to cysteine, would affect PON2 lactonase activity. Lysates from CHO cells expressing both PON2 Cys311 and Ser311 inactivated 3OC12-HSL over time, but PON2 Cys311 lysates had an impaired ability compared with PON2 Ser311 lysates (Fig. 1A). These data suggest that cysteine substitution at amino acid 311 impairs PON2 function through mechanisms other than alternative splicing, linkage disequilibrium, or promoter differences. To test whether the PON2 Cys311 variant expresses similar PON2 protein levels as PON2 Ser311, PON2 immunoblotting was performed in CHO cell lysates 48 h following transfection. Fig. 1B shows that in PON2 Cys311 samples we observed a single immunoreactive band migrating slightly higher than 37 kDa, a molecular mass consistent with PON2. In PON2 Ser311 samples, we also observed a similarly sized upper band as in PON2 Cys311, but noted an additional band with greater electrophoretic mobility several kDa lower than the upper PON2 band. Densitometric analysis and quantification showed no difference in total PON2 protein levels between PON2 Cys311 and Ser311 groups (Fig. 1C). These findings suggest that the 311 amino acid change does not alter PON2 protein stability. The smaller, second band could result from alternative splicing (although this seems highly unlikely to occur using cDNA) (2.Mochizuki H. Scherer S.W. Xi T. Nickle D.C. Majer M. Huizenga J.J. Tsui L.C. Prochazka M. Gene. 1998; 213: 149-157Crossref PubMed Scopus (151) Google Scholar), from PON2 aggregation (24.Josse D. Ebel C. Stroebel D. Fontaine A. Borges F. Echalier A. Baud D. Renault F. Le Maire M. Chabrieres E. Masson P. J. Biol. Chem. 2002; 277: 33386-33397Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), or most likely from differential post-translational modification or proteolysis. PON2 has four putative N-linked glycosylation sites at asparagine residues. Purified PON1 is ∼15.8% carbohydrate by weight (25.Gan K.N. Smolen A. Eckerson H.W. La Du B.N. Drug Metab. Dispos. 1991; 19: 100-106PubMed Google Scholar). To investigate the glycosylation of PON2 and determine whether PON2 Cys311 and Ser311 are differentially glycosylated, we first expressed PON2 Cys311 or Ser311 in CHO cells and then treated with tunicamycin, an antibiotic from the bacterium Streptomyces iysosuperficus that inhibits GlcNAc phosphotransferase, one of the initial steps required for glycoprotein synthesis. Two days later, cell lysates were harvested, and Western blot analysis showed that, in PON2 Cys311 samples, the predominant immunoreactive band following tunicamycin treatment had a greater electrophoretic mobility compared with the PON2 band in untreated cells (Fig. 2A). This finding is consistent with N-linked core glycosylation of PON2. Similar findings were observed with PON2 Ser311 (Fig. 2B). After tunicamycin treatment there was no difference in the appearance of the PON2 band between Cys311 and Ser311 variants. This suggests that the lower band in PON2 Ser311 samples represents differential or incomplete glycosylation compared with the upper band in PON2 Cys311 and Ser311. Because tunicamycin has a number of effects on the cell including induction of the unfolded protein response, we also treated cell lysates with PNGase F at 48 h following transfection. PNGase F is an enzyme that cleaves N-linked glycoproteins between the innermost GlcNAc and asparagine residues. N-Linked deglycosylation with PNGase F caused both PON2 Cys311 and Ser311 migration patterns to change as observed with tunicamycin treatment, and after PNGase F treatment there was no difference between the Cys and Ser variants (Fig. 2). PON2 was also sensitive to treatment with Endo H, an endoglycosidase that cleaves the chitobiose core of high mannose and oligosaccharides from N-linked glycoproteins. When proteins are correctly processed through the endoplasmic reticulum and Golgi complex, they become resistant to Endo H. Therefore sensitivity to Endo H indicates the presence of proteins that have not been processed beyond the ER. We observed qualitatively similar results when comparing Endo H, tunicamycin, and PNGase F treatments in both PON2 Cys311 and Ser311 samples (Fig. 2). From these results, we conclude that PON2 undergoes N-linked core glycosylation, and incomplete glycosylation is likely responsible for the additional band observed when PON2 Ser311 is expressed in CHO cells. Based on the two different bands of glycosylated PON2, we predicted that at least two of the four putative N-linked glycosylation sites are glycosylated in PON2. To determine which asparagine residues of the Asn-Xaa-(Ser/Thr) consensus sequence are N-glycosylated in PON2 Cys311 and Ser311 variants, putative N-linked glycosylation sites were disrupted by substituting glutamine for asparagine individually and also in combination (quadruple mutant). Western blotting for PON2 revealed one immunoreactive band in wild-type PON2 Cys311 and the N226Q and N269Q mutants. Cell lysates from the N254Q and N323Q mutants also showed one PON2 immunoreactive band, but with a greater electrophoretic mobility than wild-type PON2 Cys311 (Fig. 3A). Lysate from the quad mutant had a PON2 immunoreactive band with a smaller molecular mass than either N254Q or N323Q. This shows that, for PON2 Cys311, two of the four putative N-linked glycosylation sites undergo oligosaccharide addition: asparagine residues 254 and 323. We next hypothesized that the upper band in PON2 Ser311 also represents glycosylation at both asparagine 254 and 323, whereas the lower electrophoretic mobility band could be glycosylation at either one of these asparagine residues or a mixture of individually glycosylated PON2 proteins. Alternatively, PON2 Ser311 could use different asparagine residues for core glycosylation. Similar to the PON2 Cys311, only asparagine 254 and 323 were glycosylated (Fig. 3B). The immunoreactive band in both PON2 Cys311 and Ser311 quad mutants had a similar molecular mass compared with PON2 treated with PNGase F or Endo H (Fig. 3, C and D). These substitution experiments allowed us to investigate which site is glycosylated in the lower band of PON2 Ser311. The N254Q mutant had an unglycosylated band as well as an intermediate glycosylated band, suggesting that the lower PON2 Ser311 band is glycosylated at asparagine 254. This is confirmed by the finding that the N323Q substitution results in a single intermediate band (Fig. 3B). Substitution at asparagine 323 caused loss of the upper band, but the lower band remained in place. These data show that the upper band in PON2 Cys311 and Ser311 is glycosylated at asparagine 254 and 323, whereas the lower band in PON2 Ser311 is only glycosylated at asparagine 254. We hypothesized that differential glycosylation of PON2 Cys311 and Ser311 accounts for the differences in lactonase activity between the 311 variants, so we initially asked whether glycosylation is important for PON2 inactivation of 3OC12-HSL. Lysates from CHO cells transfected with the PON2 Cys311 or Ser311 with the N254G mutation had preserved 3OC12-HSL lactonase activity (Fig. 4, A and B). We expected that if the lower electrophoretic band, only glycosylated at asparagine 254, in wild-type PON2 Ser311 accounts for its enhanced 3OC12-HSL inactivation, then glycosylation at asparagine 323 must inhibit PON2 lactonase activity. In contrast, we found that the N323Q mutant had impaired lactonase activity, showing that glycosylation at asparagine 323 is important for PON2 function. Similarly, lysates from CHO cells transfected with the PON2 Cys311 or Ser311 quad mutant (N226Q/N254Q/N269Q/N323Q) were unable to degrade 3OC12-HSL, also showing that glycosylation might be important for PON2 3OC12-HSL inactivation. Mutation of asparagine residues to glutamine could have other effects on PON2 structure and activity besides disrupting N-linked glycosylation. Therefore, we constructed additional mutants of the PON2 Cys311 and Ser311 variants by replacing asparagine with alanine individually at positions 254 and 323, as well as a double mutant, N254A/N323A. Compared with the glutamine mutants, we found similar patterns of PON2 immunoreactive bands in the N254A, N323A, and N254A/N323A mutants (Fig. 4C). In addition, the N323A and N254A/N323A mutants had an impaired ability to inactivate 3OC12-HSL (Fig. 4, D and E). From these data we conclude that: 1) PON2 undergoes N-linked core glycosylation at asparagine residues 254 and 323; 2) the upper band in PON2 Ser311 is glycosylated at asparagine 254 and 323, whereas the lower band is only glycosylated at asparagine 254; 3) glycosylation at asparagine 323 is required for PON2 lactonase activity; and 4) differential glycosylation between the PON2 Cys311 and Ser311 variants does not account for greater lactonase activity by PON2 Ser311. Our data show that PON2 is Endo H-sensitive and does not undergo complex glycosylation, 4D. A. Stoltz, T. J. Recker, and J. Zabner, unpublished data. suggesting that PON2 resides in the ER. Moreover, recent work by Horke et al. (26.Horke S. Witte I. Wilgenbus P. Krüger M. Strand D. Förstermann U. Circulation. 2007; 115: 2055-2064Crossref PubMed Scopus (198) Google Scholar) demonstrated PON2 localization to the ER in endothelial cells. To test for changes in PON2 cellular localization as a mechanism of disrupted lactonase activity between PON2 Cys311 and Ser311 variants, immunohistochemical staining for PON2 was performed in CHO cells following transfection with either PON2 Cys311- or Ser311-expressing plasmids. A similar pattern of staining for both PON2 Cys311 and Ser311 was observed with the most intense staining corresponding to an ER location (Fig. 5). To test whether the PON2 311 SNP also affects lactonase activity in vivo, we asked whether naturally occurring PON2 mutations at position 311 would affect the ability of primary human airway epithelial cells to degrade 3OC12-HSL. We first isolated DNA from 200 human donor lung samples and performed genotyping by polymerase chain reaction allele-specific oligonucleotide hybridization assays for the PON2 S311C polymorphism and a number of other common PON1, PON2, and PON3 allelic variants including coding (PON1 Q192R, PON1 L55M, PON3 C133A, and PON3 G99A) and promoter (PON1 G-907C) polymorphisms (supplemental Table S1). For PON2, we found that 62% of human donor samples were homozygous for Ser/Ser, 35% heterozygous for Ser/Cys, and 3% homozygous for Cys/Cys at amino acid 311 (Fig. 6A). These findings are consistent with the HapMAP-CEU European SNP data base showing 57% of individuals homozygous for Ser/Ser, 38% heterozygous for Ser/Cys, and 5% homozygous for Cys/Cys. To test whether differences in the common PON2 311 variant affect airway epithelia inactivation of 3OC12-HSL, human airway epithelial cells from four individuals homozygous for cysteine, five homozygous for serine, and two heterozygous for cysteine/serine at amino acid position 311 were grown at the air-liquid interface. We chose these donors based upon their PON2 311 SNP and similarities in other described SNPs for PON1 and PON3. Lysates from airway epithelial cells homozygous for serine degraded 3OC12-HSL with ∼25% active 3OC12-HSL remaining after 60 min of exposure (Fig. 6B). In contrast, airway epithelial cell samples from donors homozygous for cysteine had an impaired ability to inactivate 3OC12-HSL, and samples from heterozygotes (Ser/Cys) demonstrated an int" @default.
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• W2108475283 title "A Common Mutation in Paraoxonase-2 Results in Impaired Lactonase Activity" @default.
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