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The Journal of Immunology, 2005, 174: 2314-2317.
Copyright © 2005 by The American Association of Immunologists

Preferential Recognition of Undisruptable Dimers of Inducible Nitric Oxide Synthase by a Monoclonal Antibody Directed against an N-Terminal Epitope1

Tuhina Mazumdar and N. Tony Eissa

Pulmonary and Critical Care Section, Department of Medicine, Baylor College of Medicine, Houston, TX 77030


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Overproduction of NO by inducible NO synthase (iNOS) has been implicated in the pathogenesis of many diseases. iNOS is active only as a homodimer in which the subunits align in a head-to-head manner, with the N-terminal oxygenase domains forming the dimer interface and a zinc metal center stabilizing the dimer. Thus, dimerization represents a critical locus for therapeutic interventions for regulation of NO synthesis. We have recently shown that intracellular iNOS forms dimers that are "undisruptable (UD)" by heat, SDS, strong denaturants, and/or reducing agents. Our data further suggest that the zinc metal center plays a role in forming and/or stabilizing iNOS undisruptable dimers (UD-dimers). In this study, we show that a mAb directed against a unique epitope at the oxygenase domain of human iNOS preferentially recognizes UD-dimers. This observation has implications for the mechanism of formation and regulation of dimer formation of iNOS. Our data suggest that UD-dimers of iNOS, in spite of SDS-PAGE denaturation, still maintain features of the quaternary structure of iNOS particularly at its N-terminal end and including head-to-head contact of the oxygenase domains.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Nitric oxide is an important signaling and cytotoxic molecule that is synthesized from L-arginine by isoforms of NO synthase (NOS)3 (1, 2, 3). As an agent of inflammation and cell-mediated immunity, NO is generated by a Ca2+-independent cytokine-inducible NOS (iNOS) that is widely expressed in diverse cell types under transcriptional regulation by inflammatory mediators (2, 3, 4). The human iNOS gene encodes a protein of 131 kDa (5). Human iNOS, like all NOSs, has three domains: 1) an N-terminal oxygenase domain (residues 1–504) that binds heme, tetrahydrobiopterin, and L-arginine; 2) a C-terminal reductase domain (residues 537–1153) that binds flavin mononucleotide, flavin adenine dinucleotide, and NADPH; and 3) an intervening calmodulin-binding domain (residues 505–536) that regulates electron transfer between the oxygenase and reductase domains (6, 7, 8). Whereas calmodulin binds to the constitutive isoforms in response to an increase in calcium, it is tightly coupled to iNOS at basal calcium levels (9). Therefore, iNOS is notably distinguished from the constitutive isoforms by its sustained production of a relatively large amount of NO. The high output production of NO, while suited for iNOS function as a host-defense agent, could cause inflammation and tissue injury. iNOS has been implicated in the pathogenesis of many diseases including Alzheimer, asthma, lung cancer, transplant rejection, cerebral infarct, glaucoma, inflammatory bowel disease, arthritis, and septic shock (10, 11). Understanding the regulation of NO synthesis by iNOS will assist in efforts to modulate its levels.

For the synthesis of NO, iNOS is active only as a homodimer in which the subunits align in a head-to-head manner, with the oxygenase domains forming a dimer and the reductase domains existing as independent monomeric extensions (6). Posttranslational assembly into active form, including subunit dimerization and substrate and cofactors binding, represents an important critical locus for therapeutic interventions for regulation of NO synthesis (12, 13, 14). The crystal structure of the oxygenase domain of all NOSs revealed a conserved zinc ion center tetrahedrally coordinated to pairs of symmetry-related Cys residues at the dimer interface. Cys110 and Cys115 in human iNOS form the ligand for the zinc center. The conserved motif and its strategic location suggest a structural role in maintaining the intersubunit contacts (15, 16). We have recently shown that intracellular iNOS forms dimers that are "undisruptable" by heat, SDS, strong denaturants, and/or reducing agents. These dimers are clearly distinguishable from the easily dissociated dimers formed by iNOS in vitro. The mutation of Cys115 (critical for zinc binding) greatly affects the formation of undisruptable dimers (UD-dimers). Our data suggest that the zinc metal center plays a role in either forming or stabilizing iNOS UD-dimers (17). In this study, we show that a mAb directed against an epitope at the oxygense domain of human iNOS preferentially recognizes UD-dimers. This observation has important implications for the mechanism of formation and regulation of dimer formation of iNOS.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and transfection

Human embryonic kidney (HEK)293, were cultured in IMEM supplemented with 2 mM glutamine, and 10% heat-inactivated, filtered (40-nm filter) FBS (HyClone Laboratories) at 37°C in 5% CO2. Human iNOS cDNA, inserted into the expression vector pRc/CMV (Invitrogen Life Technologies) under the control of the CMV promoter, was used. Cationic lipid-mediated transient transfection was done using Lipofectamine and a transfection enhancing Plus reagent (Invitrogen Life Technologies) following the manufacturer’s instructions (18, 19).

Antibodies

Four mAbs raised against human iNOS were used (Research and Diagnostic Antibodies). The Abs were reported by the manufacturers to bind to defined regions of human iNOS sequence. They are as follows: clone 21C10-1D10 (abN; region 25–54), clone 2D2-B2 (region 781–798), clone 2A12-A4 (region 985–1002), and clone 1E8-B8 (abC; region 985–1002).

Western analysis

After gentle rinsing twice with PBS, the cell layer was lysed on ice for 45 min in 40 mM Bis-Tris propane buffer, pH 7.7, 150 mM NaCl, 10% glycerol with 20 mM of the detergent sodium taurocholate, and in the presence of protease inhibitors (PMSF (1 mM), pepstatin A (10 µg/ml), leupeptin (10 µg/ml), aprotinin (10 µg/ml), phenanthroline (10 µg/ml), and benzamidine HCl (16 µg/ml); BD Pharmingen). Lysates were centrifuged (16,000 x g, 5 min, 4°C), and supernatants were stored at –80°C. Total protein concentrations were determined by bicinchoninic acid reagent (Pierce). Cell lysates (50 µg) were mixed with one-third volume of 4x Laemmli sample buffer (200 mM Tris-HCl, pH 6.8, 8% SDS, 0.004% bromophenol blue, 40% glycerol, 400 mM DTT), boiled at 95°C for 5 min, and then subjected to SDS-PAGE. After SDS-PAGE, immunoblotting was done with anti-iNOS Ab. An ECL system was used for detection (SuperSignal West Pico; Pierce). Images were acquired using a cooled charged couple device camera (Eagle Eye II Still Video System; Stratagene).

Peptide scan

Sets of synthetic peptides were synthesized and arrayed on a solid phase (Jerini Biotools). Peptides were covalently linked at the C terminus to a cellulose support. Bound Ab was detected with goat-anti-mouse Ig coupled to HRP and chemiluminescence (20, 21).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Preferential detection of iNOS UD-dimers

We evaluated the presence of iNOS UD-dimers in mammalian cells by Western analysis using various anti-iNOS Abs. We noted that one mAb preferentially detected iNOS UD-dimers. This mAb clone abN had been declared by the manufacturer to bind to region 25–54 of human iNOS, suggesting that it binds to an epitope near the N-terminal end. Lysates of HEK293 transfected with human iNOS cDNA and subjected to Western analysis using anti-iNOS Ab abN or abC. The latter is mAb directed against an epitope in the region 985–1002 of iNOS. The results indicated that although abN was generally much less sensitive than abC in detecting iNOS, it preferentially detected iNOS UD-dimers over monomers (Fig. 1). To further confirm this observation, we examined HEK293 cells transfected with progressively increasing amounts of human iNOS cDNA. Cell lysates were subjected to Western analysis for iNOS using either abN or a mixture of three mAbs directed against different iNOS regions (781–798 and 985–1002) (Fig. 2). The results confirmed that abN has greater ability to detect iNOS UD-dimers than other Abs. abN by itself has a low affinity for iNOS compared with other Abs. This is illustrated by its weak detection of iNOS monomers, which constitute the majority of iNOS detected by Western analysis (17). Thus, it appears that the dimeric form of iNOS stabilizes or increases the affinity of abN.



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  		FIGURE 1. Preferential detection of iNOS UD-dimers by abN. HEK293 cells transfected with human iNOS cDNA were harvested by lysis on ice for 45 min, in 40 mM Bis-Tris propane buffer, pH 7.7, 150 mM NaCl, and 10% glycerol with 20 mM sodium taurocholate. Cell lysates (50 µg) were subjected to SDS-PAGE followed by immunoblotting with anti-iNOS Ab, abN or abC. Results are shown in duplicate. Letters D and M denote dimer and monomer, respectively.

 


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  		FIGURE 2. Detection of iNOS UD-dimers by abN compared with a mixture of anti-iNOS mAbs (abMix). HEK293 cells were transfected in duplicates with increasing concentrations of human iNOS cDNA. Twenty-four hours after transfection, cells were harvested by lysis as described for Fig. 1. Cell lysates were subjected to SDS-PAGE, followed by immunoblotting with anti-iNOS Ab, abN (A), or a mixture of three mAbs, abMix (B).

 
abN does not recognize murine iNOS

We aimed to investigate whether abN has similar affinity to UD-dimers formed by murine iNOS. Murine iNOS has 80% sequence identity to human iNOS at the amino acid level (5). Therefore, anti-iNOS Abs often recognize both isoforms. We transfected HEK293 cells with either human or murine iNOS and evaluated cell lysates by Western analysis. As in prior experiments, abN preferentially recognized UD-dimers of human iNOS (Fig. 3A). However, abN did not recognize murine iNOS. The expression of murine iNOS in HEK293 cells was confirmed using abC which recognized both human and murine iNOS (Fig. 3B). Additional experiments, using the murine macrophage cell line RAW264.6 after their stimulation with IFN-{gamma}, and LPS to induce iNOS, confirmed that abN could not recognize murine iNOS (data not shown). These results indicated that abN recognizes a unique epitope in human iNOS that is not shared with the murine isoform.



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  		FIGURE 3. abN does not recognize murine iNOS. HEK293 cells were transfected with human (lane 1) or murine (lane 2) iNOS cDNA. Twenty-four hours after transfection, cells were harvested by lysis as described for Fig. 1. Cell lysates (50 µg) were subjected to SDS-PAGE followed by immunoblotting with anti-iNOS Ab abN (A) or abC (B). The expected molecular masses of murine and human iNOS are 130 and 131 kDa, respectively.

 
Epitope mapping of abN—determination of the consensus binding site

Initial information provided by the manufacturer indicated that the abN binds to a region containing residues 25–54 of human iNOS. To delineate the consensus binding sequence, a set of overlapping synthetic peptides was synthesized, each corresponding to a small segment of the sequence of iNOS and arrayed on a solid phase (20, 21). The consensus binding sequence forming the antigenic epitope recognized by the Ab was determined using gridded array of synthetic peptides containing the preliminary binding sequences and sequentially shifting in steps of two amino acids (Fig. 4). Peptides were covalently linked at the C terminus to a cellulose support. Bound Ab was detected with goat-anti-mouse Ig coupled to HRP and chemiluminescence. The residues that bound to the Ab (consensus sequence) were determined to be C33ATSSPVTQDDLQ45.



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  		FIGURE 4. Determination of the consensus binding site for abN. Twelve peptides (13 residues each) sequentially shifting in steps of 2 residues were synthesized and covalently linked to a nitrocellulose membrane. Ab binding was determined by chemiluminescence. The residues that bound to the Ab (consensus sequence) were determined to be C33ATSSPVTQDDLQ45.

 
Determination of the critical residues required for abN binding

The minimal amino acid sequence needed for Ab binding was determined using a similar approach to the one described above except peptide scan contained series of peptides with progressive N-terminal or C-terminal deletions of the consensus binding sequence (Fig. 5A) (20, 21). A surface epitope of six contiguous residues VTQDDL (residues 39–44) was identified. Sequence homology alignment of the critical residues revealed that the Ab binds to a unique iNOS epitope not shared by the other NOS isforms (Fig. 5B).



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  		FIGURE 5. Determination of the critical residues for abN binding. A, Using progressive N-terminal or C-terminal deletions of the consensus binding sequence, peptide scan was repeated with 26 truncated peptides. A surface epitope of 6 contiguous residues VTQDDL (39–44) was identified. B, Sequence alignment of the identified epitope residues of human iNOS with that of mouse iNOS and human endothelial and neuronal NOS.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Implications for our understanding of iNOS UD-dimers

The exact mechanism by which iNOS UD-dimers survive denaturation is not yet clear (17). Because dimerization is required for iNOS activity, understanding the regulation of its dimerization is critical to our effort to modulate its NO production (3, 12, 13, 14). The findings in this study provide us with an additional glimpse of the potential structure of iNOS UD-dimers. The diagram in Fig. 6 illustrates a potential model for iNOS UD-dimers based on their binding preference to abN. The model predicts that UD-dimers of iNOS, in spite of SDS-PAGE denaturation, still maintain features of the quaternary structure of iNOS. This structure predicts iNOS to form a head-to-head dimer through its N-terminal oxygenase domains (6, 7). The zinc metal center helps to stabilize the dimer (15, 16, 17). In this model, Abs binding to epitopes near the N terminus of one subunit will be situated in close proximity to Abs bound to the cognate epitope on the other subunit. This close proximity allows for high avidity binding of secondary Abs, and thus enhances signal detection (21). Our previous data suggested that the Zn metal center might play a role in maintaining iNOS UD-dimers (17). The findings of this study are consistent with this hypothesis and provide further evidence that iNOS UD-dimers are tightly associated and potentially maintain features of iNOS quaternary structure, particularly at its N-terminal end.



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  		FIGURE 6. Model for binding of iNOS UD-dimers to abN and abC. The diagram illustrates a potential model for iNOS UD-dimers based on their binding preference to abN. The model predicts that UD-dimers of iNOS, in spite of SDS-PAGE denaturation, maintain features of the quaternary structure of iNOS. This structure predicts iNOS to form a head-to-head dimer through its N-terminal oxygenase domains. The zinc metal center helps to stabilize the dimer. In this model, Abs binding to an epitope near the N terminus of one subunit will be situated in close proximity to Abs bound to the cognate epitope on the other subunit. This close proximity allows for high avidity binding of secondary Abs and thus enhances signal detection (21 ).

 
Although all the NOS isforms are active only in the dimer form, the presence of UD-dimers has been described only for iNOS (17). Previous studies have shown that neuronal isoform, nNOS, can form SDS-resistant dimers, which are detected using low temperature partially denaturing SDS-PAGE (22). In that procedure, the samples are preincubated in the presence of L-arginine and tetrahydrobiopterin before their dilution in Laemmli buffer. Samples were not heated before gel electrophoresis, which was performed at 4–15°C. These SDS-resistant dimers seemed to be stabilized by the presence of increasing concentrations of L-arginine and tetrahydrobiopterin. However, in contrast to iNOS UD-dimers, nNOS dimers were completely dissociated at a temperature of 40–50°C. Subsequent studies have shown the presence of endothelial NOS SDS-resistant dimers that also were completely dissociated at high temperature (23). The possibility that UD-dimers might be unique to the iNOS isoform suggests that they may be related to the characteristic high output of NO by iNOS which further distinguish iNOS from the other isoforms. Investigation of the potential regulatory role of UD-dimers on NO production will help increase our understanding of the mechanism of NO synthesis by iNOS and advance the potential for pharmacologic approaches to blocking NO synthesis.


   Acknowledgments
 
We thank Robert Webber for useful discussions.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by The American Lung Association, American Heart Association, National Heart Lung and Blood Institute, and National Institute of Allergy and Infectious Diseases. Back

2 Address correspondence and reprint requests to Dr. N. Tony Eissa, Baylor College of Medicine, One Baylor Plaza, BCM 285, Suite 672E, Houston, TX 77030. E-mail address: teissa{at}bcm.tmc.edu Back

3 Abbreviations used in this paper: NOS, NO synthase; iNOS, inducible NOS; UD-dimers, undisruptable dimers. Back

Received for publication August 20, 2004. Accepted for publication November 30, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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