Introduction

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Nitric oxide (NO), an important molecule in various biologic processes that include airway inflammation (1, 2) is synthesized from L-arginine by isoforms of NO synthase (NOS) (3). In regulating signaling pathways, NO is produced by two constitutive calcium (Ca²⁺)-dependent isoforms, neuronal NOS (nNOS) and endothelial NOS (eNOS). Ca²⁺-activated calmodulin binds to and transiently activates constitutive NOS dimers by facilitating electron transfer between the reductase and oxygenase domains (6). Due to the transient nature of elevated Ca²⁺ levels, the activity of NO produced is short-lived. As an agent of inflammation and cell-mediated immunity, NO is produced by a Ca²⁺-independent cytokine-induced NOS (inducible NOS [iNOS]). Calmodulin is tightly bound to iNOS even at basal Ca²⁺ levels (7), and therefore iNOS is notably distinguished from the constitutive isoforms by its prolonged production of relatively much larger amounts of NO. The high level of production of NO by iNOS is suited for its function in host defense.

Although the capacity of human lung macrophages to produce NO is controversial, there is no doubt that the respiratory epithelium produces NO via iNOS (8, 9). As is the case elsewhere in the body, NO in the airways is implicated in nonspecific defense against inhaled microbes (10). It is well recognized, however, that overproduction of NO by iNOS could cause tissue damage that outweighs its potential benefit for host defense (10, 11). One such example is the surprising observation that genetic deficiency of iNOS substantially protects mice from death caused by influenza A virus. In this infection, the inflammatory response appears to be a more important cause of mortality than the cytopathic effects of the virus, and iNOS appears to contribute substantially to the inflammation (12). Overproduction of NO by iNOS has been implicated in the pathogenesis of several diseases, including asthma, transplant rejection, cerebral infarct, inflammatory bowel disease, rheumatoid arthritis, glaucoma, and septic shock (10, 13). Although iNOS induction may not be a primary event in all of these diseases, the cytotoxic and proinflammatory effects of NO generated by iNOS contribute to their pathophysiology. Therefore, inhibition of iNOS is potentially beneficial, particularly in those diseases, such as asthma, where inflammation is not the consequence of infection (10, 13).

NO synthesis by NOSs can be inhibited using L-arginine analogues (13, 14). A major difficulty in the use of these compounds as therapeutic agents, however, is their lack of specificity for the iNOS isoform. For the synthesis of NO, all NOS enzymes are active only as homodimers (3, 4). Therefore, for the regulation of NO synthesis, post-translational subunit dimerization represents a potential critical locus for therapeutic interventions aimed at regulating iNOS activity. Understanding the structural requirements for iNOS dimerization and activity will provide the groundwork for such strategies. Selective inhibitors of iNOS have been advocated as a novel therapeutic approach for asthma and other inflammatory diseases (2, 13). Design of selective inhibitors of the iNOS isoform awaits better understanding of the structure-function relationship in the enzyme molecules.

The human iNOS gene, containing 26 exons, encodes a protein of 131 kD (15, 16). Like other NOSs, iNOS has three domains: (1) an amino-terminal oxygenase domain (residues 1-504) that binds heme, tetrahydrobiopterin (H₄B), and L-arginine; (2) a carboxy-terminal reductase domain (residues 537-1153) that binds flavin mononucleotide, flavin adenine dinucleotide, and nicotinamide adenine dinucleotide phosphate (NADPH); and (3) an intervening calmodulin-binding domain (residues 505-536) that regulates electron transfer between the oxygenase and reductase domains (6, 17). The reaction catalyzed by NOS uses L-arginine as a substrate. It requires molecular oxygen and reducing equivalents, in the form of NADPH, to produce NO and citrulline (3, 4).

We have cloned and characterized several splice variants of human iNOS using bronchial epithelial cells from normal subjects (18). Functional characterization of these isoforms led to the identification of a domain in human iNOS, encoded by exons 8 and 9, that is critical for enzyme dimerization and NO synthesis (19). It is important to note that all NOS active as dimers have conserved amino acids in this region. In this study, we report data identifying, in the region encoded by exons 8 and 9, specific residues that are critical for iNOS activity and dimerization. The identified residues are conserved among all NOS isforms across species. These data increase our understanding of the structural elements critical for NO synthesis and thus lay the groundwork for future studies aimed at downregulation of iNOS activity.

	Materials and Methods

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Cell Culture

Human embryonic kidney (HEK) 293 cells (American Type Culture Collection, Rockville, MD), were cultured at 37°C in 5% CO₂ in Improved Minimum Essential Medium (Biofluids, Inc., Rockville, MD) supplemented with 2 mM glutamine, 25 µg/ml gentamycin, and 10% heat-inactivated, filtered (40 nm-filter) fetal bovine serum (Hyclone Laboratories, Inc., Logan, UT).

Transfection and Cell Lysis

iNOS complementary DNA (cDNA) was inserted into the expression vector pRc/CMV (Invitrogen Corp., San Diego, CA) under the control of the cytomegalovirus (CMV) promoter. Cationic lipid-mediated transient transfection was performed in 100-mm-diameter tissue culture plates using 4 µg of the desired DNA, 24 µl of Lipofectamine, and 20 µl of a transfection-enhancing Plus reagent (Life Technologies, Inc., Rockville, MD), following manufacturer's instructions. After 23 h, medium was collected for nitrite measurements. After gentle rinsing with phosphate-buffered saline (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, 25 mM sodium taurocholate and the protease inhibitors (phenylmethylsulfonyl fluoride [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]) (PharMingen, San Diego, CA). Lysates were centrifuged (16,000 × g, 5 min, 4°C) and supernatants stored at 80°C. Total protein concentrations were determined by bicinchoninic acid reagent (Pierce, Rockford, IL) using bovine serum albumin as a standard.

Low-Temperature (Partially Denaturing) Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis/ Western Analysis

Lysates (50 µg) from transfected HEK 293 cells were incubated at 37°C for 30 min in the presence of 2 mM L-arginine and 0.1 mM H₄B. Lysates were then mixed with one-third volume of 4× Laemmli sample buffer stock solution (200 mM Tris-HCl, pH 6.8/ 8% sodium dodecyl sulfate [SDS]/0.004% bromophenol blue/40% glycerol/400 mM dithiothreitol), and immediately subjected to SDS-polyacrylamide gel electrophoresis (PAGE) at 4°C (19). Precast 4% gels (Novex, San Diego, CA) were used and electrophoresis was performed at a constant 200 V. Temperature throughout electrophoresis was thermostatically controlled by circulating the running buffer through a high-efficiency heat exchanger at a high flow rate (ThermoFlow System; Novex) connected to an external recirculating chiller (Isotemp 1016; Fisher Scientific, Pittsburgh, PA). Prestained molecular mass standards (Bio-Rad Laboratories, Hercules, CA) were used. After SDS-PAGE, proteins were transferred to nitrocellulose membranes (0.2-m pore size; Schleicher & Schuell, Keene, NH) using a semidry transfer method (TRANS-BLOT SD; Bio-Rad). The membrane was blocked in 5% goat milk in PBS/0.1% Tween 20 and incubated with 1E8-B8, a monoclonal antibody raised against purified recombinant human iNOS and specific for the iNOS isoform (Research and Diagnostic Antibodies, Berkeley, CA). A goat antimouse immunoglobulin G conjugated to horseradish peroxidase (Transduction Laboratories, Lexington, KY) was used as a secondary antibody. An enhanced chemiluminescence system was used for detection (Amersham, Arlington Heights, IL) (19).

NOS Activity

NOS activity was determined by measuring nitrite accumulation in culture medium. A 400-µl sample of culture medium was mixed with 400 µl of Griess reagent (1:1 mixture of 1% sulfanilamide in 5% H₃PO₄ and 0.1% naphthylethylenediamine dihydrochloride in water) for 10 min at room temperature, and absorbance at 543 nm was recorded (19, 22). Serial dilutions of sodium nitrite were used as standards.

Mutagenesis

Site-specific oligonucleotide-directed mutagenesis was performed using the QuikChange mutagenesis system (Stratagene, La Jolla, CA). All products were confirmed by DNA sequence analysis (19).

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

The crystal structure for mouse iNOS residues 115-498 of the oxygenase domain corresponding to residues 121-504 of human iNOS has been determined (17, 23). It is an elongated, curved molecule with an unusual nonmodular, single-domain - fold that resembles a baseball catcher's mitt for the left hand. The structures reveal that the mouse residues, corresponding to residues 242-335 of human iNOS, partially contribute to form the "palm and fingers" of the catcher's mitt through the formation of six-stranded  wings. Long helical hairpins cap both ends of the winged  sheet, and a long  helix runs lengthwise between these hairpins. These structures contribute partially to both the distal pocket of the heme site and the proposed dimer interface. The region encoded by exons 8 and 9 contains 94 amino acids in human iNOS (16). Analysis of iNOS crystal structures (17, 23) was used to select target residues for alanine scanning mutagenesis (24). A total of 16 conserved amino acids, judged to be involved in catalytic activity and/ or dimer formation, were selected for the initial analysis (Figure 1).

View larger version (63K): [in this window] [in a new window]   Figure 1.   Sequence alignment of residues 241-335 encoded by exons 8 and 9 of human iNOS and the corresponding residues of nNOS and eNOS. Above the sequences, black arrows show  strands and white boxes show  helices representing the protein secondary structures of the mouse residues, corresponding to residues 242-335 (exons 8 and 9) of human iNOS (17). 3 strand is at the immediate proximity of the heme site. 4a, b, and c strands, 5 strand, and 5 helix contribute to the distal heme pocket. 6a and b and 7a and b strands contribute to a winged  sheet that engenders a curved - fold characteristic of iNOS oxygenase domain structure. The 16 amino acids chosen for preliminary analysis of human iNOS are boxed.

Using full-length human iNOS cDNA as a template, site-directed mutagenesis was used to construct 16 iNOS mutants, each with an alanine replacing one of the 16 selected amino acids. The resulting iNOS mutants were characterized after their expression in the HEK 293 epithelial cell line, a line that does not express any NOS genes and has been extensively used for gene transfer experiments (19, 21). All iNOS mutants yielded iNOS protein as detected by immunoblotting with specific iNOS antibody. Four iNOS mutants with alanine replacing Trp²⁶⁰, Asn²⁶¹, Tyr²⁶⁷, or Asp²⁸⁰ did not generate NO as measured by nitrite accumulation in culture media (Figure 2A). The remaining 12 iNOS mutants generated NO (Figure 2B). However, some iNOS mutants exhibited reduced activity, particularly those mutants with alanine replacing Ile²⁴⁴, Arg²⁶⁶ or Phe²⁸⁶.

View larger version (17K): [in this window] [in a new window]   View larger version (34K): [in this window] [in a new window]   Figure 2.   NO production of wild-type iNOS and iNOS mutants. HEK 293 epithelial cells, a cell line that does not express any NOS genes, were transfected with plasmids containing vector only, wild-type iNOS, or iNOS mutant. Four iNOS mutants with alanine substituted for Trp260, Asn261, Tyr267, or Asp280 did not generate NO as measured by nitrite accumulation in culture media (A). The remaining 12 iNOS mutants generated NO (B). Nitrite accumulation in the culture medium was assessed 72 h later. Data are means ± standard deviation of three independent experiments, each done in duplicate.

The ability of iNOS mutants to form dimers, after their expression in the HEK 293 cell line, was assessed using low-temperature (partially denaturing) SDS-PAGE/Western analysis. It had been shown that incubation of iNOS-containing cell lysates with L-arginine and H₄B followed by electrophoresis under partially denaturing conditions resulted in migration of iNOS as both monomers (131 kD) and dimers (262 kD). Both forms were detected by immunoblotting using iNOS antibodies (19). Wild-type iNOS and all 12 NO-producing iNOS mutants migrated as both monomers and dimers (Figures 3A-3D). However, iNOS mutants with alanine replacing Trp²⁶⁰, Asn²⁶¹, or Tyr²⁶⁷ migrated only as monomers, suggesting that their inability to produce NO was related to a defect in dimer formation (Figure 3E). Interestingly, the inactive Asp²⁸⁰ iNOS mutant retained the ability to dimerize, indicating that it represents an inactive form of an iNOS dimer (Figure 3F). The four residues identified in this studyTrp²⁶⁰, Asn²⁶¹, or Tyr²⁶⁷ and Asp²⁸⁰are among the residues involved in the formation of the substrate, L-arginine, binding pocket (17, 23). Our study defines the critical residues in this region. It is interesting to note that these four residues are strictly conserved among all NOS isoforms cloned to date from various species (Figure 4). Asp²⁸⁰ is located at the entrance of the 35-Å-deep active center channel close to the substrate-binding site (see Figure 4 in Ref. 23). Our data are consistent with the hypothesis that the replacement of Asp²⁸⁰ alters or interferes with substrate binding, rendering the enzyme inactive.

View larger version (67K): [in this window] [in a new window]   Figure 3.   Dimer formation of iNOS and iNOS mutants. HEK 293 cells were transfected with vector only, iNOS, or iNOS mutant. Solutions of lysates (50 µg) of transfected HEK 293 cells were incubated at 37°C for 30 min with 2 mM L-arginine and 0.1 mM H4B, and subjected to SDS-PAGE in 4% gel at 4°C and immunoblotting with antihuman iNOS antibody. Positions of monomer and dimer are indicated. Wild-type iNOS and all 12 NO-producing iNOS mutants migrated as both monomers and dimers (A-D). However, iNOS mutants with alanine replacing Trp260, Asn261, or Tyr267 migrated only as monomers, suggesting that their inability to produce NO was related to a defect in dimer formation (E). The inactive Asp280 iNOS mutant retained the ability to dimerize, indicating that it represents an inactive form of an iNOS dimer (F ). Experiments were repeated three times with similar results.

View larger version (56K): [in this window] [in a new window]   Figure 4.   Sequence alignment depicting the residues 260-280 of human iNOS and the corresponding residues in NOS isoforms of different species. The strict conservation of residues 260, 261, 267, and 280 of human iNOS is observed. Single-letter abbreviations for the amino acid residues are as follows: A, alanine; D, aspartic acid; E, glutamic acid; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; and Y, tyrosine.

iNOS is active for the synthesis of NO only as a homodimer in which the subunits are aligned head-to-head, with the oxygenase domains forming a dimer and the reductase domains existing as independent monomeric extensions (25). The substrate L-arginine and the cofactors heme and H₄B appear to promote formation of iNOS dimers (25, 26). Cys²⁰⁰ (21), Glu³⁷⁷ (27), and Gly⁴⁵⁶ plus Ala⁴⁵⁹ (28) are involved, respectively in the binding of heme, L-arginine, and H₄B. However, the mechanism of dimer formation/dissociation, the exact requirements of dimerization, the roles of cofactors, and critical residues involved remain to be further elucidated.

Post-translational subunit dimerization of iNOS represents a potential critical locus for therapeutic interventions aimed at controlling its activity. Such a strategy would be based upon understanding the specific requirements for iNOS dimerization. We have demonstrated that in human iNOS, a domain encoded by exons 8 and 9 is critical for dimer formation and NO production (19). Residues 242- 335, encoded by exons 8 and 9, lie between residues involved in heme binding and those involved in binding of L-arginine and H₄B. On the basis of the crystal structures of the oxygenase domain of mouse iNOS (17, 23), the mouse residues corresponding to residues 242-335 (exons 8 and 9) of human iNOS partially contribute both to the distal pocket of heme site and to the proposed dimer interface. These findings are consistent with the strategic structural/functional importance of the exon 8-9 domain indicated in our studies (19). It is important to note that all NOS isoforms have conserved amino acids in this region. This study reports data that identify, in the region encoded by exons 8 and 9, specific residues critical for iNOS activity and dimerization. The four residues identified in this study are strictly conserved among all the NOS isoforms cloned of various species.