Generation and Characterization of Novel Human IRAS Monoclonal Antibodies

Imidazoline receptors were first proposed by Bousquet et al., when they studied antihypertensive effect of clonidine. A strong candidate for I1R, known as imidazoline receptor antisera-selected protein (IRAS), has been cloned from human hippocampus. We reported that IRAS mediated agmatine-induced inhibition of opioid dependence in morphine-dependent cells. To elucidate the functional and structure properties of I1R, we developed the newly monoclonal antibody against the N-terminal hIRAS region including the PX domain (10–120aa) through immunization of BALB/c mice with the NusA-IRAS fusion protein containing an IRAS N-terminal (10–120aa). Stable hybridoma cell lines were established and monoclonal antibodies specifically recognized full-length IRAS proteins in their native state by immunoblotting and immunoprecipitation. Monoclonal antibodies stained in a predominantly punctate cytoplasmic pattern when applied to IRAS-transfected HEK293 cells by indirect immunofluorescence assays and demonstrated excellent reactivity in flow immunocytometry. These monoclonal antibodies will provide powerful reagents for the further investigation of hIRAS protein functions.

A strong candidate for I1R, known as imidazoline receptor antisera-selected protein (IRAS), has been cloned from human hippocampus [14]. hIRAS is a larger protein of 1504 amino acids consisting of an NH 2 -terminal phox (PX) domain, 5 putative leucine-rich repeats, a predicted coiledcoil domain, and a long COOH-terminal region. Several evidence supported the identity of native I1R and IRAS protein in tissue distributions, ligand binding properties, some cellular functions and downstream signal pathways [14][15][16][17][18]. The murine form of IRAS, Nischarin, truncated at the N-terminal 244 amino acids including the PX domain compared with the hIRAS, was a soluble cytosolic protein involved in cytoskeletal organization [19]. It has been shown that decreasing the expression of rat IRAS or Nischarin in PC12 rat pheochromocytoma cells could attenuate the activation of extracellular signal-regulated kinase (ERK) or reduce the radioligand binding to I1R, which further supported that hIRAS or Nischarin might serve as I1R itself, or at least a functional subunit of I1R [20]. Recently, Molderings et al. have reported that the "I1-imidazoline receptors" mediating effects of clonidine and moxonidine in PC12 and the transfected HEK293 cells belonged to the S1P-receptor family, in particular, representing a mixture of sphingosine-1-phosphate (S1P)1-and S1P3-receptors and/or heterodimers of both. It was then proposed that an increased expression of IRAS or Nischarin may improve the receptor-trafficking from cytosolic S1P-receptors to the cell membrane and thereby increase the number of binding sites in the plasma membrane for radioligand binding [21]. In our previous study, we also reported that IRAS mediated agmatine-induced inhibition of opioid dependence in morphine-dependent cells [22]. Despite intensive efforts, the molecular base of the I1R had not yet been elucidated.
To elucidate the functional and structure properties of I1R, several different epitope-specific antisera against IRAS have been raised in rabbits [23]. Because of IRAS splice variants or nonspecificity of these antisera, more sizes of IRAS (≈33, ≈85, ≈170 KDa) have been visualized in various tissue and cells, which limited their uses on western blot. IRAS was reported to target to the endosomes by a combined action of a PX domain and a coiled-coil region. The PX domain, consisting of 10-130 amino acids, was first identified from the sequence analysis of two SH3 domain-containing cytosolic components of NADPH oxidase, p47phox and p40phox [24]. Therefore, in the present study, we developed the newly monoclonal antibody against the N-terminal hIRAS region including the PX domain (10-120aa). This development has great utility for immunoblotting, indirect immunofluorescent staining, immunoprecipitation, and flow cytometry. These monoclonal antibodies will provide powerful reagents for the further investigation of hIRAS protein functions.

Antiserum Titer Determination by Indirect Enzymelinked
Immunosorbent Analysis (ELISA). The purified NusA-IRAS (10-120aa) , diluted to 10 μg/mL in 50 mM carbonate salt buffer (pH 9.6), was coated on plates at 100 μL aliquot per well, 4 • C overnight. The wells were washed three times with PBS-Tween buffer (0.05% Tween 20 in PBS). The coated wells were blocked with 200 μL of 3% BSA for 1 hour at 37 • C and then incubated with 150 μL monoclonal antibodies against NusA-IRAS (10-120aa) fusion protein with different dilutions (from 1 : 1000 to 1 : 25600). After incubation for 1 hour at 37 • C, the wells were incubated with 150 μL horseradish peroxidase-conjugated goat antimouse IgG (dilution 1 : 5000, Santa Cruz) for 1 hour at 37 • C after thorough washing. Peroxidase activity was detected using o-phenylenediamine and H 2 O 2 as enzyme substrates. Color development was stopped with 2 M of H 2 SO 4 and the absorbance was measured at 490 nm using Microplate Reader (Bio-Rad).

Production of Antihuman IRAS Monoclonal Antibodies.
The purified NusA-IRAS (10-120aa) protein (50 μg in a volume of 80 μL) was mixed with an equal volume of Freund's complete adjuvant. The antigen-adjuvant mixture was injected into 6 female BALB/c mice and was followed by three booster injections at 2-week interval in incomplete Freund's adjuvant. The mouse with the highest antibody titer tested by ELISA was boosted intraperitoneally with 100 μg NusA-IRAS (10-120aa) protein without adjuvant 3 days before the cell fusion. Feeder layer cells were prepared 1 day prior to fusion. Splenocytes from mice with the highest ELISA antibody titers were fused with murine myeloma cells SP2/0 following standard procedures [25]. Culture supernatants were collected after fusion and initially screened by ELISA with purified NusA-IRAS (10-120aa) fusion proteins as antigens. Positive hybridoma clones were selected with the limiting dilution method, and stable hybridoma clones were obtained after 3 cloning cycles. Isotypes of antibodies were identified with a mouse subisotype panel (Bio-Rad). The pristine-primed BALB/c mice were injected intraperitoneally with 1 × 10 6 hybridoma cells per mouse in order to acquire abundant mAbs. The ascitic fluid was collected and mAbs were purified with a protein A/G affinity column (Amersham Pharmacia Biotech).

Western Blotting Analysis.
Transfected cells were harvested 48 hours after transfection. Total cell lysate preparation and western blot analysis were performed according to previously described procedures [26]. In brief, cell lysates were prepared, electrophoresed on SDS-PAGE gels, and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% nonfat milk in TBST (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, and 0.05% [v/v] Tween 20) and incubated with IRAS or GFP mAbs (Cell Signaling Technology Inc). Blots were incubated with horseradish peroxidase (HRP) conjugated goat antimouse antibodies (Santa Cruz) after primary antibody incubation, and blots were developed with enhanced electrochemiluminescence (ECL, Cell Signaling Technology Inc).

Generation of Murine Monoclonal Antibodies (mAbs) against a Recombinant NusA-IRAS (10-120aa) Fusion Protein.
IRAS is a large protein comprising of 1504 amino acids. Its NH 2 -terminal phox domain is important for membrane association and cellular localization. The N-terminal of IRAS (10-120aa) covering the PX domain was cloned into the pET-43.1a(+) (Figure 1(a)) [29]. The supernatant fusion protein was purified by His-tag affinity purification and was subsequently used to generate the monoclonal antibody. The dissolved protein yielded one major band of 78 kDa expected molecular weight (Figure 1(b)) with high purity and integrity. The NusA protein used as a control generated the 66 kDa expected molecular weight. The recombinant protein was also confirmed with western blot using anti-His mAbs (right column, Figure 1(b)). BALB/c mice (n = 3) were immunized with the NusA-IRAS (10-120aa) fusion protein, and blood was collected from the mice after multiple injections. Antibody titers were tested by ELISA on plates coated with the NusA-IRAS (10-120aa) protein (data not shown). The 2 mice  with the highest titers were sacrificed and spleens from both mice were fused to myeloma cells following standard procedures. Individual hybridomas was grown and 125 hybridomas were further characterized. Supernatant from the growing hybridoma clones was screened with ELISA. Screening was performed on plates coated with NusA-IRAS (10-120aa) , NusA protein, and GST-IRAS (10-120aa) fusion protein to determine antibody specificity. A total of 5 hybridomas (DA041, DD015, BE073, BA022, and AH021) reacted selectively with the NusA-IRAS (10-120aa) protein in all 3 assays and were further evaluated. Isotype analysis revealed that all mAbs were of the IgG1 subtype.
The immunoreactivities of the 5 representative mAbs with NusA-IRAS (10-120aa) were shown in Figure 2, all of which specifically recognized a 78 kDa protein band which corresponded to the purified NusA-IRAS (10-120aa) protein, but not to the 66 kDa NusA protein. Anti-His mAbs reacted with both proteins in the same experiment (Figure 2(a)) as controls. We evaluated the specificity of mAbs in mammalian cells by inserting IRAS cDNA into expression vectors to allow the production of GFP fusion proteins under the control of a CMV promoter. The pEGFPC1 and pEGFPC1-IRAS plasmids were separately transfected into HEK293 cells. Western blot analysis with the anti-GFP antibody demonstrated that chimeric proteins were expressed and migrated separately at expected molecular masses of approximately 19 or 27 KDa (Figure 2(b)). However, the expected 190 kDa band whose molecular weight corresponded to the full-length IRAS protein was only detected with the mAbs DA041, DD015, and BE073 in GFP-IRAS expressed cells. The remaining BA022 and AH021 mAbs were negative (Figure 2(b)). The same samples were also incubated with preimmune serum with no reactivity (data not shown). Results revealed that all 5 selected mAbs specifically recognized bacterially expressed NusA-IRAS (10-120aa) proteins, but only 3 mAbs recognized recombinant IRAS in mammalian cells.

Cellular Localization and Flow Cytometry Analysis for IRAS Expression.
IRAS mAbs were used to detect the cellular localization of IRAS protein based on specificity characterized by western blot analysis. The pEGFPC1-IRAS plasmid was transfected into HEK293 cells. IRAS protein localization was tested by fluorescence confocal microscopy after 48 hours transfection. GFP-IRAS was primarily located in the cytoplasm in a punctate pattern (Figure 3(a)) with concentration onto discrete loci and spot fluorescence (panel A, E, I, Journal of Biomedicine and Biotechnology  M, and Q), as confirmed in previous studies [30]. The mAbs DA041, DD015, and BE073 were used as probes to detect a predominantly punctate cytosolic distribution as expected (panel B, F, G). However, the mAb BA022 homogenously detected the fluorescence distribution over the cytoplasm (panel N) and the mAb AH021 scored negatively (panel R). Merged images revealed that the GFP-IRAS fluorescence significantly coincided with the immunostaining with mAbs DA041, DD015, and BE073 (panel D, H, and L), but was not reflected by the mAbs BA022 and AH021 (panel P and T). Further, the punctate fluorescence patterns appeared to be unique to the mAbs DA041, DD015, and BE073. In addition, we also detected the endogenous IRAS protein with monoclonal antibody DA041 in PC12 cells, showing a strong signal associated with the membrane and faint signal in the cytoplasm (data not shown). These results suggest that the 3 mAbs specifically detect IRAS proteins by immunofluorescence, similar to immunoblotting results. Flow cytometry is a rapid, quantitative, multiparameter cellular analysis based on the measurement of visible and fluorescent light emission. We chose the representative mAb DA041 for further characterization based on the strong signals detected by immunoblotting and immunofluorescence. The IRAS cDNA was cloned into the mammalian expression vector PCMV-myc and was expressed in HEK293 cells. The mAb DA041 and the c-myc mAb (used as a positive control) resulted in significant increases in fluorescence intensity when compared to cells incubated with preimmune serum (Figure 3(b)). The binding efficiencies were 30.77% by the mAb DA041 and 48.26% by the c-myc mAb. No reactivities were detectable in preimmune sera (0.01%). These results indicate mAb DA041 is effective for detecting IRAS protein by flow cytometry.

MAbs IRAS Immunoprecipitates IRAS from Cell Lysates.
We next characterized IRAS mAb ability to recognize IRAS proteins in their native states by immunoprecipitation. Cell extracts were immunoprecipitated with the mAb DA041 and analyzed with mAb DA041 immunoblotting after 48-hour transfection with PCMV-myc-IRAS. IRAS was selectively immunoprecipitated from cell lysates that expressed the myc-IRAS protein (approximately 175 kDa), and those which corresponded to the predicted size of the full-length IRAS ( Figure 4). Immunoprecipitation of lysates with mouse normal IgG did not result in detection of protein species. Lysates were immunoprecipitated with the c-myc mAb to confirm mAb specificity, and similarly sized bands were detected. No corresponding band was visualized when the negative control c-myc mAb was used to immunoprecipitate cell lysate of the transfectant expressing the empty PCMV-myc vector. Similar results were obtained with the mAbs DD015 and BE073 under the same conditions, and the remaining mAbs BA022 and AH021 scored negatively. Therefore, the 3 isolated antibodies specifically recognized native full-length IRAS protein products.
Our results suggested that the mAbs DA041, DD015, and BE073 were reactive for both immunofluorescence and immunoblotting. Therefore, these mAbs likely recognized linear epitopes in the IRAS protein. In addition, the 3 mAbs were capable of recognition of native full-length IRAS proteins by immunoprecipitation. However, the mAbs BA022 and AH021 specifically recognized bacterially expressed IRAS immunogens and did not detect recombinant IRAS in mammalian cells. This could be related to the different IRAS folding patterns, since misfolded IRAS proteins could result in the exposure of unique immunogenic epitopes different from native proteins. The mAbs BA022 and AH021 could be used as backup reagents to safeguard against antibodyspecific artifacts. In addition, analyzing the overall homology of the amino acid sequence of the PX domain, Sorting Nexins 13 (SNX13) is the most relative protein to IRAS [31]. We found monoclonal antibody DA041 against hIRAS could not react with SNX13 by immunofluorescence assay (data not shown). It was proposed that these antibodies developed here were specific to the PX domain of IRAS.
In summary, we generated specific mAbs directed against the human IRAS N-terminal. The mAb DA041 exhibited the best performance in a variety of assays including immunoblotting, immunoprecipitation, indirect immunofluorescence stainning, and flow cytometry. Specific mAbs may provide an ideal reagent for further investigation of the function of IRAS proteins.