PsRBR1 encodes a pea retinoblastoma-related protein that is phosphorylated in axillary buds during dormancy-to-growth transition

In intact plants, cells in axillary buds are arrested at the G1 phase of the cell cycle during dormancy. In mammalian cells, the cell cycle is suppressed at the G1 phase by the activities of retinoblastoma tumor suppressor gene (RB) family proteins, depending on their phosphorylation state. Here, we report the isolation of a pea cDNA clone encoding an RB-related protein (PsRBR1, Accession No. AB012024) with a high degree of amino acid conservation in comparison with RB family proteins. PsRBR1 protein was detected as two polypeptides using an anti-PsRBR1 antibody in dormant axillary buds, whereas it was detected as three polypeptides, which were the same two polypeptides and another larger polypeptide 2 h after terminal decapitation. Both in vitro-synthesized PsPRB1 protein and lambda protein phosphatase-treated PsRBR1 protein corresponded to the smallest polypeptide detected by anti-PsRBR1 antibody, suggesting that the three polypeptides correspond to non-phosphorylated form of PsRBR1 protein, and lower- and higher-molecular mass forms of phosphorylated PsRBR1 protein. Furthermore, in vivo labeling with [32P]-inorganic phosphate indicated that PsRBR1 protein was more phosphorylated before mRNA accumulation of cell cycle regulatory genes such as PCNA. Together these findings suggest that dormancy-to-growth transition in pea axillary buds is regulated by molecular mechanisms of cell cycle control similar to those in mammals, and that the PsRBR1 protein has an important role in suppressing the cell cycle during dormancy in axillary buds.
Introduction
In intact plants, the terminal bud predominantly grows, while axillary bud growth is suppressed. This phenomenon is called apical dominance. After terminal bud decapitation, however, the axillary buds grow rapidly and develop in the same manner as the terminal bud. Apical dominance was one of the first developmental phenomena shown to be regulated by plant hormones (Thimann and Skoog 1934). The outgrowth of axillary buds is inhibited by auxin flow from the terminal bud (Booker et al. 2003), and is promoted by cytokinin biosynthesized in the stem after decapitation (Tanaka et al. 2006). Recently, a variety of experimental approaches were used to examine the molecular mechanisms that control shoot branching (Bennett and Leyser 2006; Dun et al. 2006; Shimizu-Sato and Mori 2001). Some mutants were isolated and characterized in Arabidopsis (max1, 2, 3, and 4), pea (rms1, 2, 3, 4, and 5), and petunia (dad1 and 2). Findings from these molecular genetic approaches combined with conventional physiologic studies, such as grafting experiments, suggested that novel signals regulate axillary bud dormancy. The molecular mechanisms of cell cycle control in axillary buds, however, are still not well understood.
Most cells in pea axillary buds are arrested at the G1 phase in the cell cycle, and axillary buds have the capacity to undergo multiple cycles of dormancy and growth (Devitt and Stafstrom 1995; Shimizu and Mori 1998). Arrest and progression of the cell cycle in axillary buds are controlled in response to environmental signals and by developmental programs. Thus, we hypothesized that cells in dormant axillary buds are regulated through suppression mechanisms of cell cycle control suppression mechanisms similar to those in mammals.
In mammals, arrest and progression of the cell cycle are also controlled during the G1 phase in response to environmental signals and by developmental programs (Sherr 2004). This process is governed by both positive- and negative-regulatory factors. One of the most important key regulators is the product of the retinoblastoma tumor suppressor gene (RB). The human RB gene encodes a 110-kDa nuclear phosphoprotein (pRB). pRB activities are regulated by phosphorylation in a cell cycle-dependent manner. Phosphorylation states of pRB regulate the interactions of multiple cellular proteins. In the G1 phase, hypo-phosphorylated pRB (hypo-ppRB) binds to a transcriptional factor, E2F family protein, and suppresses its transcriptional activity. E2F family proteins regulate the transcription of several genes (e.g., dihydrofolate reductase, thymidine kinase, DNA polymerase α, and proliferating cell nuclear antigen [PCNA]) whose products are required for either G1/S transition or DNA replication. Thus, by negatively regulating E2F family proteins, pRB negatively controls cell cycle progression. pRB is highly phosphorylated by several types of cyclin and cyclin-dependent kinase (CDK) complexes in response to environmental signals or developmental programs to progress the cell cycle. E2F family proteins are released from hyper-ppRB and promote the transcription of genes for G1/S transition or DNA replication. Thus, pRB regulates the arrest and progression of the cell cycle based on its phosphorylation state.
In mammals, there are pRB-related proteins, p130 and p107 (Graña et al. 1998). RB family proteins regulate the progression of the cell cycle as well as the entry/exit transition of the cell cycle. RB family proteins have the same pocket protein feature; that is, they interact with a multitude of cellular proteins in a phosphorylation-dependent manner. RB family proteins are, however, modulated by differential regulation of phosphorylation states and protein levels in quiescent cells re-entering the cell cycle. In quiescent cells and in cells at the G0/G1 transition, pRB is in a hypo-phosphorylated form, and p130 is detected in non- and low-phosphorylated forms, whereas p107 is not detectable (Lin and DeCaprio 2003). In mid-G1/S transition, RB family proteins are phosphorylated by cyclin/CDK complexes. Hypo-ppRB and non- and low-phosphorylated p130 change to hyper-ppRB and high-phosphorylated p130, respectively. p107 is synthesized de novo, followed by phosphorylation to a high-phosphorylated form. Protein levels of pRB remain relatively constant throughout the cell cycle, compared to either p130 or p107. Although there is abundant p130 in the G0/G1 phase, it is dramatically down-regulated in the mid-G1/S phase. p130 down-regulation appears to be mediated by a cell cycle stage-dependent proteasome degradation pathway (Tedesco et al. 2002). These observations suggest that RB family proteins have overlapping functions, as well as different functions in the cell cycle regulation of quiescent cells.
In plants, knowledge of cell cycle control has increased in recent years (Gegas and Doonan 2006). Some basic mechanisms that regulate the cell cycle appear to have been conserved throughout eukaryotic evolution (Dewitte and Murray 2003). To date, plant cDNAs encoding RB-related proteins have been reported in Zea mays (Ach et al. 1997; Grafi et al. 1996; Xie et al. 1996), Nicotiana tabacum (Nakagami et al. 1999), Chenopodium rubrum (Fountain et al. 1999), Arabidopsis thaliana (Kong et al. 2000), Populus tremula × Populus tremuloides (AF133675), Cocos nucifera (AY117036), and Oryza sativa (AP004592). The amino acid conservation between animal and plant RB-related proteins suggests that the proteins have similar biochemical properties. Like animal RB family proteins, plant RB-related proteins also interact with various cellular proteins, such as E2F family proteins, D-type cyclin, mammalian viral oncoproteins (SV40 large-T antigen, adenovirus E1a, and HPV E7), and the plant virus proteins (wheat dwarf virus RepA, and the tomato golden mosaic virus replication factor AL1) (Ach et al. 1997; Grafi et al. 1996; Huntley et al. 1998; Nakagami et al. 1999; Ramirez-Parra et al. 1999; Sekine et al. 1999; Xie et al. 1996).
Because the functions of the mammalian RB family proteins depend on their phosphorylation state, the phosphorylation states of plant RB-related proteins were analyzed. ZmRBR1 protein undergoes changes in phosphorylation states concomitant with endoreduplication in maize (Grafi et al. 1996). The human G1/S protein kinases cyclinD/CDK4, cyclinE/CDK2, and cyclinA/CDK2 can phosphorylate ZmRBR1 protein in vitro (Huntley et al. 1998), and NtRBR1 protein is phosphorylated by tobacco cyclinD/CDC2 in vitro (Nakagami et al. 1999). The ZmRBR1 kinase activity correlates with the proliferation state in maize leaf (Boniotti and Gutierrez 2001), and NtRBR1 kinase activity is detected only during the mid-G1/S phase in tobacco BY-2 cells (Nakagami et al. 2002). These observations also suggest that plant RB-related proteins have biochemical properties similar to those of mammalian RB family proteins. Plant RB-related proteins seem to control not only cell cycle arrest/progression, but also development and cellular differentiation in endosperm, leaf, and root (Ebel et al. 2004; Desvoyes et al. 2006; Wildwater et al. 2005). Only limited information is available, however, on the development-dependent phosphorylation states of plant RB-related protein. In this paper, we describe the isolation and functional characterization of a pea (Pisum sativum L. cv. Alaska) cDNA encoding an RB-related protein (PsRBR1), which has biochemical properties similar to those of mammalian RB family proteins. PsRBR1 protein undergoes changes in its phosphorylation state concomitant with dormancy-to-growth transition in pea axillary buds.
Materials and methods
Plant growth and tissue collection
Seeds of Pisum sativum L. cv. Alaska were soaked in running tap water for 24 h and sown in trays of rockwool. Plants were grown at 25°C in the dark for 4 days, and then in a 16 h light/8 h dark photoperiod for 3 days. Tissues studied were axillary buds at the second node in 7-day-old seedlings. Plants were decapitated 1 cm above the second node to stimulate outgrowth of axillary buds.
PCR and cloning of PsRBR1 cDNA
Degenerate oligonucleotide primers were designed for conserved regions of published amino acid sequences of the RB family proteins (sense; 5′-TT(T/C)TT(T/C)AA(T/C)C GNCA(T/C)AT(T/C/A)GA(T/C)CA-3′ and antisense; 5′-AC(T/C)TC(G/A)TT(G/A)TA(G/A)AANGT(T/G/A)AT(T/G/A)AT-3′), where the N in the parentheses indicates all four deoxyribonucleotides. Polymerase chain reaction (PCR) amplification was performed with cDNA from total RNAs of shoot apices. The amplified fragments (237 bp) were cloned into a BSII TSK-plasmid vector (Ichihara and Kurosawa 1993) and sequenced. A pea cDNA library was constructed using poly (A)+ RNA prepared from dormant axillary buds with a HybriZAP-2.1 A Two-Hybrid cDNA Gigapack Cloning Kit (Stratagene, La Jolla, CA). Approximately 1 × 106 phage recombinants derived from the pea cDNA library were screened with the selected PCR products labeled with [32P]-dCTP. Hybridization and subsequent washing were performed as described by Shimizu and Mori (1998).
DNA sequencing
The cDNAs were sequenced by the dideoxy chain termination method using an automatic DNA sequencer (LIC-4000, LI-COR Inc., Lincoln, NE) according to the manufacturer’s protocol. Both strands were entirely sequenced.
Yeast two-hybrid system
The vectors and strains were provided in the MATCHMAKER Two-Hybrid System 2 (Clontech, Palo Alto, CA). DNA manipulations and yeast methods were performed according to the manufacturer’s instructions. PsRBR1 cDNA was introduced into the pAS2-1 vector, and another cDNA was introduced into the pACT2 vector. Saccharomyces cerevisiae Y187 was transformed with combinations of these plasmids. For quantitative liquid assay, β-galactosidase activity was assayed with O-nitrophenylgalactoside as a substrate.
Antibody preparation and immunoblot analysis
For an anti-PsRBR1 antibody, full-length PsRBR1 protein was expressed in Escherichia coli BL21 pLysS as a 6xHis-tagged protein in pET32a (Novagen, Madison, WI). The antigen was purified with TALON Metal Affinity Resin (Clontech) according to the manufacturer’s protocol. The antigen was injected into a mouse using conventional procedures.
Proteins from axillary buds were extracted by grinding with aluminum oxide in extraction buffer (20 mM Tris–HCl [pH 8.0], 100 mM NaCl, 1 mM EDTA, 0.5% NP-40, 50 μM NaF, and 1 μM Na3VO4, and the proteinase inhibitor cocktail Complete [Roche, Penzberg, Germany]). Insoluble materials were removed by microcentrifugation. Protein concentrations were determined with the DC Protein Assay (Bio-Rad, Hercules, CA). Proteins were separated using sodium dodecyl sulfate—polyacrylamide gel electrophoresis (SDS-PAGE, 10% acrylamide gels [acrylamide:bis-acrylamide = 37.5:1]), or 10% low bis-acrylamide gels [acrylamide:bis-acrylamide = 142:1]), and blotted onto a nitrocellulose membrane (BA-S 85, Schleicher & Schuell, Dassel, Germany). The membrane was blocked with 5% skim milk and 0.05% Tween 20 in Tris-buffered saline (50 mM Tris–HCl [pH 8.0], and 150 mM NaCl), and incubated with the anti-PsRBR1 antibody at a dilution of 1:1000. Horseradish peroxidase-conjugated goat anti-mouse IgG (Pierce, Rockford, IL) was used as the secondary antibody at a dilution of 1:2000. The signals were detected with the SuperSignal ULTRA (Pierce).
In vitro-coupled transcription and translation
A cDNA fragment corresponding to PsRBR1 cDNA, encoding the full-length protein, was cloned into the pBluescript SK+ vector (Stratagene). The template was digested with a restriction enzyme for linearization. In vitro-coupled transcription-translation was performed with the digested template (1 μg) in a TnT® T3 Coupled Wheat Germ Extract System (Promega, Madison, WI) in the presence of [35S]-methionine according to the manufacturer’s protocol. As a control, the in vitro-coupled transcription-translation reaction was performed without the DNA template. Samples were separated by SDS-PAGE. The gel was dried and exposed to a Fuji Imaging Plate (Fuji Photo Film, Tokyo, Japan) overnight. The radiographic image was visualized with a BAS2000 Image Analyzer (Fuji Photo Film).
Dephosphorylation
Protein extracts were incubated in reaction mixture (50 mM Hepes [pH 7.5], 2 mM MnCl2, 0.1 mM EDTA, 5 mM dithiothreitol, 0.01% Brij35, and the proteinase inhibitor cocktail Complete) with 1 unit of lambda protein phosphatase (λ-PPase, New England Biolabs, Beverly, MA) for 30 min at 30°C. Samples were immunoblotted by the anti-PsRBR1 antibody as described above.
RNA extraction and blot analysis
Total RNA was isolated from the tissues by SDS-phenol extraction, followed by LiCl precipitation. Formaldehyde agarose gel electrophoresis of total RNA was performed using standard procedures. The RNAs were blotted onto a Hybond N+ membrane (Amersham Biosciences), and hybridized with PsRBR1, PCNA, and Histone H4 cDNA labeled with [32P]-dCTP, respectively. Hybridization was performed in 0.25 M NaH2PO4 buffer (pH 7.2), 0.25 M NaCl, 7% SDS, 10% dextran sulfate (Amersham Biosciences), and 1% polyvinylpyrrolidone K30 at 65°C for at least 16 h. The membrane was washed in 2xSSPE (1xSSPE is 0.15 M NaCl, 10 mM NaH2PO4, and 1 mM EDTA [pH 7.4]) and 0.1% SDS at 50°C twice for 15 min each, and visualized by autoradiography at −80°C using Kodak XAR-5 film and an intensifying screen (Kodak, Rochester, NY).
Phospholabeling and immunoprecipitation
[32P]-inorganic phosphate (Amersham Biosciences, Piscataway, NJ) was applied to axillary buds (10 μCi per bud) 1 h before collection of the buds. Protein extraction was performed as described above. Protein extracts were incubated with both preimmune serum and Protein A Sepharose 4 Fast Flow (Amersham Biosciences) for 1.5 h at 4°C, and then non-specific associated materials were removed by microcentrifugation. The anti-PsRBR1 antibody was added to the clarified extracts and incubated for 1 h on ice. Protein A Sepharose 4 Fast Flow was added, and incubated for 30 min at 4°C. The complex of immunoprecipitates was washed three times with extraction buffer with 1 mg/ml bovine serum albumin, resuspended in SDS sample buffer (20 mM Tris-HCl [pH 6.8], 40% glycerol, 2% SDS, 2% β-mercaptoethanol, and bromophenol blue), and separated by SDS-PAGE. Dried gels were placed on a Fuji Imaging Plate, and the radiographic images were analyzed using the BAS2000 Image Analyzer.
Results
Isolation of a pea cDNA encoding RB-related protein
To analyze the molecular mechanisms of cell cycle control in axillary buds, we focused on the function of plant RB-related proteins during dormancy-to-growth transition. Based on the amino acid sequences of reported RB-related proteins, we designed degenerate primers and performed reverse transcription PCR with total RNA prepared from pea shoot apices. Using the amplified DNA fragments as a probe, we screened the cDNA library prepared from pea dormant axillary buds. We obtained 14 positive clones by screening, and partially sequenced their 5′ ends. The clone that had the longest 5′ sequence (3540 bp) was used for further analysis because the DNA sequences of the other clones corresponded to those of the longest clone. This cDNA contained a single open reading frame capable of encoding a 1026-amino acid residue protein, PsRBR1. The molecular mass of PsRBR1 protein was estimated to be 114-kDa. The PsRBR1 cDNA contained a stop codon (TAG) four amino acids upstream of the putative initiation methionine codon within the reading frame, indicating that the PsRBR1 cDNA contained the entire coding region.
The alignment of the A and B conserved domains and the N-terminal leucine-rich domain of PsRBR1 protein with maize ZmRBR1 protein and human RB family proteins is shown in Fig. 1(A). The A and B conserved domains of the PsRBR1 protein share 43% identity and 71% similarity, respectively with the A and B conserved domains of the ZmRBR1 protein. The conserved cysteine residue corresponding to Cys 706 of human pRB was present as Cys 798 in PsRBR1 protein (shown by an asterisk in the B conserved domain). This conserved cysteine residue is essential for the function of mammalian and plant RB proteins (Ach et al. 1997; Grafi et al. 1996; Kaye et al. 1990).
Fig. 1Comparison of deduced amino acid sequences of pea PsRBR1 with selected homologous sequences. (A) Sequence alignments of the A and B conserved domains, and the N-terminal leucine-rich domain of PsRBR1 protein and ZmRBR1 protein with human RB family proteins. Black boxes indicate conserved amino acid residues, and dashes indicate gaps in the sequences. The asterisk in the B conserved domain denotes a conserved cysteine residue (798 of PsRBR1 protein), essential for RB function in mammalian cells. (B) Phylogenic analysis of the RB-related proteins under neighbor-joining analysis, and schematic of the salient features of RB-related proteins. The phylogeny is based on the amino acid sequences from the A conserved domain of RB-related proteins. Branch lengths are indicated above each branch. Bootstrap values from the results of 1000 replicates are shown below a node if the node is present in more than 50% of the bootstrap replicate analyses. The three conserved regions (N, N-terminal leucine-rich domain; A, A conserved domain; B, B conserved domain) between plants and mammals are indicated in boxes. Bars represent potential CDK phosphorylation sites. Numbers refer to protein length in amino acids, and numbers in parentheses show potential CDK phosphorylation sites. The accession numbers are AtRBR1 (AC069472), CnRBR1 (AY117036), CrRBR1 (AJ011681), NtRBR1 (AB015221), OsRBR1 (AP004592), PsRBR1 (AB012024), PtRBR1 (AF133675), ZmRBR1 (AF250050), p107 (L14812), p130 (X76061), and pRB (AF551763)
The phylogenic analysis of plant RB-related proteins reported so far and human RB family proteins is shown in Fig. 1(B). The scheme on the right in Fig. 1(B) shows a diagram of the characteristic features of RB-related proteins. PsRBR1 protein was most similar to Populus tremula × Populus tremuloides PtRBR1 protein. PsRBR1 protein has 16 potential phosphorylation sites (Ser-Pro or Thr-Pro) for CDKs, which are highly clustered in regions flanking the A and B conserved domains. These conserved primary structures strongly suggest that RB-related proteins, including PsRBR1 protein, have similar biochemical properties in plant and animals.
Genomic Southern blot analyses were performed to estimate the copy number and closely related genes of PsRBR1 in pea. Full-length PsRBR1 cDNA was used as a probe. Hybridization at both high- and low-stringencies yielded only signals derived from PsRBR1 (data not shown). These results suggested that PsRBR1 gene exists as a single-copy gene in pea.
Interactions of PsRBR1 protein with both plant D-type cyclins and a mammalian oncoprotein
Mammalian and plant RB proteins bind to a large number of cellular and viral proteins, such as D-type cyclin and SV40 large-T antigen. To confirm that PsRBR1 protein acts in the same manner as the mammalian and various plant RB proteins, we tested the ability of PsRBR1 protein to interact with these proteins using a yeast two-hybrid system. The genes for the tested proteins were fused to the GAL4 DNA-binding domain or the GAL4 activation domain. The data shown in Table 1 are the averages of three independent transformants. We verified that PsRBR1 protein alone did not activate the reporter gene. PsRBR1 protein interacted with both pea and Arabidopsis D-type cyclin (Pissa;CycD3;1 and Arath;CycD3;1). Both D-type cyclins contain the canonical RB-binding motif, LXCXE, in their N-terminus (Shimizu and Mori 1998; Soni et al. 1995). PsRBR1 protein interacted with SV40 large-T antigen, which is a mammalian DNA tumor virus oncoprotein. The similarities in primary structures and the coincidence of interacting partners of RB-related proteins suggest that animal and plant RB-related proteins, including PsRBR1 protein, have similar functions in cell cycle regulation.
Table 1PsRBR1 protein binding in the two-hybrid assayPlasmidaβ-galactosidase activitybPsRBR10.11 ± 0.01PsRBR1 + Pissa;CycD3;113.9 ± 0.2PsRBR1 + Arath;CycD3;110.6 ± 0.1PsRBR1 + SV 40 large-T antigen31.5 ± 0.1aSaccharomyces cerevisiae Y187 was transformed with the indicated plasmidsb1 unit of β-galactosidase is defined as the amount that hydrolyzes 1 μmol of O-nitrophenylgalactoside to O-nitrophenyl and d-galactose per min. LacZ assays were done in triplicate and the data represent mean ± standard deviation
Immunochemical detection of PsRBR1 protein in pea axillary buds
To investigate protein levels and phosphorylation states of PsRBR1 protein in axillary buds during dormancy-to-growth transition, we prepared an anti-PsRBR1 antibody. The specificity of the anti-PsRBR1 antibody against PsRBR1 protein was examined (Fig. 2A). Protein extracted from growing axillary buds was detected by immunoblotting with the anti-PsRBR1 antibody. The anti-PsRBR1 antibody cross-reacted with three polypeptides (Fig. 2A, lane 1, filled circle, empty circle, and arrowhead), with molecular masses of approximately 150, 140, and 114-kDa. The pre-immune serum did not cross-react with any polypeptides in the protein extracts (Fig. 2A, lane 2). The anti-PsRBR1 antibody was incubated with an excess of purified recombinant PsRBR1 protein as a competitor, and then used to detect PsRBR1 protein in pea extract (Fig. 2A, lane 3). In this case, no polypeptides were detected with the anti-PsRBR1 antibody. These results indicate that the anti-PsRBR1 antibody specifically cross-reacts with PsRBR1 protein in axillary bud extracts.
Fig. 2Immunochemical detection of PsRBR1 protein in pea axillary buds. (A) The terminal bud was cut 1 cm above the second node. After 24 h, the growing axillary buds were collected. Proteins in the protein extracts (30 μg) prepared from axillary buds were detected by immunoblotting with the anti-PsRBR1 antibody (lane 1), pre-immune serum (lane 2), and anti-PsRBR1 antibody preincubated with excess recombinant PsRBR1 protein (lane 3). Molecular mass markers are indicated at the left of the lane. The anti-PsRBR1 antibody cross-reacted with three polypeptides, indicated by the filled circle, empty circle, and arrowhead. (B) Comparison of molecular mass of authentic PsRBR1 protein with that of PsRBR1 synthesized by in vitro-coupled transcription-translation reaction. The plasmid DNA containing the T3 promoter and the PsRBR1 coding sequence was transcribed, and the resultant RNA was translated. The translation products labeled with [35S]-methionine (lane 2) were immunoprecipitated by the anti-PsRBR1 antibody (lane 3). Proteins in the protein extracts prepared from growing axillary buds were detected by immunoblotting with the anti-PsRBR1 antibody (lane 4). The in vitro-coupled transcription-translation reaction was performed without the plasmid DNA (lane 1). Molecular mass markers are indicated at the left of the lane. The product synthesized in vitro was a 114-kDa polypeptide, indicated by the arrowhead (lane 2), which is consistent with the calculated values based on the deduced amino acids sequences of PsRBR1 cDNA. (C) Dephosphorylation of authentic PsRBR1 protein. Protein extracts prepared from growing axillary buds were dephosphorylated with λ-PPase treatment, separated by SDS-PAGE in either a standard acrylamide gel (left) or low bis-acrylamide gel (right), and proteins were detected by immunoblotting with the anti-PsRBR1 antibody. Numbers in parentheses indicate the ratio of acrylamide to bis-acrylamide. Lane 1, untreated samples; lane 2, samples incubated without λ-PPase; lane 3, samples incubated with λ-PPase
Comparison of molecular mass of authentic PsRBR1 protein in pea with that of in vitro-synthesized PsRBR1 protein
Three polypeptides were detected in protein extracts from pea axillary buds by immunoblotting with the anti-PsRBR1 antibody. The molecular mass of PsRBR1 protein was estimated to be 114-kDa based on the deduced amino acid sequences of PsRBR1 cDNA. To clarify these differences in the molecular mass of PsRBR1 protein, we compared the molecular mass of authentic PsRBR1 protein in pea with that of in vitro-synthesized PsRBR1 protein (Fig. 2B). PsRBR1 protein was synthesized using an in vitro-coupled transcription-translation system with [35S]-methionine. In vitro-synthesized PsRBR1 protein produced a major 114-kDa polypeptide (Fig. 2B, lane 2, arrowhead). The molecular mass of the PsRBR1 protein was consistent with that predicted from the deduced amino acid sequences of PsRBR1 cDNA. The 114-kDa polypeptide was not detected in protein products prepared from the in vitro-coupled transcription-translation system without the PsRBR1 DNA template, which was used as a negative control (Fig. 2B, lane 1). To confirm whether the 114-kDa polypeptide was PsRBR1 protein, in vitro-coupled transcription-translation products were immunoprecipitated with the anti-PsRBR1 antibody (Fig. 2B, lane 3). The 114-kDa polypeptide was immunoprecipitated with the anti-PsRBR1 antibody, indicating that in vitro-synthesized PsRBR1 protein is 114-kDa and corresponds to the smallest polypeptide detected by anti-PsRBR1 antibody in growing axillary buds (Fig. 2B, lane 4). These results suggested that the smallest polypeptide (114-kDa) detected by the anti-PsRBR1 antibody was non-modified PsRBR1 protein in axillary buds, and the two larger polypeptides of PsRBR1 proteins were modified by post-translational regulation in axillary buds.
Dephosphorylation of authentic PsRBR1 protein
The activity of mammalian RB family proteins is regulated by their phosphorylation states. Plant RB-related proteins are also phosphorylated by cyclin/CDK complexes (Huntley et al. 1998; Nakagami et al. 1999). PsRBR1 protein has some putative CDK phosphorylation sites (Fig. 1B). Thus, we expected that the modification of authentic PsRBR1 protein in the axillary buds caused its phosphorylation. To examine this, we performed in vitro dephosphorylation reactions against PsRBR1 protein using (λ-PPase. Proteins were separated by two types of SDS-PAGE gels, because some phosphorylated proteins were more clearly separated by low bis-acrylamide gel (Fig. 2C, right) than by standard acrylamide gel (Fig. 2C, left) (Iwasaki et al. 2002). Immunoblotting with anti-PsRBR1 antibody indicated that there were three polypeptides corresponding to PsRBR1 protein in growing axillary buds (Fig. 2C, lane 1), but after λ-PPase treatment of the protein extract, PsRBR1 protein was detected as the smallest of the polypeptides (Fig. 2C, lane 3). On the other hand, the molecular mass of PsRBR1 protein did not change after treatment without λ-PPase added to the protein extract as a negative control (Fig. 2C, lane 2). In addition, the low bis-acrylamide gel separated the protein more clearly than did the standard acrylamide gel. These results suggested that PsRBR1 protein was controlled by post-translational modification, and exists in non-phosphorylated form of PsRBR1 protein, lower-molecular mass form of phosphorylated PsRBR1 protein (Lp-form), and higher-molecular mass form of phosphorylated PsRBR1 protein (Hp-form) in growing axillary buds.
Protein levels and phosphorylation states of PsRBR1 protein in axillary buds during dormancy-to-growth transition
Because suppression of the cell cycle is regulated by the phosphorylation states of mammalian RB family proteins, we evaluated the phosphorylation states of PsRBR1 during dormancy-to-growth transition in pea axillary buds by immunoblotting using the anti-PsRBR1 antibody (Fig. 3A). During dormancy, PsRBR1 protein was detected as two polypeptides, which are non-phosphorylated form of PsRBR1 protein and the Lp-form (Fig. 3A, 0 h after decapitation). In addition to these non-phosphorylated form of PsRBR1 protein and the Lp-form, the Hp-form was detected 2 h after decapitation (Fig. 3A, indicated by the arrow). These results suggested that PsRBR1 protein was shifted from non-phosphorylated form of PsRBR1 protein and the Lp-form to the Hp-form; that is, the PsRBR1 protein was more phosphorylated after decapitation. The protein levels of these three polypeptides were increased 12 h after decapitation. Non-phosphorylated PsRBR1 protein and the Lp-form existed during late stage (12 to 24 h) after decapitation. PsRBR1 mRNA level was increased 12 h after decapitation (see Fig. 3B). It seems that non-phosphorylated PsRBR1 protein and the Lp-form were synthesized and phosphorylated de novo after decapitation. Moreover, because the axillary bud cell cycle is not completely synchronized, the non-phosphorylated PsRBR1 protein and the Lp-form were included in the protein extracts prepared from growing axillary buds.
Fig. 3Protein levels and phosphorylation states of PsRBR1 protein in axillary buds during the dormancy-to-growth transition. (A) Proteins were extracted from axillary buds after terminal bud decapitation. Numbers below each lane indicate the hours after decapitation. Proteins in the protein extracts (30 μg) were detected by immunoblotting with the anti-PsRBR1 antibody. Molecular mass markers are indicated at the left of the lane. The bottom panel shows the Coomassie Brilliant Blue (CBB) staining around 100 kDa as a loading control. The filled circle, empty circle, and arrowhead indicate higher-molecular mass form of phosphorylated PsRBR1 protein (Hp-form), lower-molecular mass form of phosphorylated PsRBR1 protein (Lp-form) and non-phosphorylated PsRBR1 protein, respectively. The arrow shows more phosphorylated PsRBR1 protein (Hp-form) appearing 2 h after decapitation. (B) Northern blot analyses of total RNA isolated from pea axillary buds before and after decapitation. Plants were decapitated 1 cm above the second node. After decapitation, axillary buds were collected at several time points, as indicated below each lane. Total RNA (10 μg) was separated by denaturing formaldehyde gel electrophoresis, and transferred to a Hybond N+ membrane. The membranes were hybridized with the cDNA of PsRBR1, PCNA, and histone H4. The bottom panel shows the ethidium bromide-stained RNA gel as a loading control
Accumulation patterns of PsRBR1 transcripts in axillary buds during dormancy-to-growth transition
To investigate the relationship between different phosphorylation states of PsRBR1 protein with the transcriptional control of cell cycle regulators, we examined the accumulation patterns of PsRBR1, PCNA, and Histone H4 transcripts during dormancy-to-growth transition in axillary buds. The results of Northern blot analyses using PsRBR1, PCNA, and histone H4 cDNAs (Shimizu and Mori 1998) are shown in Fig. 3B. PsRBR1 transcripts were detected at 3.6 kb. The mRNA levels of the three genes were very low during dormancy in axillary buds. When the axillary buds were stimulated to grow by terminal bud decapitation, mRNA levels of these genes increased markedly. PsRBR1 and histone H4 mRNA increased 12 h after decapitation. Histone H4 was specifically expressed during the S phase. The accumulation patterns of PsRBR1 mRNA are similar to that of histone H4 mRNA, suggesting that PsRBR1 is predominantly expressed at the S phase in pea axillary buds at least during the first 24 h after terminal bud decapitation. Because PsRBR1 protein was increased 12 h after decapitation (see Fig. 3A), the expression patterns of PsRBR1 are coincident with those of PsRBR1 protein. The mRNA expression of PCNA seems to be regulated by RB-related proteins. PCNA mRNA increased significantly 4 h after decapitation, and PCNA mRNA accumulated after the appearance of the Hp-form.
Detection of phosphorylated PsRBR1 protein in axillary buds after decapitation
We confirmed that PsRBR1 protein was phosphorylated in the axillary buds just after decapitation with in vivo labeling followed by immunoprecipitation using the anti-PsRBR1 antibody (Fig. 4). [32P]-inorganic phosphate was incorporated into growing axillary buds. Many proteins were phosphorylated in growing axillary buds after decapitation (Fig. 4A, lane 1). Immunoprecipitation with the anti-PsRBR1 antibody revealed a [32P]-labeled 150-kDa phosphoprotein (Fig. 4A, lane 3, arrow). The size of the [32P]-labeled PsRBR1 protein was consistent with the Hp-form detected by immunoblotting analysis. The signal was not detected by immunoprecipitation with preimmune serum used as a negative control (Fig. 4A, lane 2).
Fig. 4Detection of phosphorylation of PsRBR1 protein in axillary buds during dormancy-to-growth transition. (A) Eight hours after decapitation of the terminal bud, [32P]-inorganic phosphate was applied to axillary buds to be incorporated in vivo. One hour after the application of [32P]-inorganic phosphate, proteins were extracted from the axillary buds. The protein extracts were immunoprecipitated with the anti-PsRBR1 antibody (lane 3) or preimmune serum (lane 2), and separated by SDS-PAGE. Total proteins without immunoprecipitation were loaded in lane 1. The dried gel was exposed to a Fuji Imaging Plate. Molecular mass markers are indicated on the left of the lane. (B) [32P]-inorganic phosphate was applied to axillary buds 1 h before collection of the buds. The axillary buds were collected at 0, 2, and 4 h after terminal bud decapitation (lanes 1 to 3, respectively), and proteins were extracted from the axillary buds. Other details are as described in (A)
To confirm whether PsRBR1 protein is more phosphorylated in axillary buds just after decapitation, we examined dormant and outgrowing axillary buds after decapitation. No signal was detected before decapitation (Fig. 4B, lane 1), suggesting that PsRBR1 protein was not highly phosphorylated de novo before decapitation. Two hours after decapitation, the 150-kDa polypeptide was detected (Fig. 4B, lane 2) and the signal was increased 4 h after decapitation (Fig. 4B, lane 3, arrow). These results suggested that the level of phosphorylation of PsRBR1 protein increased immediately after terminal bud decapitation, producing the Hp-form.
Discussion
In intact plants, the pea shoot apex inhibits growth; i.e., cell elongation and cell division of the dormant axillary buds. The cell cycle of dormant axillary buds is arrested at the G1 phase. Therefore, from the point of view of cell cycle control, the transition from dormancy to outgrowth in axillary buds seems to be from the G1 phase to the S phase. In animal cells, RB family proteins function as negative controllers in G1 arrest, and the activities of RB family proteins are controlled by their phosphorylation states. Here, we investigated phosphorylation states of the pea RB-related protein, PsRBR1, during the dormancy-to-growth transition in axillary buds. The results of the present study provide a basis for further studies of the molecular mechanisms underlying apical dominance and cell cycle control.
Mammalian and plant RB proteins have several highly conserved regions, which are the N-terminal leucine rich, A and B conserved domains, multiple potential CDK phosphorylation sites, and a conserved cysteine residue. PsRBR1 protein, like other plant RB-related proteins, has the ability to interact with both plant D-type cyclins and a mammalian oncoprotein. Plant RB-related proteins have the same properties and activities as animal RB family proteins. For example, ZmRBR1 protein binds to human and Drosophila E2F, and inhibits transcriptional activation of human E2F (Huntley et al. 1998; Ramirez-Parra et al. 1999; Sekine et al. 1999). Geminivirus DNA replication is reduced in plant cells transfected with plasmids encoding human p130 (Xie et al. 1996). Human cyclinD/CDKA phosphorylates NtRBR1 protein in vitro (Nakagami et al. 1999). Thus, plant RB-related proteins can function not only as plant factors, but also as animal factors. These results strongly suggest that the functions of plant RB-related proteins, including PsRBR1 protein, are similar to those of the mammalian RB family proteins in G1/S transition, and that plant and animal cells might use similar regulatory proteins and pathways for cell cycle control during the G1 phase. Therefore, the maintenance of this high degree of similarity throughout evolution (in plants, insects, and vertebrates), further emphasizes the critical roles of RB-related proteins.
Three human RB family proteins (pRB, p130, and p107) have individual functions in cell cycle regulation and development. Two RB-related genes were isolated from Drosophila melanogaster. To date, plant RB-related proteins have been reported in Zea mays, Nicotiana tabacum, Chenopodium rubrum, Arabidopsis thaliana, Populus tremula x Populus tremuloides, Cocos nunucifera, and Oryza sativa (Ach et al. 1997; Fountain et al. 1999; Grafi et al. 1996; Kong et al. 2000; Nakagami et al. 1999; Xie et al. 1996). Of these, Arabidopsis thaliana and Oryza sativa might contain only one copy of the RB-related gene in the whole genome based on the similarity of amino acid sequences of RB-related proteins. Here, our genomic Southern blot analysis suggested the presence of only one copy of the PsRBR1 gene in pea. The dicot RB-related gene seems to contain only one copy in the genome. By contrast, maize contains at least three RB-related genes. ZmRBR3 protein also functions to promote endosperm development (Sabelli et al. 2005), suggesting that maize RB family proteins have special roles in endosperm development.
When the protein extracts in growing axillary buds were treated with λ-PPase, the Lp- and Hp-forms should have been dephopshorylated and stoichiometrically shifted to non-phosphorylated forms. As shown in Fig. 3(C), however, it appears that there was no increase in the non-phosphorylated form (lane 3) compared to the other lanes. The reason for this might be that a 40-kDa polypeptide was also detected by immunoblotting with the anti-PsRBR1 antibody (data not shown). The 40-kDa polypeptide was probably derived from in vitro-dephosphorylated PsRBR1 protein by degradation during λ-PPase treatment. In vitro-dephosphorylated PsRBR1 protein might be more unstable than in vivo-dephosphorylated PsRBR1 protein, despite the presence of proteinase inhibitors, though the reason for this is not clear.
The results of the immunoblotting experiments indicated that the migration patterns of mammalian RB family proteins differ depending on their phosphorylation states. According to the reports (Farkas et al. 2002), the faster migrating proteins were the non- and hypo-phosphorylated forms in the G0/G1 phases. The slower migrating protein was the hyper-phosphorylated form during the G1/S transition. In plant, immunoblotting with the anti-ZmRBR1 antibody revealed the ZmRBR1 protein to be a diffuse protein band of approximately 110-kDa during endoreduplication in maize endosperm, and there are some polypeptides of approximately 110-kDa in maize leaf (Grafi et al. 1996; Huntley et al. 1998). The diffused protein bands of ZmRBR1 were confirmed by the use of λ-phosphatase to result from phosphorylation. (Grafi et al. 1996). In the present paper, using λ-PPase treatment and in vivo labeling followed by immunoprecipitation with the anti-PsRBR1 antibody, we showed that PsRBR1 protein exists in three phosphorylated forms in axillary buds. Although we could not determine the number of phosphorylation sites in the Lp- and Hp-forms, the Lp- and Hp-forms might correspond to the hypo- and hyper-phosphorylated forms of PsRBR1 protein, respectively.
Plant E2F isolated from tobacco and wheat has transcriptional activity and interacts with RB-related protein (Ramirez-Parra et al. 1999; Sekine et al. 1999). There are E2F-binding sites in the promoter region of PCNA, whose transcripts are induced in the late-G1/S phase (Kodama et al. 1991). These observations suggest that plant E2F, like that in animal, induces gene expression of PCNA. Further phosphorylation of PsRBR1 protein was induced 2 h after terminal bud decapitation, and PCNA transcripts were induced 4 h after terminal bud decapitation in pea axillary buds. Although it is unclear whether the transcriptional activation of plant PCNA is regulated by E2F, E2F released from RB-related protein might promote PCNA mRNA expression, as in mammals. This possibility is supported by the observation that ZmRBR1-associated kinase activity peaks at the G1/S transition in synchronized tobacco BY-2 cells (Boniotti and Gutierrez 2001). It is likely that increased phosphorylation states of PsRBR1 protein have an important role in the regulation of E2F-mediated gene expression involved in the G1/S transition and DNA replication during dormancy-to-growth transition in pea axillary buds.
In some plants, the gene expression of cyclins and CDK activities are regulated by plant hormones, e.g., cytokinin, auxin, and gibberellin (Miao et al. 1993; Sauter 1997; Soni et al. 1995). In Arabidopsis suspension-cultured cells, D-type cyclin mRNA is rapidly induced by the addition of cytokinin or glucose, and repressed by the addition of auxin (Soni et al. 1995). Cytokinin is biosynthesized at the stem after decapitation and moves into the axillary buds for outgrowth (Tanaka et al. 2006). It is possible that the increasing cytokinin levels caused by decapitation induces the expression of D-type cyclin. D-type cyclin forms a complex with CDKs and immediately phosphorylates RB-related protein. The Hp-form induces gene expression for the G1/S transition in axillary bud cells. It is likely that RB-related protein controls the cell cycle in axillary buds during the dormancy-to-growth transition. Further analyses will provide more insight into the regulation of dormancy-to-growth transition in axillary buds.

pea
retinoblastoma-related protein
phosphorylation
axillary buds
dormancy
cell cycle


Using the Internet for Surveys and Health Research

This paper concerns the use of the Internet in the research process, from identifying research issues through qualitative research, through using the Web for surveys and clinical trials, to pre-publishing and publishing research results. Material published on the Internet may be a valuable resource for researchers desiring to understand people and the social and cultural contexts within which they live outside of experimental settings, with due emphasis on the interpretations, experiences, and views of `real world' people. Reviews of information posted by consumers on the Internet may help to identify health beliefs, common topics, motives, information, and emotional needs of patients, and point to areas where research is needed. The Internet can further be used for survey research. Internet-based surveys may be conducted by means of interactive interviews or by questionnaires designed for self-completion. Electronic one-to-one interviews can be conducted via e-mail or using chat rooms. Questionnaires can be administered by e-mail (e.g. using mailing lists), by posting to newsgroups, and on the Web using fill-in forms. In "open" web-based surveys, selection bias occurs due to the non-representative nature of the Internet population, and (more importantly) through self-selection of participants, i.e. the non-representative nature of respondents, also called the `volunteer effect'. A synopsis of important techniques and tips for implementing Web-based surveys is given. Ethical issues involved in any type of online research are discussed. Internet addresses for finding methods and protocols are provided. The Web is also being used to assist in the identification and conduction of clinical trials. For example, the web can be used by researchers doing a systematic review who are looking for unpublished trials. Finally, the web is used for two distinct types of electronic publication. Type 1 publication is unrefereed publication of protocols or work in progress (a `post-publication' peer review process may take place), whereas Type 2 publication is peer-reviewed and will ordinarily take place in online journals.
Identifying issues for qualitative research
As the most comprehensive archive of written material representing our world and people's opinions, concerns, and desires (in industrialized countries), the Internet can be used to identify `issues' for qualitative (descriptive) research and to generate hypotheses. Material published on the Internet may be a valuable resource for researchers desiring to understand people and the social and cultural contexts within which they live--outside of experimental settings--with due emphasis on the interpretations, experiences, and views of `real world' people. Reviews of information posted by consumers on the Internet may help to identify health beliefs, common topics, motives, information, and emotional needs of patients, and point to areas where research is needed. Comparing recommendations found on the Web against evidence-based guidelines is one way to identify areas where there is a gap between opinion and evidence, or where there is a need for clinical innovation.
The accessibility of information for analysis and the anonymity of the Internet allow researchers to analyse text and narratives on Web sites, to use newsgroups as global focus groups, and to conduct interviews and surveys via e-mail, chat rooms, Web sites, or newsgroups.Topics suited to qualitative research include:
Analysis of interactive communications (e.g. e-mail).
Study of online communities (virtual self-help groups, newsgroups, mailing lists).
Investigation of communication processes between patients and professionals.
Study of consumer preferences, patient concerns, and information needs.
Exploration of the `epidemiology of health information' on the Web [1-2].
The Internet population is unrepresentative of the general population, restricting the use of the Internet for quantitative studies (i.e. studies focusing on measurement). Qualitative studies, however, do not require representative samples:`In qualitative research we are not interested in an average view of a patient population, but want to gain an in-depth understanding of the experience of particular individuals or groups; we should therefore deliberately seek out individuals or groups who fit the bill' [3]. Three different research methodologies for qualitative research on the Internet may be distinguished:
Passive analysis: For example, studying information on Web sites or interactions in newsgroups, mailing lists, and chat rooms--without researchers actively involving themselves.
Active analysis: Also called participant observation; the researcher participates in the communication process, often without disclosing their identity as researcher. For example, they may ask questions in a patient discussion group implying that she or he is a fellow patient. Such studies often involve elements of deception, unless the researcher is a sufferer him- or herself.
Interviews and surveys: See below.
Examples of these three types of qualitative research on the Internet are available elsewhere [1].
Using the Internet for surveys
Using the Internet for surveys requires an awareness of methodologies, selection bias, and technical issues.
Methodological issues
Internet-based surveys may be conducted by means of interactive interviews or by questionnaires designed for self-completion. Electronic one-to-one interviews can be conducted via e-mail or using chat rooms. Questionnaires can be administered by e-mail (e.g. using mailing lists), by posting to newsgroups, and on the Web using fill-in forms.
When e-mail is used to administer questionnaires, messages are usually sent to a selected group with a known number of participants, thus allowing calculation of the response rate. Surveys posted to newsgroups may request that the completed questionnaire is posted back to the researcher, but it is impossible to know who and how many people read the questionnaire. If Web-based forms are used, questionnaires can be placed in a password-protected area of a Web site (i.e. participation by invitation or registration only), or alternatively they may be open to the public (i.e. any site visitor can complete the survey). The latter option makes calculation of a response rate more difficult but not impossible: the number of people who access (without necessarily completing) the questionnaire is counted and used as the denominator. Web-based surveys have the advantage that the respondent can remain anonymous (as opposed to e-mail surveys, where the e-mail address of the responder is revealed). Furthermore, they are very convenient for the researcher, as responses can be directly stored in a database where they are immediately accessible for analysis.
Electronic interviews and surveys (`e-surveys') are emerging scientific research methodologies, pioneered by communication scientists, sociologists, and psychologists, although their use for health-related research is still in its infancy [4-10]. Examples of health-related research include:
A Web-based survey on the effects of ulcerative colitis on quality of life [11].
Collection of clinical data from atopy patients [12].
A Web-based survey looking at complementary and alternative medicine use by patients with inflammatory bowel disease and Internet access [13].
A survey of dentists regarding the usefulness of the Internet in supporting patient care [14,15].
Guidelines for Web-based surveys
Scenarios that may be suitable for a Web-based survey
Respondent features:
Respondents are already avid Internet users; e-mail addresses known for reminder messages.
Respondents are enthusiastic form fillers; will not require monetary incentives.
Need for respondents covering a wide geographical area (e.g. rare clinical special ties, diseases).
Respondents are known to match non-respondents and even non-Internet users on key variables.
Survey features:
Need for complex branching, interactive questionnaire or multimedia as part of the survey instrument.
Survey content will evolve fast (e.g. Delphi method surveys use repeating rounds of revised questionnaires delivered over a short period, incorporating aggregate results from previous rounds until convergence is achieved).
Intent is to document bizarre, rare phenomena whose simple occurrence is of interest.
No need for representative results: collecting ideas vs. hypothesis testing.
Investigator features:
Limited budget for mailing and data processing, but good in-house Web skills.
Precautions can be taken against multiple responses by same individual, password sharing.
Web survey forms have been piloted with representative participants and demonstrate acceptable validity and reliability with most platform, browser, and Internet access provider combinations.
Data is required fast in a readily analysed form.
Scenarios that are unsuitable for a Web-based survey
Respondent features:
Target group is under-represented on Internet; e.g. the underprivileged, elderly people.
Target group is concerned, however unreasonably, about privacy aspects.
Target group requires substantial incentives to complete the survey.
Need for a representative sample.
Survey features:
Need for very accurate timing data on participants (inaccuracies in the range of seconds are added due to network transmission times, unless JavaScript or Java applets are used; see Glossary) or observational data on participants.
An existing paper instrument has been carefully validated on target group.
Need to capture qualitative data or observations about participants.
Wish to reach the same group of participants in the same way months or years later.
Investigator features:
Limited in-house Web or Java expertise but existing desktop publishing and mailing facility.
E-surveys may be part of a qualitative research process, but results can be analysed quantitatively as long as researchers are aware of potential bias (see below). In addition to gathering data, the Internet may also be used in the course of developing questionnaires, as it allows rapid prototyping and pilot testing of instruments, e.g. to evaluate the effect of framing the questions differently [16].
Several studies have checked the validity of Web-based surveys by comparing the results of studies conducted on the Web with identical studies in the real world. These seem to suggest that the validity and reliability of data obtained online are comparable to those obtained by classical methods [4,5,17-19]. However, issues of generalizability (mainly due to selection bias, discussed in detail below) remain important considerations, and the researcher should select his or her research question and interpret the results with care.The benefits and problems of Web-based surveys have been summarized by Wyatt, who suggests guidelines for when they may be appropriate (see Box 1) [20].
Selection bias
In `open' surveys conducted via the Internet where Web users, newsgroup readers, or mailing list subscribers are invited to participate by completing a questionnaire, selection bias is a major factor limiting the generalizability (external validity) of results. Selection bias occurs due to:
The non-representative nature of the Internet population.
The self-selection of participants, i.e. the non-representative nature of respondents, also called the `volunteer effect' [21].
The non-representative nature of Internet demographics was briefly considered earlier. Considering whether the topic chosen for study is suitable for the Internet population is the first and probably the most important step in minimizing bias, thus maximizing response rates and increasing the external validity of the results [20]. For example, targeting elderly homeless alcoholics is unsuitable for an Internet survey and the results are likely to be heavily skewed by hoax responses.
Self-selection bias originates from the fact that people are more likely to respond to questionnaires if they see items which interest them, e.g. because they are affected by the items asked about, or because they are attracted by the incentives offered for participating. As people who respond almost certainly have different characteristics than those who do not, the results are likely to be biased.This kind of selection bias is more serious than the bias arising from the non-representative nature of the population, because the researcher deals with a myriad of unknown factors and has little opportunity to interpret his or her results accordingly. Such bias may be exacerbated via loaded incentives (e.g. typical `male' incentives such as computer equipment). Evidence suggests women are generally more interested in health topics and exhibit more active information-seeking behaviour [22], so are more likely to volunteer participation in health questionnaires. For Web surveys, the potential for self-selection bias can be estimated by measuring the response rate, expressed as the number of people completing the questionnaire divided by those who viewed it (cf. the participation rate, expressed as the number of site visitors viewing the questionnaire divided by the total number of site visitors).
Technical issues
Although a detailed analysis is beyond the scope of this chapter, a synopsis of important techniques and tips for implementing Web-based surveys provides some insight into the difficulties faced by survey designers (see Box 2).
Technical issues in implementing Web-based surveys
Use of `cookies'
Cookies can assign a unique identifier to every questionnaire viewer, useful for determining response and participation rates, and for filtering out multiple responses by the same person. As cookies may be regarded with suspicion, we recommend that researchers openly state that cookies will be sent (and the reasons for this); set the cookie to expire on the day that data collection ceases; and publish a privacy policy.
Measuring response time
The time needed to complete a questionnaire can be readily calculated by subtracting the time a form was called up by the browser from the time it was submitted using an automatic time-stamp. The response time may be used to exclude respondents who fill in the questionnaire too quickly: this may identify hoax responses, where respondents don't read the questions.
Avoiding missing data
Forms can be configured to automatically reject incomplete questionnaires and point out missing or contradictory items. Checks can be made on the client (p. 9) prior to submission, or following submission to the server (where incomplete responses can also be analysed, e.g. during a questionnaire pilot).
Maximizing response rate
The number of contacts, personalized contacts, and contact with participants before the actual survey are the factors most associated with higher response rates in Web surveys [23]. Incentives increase the risk of selection bias (see text), but less so if cash is offered. Perhaps the best incentive (and the easiest to deliver via the Internet) is the promise of survey results or personalized answers (e.g. a score). The option to complete questionnaires anonymously avoids wariness associated with requests for personal information (e.g. an e-mail address), but increases the risk of hoax responses. Researchers should be open about who is behind the study, what the aim is, and provide opportunities for feedback. Although postal surveys are superior to e-mail surveys with regard to response rate, online surveys are much cheaper [24,25]. Schleyer [15] estimated that the cost of their Web-based survey was 38 percent less than that of an equivalent mail survey and presented a general formula for calculating break-even points between electronic and hard-copy surveys. Jones gave figures of 92 p per reply for postal surveys, 35 p for e-mail, and 41 p for the Web [24].
Randomizing items
Scripting languages may be used to build dynamic questionnaires (as opposed to static forms) that look different for certain user groups or which randomize certain aspects of the questionnaire (e.g. the order of the items). This can be useful to exclude possible systematic influences of the order of the items upon responses.
Ethical issues
The ethical issues involved in any type of online research should not be forgotten [1,26-31]. These include informed consent as a basic ethical tenet of scientific research on human populations [32], protection of privacy, and avoiding psychological harm.
In qualitative research on the Web, informed consent is required when:
Data are collected from research participants through any form of communication, interaction, or intervention.
Behaviour of research participants occurs in a private context where an individual can reasonably expect that no observation or reporting is taking place, except when researchers do research `in public places or use publicly available information about individuals (e.g. naturalistic observations in public places, analysis of public records, or archival research)' [33].
The question therefore arises of whether researchers analysing newsgroup postings enter a `public place', or whether the space they invade is perceived as private. In the context of research, the expectation of the individual (whether he/she can reasonably expect that no observation is taking place) is crucial. Different Internet services have different levels of perceived privacy (in decreasing order of privacy: private e-mail; chat rooms; mailing lists; newsgroups;Web sites).The perceived level of privacy is a function of the number of participants, but also depends on other factors such as group norms established by the community to be studied. For example, in a controversial paper, Finn studied a virtual self-support group where the moderator was actively discouraging interested professionals who were not sexual abuse survivors from joining the group [34]. In those cases, obtaining informed consent (or seeking an ethical waiver, if the research could not practicably be carried out were informed consent to be required) is mandatory.
In practice, obtaining informed consent, especially for passive research methods, is difficult, as researchers usually cannot post an announcement to a mailing list or newsgroup saying that it will be monitored and analysed for the next few months, as this may greatly influence or even spoil the results, and because the mere posting of such a request may disrupt the community, and therefore be considered unethical. Researchers should therefore first obtain consent from a group moderator in order to explore whether even a request for permission is felt to be disruptive to the group process. If the moderator or person responsible for the list has no objections, one may then post a message to a newsgroup or mailing list explaining the purpose of the research, explaining that one will observe the community, assuring all participants of anonymity, and giving them the opportunity to withdraw from the newsgroup or mailing list or to exclude themselves from the study by writing to the researcher. The fundamental problem is that this may influence the communication process and may even destroy the community. Besides, participants who later join the group need to get the same information. An alternative would be to analyse the communication retrospectively and to write individual e-mails to all participants whose comments were to be analysed or quoted, asking for permission to use them; this technique has been used by Sharf [35].
In any case, researchers should make themselves familiar with the virtual community they are approaching; i.e. read the messages in a newsgroup for some time (`lurking'). Under no circumstances should researchers blindly spam (p. 31) or cross-post requests for research participation to various newsgroups.
Informed consent may also play a role when researchers report aggregate (collated and hence anonymous) data on usage patterns, such as a log-file analysis (reporting data on what Web sites have been accessed by a population). Crucial here is an appropriate privacy statement stating that these data may be analysed and reported in aggregate [28]. Note that aggregate data are exempt from the registration requirements of the UK's Data Protection Act of 1998.
In conducting surveys researchers may obtain informed consent by declaring the purpose of the study; disclosing which institutions are behind the study; explaining how privacy will be assured; and detailing with whom data will be shared and how it will be reported, before participants complete the questionnaire.
When reporting results, it is obvious that the total anonymity of research participants needs to be maintained. Researchers have to keep in mind that, by the very process of quoting the exact words of a newsgroup or mailing list participant, the confidentiality of the participant may already be broken as Internet search engines may be able to retrieve the original message, including the e-mail address of the sender. It is essential, therefore, to ask participants whether they agree to be quoted whenever there may be a retrievable archive, pointing out the risk that they may be identifiable. Problems can also potentially arise from just citing the name of the community (e.g. of a newsgroup), which may damage the community being studied.
Finding methods, protocols, and instruments
For laboratory `bench work', researchers often need a protocol for a specific assay method. In addition to the possibility of searching literature databases, there are also specialized services on the Web that can assist in this research, such as MethodsFinder and the `Technical tips online' database at BioMedNet:
MethodsFinder (BIOSIS):
http://www.methodsfinder.org/
BioMedNet:
http://www.bmn.com/
Sometimes asking a specific question on the right newsgroup or mailing list is also very effective. Clinical researchers may be more interested in instruments to measure patient outcomes.An excellent guide to selecting quality-of-life instruments is the Quality of Life Instruments Database at the Mapi Research Institute: http://www.qolid.org/
Online statistical analysis tools are available at the Simple Interactive Statistical Analysis (SISA) Web site, while background information is available within the online book Statistics at square one:
SISA (Daan Uitenbroek):
http://home.clara.net/sisa/
Statistics at square one (British Medical Journal Publishing Group):
http://www.bmj.com/collections/statsbk/
Protocols of clinical trials, which may be useful for researchers developing their own protocols, can be found in some of the clinical trial databases available on the Web, as described below.
Clinical trials and the Web
The Web is being used to assist in the identification and conduction of clinical trials.
Identifying trials
To prevent unintended duplication of clinical research, detect underreporting of research, and ease the work of systemic reviewing, it has been suggested that we should prospectively register clinical trials [36-39]. It is, however, unlikely that there will ever be one complete centralized multinational database. Instead, multiple resources set up by numerous different organizations will exist [40]. Internet technology will play a central role in linking these databases and making this information available to researchers and patients. Some scenarios in which a search of trial databases may be useful:
A researcher wants to conduct a randomized controlled trial and wants to know whether anyone else is already running one on the same topic.
A physician has a patient who is asking about available trials.
A patient is looking for ongoing trials.
A researcher is looking for possible participants for his trial.
A researcher doing a systematic review is looking for unpublished trials.
Information about ongoing and completed clinical trials is increasingly being published on the Internet, and searches on the Web may be a useful means of complementing traditional bibliographic searches if authors of systematic reviews wish to find ongoing or unpublished trials [41].
Researchers use their personal or department home pages to announce their interest in a certain research area or to recruit patients [42]. Journals like The Lancet have begun to publish research protocols on their Web site [43], and more and more researchers will also publish `pre-prints' (p. 239) of their findings on the Web [44].
Consumers and patient organizations also have an interest in disseminating information about ongoing trials; e.g. the National Alliance of Breast Cancer Organizations: http://www.nabco.org/
Government and funding agencies react to this need by establishing trial databases for consumers; e.g. the US National Institutes of Health searchable database [45]: http://ClinicalTrials.gov
Commercial enterprises also help researchers to recruit patients, or help patients to find clinical trials. For example:
CenterWatch Clinical Trials Listing Service (CenterWatch, Inc.):
http://www.centerwatch.com/
ClinicalTrialFinder.com (Clinical Data Technologies Ltd):
http://www.clinicaltrialfinder.com/
Current Controlled Trials (BioMed Central):
http://www.controlled-trials.com
Pharmaceutical companies and industry associations have likewise begun to recognize that openness and access to information on clinical trials and new drug developments can improve patient care and are part of social responsibility [46]. For example:
Clinical Trials Register (GlaxoSmithKline):
http://ctr.glaxowellcome.co.uk/
Search for Cures (Pharmaceutical Research and Manufacturers of America):
http://www.phrma.org/searchcures/
Finally, information or databases on ongoing clinical trials can often also be found on disease-specific sites. For example:
Canadian HIV Trials Network:
http://www.hivnet.ubc.ca/ctn.html
CancerNet (National Cancer Institute):
http://cancernet.nci.nih.gov/
Conducting trials on the Web
The Web is increasingly being used in the course of conducting large-scale multi-centre clinical trials (e.g. for remote randomization and data entry), and in the distribution of information on trial progress or protocols [47,48]. Trial centres may enter patient data using Java applets (see Glossary) that encrypt data and send it to the data centre via the Internet [49-52], where the data are stored and randomized, returning for example a study number and randomization information.
Pre-publishing and publishing research
Traditional publication is a well-defined event, whereas `publication' in the electronic age is much more of a continuum [53], reflecting and occurring during the entire research process from hypothesis formulation to data gathering, interpretation, and the presentation and discussion of the final results. In order to distinguish online collaborative `work in progress' from `final' peer-reviewed publication we may term the former `Type 1' and the latter `Type 2' electronic publication [54]. Here, peer review is not the distinguishing characteristic: in Type 1 publication a `post-publication' peer review process takes place. Type 2 publication will ordinarily take place in online journals. The following scenarios illustrate how researchers might use Type 1 electronic publication on the Internet:
Sending and discussing preliminary results on mailing lists.
Publishing drafts of scientific papers on pre-print/e-print sites (p. 239) in order to solicit comments and to improve the manuscript.
Publishing data and information in databases; e.g. nucleotide sequences in the EMBL/Genbank databases.
Publishing clinical trial protocols and raw data in a `trial bank' [55].
Current awareness services
Electronic editions of paper journals and `stand alone' e-journals typically offer subscriptions to `TOC alerts', where users receive a table of contents by e-mail as soon as a new issue appears.The more sophisticated systems allow users to specify their interests using a controlled vocabulary, enabling the system to screen each newly published article for certain keywords or citations. Examples of current awareness services include:
Customised @lerts (British Medical Journal):
http://bmj.com/cgi/customalert/
JournAlert (Doctors.net.uk):
http://www.doctors.net.uk/
Journal Watch (Massachusetts Medical Society):
http://www.jwatch.org/

internet
qualitative research
clinical trials
survey research
selection bias
data collection
informed consent
confidentiality
ethics, research
evaluation studies
patient selection
research design
research subjects


Monitoring the expression of purinoceptors and nucleotide-metabolizing ecto-enzymes with antibodies directed against proteins in native conformation

Following their release from cells, ATP and NAD, the universal currencies of energy metabolism, function as extracellular signalling molecules. Mammalian cells express numerous purinoceptors, i.e., the nucleotide-gated P2X ion channels and the G-protein-coupled P2Y receptors. Signalling through purinoceptors is controlled by nucleotide-metabolizing ecto-enzymes, which regulate the availability of extracellular nucleotides. These enzymes include ecto-nucleoside triphosphate diphosphohydrolases (ENTPD, CD39 family) and ecto-nucleotide pyrophosphatase/phosphodiesterases (ENPP, CD203 family). Investigation of these receptors and enzymes has been hampered by the lack of available antibodies, especially ones that recognize these proteins in their native conformation. This study reports the use of genetic immunization to generate such antibodies against P2X1, P2X4, P2X7, ENTPD1, ENPTD2, ENPTD5, ENPTD6, ENPP2, ENPP3, ENPP4, ENPP5, and ENPP6. Genetic immunization ensures expression of the native protein by the cells of the immunized animal and yields antibodies directed against proteins in native conformation (ADAPINCs). Such antibodies are especially useful for immunofluorescence and immunoprecipitation analyses, whereas antibodies against synthetic peptides usually function well only in Western-blot analyses. Here we illustrate the utility of the new antibodies to monitor the cell surface expression of and to purify some key players of purinergic signalling.
Introduction
Following their release from cells, the universal currencies of energy metabolism, ATP and NAD, function as extracellular signalling molecules [1–5]. Molecular cloning has identified 7 nucleotide-gated ion channels (P2X purinoceptors) and >8 nucleotide-activated G-protein-coupled receptors (P2Y purinoceptors) [6–10]. Signaling through purinoceptors is effectively controlled by nucleotide-metabolizing ecto-enzymes, which regulate the availability of extracellular nucleotides [11]. Prominent roles are played by members of two families of ecto-enzymes: the ecto-nucleoside triphosphate diphosphohydrolases (ENTPD, CD39 family) [12] and the ecto-nucleotide pyrophosphatase/phosphodiesterases (ENPP, CD203 family) [13].
The mammalian P2X family encompasses seven members. All P2X purinoceptors contain cytosolic N- and C-terminal tails, two membrane spanning domains, and an extracellular domain of approximately 280 amino acids containing the ligand-binding site and 10 conserved cysteine residues that likely form five intrachain disulfide bonds [14, 15]. The extracellular domains of the respective mouse and human P2X orthologues show high (ca. 80–90%) sequence identity; the paralogues within a given species show lower (40–50%) identity.
The mammalian ENTPD family encompasses eight members [16]. ENTPD1–4, 7 and 8 have a structure similar to that of P2X purinoceptors, i.e., cytosolic N- and C-terminal tails, two transmembrane spanning domains, and an extracellular domain of approximately 440 amino acids, encompassing the ligand-binding site and four conserved cysteines likely engaged in intrachain disulfide bridges [12]. ENTPD1–3 and 8 are expressed as cell surface enzymes, ENTPD4 and ENTPD7 as luminal enzymes of intracellular organelles. ENTPDases share structural similarities with the actin/HSP70/sugar kinase superfamily [12]. ENTPD5 and ENTPD6 lack the second transmembrane region. Moreover, in ENTPD5, the region corresponding to the first transmembrane domain of the other family members is N-terminal and functions as a signal peptide, resulting in the secretion of ENTPD5 into the lumen of the endoplasmic reticulum. ENTPD6 is a type II transmembrane protein. ENTPD orthologues show high (ca. 80–90%) sequence identity; the paralogues within a given species show lower (30–50%) identity.
The ENPP family encompasses seven members [13]. ENPP1–3 are type II transmembrane proteins with a structure analogous to that of ENTPD6, i.e., an N-terminal cytosolic tail, a single transmembrane domain and an extracellular catalytic domain of approximately 400 amino acids. ENPP4–7, in contrast, are type I membrane proteins. A hydrophobic N-terminus acts as a signal peptide, the extracellular catalytic domain is followed by a single transmembrane domain and a short C-terminal cytosolic tail. ENPPs 4–7 consist of catalytic domains only, the catalytic domains of ENPPs 1–3 are flanked by genetically fused upstream somatomedin B and downstream nuclease-like domains [13]. ENPPs share structural similarities with the alkaline-phosphatase superfamily [17, 18]. The catalytic domain of ENPP orthologues shows high (ca. 80–90%) sequence identity; the paralogues within a given species show lower (40–50%) identity.
Despite their relevance for purinergic signalling, investigation of these receptors and enzymes has been hampered by the lack of available antibodies, especially ones that recognize the enzymes in their native conformation. Antibodies raised by immunization with synthetic peptides derived from the known amino acid sequence of a protein of interest generally work well in Western-blot analyses but often fail to recognize the native protein on the cell surface (Fig. 1). Such antibodies can be used successfully for monitoring the overall expression level of the protein of interest in a population of cells but cannot be used to assess cell surface expression on individual cells. Genetic immunization, in contrast, ensures that the protein of interest is expressed in native conformation by the cells of the immunized animal and yields antibodies directed against proteins in native conformation (ADAPINCs) [19, 20]. Such antibodies function well in immunofluorescence, FACS, ELISA, and immunoprecipitation analyses, i.e., assays in which antipeptide antibodies often fail. We have previously employed this technique to generate highly specific polyclonal and monoclonal antibodies against ecto-ADP-ribsoyltransferases and against murine P2X7 [20–23]. Here we report the generation and characterization of similar antibodies against other key players of purinergic signalling and illustrate the utility of these antibodies for monitoring the cell surface expression of their cognate antigens.
Fig. 1DNA immunization vs. peptide immunization
Materials and methods
Cloning of expression vectors and cell transfections
Clones encoding full-length open-reading frames representing members of the P2X, ENTPD, and ENPP families were purchased from the mammalian genome collection (www.rzpd.de). Clones that were not already in an expression vector (pCMV-SPORT6) were subcloned into the pCDNA6 expression vector. Further expression constructs were kindly provided by James Wiley, Sydney (human P2X7), Richard J. Evans, Leicester (human P2X1), François Rassendren, Montpellier (mouse P2X4), Ruth Murrell-Lagnado, Cambridge (rat P2X4), and Astrid Kehlen, Halle (hENPP2). An expression construct for nuclear green fluorescent protein was generated by cloning the DNA-binding domain of the LKLF transcription factor [24] as a C-terminal fusion protein into the pEGFP-N1 vector (Clontech). Expression constructs were transfected into CHO and HEK cells (5 μg for purinoceptors and ecto-enzymes and/or 0.5 μg eGFP-LKLF per T25 flask containing 2 × 106 cells) with the JetPei transfection reagent (Q-Biogen). For stable transfections, plasmids were linearized with Ssp1 or Sca1 before transfection, and stable transfectants were selected by cultivating cells in the presence of 12 μg/ml blasticidin (Sigma).
Genetic immunization and purification of antibodies
Genetic immunizations were performed as described [20, 22]. In brief, rabbits or rats were immunized four times by ballistic DNA immunization with plasmid-conjugated gold particles. Serum samples were obtained 10 days post-immunization. For monoclonal antibody production, animals received a final boost with transiently transfected HEK cells (2 × 106 cells in 200 μl PBS i.v.) 3 days prior to sacrifice. Splenocytes were fused to Sp2/0 myeloma cells and screened for production of specific antibodies by immunofluorescence analysis of CHO cells that had been co-transfected 20–42 h earlier with constructs encoding eGFP-LKLF and the protein of interest. Antibodies were purified by affinity chromatography on Protein G Sepharose (Pharmacia), and conjugated to Alexa488 according to the manufacturer’s (Molecular Probes) instructions. Antibodies directed against the C-terminal peptides of P2X purinoceptors were purchased from Caltag (rat P2X1, Cal), ABCAM (rat P2X4, Abc), and Alomone (mouse P2X7, Al). The L4 anti-human P2X7 mAb was kindly provided by Dr. Ian Chessel at Glaxo Wellcome (Middlesex, UK).
Immunofluorescence analyses
CHO cells were co-transfected with expression constructs for purinoceptors or ecto-enzymes and nuclear GFP (5 and 0.5 μg/106 cells, respectively). Cells were transferred onto 96-well plates (4 × 105 cells/well) 6 h post-transfection. Then, 20 h after transfection, cells were fixed for 10 min in 2% paraformaldehyde (PFA) and then stained with serial dilutions of immune serum for 30 min at room temperature. To detect ENTPD5, which is not bound to the cell surface, cells were fixed in 4% PFA and subsequently permeabilized with 0.5% IGEPAL (Sigma) before staining. Bound antibody was detected with PE-conjugated antirat or antirabbit IgG (1:100) (Dianova). Cell nuclei were counterstained with Hoechst 33342. Cells were analyzed with a Zeiss Axiovert 25 microscope equipped with the Canon PowerShot G2 digital camera and appropriate filters for visualizing Hoechst stain (Zeiss filter set 01), green fluorescent protein (Zeiss filter set 10) and PE fluorochrome (Zeiss filter set 15). Photographs were taken at 40× magnification with fixed camera settings (digital zoom: 4.3×, aperture: 5.0, exposure time: 2 s for GFP and PE, 1/8 s for Hoechst 33342). GFP and PE images were merged using Adobe Photoshop software.
FACS analyses
For FACS analyses, cells were harvested by brief trypsinization (5 min at room temperature) and washed in serum-containing medium. Cells (5 × 105/100 μl) were stained either directly with Alexa-488-conjugated antibodies (1 μg) or with serially diluted immune serum followed by PE-conjugated antirat or antirabbit IgG (1:100, Dianova). Gating was performed on living cells on the basis of propidium iodide exclusion.
Immunoprecipitation analyses
Antibodies were conjugated to Protein G Sepharose beads (Pharmacia) (1 μg antibody/20 μL beads in 200 μL PBS containing 1% Triton X-100) by incubation on a roller for 30 min at room temperature. Beads were washed twice in PBS/1% Triton X-100 before use for immunoprecipitation. Cells (107/ml) were lysed in PBS, 1% Triton-X100, 1 mM AEBSF (Sigma) for 20 min at 4°C. Insoluble material was pelleted by centrifugation, and the cleared lysates (2 × 106 cells in 200 μL) were subjected to immunoprecipitation using 20 μL antibody-conjugated beads by incubation on a roller for 30 min at room temperature. Beads were washed four times in PBS/1% Triton X-100. Proteins were eluted from the beads by incubation in SDS-PAGE sample buffer for 15 min at 70°C. Eluted proteins were size-fractionated on precast SDS-PAGE gels (Invitrogen) and blotted onto PVDF membranes. Purinoceptors and ecto-enzymes were detected with serially diluted serum followed by peroxidase (PO)-conjugated antirabbit IgG (1:5,000) using the ECL system (Amersham).
Immunoblot analyses
Cells (107/ml) were pelleted by centrifugation and lysed by resuspension in SDS-PAGE sample buffer (Invitrogen). DNA was sheared by brief ultrasonication on ice (two 15-s pulses with an MSE ultrasonicator at maximum power setting). Insoluble material was pelleted by centrifugation, and soluble proteins (5 × 105 cell equivalents/lane) were size-fractionated on precast SDS-PAGE gels (Invitrogen) under reducing conditions and blotted onto PVDF membranes. Purinoceptors and ecto-enzymes were detected with serially diluted serum followed by PO-conjugated antirabbit IgG (1:5,000) using the ECL system (Amersham).
Results and discussion
Use of transiently transfected CHO cells to assess the specificity and titres of antisera against purinoceptors and ecto-enzymes
Antibodies raised by genetic immunization of rabbits and rats with cDNA expression constructs for purinoceptors, ENTPDs, and ENPPs were assayed for reactivity and specificity by indirect immunofluorescence staining of CHO cells, 24 h after cotransfection with a construct encoding nuclear GFP and the cDNA expression construct used for immunization (Fig. 2 and Table 1). By this time, transfected cells were clearly distinguishable from untransfected cells by virtue of their green fluorescent nuclei (Fig. 2). Specific antibodies were detected on the basis of bright cell surface staining of transfected cells and lack of reactivity with untransfected cells. Antipeptide antisera against the same antigens, in contrast, did not show any detectable reactivity in this assay (Table 1).
Fig. 2Immunofluorescence analyses of CHO cells following transient cotransfection with expression constructs for nuclear green fluorescent protein (GFP) and purinoceptors (a), CD39-like ENTPDs (b), and CD203-like ENPPs (c). CHO cells were cotransfected with nuclear GFP and expression constructs for the indicated purinoceptors or ecto-enzymes. Twenty hours after transfection, cells were stained with serial dilutions of the indicated antisera followed by PE-conjugated antirabbit IgG. To detect non-membrane-bound ENTPD5, intracellular staining was performed by fixing and permeabilizing cells. Cell nuclei were counterstained with Hoechst 33342. Cells were analyzed with a Zeiss Axiovert 25 microscope equipped with filters for visualizing Hoechst dye, GFP, and PE fluorochrome. Panels show merged images for Hoechst dye and GFP (b, c, left panels) or for GFP and PE (a–c, right panels)Table 1Titres, specificities, and applications of antibodies against members of the families of P2X purinoceptors, CD39-like ENTPDs, and CD203-like ENPPsAntibodyHostTitreSpecificityFACS/IFTWestern blotP2X family9316rb1:800hP2X1+++(+)RG2rt1:800hP2X1+++++CR29rb1:1600hP2X4+++++CR30rb1:1600mP2X1+++++116rb1:800mP2X4RG22rt1:1600mP2X4++++++K1Grb1:1600mP2X7+++−RH23rt1:1600mP2X7++−Hano43rtmAbmP2X7++−3428rb1:1600rP2X7++−Caltrb1:2000rP2X1−+++Abcrb1:2000rP2X4−+++Alrb1:5000mP2X7−+++ENTPD family5441rb1:1600hENTPD2+++−RG10rt1:1000h/mENTPD2+++ndRG10-B28rtmAbhENTPD2+++nd5446rb1: 400hENTPD5+++−CR44rb1:6,400mENTPD1++++++RG9rt1:1600mENTPD1+++ndRG9-A59rtmAbmENTPD1+++−CR45rb1:1600mENTPD2++++CR63rb1:1600mETNTPD2+++ndCR46rb1:400mENTPD5+++−CR47rb1:200mENTPD6+++−ENPP familyK2Grb1:200hENPP2+++nd7619rb1:800hENPP2+++ndCR65rb1:800mENPP4+++ndCR66rb1:400mENPP5+++ndCR67rb1:800mENPP6+++nd7620rb1:3200rENPP3+++nd
Monitoring cell surface expression of purinoceptors and ecto-enzymes by flow cytometry
The utility of the ADAPINC antisera for monitoring cell surface expression was assessed further by flow cytometry (FACS analyses) of transiently transfected HEK cells (Fig. 3). The results confirm that the antisera react specifically with appropriately transfected cells and that they do not react with untransfected HEK cells. Importantly, the antisera recognize the respective purinoceptor/ecto-enzyme on the cell surface of living cells. In contrast, antipeptide antisera against the same antigens did not show any detectable reactivity with the transfected cells in these assays. Further, the use of the ADAPINC antisera for FACS analyses permits an assessment of the relative expression level of the cognate protein on the cell surface of individual cells as well as an assessment of the number of transfected vs. untransfected cells in the cell population. These parameters cannot be quantified appropriately by Western-blot analyses, since for these analyses, lysates are prepared from cell populations. We further used the FACS-based assay system to test whether the antisera crossreact with other members (paralogues) of the receptor/enzyme family and/or the same receptor/enzyme (orthologue) in other species (human, rat, or mouse). The results reveal that most antisera are specific for the receptor/enzyme used for immunization and do not cross-react with other members of the same protein family. Some antisera, however, did react with the orthologous protein, in particular at low dilutions, e.g., antiserum CR30 raised against mouse P2X1 shows some reactivity with the human P2X1 orthologue (Fig. 3a).
Fig. 3FACS analyses of purinoceptor (a) and ecto-enzyme (b) expression by transfected HEK cells. HEK cells were transfected with expression constructs for the indicated purinoceptors and ecto-enzymes. Then, 24 h post-transfection, cells were harvested by brief trypsinization and single cell suspensions were stained with serial dilutions of the indicated antisera followed by PE-conjugated antirabbit IgG before FACS analyses. Control stainings were performed with untransfected cells (shaded histograms in a, dotted lines in b) and with cells transfected with the species orthologue (red lines in a)
Some of the antisera recognize the denatured cognate antigen in Western-blot analyses
We next tested the utility of the ADAPINC antisera for monitoring overall protein expression levels in immunoblot analyses (Fig. 4). To this end, we size-fractionated the proteins from lysates of untransfected and of transiently transfected HEK cells by SDS-PAGE followed by immunoblot analyses. The results show that only some of the ADAPINC antisera detected bands of the expected size. For comparison, we again performed parallel analyses with antisera raised against the same proteins by peptide immunization. The results show that the antipeptide sera generally detected bands of the expected size. The C-terminal peptide is commonly used for peptide immunization. In the case of P2X7, this sequence is highly conserved between rat, mouse and human orthologues, and the corresponding antipeptide serum recognized the respective orthologues (not shown). In contrast, in the case of P2X4, the mouse and human sequences differ in two and five positions, respectively, from the rat peptide used for immunization, and this antipeptide serum shows only weak cross-reactivity with these orthologues (Fig. 4a, Abc α-mP2X4).
Fig. 4Immunoblot analyses of purinoceptor (a) and ecto-enzyme (b) expression by transfected HEK cells. Untransfected (u) or HEK cells transfected with mouse (m) or human (h) purinoceptors or ecto-enzymes were solubilized 24 h post-transfection with SDS-PAGE sample buffer. Proteins in cell lysates were size-fractionated by SDS-PAGE and subjected to immunoblot analyses using the indicated antisera. Bound antibodies were detected with peroxidase-conjugated secondary antibody and the ECL system
Affinity purification of purinoceptors and ecto-enzymes
Finally, we tested the utility of the ADAPINC antisera for immunoprecipitation of the cognate proteins from cell lysates (Fig. 5). To this end, we prepared lysates of untransfected and of transiently transfected HEK using the nondenaturing nonionic detergent Triton-X-100. Lysates were incubated with ADAPINC antibodies immobilized on protein-G sepharaose beads, and proteins bound to washed beads were subsequently analyzed by SDS-PAGE followed by immunoblot analyses with antipeptide antibodies. The results show that all of the ADAPINC antisera efficiently precipitated proteins of the expected size from cell lysates. This underscores the utility of these antibodies for purifying native receptors and ecto-enzymes from lysed cells.
Fig. 5Immunoprecipitation of purinoceptors by antibodies raised via genetic immunization. Untransfected (u) and transfected (t) HEK cells were solubilized 24 h post-transfection with PBS/1% Triton X-100. Purinoceptors were immunoprecipitated from cell lysates by incubation with the indicated antisera bound to Protein G Sepharose. Precipitates were subjected to immunoblot analysis as in Fig. 4, using the indicated antipeptide antisera for detection
Conclusion
Using genetic immunization, we have raised highly specific polyclonal and monoclonal antibodies against key players of purinergic signalling, i.e., P2X1, P2X4, and P2X7 purinoceptors and ENTPD1, ENPTD2, ENPTD5, ENPTD6, ENPP2, ENPP3, ENPP4, ENPP5, and ENPP6 enzymes. Our findings underscore the utility of genetic immunization for generating highly specific polyclonal and monoclonal antibodies directed against proteins in native conformation (ADAPINCs) [19, 20]. These antibodies are valuable tools for assessing the expression levels of the native protein by immunofluorescence analyses and flow cytometry, i.e., assays in which antipeptide antibodies often fail. The antibodies described here provide useful tools for further characterization of the structure and function of these purinoceptors and ecto-enzymes.

purinoceptor
genetic immunization
monoclonal antibodies
ecto-nucleotidases