Use of genetic immunization to raise antibodies recognizing toxin-related cell surface ADP-ribosyltransferases in native conformation

ADP-ribosyltransferases (ARTs) transfer ADP-ribose from NAD to arginine, asparagine, or cysteine residues in target proteins. This post-translational protein modification is the mechanism by which cholera-toxin and other bacterial toxins cause pathology in human host cells. Molecular cloning has identified five toxin-related GPI-anchored cell surface ARTs in the mouse (ART1, ART2.1, ART2.2, ART3, and ART4) and three in the human (ART1, ART3, and ART4). ART2-which has sparked interest because of its ability to activate the cytolytic P2X7 purinergic receptor by ADP-ribosylation-is encoded by two functional gene copies in the mouse genome while the human genome carries two inactivated ART2 pseudogenes. We generated stable transfectants for FLAG-tagged versions of each of the functional human and mouse ARTs. Using genetic immunization we raised monoclonal antibodies that recognize the native human ARTs on the surface of living cells. Some of these mAbs recognize an epitope shared with the mouse ART orthologue but not with more distant ART paralogues. Screening of primary cells and established cell lines by FACS revealed expression of ART1 by monocytes, neutrophils and myeloid leukemia cell lines but not by cell lines derived from solid tumors. ART1 and ART4 have been assigned the designations: CD296, and CD297, respectively.     

A new monoclonal antibody detects a developmentally regulated mouse ecto-ADP-ribosyltransferase on T cells: subset distribution, inbred strain variation, and modulation upon T cell activation

ADP-ribosylation of membrane proteins on mouse T cells by ecto-ADP-ribosyltransferase(s) (ARTs) can down-regulate proliferation and function. The lack of mAbs against mouse ARTs has heretofore prevented analysis of ART expression on T cell subsets. Using gene gun technology, we immunized a Wistar rat with an Art2b expression vector and produced a novel mAb, Nika102, specific for ART2.2, the Art2b gene product. We show that ART2.2 is expressed as a GPI-anchored protein on the surface of mature T cells. Inbred strain-dependent differences in ART2.2 expression levels were observed. C57BL/6J and C57BLKS/J express the Ag at high level, with up to 70% of CD4+ and up to 95% of CD8+ peripheral T cells expressing ART2.2. CBA/J and DBA/2J represent strains with lowest expression levels. T cell-deficient mice and NZW/LacJ mice with a defective structural gene for this enzyme were ART2.2 negative. In the thymus, ART2.2 expression is restricted to subpopulations of mature cells. During postnatal ontogeny, increasing percentages of T cells express ART2.2, reaching a peak at 6-8 wk of age. Interestingly, ART2.2 and CD25 are reciprocally expressed: activation-induced up-regulation of CD25 is accompanied by loss of ART2.2 from the cell surface. Nika102 thus defines a new differentiation/activation marker of thymic and postthymic T cells in the mouse and should be useful for further elucidating the function of the ART2.2 cell surface enzyme.     

Metalloprotease-mediated shedding of enzymatically active mouse ecto-ADP-ribosyltransferase ART2.2 upon T cell activation

T cells proteolytically shed the ectodomains of several cell surface proteins and, thereby, can alter their responsiveness and can release soluble intercellular regulators. ART2.2 is a GPI-anchored ecto-ADP-ribosyltransferase (ART) related to ADP-ribosylating bacterial toxins. ART2.2 is expressed exclusively by mature T cells. Here we show that ART2.2 is shed from the cell surface in enzymatically active form upon activation of T cells. Shedding of ART2.2 resembles that of L-selectin (CD62L) in dose response, kinetics of release, and sensitivity to the metalloprotease inhibitor Immunex Compound 3, suggesting that ART2.2, like CD62L, is cleaved by TNF-alpha-converting enzyme or by another metalloprotease. ART2.2 shed from activated T cells migrates slightly faster in SDS-PAGE analyses than does ART2.2 released upon cleavage of the GPI anchor. This indicates that shedding of ART2.2 is mediated by proteolytic cleavage close to its membrane anchor. Shed ART2.2 is enzymatically active and ADP-ribosylates several substrates in vitro. Thus, shedding of ART2.2 releases a potential intercellular regulator. Finally, using a new FACS assay for monitoring ADP-ribosylation of cell surface proteins, we demonstrate that shedding of ART2.2 correlates with a reduced sensitivity of T cell surface proteins to ADP-ribosylation. Our findings suggest that by shedding ART2.2 the activated T cell not only releases a potential intercellular regulator but also may alter its responsiveness to immune regulation by ART2.2-mediated ADP-ribosylation of cell surface proteins.     

Rapid induction of naive T cell apoptosis by ecto-nicotinamide adenine dinucleotide: requirement for mono(ADP-ribosyl)transferase 2 and a downstream effector.

Lymphocytes express a number of NAD-metabolizing ectoenzymes, including mono(ADP-ribosyl)transferases (ART) and ADP ribosylcyclases. These enzymes may regulate lymphocyte functions following the release of NAD in injured or inflammatory tissues We report here that extracellular NAD induces apoptosis in BALB/c splenic T cells with an IC(50) of 3-5 microM. Annexin V staining of cells was observed already 10 min after treatment with NAD in the absence of any additional signal. Removal of GPI-anchored cell surface proteins by phosphatidylinositol-specific phospholipase C treatment rendered cells resistant to NAD-mediated apoptosis. RT-PCR analyses revealed that resting BALB/c T cells expressed the genes for GPI-anchored ART2.1 and ART2.2 but not ART1. ART2-specific antisera blocked radiolabeling of cell surface proteins with both [(32)P]NAD and NAD-mediated apoptosis. Further analyses revealed that natural knockout mice for Art2.a (C57BL/6) or Art2.b (NZW) were resistant to NAD-mediated apoptosis. Labeling with [(32)P]NAD revealed strong cell surface ART activity on T cells of C57BL/6 and little if any activity on cells of NZW mice. T cells of (C57BL/6 x NZW)F(1) animals showed strong cell surface ART activity and were very sensitive to NAD-induced apoptosis. As in BALB/c T cells, ART2-specific antisera blocked cell surface ART activity and apoptosis in (C57BL/6 x NZW)F(1) T cells. The fact that T cells of F(1) animals are sensitive to rapid NAD-induced apoptosis suggests that this effect requires the complementation of (at least) two genetic components. We propose that one of these is cell surface ART2.2 activity (defective in the NZW parent), the other a downstream effector of ADP-ribosylation (defective in the C57BL/6 parent).     

CD38 controls ADP-ribosyltransferase-2-catalyzed ADP-ribosylation of T cell surface proteins

ADP-ribosyltransferase-2 (ART2), a GPI-anchored, toxin-related ADP-ribosylating ectoenzyme, is prominently expressed by murine T cells but not by B cells. Upon exposure of T cells to NAD, the substrate for ADP-ribosylation, ART2 catalyzes ADP-ribosylation of the P2X7 purinoceptor and other functionally important cell surface proteins. This in turn activates P2X7 and induces exposure of phosphatidylserine and shedding of CD62L. CD38, a potent ecto-NAD-glycohydrolase, is strongly expressed by most B cells but only weakly by T cells. Following incubation with NAD, CD38-deficient splenocytes exhibited lower NAD-glycohydrolase activity and stronger ADP-ribosylation of cell surface proteins than their wild-type counterparts. Depletion of CD38(high) cells from wild-type splenocytes resulted in stronger ADP-ribosylation on the remaining cells. Similarly, treatment of total splenocytes with the CD38 inhibitor nicotinamide 2'-deoxy-2'-fluoroarabinoside adenine dinucleotide increased the level of cell surface ADP-ribosylation. Furthermore, the majority of T cells isolated from CD38-deficient mice "spontaneously" exposed phosphatidylserine and lacked CD62L, most likely reflecting previous encounter with ecto-NAD. Our findings support the notion that ecto-NAD functions as a signaling molecule following its release from cells by lytic or nonlytic mechanisms. ART2 can sense and translate the local concentration of ecto-NAD into corresponding levels of ADP-ribosylated cell surface proteins, whereas CD38 controls the level of cell surface protein ADP-ribosylation by limiting the substrate availability for ART2.     

Generation and characterization of ecto-ADP-ribosyltransferase ART2.1/ART2.2-deficient mice

This is the first study reporting the inactivation of a member of the mouse gene family of toxin-related ecto-ADP-ribosyltransferases (ARTs). Transfer of the ADP-ribose moiety from NAD onto extracellular arginine residues on T-cell membrane proteins is mediated by glycosylphosphatidylinositol-linked cell surface ARTs. Exposure of T cells to ecto-NAD blocks T-cell activation and induces T-cell apoptosis. To determine a possible role of ecto-ART2.1 and ART2.2 in these processes, we generated ART2.1/ART2.2 double-knockout mice. ART2-deficient mice were healthy and fertile and showed normal development of lymphoid organs. ART2-deficient T cells showed a dramatically reduced capacity to ADP-ribosylate cell surface proteins, indicating that most if not all ART activity on the T-cell surface can be attributed to the ART2s. Moreover, ART2-deficient T cells were completely resistant to NAD-induced apoptosis and partially resistant to NAD-mediated suppression of proliferation. These results demonstrate that the ART2 ectoenzymes are an essential component in the regulation of T-cell functions by extracellular NAD, e.g., following release of NAD upon lysis of cells in tissue injury and inflammation.     

K-ras transformation greatly increases the toxin-dependent ADP-ribosylation of GTP binding proteins in thyroid cells. Involvement of an inhibitor of the ADP-ribosylation reaction.

The GTP binding (G) proteins of normal (FRTL5) and ras-transformed thyroid cells (KiKi) were characterized by cholera and pertussis toxin-induced ADP-ribosylation and immunoblot analysis. Two pertussis toxin substrates with molecular masses of 40 and 41 kDa were identified in normal cells as the alpha i2 and alpha i3 subunits. The molecular masses of the cholera toxin substrates were 42 and 45 kDa. The same cholera and pertussis toxin substrates were present in the K-ras-transformed cell line. However, the toxin-dependent ADP-ribosylation was markedly higher in KiKi than in normal cell membranes (more than 50-fold). The reason for this difference was investigated; it could not be explained by the relative amounts of G proteins in the two cell systems, since the levels of alpha i2 subunit as measured by quantitative immunoblot in K-ras-transformed cells were only slightly (65%) higher than in normal cells. The difference in ADP-ribosylation was not due to poly-ADP-ribosylation nor to a different degree of subunit dissociation of G proteins in the two cell lines. Rather, the enhanced ADP-ribosylation in K-ras-transformed cells appears to be due to the loss of an inhibitory factor present in the normal cells. Partial characterization indicates that such a factor is a peripheral membrane protein of less than 25 kDa capable of directly interfering with the ADP-ribosylation reaction.     

Basal and inducible expression of the thiol-sensitive ART2.1 ecto-ADP-ribosyltransferase in myeloid and lymphoid leukocytes

ADP-ribosylation of cell surface proteins in mammalian cells is a post-translational modification by which ecto-ADP-ribosyltransferases (ARTs) transfer ADP-ribose from extracellular NAD to protein targets. The ART2 locus at murine chromosome 7 encompasses the tandem Art2a and Art2b genes that encode the distinct ART2.1 and ART2.2 proteins. Although both ecto-enzymes share 80% sequence identity, ART2.1 activity is uniquely regulated by an allosteric disulfide bond that is reducible in the presence of extracellular thiols, such as cysteine and glutathione, that accumulate in hypoxic and ischemic tissues. Previous studies have characterized the expression of ART2.1 and ART2.2 in murine T lymphocytes but not in other major classes of lymphoid and myeloid leukocytes. Here, we describe the expression of ART2.1 activity in a wide range of freshly isolated or tissue-cultured murine myeloid and lymphoid leukocytes. Spleen-derived macrophages, dendritic cells (DC), and B cells constitutively express ART2.1 as their predominant ART while spleen T cells express both ART2.1 and the thiol-independent ART2.2 isoform. Although bone-marrow-derived macrophages (BMDM) and dendritic cells (BMDC) constitutively express ART2.1 at low levels, it is markedly up-regulated when these cells are stimulated in vitro with IFNβ or IFNγ. ART2.1 expression and activity in splenic B cells is modestly up-regulated during incubation in vitro for 24 h, a condition that promotes B cell apoptosis. This increase in ART2.1 is attenuated by IL-4 (a B cell survival factor), but is not affected by IFNβ/γ, suggesting a possible induction of ART2.1 as an ancillary response to B cell apoptosis. In contrast, ART2.1 and ART2.2, which are highly expressed in freshly isolated splenic T cells, are markedly down-regulated when purified T cells are incubated in vitro for 12–24 h. Studies with the BW5147 mouse thymocyte line verified basal expression of ART2.1 and ART2.2, as in primary spleen T cells, and demonstrated that both isoforms can be up-regulated when T cells are maintained in the presence of IFNs. Comparison of the surface proteins which are ADP-ribosylated by ART2.1 in the different leukocyte subtypes indicated both shared and cell-specific proteins as ART2.1 substrates. The LFA-1 integrin, a major target for ART2.2 in T cells, is also ADP-ribosylated by the ART2.1 expressed in macrophages. Thus, ART2.1, in contrast to ART2.2, is expressed in a broad range of myeloid and lymphoid leukocytes. The thiol redox-sensitive nature of this ecto-enzyme suggests an involvement in purinergic signaling that occurs in the combined context of inflammation and hypoxia/ischemia.     

The family of toxin-related ecto-ADP-ribosyltransferases in humans and the mouse

ADP-ribosyltransferases including toxins secreted by Vibrio cholera, Pseudomonas aerurginosa, and other pathogenic bacteria inactivate the function of human target proteins by attaching ADP-ribose onto a critical amino acid residue. Cross-species polymerase chain reaction (PCR) and database mining identified the orthologs of these ADP-ribosylating toxins in humans and the mouse. The human genome contains four functional toxin-related ADP-ribosyltransferase genes (ARTs) and two related intron-containing pseudogenes; the mouse has six functional orthologs. The human and mouse ART genes map to chromosomal regions with conserved linkage synteny. The individual ART genes reveal highly restricted expression patterns, which are largely conserved in humans and the mouse. We confirmed the predicted extracellular location of the ART proteins by expressing recombinant ARTs in insect cells. Two human and four mouse ARTs contain the active site motif (R-S-EXE) typical of arginine-specific ADP-ribosyltransferases and exhibit the predicted enzyme activities. Two other human ARTs and their murine orthologues deviate in the active site motif and lack detectable enzyme activity. Conceivably, these ARTs may have acquired a new specificity or function. The position-sensitive iterative database search program PSI-BLAST connected the mammalian ARTs with most known bacterial ADP-ribosylating toxins. In contrast, no related open reading frames occur in the four completed genomes of lower eucaryotes (yeast, worm, fly, and mustard weed). Interestingly, these organisms also lack genes for ADP-ribosylhydrolases, the enzymes that reverse protein ADP-ribosylation. This suggests that the two enzyme families that catalyze reversible mono-ADP-ribosylation either were lost from the genomes of these nonchordata eucaryotes or were subject to horizontal gene transfer between kingdoms.     

A second Epstein-Barr virus early antigen gene in BamHI fragment M encodes a 48- to 50-kilodalton nuclear protein

We used antiserum raised against the bacterially synthesized product of one of the open reading frames in Epstein-Barr virus (EBV) BamHI fragment M to demonstrate that this reading frame (BMRF1) codes for a nuclear protein of the diffuse early antigen (EA) class. In indirect immunofluorescence assays, the rabbit anti-BMRF1 antiserum gave nuclear staining in approximately 5% of Raji cells which had been treated with sodium butyrate, and positive fluorescence was observed in both acetone- and methanol-fixed cells. Uninduced Raji cultures contained less than 0.1% positive cells regardless of whether indirect immunofluorescence or anti-complement immunofluorescence was used. In immunoblot analyses, the rabbit serum identified a family of polypeptides of 46 to 55 kilodaltons (kDa) in total protein extracts from B95-8 cells or from butyrate-induced Raji cells. In both cell types, the dominant polypeptides were the 48- and 50-kDa species. This same family of polypeptides was identified when the immunoblots were reacted with the R3 monoclonal antibody, and we concluded that this antibody also recognized the product of the BMRF1 open reading frame. Fibroblast cell lines containing EBV BamHI fragment M were established by cotransfection of baby hamster kidney cells with BamHI-M and the gene for neomycin resistance. Aminoglycoside G418-resistant colonies which showed evidence for EBV antigen expression in immunofluorescence assays were selected, and clonal cell lines were established. After 3 to 4 months of passaging, constitutive synthesis of EA was no longer detectable in these cell lines either by immunofluorescence or by immunoblot analysis. However, in the one cell line examined, synthesis of the 48- to 50-kDa EA was induced by treatment of the culture with sodium butyrate. Thus, the regulation of expression of this EA in transfected fibroblasts is analogous to that seen in Raji lymphoblasts. We showed previously that BamHI fragment M also contains the coding sequences for a 60-kDa nuclear EA, and hence BamHI-M encodes two separate components of the diffuse EA complex.     

Lipopolysaccharide, IFN-gamma, and IFN-beta induce expression of the thiol-sensitive ART2.1 Ecto-ADP-ribosyltransferase in murine macrophages

Nicotinamide adenosine dinucleotide (NAD) can act as a modulator of multiple immune and inflammatory responses when released into extracellular compartments. These actions of extracellular NAD are largely mediated by a family of mammalian ecto-ADP-ribosyltransferases (ARTs) that covalently modify target extracellular or cell surface proteins by transferring ADP-ribose to arginine or cysteine residues. In this study, we report that bone marrow-derived macrophages (BMDM) from BALB/c mice lack constitutive expression of any of the six murine ecto-ART subtypes, but selectively up-regulate ART2.1 in response to multiple proinflammatory mediators including agonists for TLR and type I and type II IFN. Stimulation of BMDM with LPS, IFN-beta, or IFN-gamma induced high expression of ART2.1, but not ART2.2, as a GPI-anchored cell surface ectoenzyme. ART2.1 expression in response to LPS was potentiated by inhibition of ERK1/2 signaling, but inhibited by blockade of the NF-kappaB, PI3K, and JAK-STAT pathways or the presence of neutralizing anti-IFN-beta. The catalytic function of the induced cell surface ART2.1 was strictly dependent on the presence of extracellular thiol-reducing cofactors, suggesting that in vivo activity of ART2.1-expressing macrophages may be potentiated in hypoxic or ischemic compartments. Consistent with the mutated art2a gene in C57BL/6 mice, LPS- or IFN-stimulated BMDM from this strain lacked expression of cell surface ART2 activity in the presence or absence of extracellular thiol reductants. Collectively, these studies identify ART2.1 as a new candidate for linking autocrine/paracrine activation of inflammatory macrophages to the release of NAD, a critical intracellular metabolite.     

Extracellular NAD and ATP: Partners in immune cell modulation

Extracellular NAD and ATP exert multiple, partially overlapping effects on immune cells. Catabolism of both nucleotides by extracellular enzymes keeps extracellular concentrations low under steady-state conditions and generates metabolites that are themselves signal transducers. ATP and its metabolites signal through purinergic P2 and P1 receptors, whereas extracellular NAD exerts its effects by serving as a substrate for ADP-ribosyltransferases (ARTs) and NAD glycohydrolases/ADPR cyclases like CD38 and CD157. Both nucleotides activate the P2X7 purinoceptor, although by different mechanisms and with different characteristics. While ATP activates P2X7 directly as a soluble ligand, activation via NAD occurs by ART-dependent ADP-ribosylation of cell surface proteins, providing an immobilised ligand. P2X7 activation by either route leads to phosphatidylserine exposure, shedding of CD62L, and ultimately to cell death. Activation by ATP requires high micromolar concentrations of nucleotide and is readily reversible, whereas NAD-dependent stimulation begins at low micromolar concentrations and is more stable. Under conditions of cell stress or inflammation, ATP and NAD are released into the extracellular space from intracellular stores by lytic and non-lytic mechanisms, and may serve as ‘danger signals–to alert the immune response to tissue damage. Since ART expression is limited to naïve/resting T cells, P2X7-mediated NAD-induced cell death (NICD) specifically targets this cell population. In inflamed tissue, NICD may inhibit bystander activation of unprimed T cells, reducing the risk of autoimmunity. In draining lymph nodes, NICD may eliminate regulatory T cells or provide space for the preferential expansion of primed cells, and thus help to augment an immune response.     

Artesunate inhibits cell proliferation and decreases growth hormone synthesis and secretion in GH3 cells

To determine the effect of artesunate (ART) on the rat pituitary adenoma GH3 cell line to evaluate its potential as a novel agent in growth hormone (GH) adenoma and to investigate its underlying mechanisms of action. The MTT assay was used to assess cell proliferation. DAPI staining was used to visualise apoptotic changes in the nucleus. We also analyzed cell apoptosis and cell cycle stage by flow cytometry, semi-quantitative RT-PCR analysis for the expression of GH mRNA and apoptosis-induced factor (AIF) mRNA, analysis of GH protein by western blot, ELISA detection of secreted GH, and the caspase inhibition assay. We found that ART inhibited the proliferation of GH3 cells in a dose- and time-dependent manner, with an IC50 of 9.53 ± 4.12 μM. The IC50s of ART against of two normal cell lines (mouse embryonic fibroblasts, and rat bone mesenchymal cells) were much higher than the IC50 recorded for the GH3 cells. ART induced apoptosis and blocked GH3 at G2/M arrest. The pan caspase inhibitor V-ZAD-FMK partly attenuated the inhibitory effect of ART. ART increased the expression of AIF mRNA and reduced GH mRNA levels, GH synthesis and the secretion of GH level in GH3 cells. ART can inhibit proliferation and induce apoptosis in GH3 cells by caspase-dependent pathways. Additionally, ART can inhibit GH synthesis and secretion. Thus, we propose ART as a probably anti-tumour candidate drug in the treatment of GH adenoma.     

Changes in the level of poly ADP-ribosylation during a cell cycle

In order to analyze the fluctuation of the poly ADP-ribosylation level during the cell cycle of synchronously growing He La S3 cells, we have developed three different assay systems; intact and disrupted nuclear systems, and poly(ADP-ribose) polymerase in vitro system. The optimum conditions for poly ADP-ribosylation in each assay system were similar except the pH optimum. Under the conditions favoring poly ADP-ribosylation, little radioactivity incorporated into poly(ADP-ribose) was lost after termination of the poly ADP-ribosylation by addition of nicotinamide which inhibits the reactions by more than 90% in any system. In the intact nuclear system, the level of poly ADP-ribosylation increased slightly subsequent to late G2 phase with a peak at M phase. The high level of poly ADP-ribosylation in M phase was also confirmed by using selectively collected mitotic cells which were arrested in M phase by Colcemid. The level in mitotic chromosomes was 5.1-fold higher than that in the nuclei from logarithmically growing cells. Colcemid has no effect on the poly ADP-ribosylation. In the disrupted nuclear system, a relatively high level of poly ADP-ribosylation was observed during mid S-G2 phase. When poly(ADP-ribose) polymerase was extracted from the nuclei with a buffer solution containing 0.3 M KCl, more than 90% of the enzyme activity was recovered. The poly(ADP-ribose) polymerase in vitro system was dependent on both DNA and histone—10 μg each. In the enzyme system, enzyme activity was detected throughout the cell cycle and was observed to be highest in G2 phase. The high level at M phase observed in the intact nuclear system was not seen in the other two systems. Under the assay conditions, little influence of poly(ADP-ribose) degrading enzymes was noted on the level of poly ADP-ribosylation in any of the three systems. This was confirmed at various stages during the cell cycle through pulse-labeling and “chasing” by adding nicotinamide.     

Nucleolin: acharan sulfate-binding protein on the surface of cancer cells

Glycosaminoglycans (GAGs) are complex polysaccharides that participate in the regulation of physiological processes through the interactions with a wide variety of proteins. Acharan sulfate (AS), isolated from the giant African snail Achatina fulica, primarily consists of the repeating disaccharide structure alpha-D-N-acetylglucosaminyl (1-->4) 2-sulfoiduronic acid. Exogenous AS was injected subcutaneously near the tumor tissue in C57BL/6 mice that had been implanted with Lewis lung carcinoma cells (LLCs). The location of AS in the tumor was assessed by staining of sectioned tissues with alcian blue and periodic acid-Schiff (PAS) reagent. In vitro assays indicated binding of cells to 50 microg/ml AS (or heparin) after a 5-h incubation. Immunofluorescence assays, using anti-AS antibody, detected AS at the cell surface. The outer-surface of LLCs were next biotinylated to identify the AS-binding proteins. Biotinylated cells were lysed, and the lysates were fractionated on the AS affinity column using a stepwise salt gradient (0, 0.1, 0.3, 0.5, 0.7, 1.0, and 2.0 M). The fractions were analyzed by SDS-PAGE with silver staining and western blotting. We focused on the proteins with high affinity for AS (eluting at 1 M NaCl) and detected only two bands by western blotting. ESI Q-TOF MS analysis of one of these bands, molecular weight approximately 110 kDa, showed it to be nucleolin. A phosphorylated form of nucleolin on the surface of cells acts as a cell surface receptor for a variety of ligands, including growth factors (i.e., basic fibroblast growth factor) and chemokines (i.e., midkine). These results show that nucleolin is one of several AS-binding proteins and suggest that AS might demonstrate its tumor growth inhibitory activity by binding the nucleolin receptor protein on the surface of cancer cells.