"Numatrin," a nuclear matrix protein associated with induction of proliferation in B lymphocytes

To detect nuclear proteins that might be involved in induction of cellular mitogenesis, we examined the effect of various mitogens on early changes in synthesis of nuclear proteins in murine B lymphocytes. Using two-dimensional gel electrophoresis, we found that activation of B cells by mitogens (anti-immunoglobulin antibody, lipopolysaccharide, phorbol 12-myristate 13-acetate (PMA)/A23187) was associated with a rapid and prominent (5-20-fold) increase in the synthesis of a 40-kDa/pI 5.0 nuclear protein, here termed numatrin. Numatrin was found to be absent from the cytosol (soluble fraction) of resting as well as activated B cells and was markedly resistant to DNase/RNase digestion and 2 N NaCl extraction, indicating that this protein is tightly bound to the nuclear matrix. Kinetic studies showed that the increase in synthesis of numatrin was detected 60-120 min following mitogen activation, reached a peak at 16 h, and declined to almost control level by 48 h, correlating with the peak of cellular DNA synthesis. The increase in synthesis of numatrin in normal B cells was found to be associated exclusively with cellular commitment for mitogenesis because activation of B cells by stimuli such as B cell stimulating factor 1, PMA alone, and calcium ionophore A23187, which do not stimulate an increase in DNA synthesis, also failed to induce an increase in the synthesis of numatrin. Inhibition of anti-Ig-induced proliferation (by PMA pretreatment) was associated with a 63% inhibition in the synthesis of numatrin. Addition of 8-mercaptoguanosine to these PMA-treated cells was associated with restoration of the increase in synthesis of numatrin, concomitant with induction of proliferation. Elevated synthesis of numatrin was also detected in the malignant B lymphoma cells: Raji, BAL-17, and WEHI-231. Taken collectively, these results suggest that numatrin, a tightly bound nuclear matrix protein, is a growth-regulated protein which might have an important role in regulation of cellular mitogenesis in normal and malignant B lymphocytes.     

The nuclear matrix protein, numatrin (B23), is associated with growth factor-induced mitogenesis in Swiss 3T3 fibroblasts and with T lymphocyte proliferation stimulated by lectins and anti-T cell antigen receptor antibody

Numatrin is a tightly bound nuclear matrix protein (40 kD/pI-5) whose synthesis is markedly and promptly increased in association with cellular commitment for mitogenesis in B lymphocytes. (Feuerstein, N., and J.J. Mond. 1987. J. Biol. Chem. 262:11389-11397). To study whether this event is exclusively associated with proliferation of B lymphocytes, we examined the synthesis of numatrin in T lymphocytes (murine and human) activated by lectins or by anti-T cell antigen receptor monoclonal antibody and in Swiss 3T3 fibroblasts stimulated by growth factors. We showed a close correlation between induction of DNA synthesis and induction of numatrin synthesis in T lymphocytes stimulated by concanavalin A, anti-T cell antigen receptor monoclonal antibody, and IL-2 in murine T cells. Similar results were observed in Swiss 3T3 fibroblasts, thus only combinations of growth factors (insulin/EGF or insulin/B subunit of cholera toxin) or serum, which induced a significant increase in DNA synthesis, were also associated with a significant increase in synthesis of numatrin. Similar to B cells, the increase in numatrin synthesis in fibroblasts was found to occur at early G1 phase. The calcium ionophores, A23187 and ionomycin, previously shown to induce an increase in c-myc and c-fos mRNA levels in fibroblasts, induced a marked increase in the synthesis of a nuclear protein at 80 kD/pI-5 but failed to induce an increase in the synthesis of numatrin indicating that an increase in intracellular Ca++ level is not sufficient for induction of the synthesis of numatrin. This further indicates that the increase in synthesis of numatrin may be more closely correlated with cellular commitment for mitogenesis as compared with other biochemical parameters. Using a polyclonal numatrin antibody we demonstrated that mitogen stimulation is also associated with a marked increase in numatrin abundance, which reached a peak at the onset of S phase and declined at the end of S phase. Evidence is presented to show that numatrin synthesis and abundance is elevated in various lymphoma cell lines. Using indirect immunofluorescence assays we showed that numatrin is abundant in other malignant cells: KB, epidermoid carcinoma, and Hep2 human hepatoma. Immunofluorescence studies further showed that mitogen stimulation of B lymphocytes induced a marked accumulation of numatrin in the nucleoli. This observation is in accord with the recent finding of identity of numatrin with the nucleolar protein B23 (Feuerstein et al. 1988. J. Biol. Chem. 263:10608-10612).(ABSTRACT TRUNCATED AT 400 WORDS)     

Evidence for DNA binding activity of numatrin (B23), a cell cycle-regulated nuclear matrix protein

Stimulation of various cell types with growth factors is associated with a rapid induction in the synthesis of a nuclear matrix protein, termed 'numatrin' which was shown to be identical to the nucleolar protein B23. The abundance of numatrin was shown to be correlated with entry and progression through the S-phase. Thus, experiments were undertaken to examine whether numatrin also has DNA binding activity. Using whole nuclear extract, we showed that numatrin binds to both double-stranded (DS) DNA and to single-stranded (SS) DNA cellulose columns. Purified numatrin, which was extracted either under native conditions (in oligomeric form) or under urea conditions (in monomeric form), demonstrated significant binding to either [3H]DS-DNA or [3H]DS-DNA as shown by nitrocellulose filter binding assay. However, numatrin binding to DS-DNA was qualitatively and quantitatively different from its binding to SS-DNA. Thus, the binding of numatrin was several fold higher to DS-DNA as compared to SS-DNA. The binding to DS-DNA was reduced by 77% in the presence of 0.5 M NaCl, while the binding to SS-DNA was not affected under this condition. Furthermore, treatment of the native numatrin under conditions which caused monomerization of the protein resulted in a significant enhancement of numatrin binding to SS-DNA but did not affect the binding to DS-DNA. Following heparin-Sepharose chromatography purification (under native conditions), numatrin at picomole amounts showed significant binding to both DS-DNA and SS-DNA. Finally, numatrin was found to copurify with the complex of DNA polymerase alpha primase together with other proteins required for SV-40 in vitro replication activity. These results demonstrate that numatrin has DNA binding activity, and imply a possible role for numatrin/B23 in DNA-associated processes.     

In vivo and in vitro phosphorylation studies of numatrin, a cell cycle regulated nuclear protein, in insulin-stimulated NIH 3T3 HIR cells

Numatrin, a nuclear matrix protein has been implicated to be involved in mitogenesis of normal and malignant cells (Feuerstein and Mond, J. Biol. Chem. 262, 11389, 1987) and was later found to be identical to the nuclear phosphoprotein, B23. To study whether phosphorylation of numatrin is regulated by mitogenic stimulation, we examined the effect of phosphorylation of numatrin in the insulin-responsive cells, NIH 3T3 HIR. We found that an increase in phosphorylation of numatrin was associated with stimulation of the cells with insulin for 4 h and that the level of phosphorylation remained elevated after 8 h. By this time there was no increase in numatrin abundance as shown by Coom-massie blue stain and Western blot analysis. The induction in phosphorylation of numatrin could not be detected after 30 min stimulation with insulin, thus, indicating that the increase in phosphorylation of numatrin is not a rapid event. Analysis of the phosphopeptides by thin layer chromatography indicated four peptides that were phosphorylated in numatrin (one major and three minor). Stimulation with insulin was associated primarily with an increase in phosphorylation of the minor phosphopeptides. The phosphopeptide map of numatrin was identical after 4, 8, 17, 24, and 32 h stimulation with insulin, indicating that identical sites are phosphorylated at different phases of the cell cycle. In a search for the protein kinase which is involved in phosphorylation of numatrin we found that numatrin is a most prominent substrate for the cell cycle regulated cdc2 (p 34) kinase. However, the major phosphopeptides which were phosphorylated by this kinase did not comigrate with either of the phosphopeptides phosphorylated in insulin-stimulated intact cells. This may indicate that it is unlikely that cdc2 kinase may account for the mechanism(s) associated with phosphorylation of numatrin by insulin under physiological conditions.     

ADP-ribosylation of nucleolar proteins in HeLa tumor cells

ADP-ribosylation reactions in nucleoli of exponentially growing HeLa cells were studied. Isolated nuclei or nucleoli were labeled with ³²P-NAD; then the nucleolar proteins were analyzed by 1-dimensional and 2-dimensional polyacrylamide gel electrophoresis (PAGE) and modified proteins were detected by autoradiography. The labeled nucleolar proteins were also chromatographically fractionated on DEAE-cellulose. Electrophoretic analysis of total nucleolar and chromatographically purified proteins revealed that besides nuclear ADP-ribosyltransferase and histones two characteristic nucleolar phosphoproteins numatrin/B23 and nucleolin/C23 were modified by ADP-ribosylation.     

In vivo and in vitro phosphorylation studies of numatrin, a cell cycle regulated nuclear protein, in insulin-stimulated NIH 3T3 HIR cells

Numatrin, a nuclear matrix protein has been implicated to be involved in mitogenesis of normal and malignant cells (Feuerstein and Mond, J. Biol. Chem. 262, 11389, 1987) and was later found to be identical to the nuclear phosphoprotein, B23. To study whether phosphorylation of numatrin is regulated by mitogenic stimulation, we examined the effect of phosphorylation of numatrin in the insulin-responsive cells, NIH 3T3 HIR. We found that an increase in phosphorylation of numatrin was associated with stimulation of the cells with insulin for 4 h and that the level of phosphorylation remained elevated after 8 h. By this time there was no increase in numatrin abundance as shown by Coommassie blue stain and Western blot analysis. The induction in phosphorylation of numatrin could not be detected after 30 min stimulation with insulin, thus, indicating that the increase in phosphorylation of numatrin is not a rapid event. Analysis of the phosphopeptides by thin layer chromatography indicated four peptides that were phosphorylated in numatrin (one major and three minor). Stimulation with insulin was associated primarily with an increase in phosphorylation of the minor phosphopeptides. The phosphopeptide map of numatrin was identical after 4, 8, 17, 24, and 32 h stimulation with insulin, indicating that identical sites are phosphorylated at different phases of the cell cycle. In a search for the protein kinase which is involved in phosphorylation of numatrin we found that numatrin is a most prominent substrate for the cell cycle regulated cdc2 (p 34) kinase. However, the major phosphopeptides which were phosphorylated by this kinase did not comigrate with either of the phosphopeptides phosphorylated in insulin-stimulated intact cells. This may indicate that it is unlikely that cdc2 kinase may account for the mechanism(s) associated with phosphorylation of numatrin by insulin under physiological conditions.     

The structure and functions of NPM1/Nucleophsmin/B23, a multifunctional nucleolar acidic protein

NPM1/Nucleophosmin/B23, also termed NO38 or numatrin, is an acidic nucleolar protein that plays multiple roles in cell growth and proliferation. In general, the expression level of B23 is proportional to the cell growth rate, suggesting that it plays a positive role(s) in cell growth and proliferation. It is important to note that the deletion of the B23 gene and expression of an aberrant type of this gene--caused by gene conversion via translocation or reading-frame shift via nucleotides insertion-have been observed in diverse haematopoietic malignancies. Thus, it is important to understand the function of B23 in the regulation of cell growth and proliferation. In addition, B23 has been reported to undergo a variety of post-translational modifications such as phosphorylation, ubiquitination, SUMOylation, acetylation and poly-(ADP-ribosyl)ation. In this review, the basic structure and functions of B23 as well as the regulation of these functions are summarized.     

Nuclear substrates of protein kinase C.

Starting from the finding that, for neuronal cells, the nuclear-membrane-associated protein kinase C (PKC) is the so-called 'membrane inserted', constitutively active form, we attempted to identify substrates of this nuclear PKC. For this purpose, nuclear membranes and other subcellular fractions were prepared from bovine brain, and in-vitro phosphorylation was performed. Several nuclear membrane proteins were found, the phosphorylation of which was inhibited by specific PKC inhibitors and effectively catalyzed by added PKC. Combining the methods of two-dimensional gel electrophoresis, in-situ digestion, reverse-phase HPLC and microsequencing, two of these nuclear PKC substrates were identified; the known PKC substrate Lamin B2, which serves as a control of the approach and the nucleolar protein B23. Our data suggest, that, for B23, Ser225 is a site of phosphorylation by PKC.     

Nucleophosmin/B23 is a proliferate shuttle protein associated with nuclear matrix

It has become obvious that a better understanding and potential elucidation of the nucleolar phosphoprotein B23 involving in functional interrelationship between nuclear organization and gene expression. In present study, protein B23 expression were investigated in the regenerative hepatocytes at different periods (at days 0, 1, 2, 3, 4, 7) during liver regeneration after partial hepatectomy on the rats with immunohistochemistry and Western blot analysis. Another experiment was done with immunolabeling methods and two-dimensional (2-D) gel electrophoresis for identification of B23 in the regenerating hepatocytes and HepG2 cells (hepatoblastoma cell line) after sequential extraction with detergents, nuclease, and salt. The results showed that its expression in the hepatocytes had a locative move and quantitative change during the process of liver regeneration post-operation. Its immunochemical localization in the hepatocytes during the process showed that it moved from nucleoli of the hepatocytes in the stationary stage to nucleoplasm, cytoplasm, mitotic spindles, and mitotic chromosomes of the hepatocytes in the regenerating livers. It was quantitatively increased progressively to peak level at day 3 post-operation and declined gradually to normal level at day 7. It was detected in nuclear matrix protein (NMP) composition extracted from the regenerating hepatocytes and HepG2 cells and identified with isoelectric point (pI) value of 5.1 and molecular weight of 40 kDa. These results indicated that B23 was a proliferate shuttle protein involving in cell cycle and cell proliferation associated with nuclear matrix.     

A constitutive nucleolar protein identified as a member of the nucleoplasmin family.

Using monoclonal antibodies we have localized a polypeptide, appearing on gel electrophoresis with a Mr of approximately 38,000 and a pI of approximately 5.6, to the granular component of the nucleoli of Xenopus laevis oocytes and a broad range of cells from various species. The protein (NO38) also occurs in certain distinct nucleoplasmic particles but is not detected in ribosomes and other cytoplasmic components. During mitosis NO38-containing material dissociates from the nucleolar organizer region and distributes over the chromosomal surfaces and the perichromosomal cytoplasm; in telophase it re-populates the forming nucleoli. With these antibodies we have isolated from a X. laevis ovary lambda gt11 expression library a cDNA clone encoding a polypeptide which, on one- and two-dimensional gel electrophoresis, co-migrates with authentic NO38. The amino acid sequence deduced from this clone defines a polypeptide of 299 amino acids of mol. wt 33,531 which is characterized by the presence of two domains exceptionally rich in aspartic and glutamic acid, one of them flanked by two putative karyophilic signal heptapeptides. Comparison with other protein sequences shows that NO38 is closely related to the histone-binding, karyophilic protein nucleoplasmin: the first 124 amino acids have 58 amino acid positions in common. Protein NO38 also shows striking homologies to the phosphopeptide region of rat nucleolar protein B23 and the carboxyterminal region of human B23. We propose that protein NO38, which forms distinct homo-oligomers of approximately 7S and Mr of approximately 230,000, is a member of a family of karyophilic proteins, the 'nucleoplasmin family'. It is characterized by its specific association with the nucleolus and might be involved in nuclear accumulation, nucleolar storage and pre-rRNA assembly of ribosomal proteins in a manner similar to that discussed for the role of nucleoplasmin in histone storage and chromatin assembly.     

Antibodies to a nucleolar protein are localized in the nucleolus after red blood cell-mediated microinjection

To determine whether red blood cell-mediated microinjection of antibodies can be used to study nuclear protein localization and function, we microinjected antibodies that have been shown to react specifically with nucleolar acidic phosphoprotein C23 into Walker 256 cells. The intracellular distribution of microinjected anti-C23 antibodies and preimmune immunoglobulins were determined by immunofluorescence. At 3 h after microinjection, affinity-purified anti- C23 antibodies were localized in the cytoplasm and nucleolus. At 17 h after microinjection, the affinity-purified antibody was localized to those nucleolar structures previously shown to contain protein C23. Furthermore, the antibody remained localized in the nucleolus for at least 36 h after microinjection. In contrast to the results obtained with specific antibodies, preimmune immunoglobulins remained in the cytoplasm 36 h after microinjection. These results indicate that red blood cell-mediated microinjection of antibodies can be used to study nucleolar and nuclear antigens.     

The protein composition of Friend cell nuclear matrix stabilized by various treatments. Different recovery of nucleolar proteins B23 and C23 and nuclear lamins

Summary— Using two-dimensional polyacrylamide gels stained with Coomassie blue we have studied the protein composition of the nuclear matrix obtained from mouse erythroleukemic nuclei kept at O°C throughout the isolation procedure to prepare the high ionic strength resistant fraction (control matrix) or stabilized in vitro or in vivo by different procedures prior to subfractionation (ie 37°C incubation of isolated nuclei; sodium tetrathionate exposure of purified nuclei; heat shock of intact cells). When the matrix obtained from 37°C incubated nuclei was compared with the control matrix, striking differences in the polypeptide pattern were seen if the protein was obtained in both cases from an equivalent number of nuclei. On the other hand, if the same amount of protein for both the samples was applied to the gels the differences were less evident. Sodium tetrathionate stabilization of isolated nuclei and heat shock of intact cells produced a matrix protein pattern that was very similar and differed from that of the in vitro heat-exposed matrix. Using specific polyclonal antisera, we demonstrate that nucleolar proteins B23/numatrin and C23/nucleolin were very abundant in the matrix obtained from chemically-treated nuclei or in vivo heat-stabilized nuclei but were recovered in very small amounts (B23) or completely absent (C23) in the matrix prepared from nuclei heated to 37°C in vitro. Differences were seen also in the recovery of nuclear lamins, and especially lamin B, that was poorly represented in the sodium tetrathionate-stabilized matrix. The results demonstrate that in mouse erythroleukemia cells the increased recovery of nuclear matrix protein that is seen after in vitro heating of isolated nuclei is predominantly due to an additional recovery of the same types of polypeptides that are detected also in the absence of such a treatment. The data also indicate that in vivo heat shock of intact cells produces a nuclear matrix protein pattern that is more similar to the pattern seen after stabilization of purified nuclei with sodium tetrathionate and differs significantly from that obtained by exposing nuclei to 37°C in vitro, unlike to that what previous reports have indicated.     

Nuclear matrix proteins specific for subtypes of human hematopoietic cells

Nuclear matrices were prepared from isolated subtypes of human hematopoietic cells and from cultured leukemia cells. The nuclear matrix proteins were analyzed by high-resolution two-dimensional gel electrophoresis and computer-assisted image analysis. While more than 200 protein spots were shared among the cells, about 50 distinct spots were found characteristic for individual cells or groups of related cells. This allowed to differentiate between hematopoietic cells and nonhematopoietic cells, lymphocytes and myeloid cells, monocytes, neutrophils, and promyelocytic leukemia cells. B and T lymphocytes could not be differentiated. Myeloid cells with their polymorph nuclei were characterized by the presence of 13 and by the absence of seven distinct spots, as well as by low concentrations of nuclear lamins and of heterogeneous nuclear ribonucleoproteins. Neutrophils with multilobular nuclei displayed six additional spots, while lacking 18 nuclear matrix protein spots. The nuclear matrix of proliferating cells showed three distinct spots in addition to proliferating cell nuclear antigen, increased concentrations of numatrin (B23), and heterogeneous nuclear ribonucleoproteins. The described cell-specific nuclear matrix proteins may represent new markers for hematopoietic cells.     

hnRNP proteins and B23 are the major proteins of the internal nuclear matrix of HeLa S3 cells

The nuclear matrix is the structure that persists after removal of chromatin and loosely bound components from the nucleus. It consists of a peripheral lamina-pore complex and an intricate internal fibrogranular structure. Little is known about the molecular structure of this proteinaceous internal network. Our aim is to identify the major proteins of the internal nuclear matrix of HeLa S3 cells. To this end, a cell fraction containing the internal fibrogranular structure was compared with one from which this structure had been selectively dissociated. Protein compositions were quantitatively analyzed after high-resolution two-dimensional gel electrophoresis. We have identified the 21 most abundant polypeptides that are present exclusively in the internal nuclear matrix. Sixteen of these proteins are heterogeneous nuclear ribonucleoprotein (hnRNP) proteins. B23 (numatrin) is another abundant protein of the internal nuclear matrix. Our results show that most of the quantitatively major polypeptides of the internal nuclear matrix are proteins involved in RNA metabolism, including packaging and transport of RNA. © 1996 Wiley-Liss, Inc.     

Characterization of the nucleolar protein, B-36, using monoclonal antibodies

A panel of nine monoclonal antibodies has been produced against a major nuclear protein, B-36, purified from the slime mold Physarum polycephalum. B-36, a 34 kD protein biochemically similar to the major structural proteins of mammalian hnRNP particles, was previously shown to be largely associated with the nucleolus. Eight of the monoclonal antibodies are specific for B-36 protein in Physarum and at least three different epitopes are represented among these eight. Using the monoclonal antibodies B-36 has been shown to be localized exclusively to the nucleolus in actively-growing Physarum cultures. The nucleolar localization of B-36 is dependent on the presence of intact RNA, but not DNA, supporting the hypothesis that B-36 is associated with nucleolar RNA, possibly in some analogous manner to the interaction of the related proteins within heterogeneous nuclear ribonucleoproteins (hnRNP) particles. B-36 is apparently a highly conserved nucleolar protein in eukaryotes as all eight of the monoclonal antibodies specific for B-36 in Physarum are also specific for a 34.5 kD nucleolar protein in rat liver. This indicates that a minimum of three distinct epitopes are conserved in B-36 protein from slime mold to rat.     

Analysis of the proliferative activity of a cell using new monoclonal antibodies to nucleolar protein B23/nucleophosmin

Nowadays, antinucleolar antibodies are widely used for exploration of the nucleolar organization and molecular mechanisms of ribosome production. Here we have described a new monoclonal antibody against the major nucleolar phosphoprotein B23/nucleophosmin (3C9) that is involved in the terminal stages of ribosome production. It is used to examine immunocytochemical peculiarities of the nucleolus in terms of the cell proliferative status and also during mitosis. In human peripheral blood lymphocytes, activated for proliferation with phytohaemagglutinin (PHA), PHA stimulation of lymphocytes was shown to result in accumulation of protein B23 in augmentative nucleoli. A comparative study of 3C9 and two other anti-B23 antibodies 20B2 and anti-B23 by Western blots and indirect immunofluorescence favored the idea that 3C9 cross-reacted with the major isoform of B23, B23.1, that have an apparent molecular weight of 40 kDa.     

Alterations in immunolocalization of the phosphoprotein B23 in HeLa cells during serum starvation

Bright nucleolar immunofluorescence was observed in HeLa S3 cells by immunostaining with a monoclonal antibody to the nucleolar phosphoprotein B23 (MW 37 kD/pI 5.1). After 48 h of incubation in a serum-free medium, the nucleolar fluorescence was diminished and a general nuclear immunofluorescence was observed. This change in localization of fluorescence indicated that protein B23 had migrated out of the nucleoli. No gross morphological change in nucleoli was observed by light microscopy and the immunolocalization of another nucleolar phosphoprotein, C23, was unaffected by serum deprivation. Relocation of protein B23 in nucleoli was observed after refeeding with serum-containing medium. This re-entry process was not observed after treatment with actinomycin D (50 ng/ml-5 micrograms/ml), but the process was unaffected by cycloheximide (0.2 mM). Quantitation of protein B23 in the nucleoli of the control (fed) or starved HeLa cells was done by ELISA immunoassay. A marked decrease in the amount of protein B23 occurred in the nucleoli of the starved cells (11.8 micrograms B23/mgDNA) as compared with the control nucleoli (20.8 micrograms B23/mgDNA). The amount of protein B23 in the nucleoplasm (excluding nucleoli) was 70% higher in the starved cells. Protein B23 was analysed by one- and two-dimensional PAGE. Three components of protein B23 with slightly different molecular weights and pIs (37 kD/5.1, 35 kD/5.1 and 35 kD/5.3) were observed in nucleoli. The lower molecular weight components were predominantly found in the nucleoplasm.     

Cellular localization of the galactose-binding lectin from human serum

An SL2 lectin was isolated from human serum and characterized previously; cellular localization of the lectin was studied using polyclonal rabbit antibodies. According to cytofluorimetry, anti-SL2 antibodies bound only to lymphocytes and monocytes but not to other blood cells. Antibodies bound to Jurkat T cell lymphoma but did not interact with IM-9 cells of B cell origin. Moreover, the Jurkat cells bound oligosaccharides having the highest affinity to SL2 (GalNAcalpha_and Fucalpha1-2Gal), and this interaction was inhibited by anti-SL2 antibodies. Lysis of the Jurkat cells with subsequent electrophoresis and Western blotting indicates that anti-SL2 antibodies recognized a 14-kD protein.     

Human eosinophil-derived neurotoxin and eosinophil protein X are likely the same protein

The human eosinophil granule contains a series of cationic proteins. Two of these, eosinophil-derived neurotoxin (EDN) and eosinophil protein X (EPX), are reported to have similar m.w. and both possess neurotoxic and helminthotoxic activities. Therefore, the properties of these molecules were analyzed to determine whether they differ. EDN was purified from eosinophils of patients with the hypereosinophilic syndrome and EPX from the buffy coat cells of normal individuals. By SDS-PAGE, both proteins showed a major band at 18.7 kDa and a minor band at 21.4 kDa. By two-dimensional non-equilibrium gel electrophoresis the proteins migrated identically. With radiolabeled proteins in reverse phase HPLC, both proteins eluted at the same concentration of acetonitrile and showed identical tryptic maps. Both proteins possessed comparable ribonuclease activity and both were comparably neurotoxic in the rabbit. By immunodiffusion the two proteins showed a reaction of identity; by RIA, with both polyclonal and monoclonal antibodies, the proteins had very similar inhibitory activities. These results indicate that EDN and EPX have virtually identical properties and are probably the same protein.