Affinity Purification and cDNA Cloning of Rat Neural Differentiation and Tumor Cell Surface Antigen gp130RB13-6 Reveals Relationship to Human and Murine PC-1 (∗)

  • Helmut Deissler
    Affiliations
    From the Institute of Cell Biology (Cancer Research), University of Essen Medical School, Hufelandstrasse 55, D-45122 Essen, and the
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  • Friedrich Lottspeich
    Affiliations
    Max-Planck-Institute of Biochemistry, Am Klopferspitz 18a, D-82152 Martinsried, Germany
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  • Manfred F. Rajewsky
    Correspondence
    To whom correspondence and reprint requests should be addressed. Tel.: 49-201-723-2802; Fax: 49-201-723-5905
    Affiliations
    From the Institute of Cell Biology (Cancer Research), University of Essen Medical School, Hufelandstrasse 55, D-45122 Essen, and the
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  • Author Footnotes
    ∗ This work was supported by Grant W87-92-Ra5 from the Dr. Mildred Scheel Foundation for Cancer Research, by Grant Ra119-16-1 from the Deutsche Forschungsgemeinschaft, and by the National Foundation for Cancer Research through Krebsforschung International, Germany. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) Z47987.
      Monoclonal antibody RB13-6 recognizes a subset of rat brain glial precursor cells that are highly susceptible to malignant conversion by the carcinogen N-ethyl- N-nitrosourea. The corresponding cell surface antigen was identified as a membrane glycoprotein (gp130RB13-6) and purified by immunoaffinity chromatography from the tumorigenic neuroectodermal rat cell line BT4Ca. Sequencing of 5 endoproteinase-generated peptides of the purified antigen permitted the specific amplification of a cDNA fragment by reverse transcription-polymerase chain reaction and subsequent isolation of the complete coding sequence from a fetal rat brain cDNA library. The derived amino acid sequence indicates that the RB13-6 antigen is related to the human and murine plasma cell membrane protein PC-1, a nucleotide pyrophosphatase/alkaline phosphodiesterase and ectoprotein kinase. Similarly, purified gp130RB13-6possesses 5′-nucleotidase activity that can be inhibited with EDTA. Different from PC-1, gp130RB13-6isolated from BT4Ca cells is not a disulfide-linked dimer and contains an RGD-tripeptide sequence which, together with other structural features, suggests a possible function in cell adhesion and its subversion in malignant phenotypes.

      INTRODUCTION

      The induction of malignant tumors of the central and peripheral nervous system of the rat by pulse exposure to the alkylating N-nitroso carcinogen ethylnitrosourea (EtNU1;
      The abbreviations used are: EtNU
      N-ethyl- N-nitrosourea
      mAb
      monoclonal antibody/antibodies
      gp
      glycoprotein
      bp
      base pair(s)
      PAGE
      polyacrylamide gel electrophoresis
      PBS
      phosphate-buffered saline
      HPLC
      high pressure liquid chromatography
      PCR
      polymerase chain reaction
      RT-PCR
      reverse transcription-PCR.
      • Druckrey H.
      • Ivankovic S.
      • Preussmann R.
      ) provides a model for the molecular analysis of cell type-specific carcinogenesis (
      • Rajewsky M.F.
      • Augenlicht L.H.
      • Biessmann H.
      • Goth R.
      • Hülser D.F.
      • Laerum O.D.
      • Lomakina L.Ya.
      ;
      • Rajewsky M.F.
      ). Specific DNA-alkylation products formed by this carcinogen in proliferation-competent target cells may lead to mutations in genes critically associated with cell differentiation and malignant transformation. Thus, a specific mutation of the neu/erbB-2 gene, whose expression is diagnostic for EtNU-induced malignant schwannomas in the peripheral nervous system, may be traced back to a small number of mutant Schwann precursor cells with uncontrolled proliferation which first become detectable shortly after carcinogen exposure (
      • Nikitin A.Yu.
      • Ballering L.A.P.
      • Lyons J.
      • Rajewsky M.F.
      ). In the central nervous system, target genes of similar relevance to malignant conversion by chemical carcinogens have not yet been identified.
      The neuro-oncogenic effect of EtNU in the rat is strongly dependent on the developmental stage at the time of carcinogen exposure, with maximum susceptibility during the late prenatal to early postnatal period. To characterize cell subpopulations in the developing central nervous system in terms of cell lineage relationships, phenotypic differentiation, and relative risk of malignant conversion by EtNU, monoclonal antibodies (mAb) have been produced after immunization with intact fetal brain cells isolated at different stages of development (
      • Kindler-Röhrborn A.
      • Ahrens O.
      • Liepelt U.
      • Rajewsky M.F.
      ). One of these antibodies (mAb RB13-6) recognizes a cell surface antigen expressed by a small subpopulation of neural precursor cells, but not by cells of the mature brain. In contrast, sustained expression of the RB13-6 antigen is observed in the cells of most EtNU-induced brain tumors analyzed thus far and in all tumorigenic neuroectodermal cell lines derived from such tumors or from fetal brain cells that underwent malignant transformation in culture after exposure to EtNU in vivo (e.g. cell line BT4Ca; Kindler-Röhrborn et al. (1994)).2
      S. Blass-Kampmann, T. Bilzer, and M. F. Rajewsky, manuscript in preparation.
      Expression of the RB13-6 antigen, as observed in brain tumors, could thus specify differentiating neural precursor cells particularly sensitive to malignant conversion by EtNU.
      Prior to biochemical characterization and molecular cloning of the RB13-6 antigen, the rate and equilibrium constants for binding of mAb RB13-6 to the surface of intact cells were determined and the number of antigenic determinants/cell was calculated to be ∼6,000/fetal brain cell and ∼30,000/BT4Ca cell (
      • Dux R.
      • Kindler-Röhrborn A.
      • Lennartz K.
      • Rajewsky M.F.
      ). Initial biochemical analyses suggested that the RB13-6 antigen was a membrane glycoprotein with an apparent molecular mass of 125 kDa (

      Cleeves, V., 1991, Biochemische Charakterisierung Antikörper-definierter Zelloberflächenmoleküle Normaler und Maligner Neuraler Zellen der Ratte. Ph.D. dissertation, University of Essen, Essen, Germany.

      ). Here we describe the characterization of the membrane glycoprotein recognized by mAb RB13-6 and its large scale purification by immunoaffinity chromatography, followed by sequencing of endoproteinase-generated peptides, and isolation of the cDNA encoding gp130RB13-6from a fetal rat brain cDNA library. Potential structural and functional features of gp130RB13-6are discussed on the basis of its significant sequence homology to the human and murine plasma cell membrane glycoprotein PC-1 (
      • van Driel I.R.
      • Goding J.W.
      ;
      • Buckley M.F.
      • Loveland K.A.
      • McKinstry W.J.
      • Garson O.M.
      • Goding J.W.
      ).

      EXPERIMENTAL PROCEDURES

       Cell Lines and Antibodies

      Malignant neuroectodermal rat cell line BT4Ca (
      • Laerum O.D.
      • Rajewsky M.F.
      • Schachner M.
      • Stavrou D.
      • Haglid K.G.
      • Haugen Å.
      ) and the isotype-matched control antibody EM-21 (directed against O6-ethyl-2′-deoxyguanosine;

      Eberle, G., 1989, Monoklonale Antikörper gegen Kanzerogen-DNS Adukte. Ph.D. dissertation, University of Essen, Essen, Germany.

      ) were from the collection of the Institute of Cell Biology (Cancer Research), Essen.

       Immunoprecipitation

      Cells were metabolically labeled with 500 μCi of [35S]methionine (Amersham) in 6 ml of Dulbecco's modified Eagle's medium per 144-cm2Petri dish for 4 h and lysed at a concentration of 108cells/ml of solubilization buffer (SBT: 50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 1% Triton X-100, 1 μg/ml aprotinin, 1 mg/ml leupeptin, 0.5 mM phenylmethanesulfonyl fluoride) for 1 h at 4°C. After centrifugation for 30 min at 100,000 × g, the supernatant was incubated with purified antibody (20 μg/ml solubilisate) for 10 min. The immunocomplex was precipitated with Protein G-Sepharose (Pharmacia Biotech Inc.) for 5 min at room temperature, and the matrix was washed with SBT and SBT containing 500 mM NaCl, 0.5% deoxycholate, and 0.05% SDS. Bound protein was released at 50°C by incubation with sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 5% glycerol) for SDS-PAGE analysis which was supplemented with 0.5%β-mercaptoethanol for reduction of samples.

       Preparation of Plasma Membranes

      Adherent BT4Ca cells were grown to confluence (∼3 × 105cells/cm2) on Petri dishes in Dulbecco's modified Eagle's medium (
      • Frank W.
      • Ristow H.J.
      • Schwalb S.
      ) containing 2% fetal calf serum. For expansion of cultures, 1/20 of confluent cell layers were cultivated on new dishes. After washing with PBS, cells were collected in PBS and resuspended in homogenization buffer (20 mM sodium phosphate, pH 7.4, 1 mM MgCl2, 250 mM sucrose, and protease inhibitors: 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 mM phenylmethanesulfonyl fluoride) at a concentration of 3 × 107cells/ml. The suspension was homogenized with a Polytron PT35 (Kinematica) and centrifuged at 1,000 × g for 10 min. Crude membranes were collected from the resulting turbid supernatant by centrifugation at 100,000 × g for 60 min, resuspended in PBS, and centrifuged at 30,000 × g for 30 min. After washing with PBS, the pellet was resuspended in PBS, 1 mM MgCl2, 1 mM CaCl2and stored at −80°C. Membrane protein contents were determined according to
      • Lowry O.H.
      • Rosebrough N.J.
      • Farr A.L.
      • Randall R.J.
      after solubilization in 0.7% SDS.

       Purification of the RB13-6 Antigen

      Monoclonal antibody RB13-6, purified from hybridoma supernatant by ion exchange chromatography on DEAE-Sepharose CL6B (Pharmacia), was coupled to Tresyl-activated Affinica agarose (Schleicher & Schüll) at a density of 5 mg/ml matrix by incubation in 100 mM NaHCO3, pH 8.0, 500 mM NaCl for 16 h at 4°C. The matrix was blocked with 100 mM ethanolamine, pH 8.5, and washed several times with PBS. Plasma membranes from BT4Ca cells were solubilized for 90 min at a protein concentration of 4.2 mg/ml in 40 mM Tris-HCl, pH 7.5, 200 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 50 mM n-octyl-β-D-glucoside (U. S. Biochemical Corp.), 0.14% deoxycholate plus protease inhibitors as mentioned above. The insoluble membrane fraction was removed by centrifugation at 100,000 × g, and the supernatant was incubated for 3 h with affinity matrix (20-100 μg of coupled antibody/mg of membrane protein). After washing with the buffer used for solubilization, this buffer containing 500 mM NaCl, and with 10 mM sodium phosphate, pH 8.0, 1 mM n-octyl-β-D-glucoside, the antigen was eluted from the matrix with 100 mM diethylamine, 1 mM n-octyl-β-D-glucoside. Individual fractions were immediately neutralized by adding 1 M sodium phosphate, pH 6.8, and analyzed by SDS-PAGE. After large scale preparation, the eluted protein was concentrated in a vacuum concentrator without neutralization and mixed with sample buffer for SDS-PAGE.

       Deglycosylation of Purified RB13-6 Antigen

      Purified protein was incubated at 95°C for 5 min at a concentration of 50 μg/ml in an aqueous solution of 30 mM sodium phosphate, pH 7.2, 0.1% Triton X-100, 0.6% n-octyl-β-D-glucoside, 3 mM EDTA. After cooling to room temperature, 0.4 unit of N-glycosidase F (Boehringer Mannheim) was added and the mixture was incubated at 37°C for 18 h.

       Assay for 5′-Nucleotidase Activity

      The standard reaction mixture contained 100 μl of aqueous substrate solution (7 mM thymidine 5′-monophosphate nitrophenyl ester, Sigma), 870 μl of buffer (100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 2 mM MgCl2), and 30 μl of purified gp130RB13-6diluted in 2 mM n-octyl-β-D-glucoside.

       Endoproteinase Cleavage, Peptide Analysis, and Microsequencing

      After SDS-PAGE on a 7% gel, the band representing the purified antigen was excised and cleaved directly in the gel with endoproteinase Lys-C (Boehringer Mannheim; enzyme/protein ratio 1:10) essentially according to
      • Eckerskorn C.
      • Lottspeich F.
      . Resulting peptide fragments were eluted with 60% acetonitrile, 40% H2O, 0.1% trifluoroacetic acid and subjected to reversed-phase HPLC on a Smart System (Pharmacia; detection wavelength 206 nm). Using 0.1% trifluoroacetic acid in H2O as solvent A and 0.1% trifluoroacetic acid in acetonitrile as solvent B, a gradient of 0 to 60% B was applied. Peptide-containing fractions were sequenced on a 473A gas phase sequencer (Applied Biosystems) according to the instructions of the manufacturer.

       RT-PCR with Degenerate Inosine-containing Primers

      The sequences of protease-generated peptides K15, K45, and K37 (see Fig. 5) were used to design the following oligonucleotides: 5′-(AATCTGCAG)ACICACGGITACAA(T/C)AA(T/C)GA(G/A)TT-3′, 5′-(AATGAGCTC)ATGGAIGCIATITTC(C/T)TIGCICA(C/T)GG-3′, and 5′-(AATGGATCC)TTCTGIATIACIC(T/G)IGGIC(T/G)ICC(G/A)AA-3′ (opposite strand). These primers are degenerate in the 3′-region and contain inosine or the most likely nucleotide according to rat codon usage in other ambiguous positions. At the 5′-end, primers contain cleavage sites for restriction enzymes and three additional nucleotides.
      Figure thumbnail gr5
      Figure 5:Nucleotide sequence and deduced amino acid sequence of gp130RB13-6. Termination codons and polyadenylation signal in the 3′-noncoding region are double-underlined. The putative transmembrane domain is marked by a dashed line. Cysteines of two somatomedin B-like domains conserved between human and murine PC-1 and gp130RB13-6are circled. Sequences previously determined by peptide sequencing are underlined.
      Poly(A)+RNA from BT4Ca cells was prepared using Oligotex-dT latex beads (Qiagen) according to the protocol supplied by the manufacturer. After (dT)12-18-primed reverse transcription with Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.), the cDNA synthesized from 30 ng of RNA was used in a standard PCR reaction (
      • Sambrook J.
      • Fritsch E.F.
      • Maniatis T.
      ) with 35 pmol of each primer and a cycle profile of 1 min at 94°C, 1 min at 40°C, and 1 min at 72°C. For sequence analysis, PCR products purified by gel electrophoresis were cloned into the SrfI site of the plasmid pCRScript SK(+) (Stratagene).

       Isolation of cDNA Clones Encoding the RB13-6 Antigen

      Twenty sublibraries were generated from a λgt11 fetal rat brain library (Clontech) by plating 105phages/dish and preparation of plate lysate stocks (
      • Sambrook J.
      • Fritsch E.F.
      • Maniatis T.
      ). Sublibraries were then screened for the presence of clones encoding the RB13-6 antigen by standard PCR with 35 pmol of primers F1 (5′-TTCTGCACATCCAACCGGCAC-3′) and R1 (5′-AGTCTGCAGTGCGAGGCAGGAG-3′), using 1 μl of phage suspension containing 1-5 × 107phages. Sublibraries giving a PCR product of the expected size were plated at a density of 300 phages/cm2and screened by hybridization with the RB13-6 antigen cDNA fragment obtained by RT-PCR and labeled with α-[32P]dCTP using a multiprime labeling kit (Amersham). Standard protocols (
      • Sambrook J.
      • Fritsch E.F.
      • Maniatis T.
      ) were used for plating of phages, screening, and isolation of pure cDNA clones. Insert DNA of positive clones was excised with restriction endonuclease EcoRI and cloned in pBluescript SK(+) (Stratagene) for sequence analysis. Starting with 108phages, the 5′-end of the cDNA encoding the RB13-6 antigen was amplified from the complete phage library by sequential standard PCR with primer pairs LF (5′-GACTCCTGGAGCCCGT-3′)/R3 (5′-CCTGGGATGAGGCACAGGCTT-3′) and LF/R2 (5′-ATCCGCACAGGAACAGAGGGC-3′). For the second PCR (25 cycles: 1 min at 94°C, 1 min at 55°C, and 1 min at 72°C), 1/100 of the primary PCR mixture (5 min at 95°C; 34 cycles as described above) was used.

       DNA Sequencing and Sequence Analysis

      DNA fragments cloned in plasmid vectors were sequenced with the AutoRead Sequencing Kit using an A.L.F. DNA sequencer (both from Pharmacia) and standard sequencing primers. For sequencing of long DNA fragments cloned in pBluescript SK(+), sequential nested deletions were generated with exonuclease III and mung bean nuclease (Stratagene) according to the supplier's protocol after SacI and BamHI cleavage of the plasmid. Sequences of cloned PCR products were confirmed by sequencing several independent clones. Sequence analysis and data base searching were performed with the program package HUSAR (Heidelberg Unix Sequence Analysis Resources) used within the GENIUSnet (a service of the German Cancer Research Center, Heidelberg).

      RESULTS

       Biochemical Characterization and Purification of the RB13-6 Antigen for Microsequencing

      mAb RB13-6 binds to a plasma membrane protein with an apparent molecular mass (SDS-PAGE) of 130 kDa, as shown by immunoprecipitation from Triton X-100 solubilisates of metabolically labeled BT4Ca cells (Fig. 1). Without reduction of the protein prior to electrophoresis, a decreased mobility between 140 kDa and 180 kDa was observed, depending on the pore size of the gel. Improved solubilization of plasma membranes isolated from antigen-positive BT4Ca cells with 50 mM n-octyl-β-D-glucoside and 0.2% deoxycholate instead of Triton X-100 permitted us to detect the specifically immunoprecipitated 130-kDa protein by conventional silver staining of the SDS-polyacrylamide gel. Deglycosylation of the purified protein by cleavage with N-glycosidase F resulted in a protein core with an apparent molecular mass of 115 kDa (Fig. 2 B).
      Figure thumbnail gr1
      Figure 1:Immunoprecipitation with monoclonal antibody RB13-6 (lanes Ab) from Triton X-100 solubilisates of metabolically [35S]methionine-labeled BT4Ca cells. Precipitations with an isotype-matched control antibody are shown in lanes C. Proteins were analyzed by SDS-PAGE (10.5% gel), without (A) and with (B) reduction of samples prior to electrophoresis and autoradiography.
      Figure thumbnail gr2
      Figure 2:Analysis of gp130RB13-6 purified for microsequencing (A) and deglycosylation of purified protein with N-glycosidase F (B). Untreated protein was loaded on lane 1, deglycosylated antigen on lane 2. SDS-polyacrylamide gels were stained with Coomassie R-250.
      The RB13-6 antigen was purified from BT4Ca cells by immunoaffinity chromatography with immobilized mAb RB13-6. Several parameters had to be optimized for successful purification (see “Experimental Procedures”): coupling of the antibody to the matrix resulting in intermediate surface density, solubilization of plama membranes with both n-octyl-β-D-glucoside and deoxycholate, the presence of divalent cations (Ca2+, Mg2+), and high excess of coupled mAb over solubilized antigen. The antibody-antigen complex formed at the affinity matrix could be dissociated with alkaline solutions (pH > 11) or 6 M urea, but not with an acidic (pH 2) buffer. Based on the approximate number of antigenic determinants on BT4Ca cells (30,000; Dux et al. (1991a)), the yield of antigen purified according to the optimized protocol was calculated to be ∼25%.
      Since the N terminus of purified antigen was not accessible to Edman degradation, large scale purification was necessary to obtain an amount of protein sufficient for protease cleavage. Plasma membranes from 4.2 × 1010BT4Ca cells containing 800 mg of protein yielded ∼50 μg of homogeneous gp130RB13-6(Fig. 2 A) which was treated with endoproteinase Lys-C to generate peptides for microsequencing. After purification by HPLC, the sequences of 5 peptides (K15: KGSSNXEGGTHGYNNEF; K17: KSGPVSAGV; K29: KAPFYQPSHAEELSK; K37: KT/GNLPFGRPRVIQK; and K45: KSMEAIFLAHGPSFK) were determined by automated Edman degradation.

       cDNA Cloning Based on gp130RB13-6Peptide Sequences

      The significant degree of homology of all gp130RB13-6peptides to the sequences of human and murine PC-1 proteins (29-64% identical amino acids) suggested a certain arrangement of the sequenced peptides in the protein (see Fig. 5). Based on their assumed positions, degenerate, inosine-containing oligonucleotides were designed from the peptide sequences K15, K45 (forward primers), and K37 (reverse primer) (see “Experimental Procedures”) and used for RT-PCR with mRNA from BT4Ca cells (Fig. 3). The amplified 400-bp cDNA fragment was then used as a probe for screening of a fetal rat brain cDNA library, resulting in a λgt11 clone with a 2.5-kilobase insert containing an open reading frame of 2420 bp. The 1.5-kilobase insert of a second isolated λ clone was found to be identical with the 3′ part of this λ insert. Completion of the 5′ region was achieved by sequential PCR amplification of a predominant 420-bp fragment from the whole phage library, using one λ-specific primer (LF) and two primers (R3, R2) from the terminal part of the known sequence (Fig. 4). The complete sequence (Fig. 5) contains an open reading frame of 2664 bp coding for 888 amino acids with a single potential initiation codon near the 5′ end. The sequence context of this ATG coding for the first Met (ACAATGG) is in accordance with the Kozak rules for functional initiation codons of vertebrate mRNAs (
      • Kozak M.
      ). Two consecutive stop codons at positions 2666 and 2678 terminate the open reading frame which is followed by a 97-bp noncoding sequence with a polyadenylation signal 30 bp from eight terminal adenosines. All sequenced gp130RB13-6peptides were found to be part of the derived amino acid sequence; this was considered as evidence for the authenticity of the isolated sequence.
      Figure thumbnail gr3
      Figure 3:RT-PCR with degenerate, inosine-containing primers based on gp130RB13-6 peptide sequences (see “Experimental Procedures”). BT4Ca mRNA was used in reactions with primers K15F/K37R (lane A) and K45F/K37R (lane B).
      Figure thumbnail gr4
      Figure 4:cDNA fragments and sequencing strategy. Positions and lengths of cDNA fragments isolated by screening of a fetal rat brain cDNA library and by PCR techniques are shown schematically. Primary sequencing data that were combined to yield the total cDNA sequence are shown in the lower part of the graph.

       Relationship of gp130RB13-6to the PC-1 Glycoprotein

      Comparison of the gp130RB13-6cDNA and the derived amino acid sequence with sequences submitted to data bases (GenBank, EMBL Nucleotide Sequence Library) confirmed the homologies to murine and human PC-1 proteins which were found to be 49% and 51% identical amino acids, respectively. The degree of homology between human and murine PC-1 sequences is 81%. The entire gp130RB13-6sequence can be divided into parts of high and low homology, suggesting functional domains (Fig. 6). Sequence-based calculations of the hydrophobicity profiles (
      • Kyte J.
      • Doolittle R.F.
      ) of gp130RB13-6and of the PC-1 proteins confirmed their relationship and are indicative of a similarly located transmembrane domain (amino acids 23-45) followed by a short intracellular domain. Further analysis revealed several putative structural features of gp130RB13-6which are summarized in Table I. The putative catalytic domain is highly conserved between gp130RB13-6, human and murine PC-1, and a bovine intestinal nucleotide phosphodiesterase (Fig. 7).
      Figure thumbnail gr6
      Figure 6:Amino acid sequence homology between murine PC-1 and gp130RB13-6: comparison with the homology between murine and human PC-1. Sequences were divided into segments of high and low homology on the basis of identical amino acids. Segment A contains the intracellular and transmembrane domains, segment B the somatomedin B-like domains, segment C the catalytic domain, and segment H the EF-hand motif (see Table I).
      Figure thumbnail gr7
      Figure 7:Catalytic domain of bovine intestinal 5′-nucleotide phosphodiesterase and corresponding regions of gp130RB13-6 and PC-1. The threonine marked with an asterisk was identified as the active site residue (
      • Culp J.S.
      • Blytt H.J.
      • Hermodson M.
      • Butler L.G.
      ). Amino acids identical with those of the bovine enzyme are represented by dashes.
      Purified gp130RB13-6catalyzes the cleavage of thymidine 5′-monophosphate nitrophenyl ester in accordance with the enzymatic activity of PC-1. The pH optimum of this reaction was found to be 9.7; the Michaelis constant at pH 9.5 was determined to be Km= 0.16 mM. The absence of Mg2+in the assay did not affect this reaction, whereas addition of 1 mM EDTA inhibited the enzymatic cleavage.

      DISCUSSION

      The surface antigen recognized by mAb RB13-6 on cells of the malignant neuroectodermal rat cell line BT4Ca was identified by affinity purification and cDNA cloning as a glycoprotein (gp130) related to the human and murine PC-1 proteins. PC-1 was originally described as a cell surface antigen of differentiated, antibody-secreting B cells (
      • Takahashi T.
      • Old L.J.
      • Boyse E.A.
      ), but later found to be expressed in other tissues also (
      • Harahap A.R.
      • Goding J.W.
      ). The class II transmembrane protein PC-1 is a homodimer consisting of two disulfide-linked 115-kDa polypeptides. cDNA cloning of murine and human PC-1 had neither revealed homology to other proteins nor possible biological functions (
      • van Driel I.R.
      • Goding J.W.
      ;
      • Buckley M.F.
      • Loveland K.A.
      • McKinstry W.J.
      • Garson O.M.
      • Goding J.W.
      ). Rebbe et al. (1991) reported that nucleotide pyrophosphatase/alkaline phosphodiesterase I activity is associated with murine PC-1 which was confirmed by expression of enzymatically active PC-1 in CHO cells (
      • Rebbe N.F.
      • Tong B.D.
      • Hickman S.
      ). PC-1 isolated from bovine liver can be phosphorylated at a threonine residue, and it has been suggested that it may act as a threonine-specific ectoprotein kinase (
      • Oda Y.
      • Kuo M.-D.
      • Huang S.S.
      • Huang J.S.
      ,
      • Oda Y.
      • Kuo M.-D.
      • Huang S.S.
      • Huang J.S.
      ). Recently, evidence for the existence of a soluble form of PC-1 generated by proteolytic cleavage has been published (
      • Belli S.I.
      • van Driel I.R.
      • Goding J.W.
      ).
      The protein purified from BT4Ca cells most probably corresponds to the antigenic determinant detected by the same antibody on a subpopulation of neural precursor cells of rat brain and in EtNU-induced brain tumors of the rat (
      • Kindler-Röhrborn A.
      • Ahrens O.
      • Liepelt U.
      • Rajewsky M.F.
      , 1994). Direct proof of this assumption by biochemical analysis of different cell types of the immature rat brain has thus far not been possible due to the relatively weak antigen expression and the small number of antigen-positive fetal brain cells (
      • Dux R.
      • Kindler-Röhrborn A.
      • Lennartz K.
      • Rajewsky M.F.
      ;
      • Kindler-Röhrborn A.
      • Blass-Kampmann S.
      • Lennartz K.
      • Liepelt U.
      • Minwegen R.
      • Rajewsky M.F.
      ).
      The EtNU-induced, malignant cell line BT4Ca was chosen for biochemical analysis and purification of gp130RB13-6because it combines a high rate of cell proliferation with high antigen expression. Several parameters of the purification procedure had to be defined and optimized before sufficient amounts of gp130RB13-6could be isolated, probably due to an unstable epitope recognized by the antibody, retaining its native structure only under certain conditions. The unusually high excess of affinity matrix required may be a consequence of the moderate binding affinity of the antibody (K = 1.87 × 108liters/mol;
      • Dux R.
      • Kindler-Röhrborn A.
      • Lennartz K.
      • Rajewsky M.F.
      ) and the low concentration of solubilized antigen.
      The electrophoretic mobility of unreduced gp130RB13-6isolated from BT4Ca cells indicates that it is not a disulfide-linked homodimer, in contrast to the related PC-1 glycoproteins which migrate in SDS-polyacrylamide gels in accordance with the molecular mass expected for a dimer (
      • Goding J.W.
      • Shen F.-W.
      ). It will be of interest to investigate different cell types, including transformed phenotypes, for the presence of dimeric gp130RB13-6and with respect to the monomer-dimer equilibrium. This analysis will require the production of a new anti-gp130RB13-6antibody suitable for Western blot analysis.
      The sequencing of endoproteinase-generated gp130RB13-6peptides has facilitated the isolation of the corresponding cDNA which was aided by inclusion of an RT-PCR step to generate a homologous probe for stringent screening of a cDNA library. Assuming the first ATG in-frame to be the translation initiation site, as also concluded from the similarity to the PC-1 proteins, the molecular mass calculated from the sequence (100 kDa) is lower than the 115 kDa expected from the electrophoretic mobility of N-deglycosylated gp130RB13-6. This difference may be explained by additional protein modifications and/or bound nonionic detergent molecules used for solubilization, which influence SDS binding and mobility. However, it cannot be excluded and will be subject to further investigation that the isolated cDNA represents a shorter splice variant of gp130RB13-6, although longer cDNA sequences were not detected in the fetal brain cDNA library. The existence of three phosphorylated rat liver proteins reacting with an anti-PC-1 antibody described by
      • Uriarte M.
      • Stalmans W.
      • Hickman S.
      • Bollen M.
      invites speculations on the existence of such variants of the related rat protein. Apart from such speculation, the isolated cDNA must be regarded as coding for the isolated protein because all sequenced peptides obtained from the purified protein occur in the derived amino acid sequence.
      The limited sequence homology between gp130RB13-6and PC-1 compared to the high degree of conservation between human and murine PC-1 sequences (Fig. 6) suggests that gp130RB13-6may not be the rat homolog of PC-1, but rather a different member of a larger protein family. This will have to be confirmed by cloning of the human gp130RB13-6and rat PC-1 homologs, respectively.
      The sequence motifs of gp130RB13-6and its homology to PC-1 point to several possible functions of this molecule in normal and transformed cells. 5′-Nucleotide pyrophosphatase activity, as expected from the presence of a highly conserved catalytic domain (see Fig. 7), was shown for PC-1 (
      • Rebbe N.F.
      • Tong B.D.
      • Finley E.M.
      • Hickman S.
      , 1993) and could be confirmed for purified gp130RB13-6. The biological role of a molecule with this primary function could be: (i) hydrolysis of nucleoside triphosphates to promote uptake of metabolically used nucleosides and (ii) control of the concentration of extracellular adenosine which can influence various important cellular functions (
      • Arch J.R.S.
      • Newsholme E.A.
      ). Ectonucleotidase activity was also found as an accompanying function of cell adhesion molecules C-CAM (
      • Aurivillius M.
      • Hansen O.C.
      • Lazrek M.B.S.
      • Bock E.
      • brink B.
      ) and N-CAM (
      • Dzhandzhugazyan K.
      • Bock E.
      ). Ectoprotein kinase activity, as reported for PC-1, must also be considered for the RB13-6 antigen, although the in vitro data for PC-1 (
      • Oda Y.
      • Kuo M.-D.
      • Huang S.S.
      • Huang J.S.
      , 1993) are only circumstantial evidence for a similar function in vivo. The predicted intracellular domain contains a potential serine phosphorylation site for a cAMP-dependent protein kinase in a context (KKDSLKR) reminiscent of a consensus sequence of integrin α subunits (K XGFFKR) responsible for calreticulin binding (
      • Rojiani M.V.
      • Finlay B.B.
      • Gray V.
      • Dedhar S.
      ). In integrin α subunits, this consensus sequence is also found immediately adjacent to the transmembrane domains. Two somatomedin B-like domains (
      • Patthy L.
      ;
      • Jenne D.
      ) were detected in the cDNA-derived amino acid sequence of gp130RB13-6. Such domains have thus far only been found in PC-1, in two proteins with unknown function (placental protein 11,
      • Grundmann U.
      • Romisch J.
      • Siebold B.
      • Bohn H.
      • Amann E.
      ; Tcl-30,
      • Baughman G.
      • Lesley J.
      • Trotter J.
      • Hyman R.
      • Bourgeois S.
      ), and in the cell-substrate adhesion molecule vitronectin. The serum peptide somatomedin B is released by proteolytic cleavage from vitronectin which interacts with integrin receptors via an RGD tripeptide (
      • Suzuki S.
      • Oldberg Å.
      • Hayman E.G.
      • Pierschbacher M.D.
      • Ruoslahti E.
      ). The RGD sequence of gp130RB13-6invites the hypothesis that it also binds to integrins, as other membrane proteins and components of the extracellular matrix containing this tripeptide (
      • D'Souza S.E.
      • Ginsberg M.H.
      • Plow E.F.
      ). As speculated on the basis of data indicating the existence of soluble PC-1 (
      • Belli S.I.
      • van Driel I.R.
      • Goding J.W.
      ), proteolytic cleavage of gp130RB13-6could release a soluble protein fragment capable of blocking integrin receptors. In view of the putative structural features deduced from the presence of sequence motifs, involvement of gp130RB13-6in cell-cell or cell-substrate adhesion seems possible and would be in accordance with an expected function during physiological brain development and its subversion by malignant transformation. Future experiments will be focused on the elucidation of the main function of gp130RB13-6in normal and malignant neural cells. The purification and cDNA cloning of gp130RB13-6provides the basis for such functional characterization and the search for alterations in malignant phenotypes.

      Acknowledgments

      We thank Drs. Heidrun Krüger and Andrea Kindler-Röhrborn for helpful discussions. We also thank Ulla Schmücker for DNA sequencing and Dr. R. Wagner (GBF, Braunschweig, Germany) for large scale fermentation of BT4Ca cells. Addendum-After completion of this work, a publication of
      • Murata J.
      • Lee H.Y.
      • Clair T.
      • Krutzsch H.C.A.A.
      • Sobel M.E.
      • Liotta L.A.
      • Stracke M.L.
      described the cDNA cloning of autotaxin, a secreted human tumor motility-stimulating protein which is related to PC-1 and gp130RB13-6.

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