From the
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 (gp130
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 (EtNU
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 et al., 1985). 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)).
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
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
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 et al., 1970), but later
found to be expressed in other tissues also (Harahap and Goding, 1988).
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 and Goding, 1987; Buckley et
al., 1990). 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 et al., 1993). 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 et al., 1991, 1993). Recently, evidence for the
existence of a soluble form of PC-1 generated by proteolytic cleavage
has been published (Belli et al., 1993).
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 et al., 1985, 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 et al., 1991a;
Kindler-Röhrborn et al., 1994).
The EtNU-induced,
malignant cell line BT4Ca was chosen for biochemical analysis and
purification of gp130
The
electrophoretic mobility of unreduced gp130
The sequencing of endoproteinase-generated
gp130
The limited sequence homology between
gp130
The sequence motifs of
gp130
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank/EMBL Data Bank with accession number(s) Z47987.
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
et al. (1994) described the cDNA cloning of autotaxin, a
secreted human tumor motility-stimulating protein which is related to
PC-1 and gp130
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
) 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 gp130
possesses 5`-nucleotidase activity that can be inhibited with
EDTA. Different from PC-1, gp130
isolated 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.
;
(
)
Druckrey et al., 1966) provides a model for the
molecular analysis of cell type-specific carcinogenesis (Rajewsky
et al., 1977; Rajewsky, 1985). 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 et al., 1991). In the
central nervous system, target genes of similar relevance to malignant
conversion by chemical carcinogens have not yet been identified.
(
)
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.
6,000/fetal brain
cell and
30,000/BT4Ca cell (Dux et al., 1991a). Initial
biochemical analyses suggested that the RB13-6 antigen was a membrane
glycoprotein with an apparent molecular mass of 125 kDa (Cleeves,
1991). 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
gp130
from a fetal rat brain cDNA library. Potential
structural and functional features of gp130
are
discussed on the basis of its significant sequence homology to the
human and murine plasma cell membrane glycoprotein PC-1 (van Driel and
Goding, 1987; Buckley et al., 1990).
Cell Lines and Antibodies
Malignant
neuroectodermal rat cell line BT4Ca (Laerum et al., 1977) and
the isotype-matched control antibody EM-21 (directed against
O-ethyl-2`-deoxyguanosine; Eberle (1989)) were
from the collection of the Institute of Cell Biology (Cancer Research),
Essen.
Immunoprecipitation
Cells were metabolically
labeled with 500 µCi of [S]methionine
(Amersham) in 6 ml of Dulbecco's modified Eagle's medium
per 144-cm
Petri dish for 4 h and lysed at a concentration
of 10
cells/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%
-mercaptoethanol for
reduction of samples.
Preparation of Plasma Membranes
Adherent BT4Ca
cells were grown to confluence (3
10
cells/cm
) on Petri dishes in Dulbecco's
modified Eagle's medium (Frank et al., 1972) 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 MgCl
,
250 mM sucrose, and protease inhibitors: 1 µg/ml
aprotinin, 1 µg/ml leupeptin, 1 mM phenylmethanesulfonyl
fluoride) at a concentration of 3
10
cells/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 MgCl
,
1 mM CaCl
and stored at
80 °C. Membrane
protein contents were determined according to Lowry et al. (1951) 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
NaHCO, 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
CaCl
, 1 mM MgCl
, 50 mM n-octyl-
-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-
-D-glucoside, the antigen was eluted
from the matrix with 100 mM diethylamine, 1 mM n-octyl-
-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--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 MgCl), and 30 µl of purified
gp130
diluted in 2 mM n-octyl-
-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 and Lottspeich (1989).
Resulting peptide fragments were eluted with 60% acetonitrile, 40%
HO, 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 H
O 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 5:
Nucleotide sequence and deduced amino acid
sequence of gp130
. 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 gp130
are
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)
-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 et al., 1989) 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 gt11
fetal rat brain library (Clontech) by plating 10
phages/dish and preparation of plate lysate stocks (Sambrook
et al., 1989). 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
10
phages. Sublibraries giving
a PCR product of the expected size were plated at a density of 300
phages/cm
and screened by hybridization with the RB13-6
antigen cDNA fragment obtained by RT-PCR and labeled with
-[
P]dCTP using a multiprime labeling kit
(Amersham). Standard protocols (Sambrook et al., 1989) 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 10
phages, 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).
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--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 1:
Immunoprecipitation with
monoclonal antibody RB13-6 ( lanes Ab) from Triton X-100
solubilisates of metabolically
[S]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 2:
Analysis of gp130
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--D-glucoside and deoxycholate, the
presence of divalent cations (Ca
,
Mg
), 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%.
10
BT4Ca cells containing 800 mg of protein yielded
50 µg of homogeneous gp130
(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 gp130
The significant degree of homology of all
gp130Peptide
Sequences
peptides 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
gt11 clone with a 2.5-kilobase insert containing an
open reading frame of 2420 bp. The 1.5-kilobase insert of a second
isolated
clone was found to be identical with the 3` part of this
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
-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, 1987). 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 gp130
peptides were found to be part of the
derived amino acid sequence; this was considered as evidence for the
authenticity of the isolated sequence.
Figure 3:
RT-PCR
with degenerate, inosine-containing primers based on
gp130 peptide sequences (see ``Experimental
Procedures''). BT4Ca mRNA was used in reactions with primers
K15F/K37R ( lane A) and K45F/K37R ( lane
B).
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 gp130
Comparison of the gp130to the PC-1
Glycoprotein
cDNA
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
gp130
sequence can be divided into parts of high and
low homology, suggesting functional domains (Fig. 6).
Sequence-based calculations of the hydrophobicity profiles (Kyte and
Doolittle, 1982) of gp130
and 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 gp130
which are summarized in
. The putative catalytic domain is highly conserved between
gp130
, human and murine PC-1, and a bovine intestinal
nucleotide phosphodiesterase (Fig. 7).
Figure 6:
Amino acid sequence homology between
murine PC-1 and gp130: 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 7:
Catalytic domain of bovine intestinal
5`-nucleotide phosphodiesterase and corresponding regions of
gp130 and PC-1. The threonine marked with an
asterisk was identified as the active site residue (Culp
et al., 1985). Amino acids identical with those of the bovine
enzyme are represented by dashes.
Purified
gp130catalyzes 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
K
= 0.16 mM. The absence
of Mg
in the assay did not affect this reaction,
whereas addition of 1 mM EDTA inhibited the enzymatic
cleavage.
because 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 gp130
could 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
10
liters/mol; Dux et al. (1991b))
and the low concentration of solubilized antigen.
isolated
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 and Shen, 1982). It will be of interest to
investigate different cell types, including transformed phenotypes, for
the presence of dimeric gp130
and with respect to the
monomer-dimer equilibrium. This analysis will require the production of
a new anti-gp130
antibody suitable for Western blot
analysis.
peptides 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 gp130
. 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 gp130
, 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 et al. (1993) 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.
and PC-1 compared to the high degree of
conservation between human and murine PC-1 sequences (Fig. 6)
suggests that gp130
may 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 gp130
and rat PC-1 homologs, respectively.
and 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 et al., 1991, 1993) and could be confirmed for
purified gp130
. 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 and Newsholme,
1978). Ectonucleotidase activity was also found as an accompanying
function of cell adhesion molecules C-CAM (Aurivillius et al.,
1990) and N-CAM (Dzhandzhugazyan and Bock, 1993). 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 et
al., 1991, 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
subunits (K XGFFKR) responsible for calreticulin
binding (Rojiani et al., 1991). In integrin
subunits,
this consensus sequence is also found immediately adjacent to the
transmembrane domains. Two somatomedin B-like domains (Patthy, 1988;
Jenne, 1991) were detected in the cDNA-derived amino acid sequence of
gp130
. Such domains have thus far only been found in
PC-1, in two proteins with unknown function (placental protein 11,
Grundmann et al. (1990); Tcl-30, Baughman et al. (1992)), 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 et al., 1985). The RGD sequence of
gp130
invites the hypothesis that it also binds to
integrins, as other membrane proteins and components of the
extracellular matrix containing this tripeptide (D'Souza et
al., 1991). As speculated on the basis of data indicating the
existence of soluble PC-1 (Belli et al., 1993), proteolytic
cleavage of gp130
could 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 gp130
in 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 gp130
in normal and malignant neural cells. The purification and cDNA
cloning of gp130
provides the basis for such
functional characterization and the search for alterations in malignant
phenotypes.
Table: 537551973p4in
Kretsinger, 1987.
.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.