From the Department of Oncology Research and the
¶ Genomics Group, Boehringer Ingelheim Pharma KG, 88397 Biberach an der Riss, Germany, the § Department of Drug
Discovery, Boehringer Ingelheim Austria GmbH, A-1121 Vienna,
Austria, and the
Research Institute of Molecular Pathology,
A-1030 Vienna, Austria
Received for publication, October 20, 2000, and in revised form, November 9, 2000
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ABSTRACT |
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Endosialin, the antigen identified with
monoclonal antibody FB5, is a highly restricted 165-kDa cell surface
glycoprotein expressed by tumor blood vessel endothelium in a broad
range of human cancers but not detected in blood vessels or other cell types in many normal tissues. Functional analysis of endosialin has
been hampered by a lack of information about its molecular structure.
In this study, we describe the purification and partial amino acid
sequencing of endosialin, leading to the cloning of a full-length
cDNA with an open reading frame of 2274 base pairs. The endosialin
cDNA encodes a type I membrane protein of 757 amino acids with a
predicted molecular mass of 80.9 kDa. The sequence matches with an
expressed sequence tag of unknown function in public data bases,
named TEM1, which was independently linked to tumor endothelium by
serial analysis of gene expression profiling. Bioinformatic evaluation
classifies endosialin as a C-type lectin-like protein, composed of a
signal leader peptide, five globular extracellular domains (including a
C-type lectin domain, one domain with similarity to the Sushi/ccp/scr
pattern, and three EGF repeats), followed by a mucin-like region, a
transmembrane segment, and a short cytoplasmic tail. Carbohydrate
analysis shows that the endosialin core protein carries abundantly
sialylated, O-linked oligosaccharides and is sensitive to
O-sialoglycoprotein endopeptidase, placing it in the group
of sialomucin-like molecules. The N-terminal 360 amino acids of
endosialin show homology to thrombomodulin, a receptor involved in
regulating blood coagulation, and to complement receptor C1qRp. This
structural kinship may indicate a function for endosialin as a tumor
endothelial receptor for as yet unknown ligands, a notion now amenable
to molecular investigation.
The endosialin antigen was identified in a survey of normal and
neoplastic human tissues conducted at the Ludwig Institute for Cancer
Research in pursuit of new targets for antibody-based cancer therapies
(37). The hallmark of monoclonal antibody
(mAb)1 FB5, the probe used to
discover endosialin (1), is its distinctive pattern of reactivity with
human tissues. Thus, in a detailed study of biopsy and surgical
specimens representing diverse cancer types, FB5 immunostaining was
found primarily in tumor blood vessels and not in malignant tumor
cells. Significantly, the antigen was not observed in all cancer
samples examined, and even in cancers showing FB5-immunoreactive
endothelial cells, the antigen was frequently detected with a
heterogeneous pattern in the tumor vascular bed. Such a mixed pattern
might be expected for a molecule involved in the reorganization of
blood vessels in tissues such as cancers, in which areas of stable
blood supply and histology are juxtaposed to regions of necrosis,
hypoxia, excessive growth, tissue invasion, and remodeling. The normal
tissues examined were unreactive with mAb FB5, including the blood
vessel endothelium present in the respective organs.
The expression of the FB5 antigen by cultured normal and tumor cells
was also investigated (1), revealing that the standard test cells for
normal endothelial differentiation markers, cultured human umbilical
vein endothelial cells, and microvascular endothelial cells derived
from bone marrow and dermis are antigen-negative. Human umbilical vein
endothelial cell cultures stimulated with a range of mediators known to
induce the expression of endothelial activation antigens (2, 3)
maintain their FB5-negative phenotype (1). Among a host of cultured
epithelial, neuroectodermal, mesenchymal, and hematopoietic cell types
tested, most were FB5 antigen-negative. Notable exceptions are short
term cultures of normal fibroblasts and neuroblastoma cell lines, which
consistently express the antigen in tissue culture (1), allowing the
chromosomal assignment of an FB5 coding gene to human chromosome
11q13-qter, based on the serologic analysis of mouse-human
neuroblastoma cell hybrids. The reason why cultured fibroblasts and
neuroblastomas are FB5 positive in vitro, yet the
corresponding cells in uncultured tissue sections are FB5 negative, is
not known. Presumably, some of these findings reflect cell
type-restricted, adaptive changes of the cell surface phenotype
triggered by tissue culture factors (38); the mechanism underlying such
induction and its relationship to FB5 antigen induction in tumor blood
vessels are not known.
Methods are not generally available to examine the biochemical nature
of antigens with restricted expression in tumor endothelium, because of
to three properties of these cells: (i) they constitute a very minor
fraction in most tumor tissues; (ii) they are not readily purified in
sufficient numbers for direct protein analysis; and (iii) no cell
culture model of tumor endothelium exists that faithfully reproduces
its distinctive in vivo phenotype. With no source for
antigen characterization in tumor endothelium, the target molecule for
mAb FB5 was studied in cultured neuroblastoma and sarcoma cells and
fibroblast cultures. In these cell types, the antigen is a unique
165-kDa glycoprotein, comprised of a core protein that migrates as a
95-kDa species on SDS gels and carries the mAb FB5-defined
epitope, and abundant, highly sialylated O-linked carbohydrate moieties. There has been no proof that the same protein carries the FB5 epitope in tumor endothelium, but in recognition of the
characteristic biochemical properties and the tumor endothelial expression pattern the molecule was designated endosialin.
In the absence of more detailed information about the molecular
structure of endosialin, no clues were available regarding its
potential function in cancer. Therefore, considering the keen interest
in angiogenesis as a determinant of cancer progression and metastasis
(4) and as a target for novel cancer therapies (5), this study aimed to
clone the endosialin gene and provide the requisite probes and
structural information to explore endosialin function in suitable model
systems and in human cancers.
Cells and Antibodies--
Cell lines LA1-5s and MF-SH were grown
in RPMI 1640 medium with 10% fetal calf serum and 20 IU/ml
penicillin/streptomycin; HeLa-S3 cells were grown in Nutrient mixture
HAM F-12 with fetal calf serum and penicillin/streptomycin (Life
Technologies, Inc.). The mAbs used were FB5 (mouse IgG2a) (1) and 9EG7
(ratIgG2a) against human Protein Purification and Edman Sequencing--
Pelleted LA1-5s
cells were lysed in RIPA buffer (150 mM NaCl, 1% Triton
X-100, 0.1% SDS, 20 mM EDTA, 50 mM Tris/HCl,
pH 7.4, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 0.1 unit/ml Northern Blot Analysis--
The SV Total RNA Isolation System
(Promega) was used for total RNA isolation according to the
manufacturer's instructions. Oligo(dT)-cellulose (Life Technologies,
Inc.) was taken for poly(A)+ RNA isolation as described
(8). For Northern blots, 4 µg of poly(A)+ RNA were
electrophoresed on a 0.8% (w/v) agarose gel containing 20 mM MOPS, 5 mM sodium acetate, pH 6.6, and
1.11% formaldehyde. The RNA was blotted in 10× SSC (1.5 M
NaCl, 0.15 M sodium citrate, pH 7.0) on a Hybond N+
membrane (Amersham Pharmacia Biotech) for 16-20 h and UV cross-linked.
To detect endosialin mRNA, a 287-bp polymerase chain reaction
fragment, comprising positions + 2237 to + 2524 of endosialin cDNA,
was used.
RACE-Polymerase Chain Reaction--
For reverse transcription 2 µg of total RNA were added to a mix of 4 µl of the display
THERMO-RT buffer (Display Systems Biotech), 2 µl of a 5 mM dNTPmix, 7 µl of 3 M betain (Sigma), 1 µl of the display THERMO-RT Initiator Mix (total volume, 20 µl),
and 1 µM of primer Est2243DN2
(ACAGGTAGCCGTCGACAGCCAGCGTGC). For cDNA synthesis the mixture was
incubated for 10 min at 65 °C, cooled to 42 °C, and incubated
with 2 µl of the display THERMO-RT terminator solution for 40 min.
The temperature was raised to 65 °C for 15 min, cooled to 37 °C,
and then 1 µl of RNase Mix (5' RACE System for Rapid Amplification of
cDNA Ends Reagent Assembly, version 2.0; Life Technologies, Inc.)
was added and incubated at 37 °C for 30 min. The cDNA was
purified via GlassMax (Life Technologies, Inc.), and the subsequent
tailing reaction was performed in the presence of 1 M
betain according to the manufacturer's instructions (Life Technologies, Inc.) using primer EST 1987DN1
(AGAGGCTGGCTGGGCCCAGTTGGTG) and the Abridged Anchor Primer (Life
Technologies, Inc.). The cycling parameters were 94 °C for 40 s, 62 °C for 40 s, and 72 °C for 105 s, for a total of
40 cycles, with a final extension cycle of 72 °C for 10 min. An
aliquot containing 1% of the specifically amplified tailed cDNA
was subjected to a second nested polymerase chain reaction using the
endosialin primer GSP4 (ACCCACCAGGCTGTCCACACGCTGG) and the Abridged
Universal Amplification Primer (Life Technologies, Inc.) under the
cycling conditions described above. The products were resolved on a 2%
(w/v) agarose gel and sequenced on an ABI PRISM 310 Genetic Analyser
(PerkinElmer Life Sciences).
Transfection and Immunocytochemistry--
Transient transfection
of HeLa-S3 cells plated in chamber slides (Becton Dickinson,
Heidelberg, Germany) was carried out with the transfection reagent
FuGENE 6 and expression vector pMH (Roche Diagnostics), containing the
complete endosialin coding sequence or as empty vector. Cells were
analyzed for antigen expression 24 h after transfection, using
immunocytochemistry with mAb FB5 or control IgG essentially as
described (9). Antibody binding was detected with Alexa Fluor
488-conjugated goat anti-mouse IgG F(ab')2 fragment
(Molecular Probes, Eugene, OR).
Immunoprecipitation Assays and Carbohydrate
Analysis--
Immunoprecipitations were performed as described (8).
For glycosylation studies, cells were metabolically labeled with 400 µCi of [35S] methionine/400 µCi of
[35S]cysteine in 1.2 ml of methionine/cysteine-free
minimal essential medium (Life Technologies, Inc.) for 16 h. Cell
lysates were precleared with protein A-Sepharose (Amersham Pharmacia
Biotech) and precipitated with mAbs or mIgG coupled to CNBr-Sepharose
(Amersham Pharmacia Biotech) at a ratio of 2.8 mg/ml of gel. The
precipitate was washed, split into equal aliquots, and incubated for
16 h at 37 °C either with 1 unit of PNGase F
(N-glycanase F) from Flavobacterium
meningosepticum, 10 milliunits sialidase from Arthrobacter
ureafaciens, or a mixture of 10 milliunits sialidase and 0.5 milliunits O-glycosidase from Diplococcus
pneumoniae (Roche Diagnostics) or with 25 µl of reaction buffer
alone (50 mM sodium phosphate, pH 7.0, 0.5% Triton X-100). Precipitates were washed, eluted in Laemmli buffer, and reduced with 50 mM dithiothreitol. Signals were detected after SDS-PAGE by
exposure to Kodak BIOMAX MR film. For O-sialoglycoprotein
endopeptidase digestion, equal aliquots of LA1-5s cell detergent
lysates were precipitated with mAb FB5 coupled to CNBr-Sepharose (2.8 mg/ml of gel) or with mAb 9EG7 against integrin Data Base Mining and Sequence Analyses--
Amino acid sequence
information for endosialin peptides was used to perform iterative
BLAST, FASTA, and Prosite pattern data base searches against public
sequence data bases. The same algorithms, including gapped BLAST and
PSI_BLAST (10), have been applied for further analysis of endosialin
cDNA and amino acid sequence. The SEG program was applied for
searching low complexity regions in protein sequences with the
following search parameters: window length, 25; trigger complexity,
3.0; and extension complexity, 3.3 (11). Known functional sequence
domains matching sequence segments in endosialin were examined in the
PFAM and SMART (13) domain libraries.
Protein Purification of Human Endosialin and Identification of the
cDNA--
We used immunocytochemistry and enzyme-linked
immunosorbent assays with mAb FB5 to confirm that human umbilical vein
endothelial cell cultures and cultures of microvascular endothelial
cells from dermis and bone marrow lack endosialin expression.
Therefore, the FB5 antigen-positive neuroblastoma cell line LA1-5s was
selected as a source for endosialin purification. Protein was isolated from detergent extracts of 6 × 109 cells using mAb
FB5 conjugated to CNBr-Sepharose as the immunoaffinity matrix. To
confirm the purity of the resulting preparation, an aliquot of the
eluted material was separated on an SDS-polyacrylamide gel, and a major
165-kDa band, consistent with the size of endosialin, was visualized by
silver staining (Fig. 1). The remaining
eluate was subjected to quantitative SDS-PAGE and yielded about 10 pmol of specific protein as determined by Coomassie staining.
Endosialin-containing gel fragments were digested with trypsin, and the
resulting peptides, separated by HPLC and analyzed by Edman degradation
and matrix-assisted laser desorption ionization-time of flight,
gave rise to six unique peptide sequences. In a Prosite pattern search,
five out of these six peptides matched with closely spaced protein
sequences deduced from the EST clone AI371224 (Fig.
2, double underlined). The complete cDNA sequence of this EST revealed a single open reading frame of 1,394 bp. No potential start and stop codons were found, prompting us to conduct an iterative data base search that identified two additional ESTs, H19258 and AA595199, respectively, which extend
the 3' end of the open reading frame to 2,239 bp with a stop codon at
position + 2272 and a polyadenylation signal at position + 2512 followed by the poly(A) tail (Fig. 2). The cDNA data were
verified by sequencing two independent EST clones, H12800 and H12557,
that cover the sequence of HS19258 and AA595199. Because Northern blots
of poly(A)+ RNA isolated from the two endosialin-expressing
cell lines LA1-5s and MF-SH show a single, major transcript of about
2,600 bp (Fig. 3), only a short 5'
segment of the endosialin mRNA appeared to be missing in the
EST-derived cDNA sequence. Using RACE, we succeeded in extending
the 5' sequence by 52 bp, including a putative start ATG. Thus, the
complete cDNA of endosialin comprises an open reading frame of
2,274 bp and codes for a protein of 757 amino acids with a predicted
molecular mass of 80.9 kDa.
Endosialin cDNA Encodes the FB5 Cell Surface
Antigen--
Transient transfection studies with the
endosialin-negative epithelial cell line HeLa-S3 were used to confirm
that the putative endosialin cDNA indeed specifies the cell surface
antigen defined by mAb FB5. Although the parental or mock-transfected
HeLa-S3 cells grown in plastic chamber slides show no detectable
binding of mAb FB5 in indirect immunofluorescence tests, the endosialin transfectants generated with an expression vector containing
full-length endosialin cDNA show strong staining with mAb FB5
(Fig. 4).
Endosialin Is a C-type Lectin-like Membrane
Receptor--
Bioinformatic analysis identifies endosialin as a
typical precursor sequence for a eukaryotic cell surface protein,
resulting evolutionarily from domain shuffling. The endosialin protein
displays a considerable number of disparate functional segments (Fig.
5A). First, there is an
N-terminal sequence of about 20 amino acids that is similar to a signal
leader peptide for export to the endoplasmic reticulum and can be
detected by the SIGNALP2 suite of Nielsen et al. (14). Next,
there are two major segments, a more N-terminal globular portion with
five distinct domains comprising amino acids 30-360, and a more
C-terminal portion comprising amino acids 361-757 and classified
almost exclusively as low complexity region by the SEG program (11). A
more detailed analysis of domain structure architecture in the
N-terminal globular portion is accomplished by comparison with hidden
Markov models in libraries of described sequence domains, such as PFAM
(12) and SMART (13), as well as sequence similarity searches in data
bases with the BLAST and PSI-BLAST tools (10) that rely on the concept
of a common evolutionary ancestor among sequentially homologous
sequences. By this approach, we found statistically significant hits
for a C-type lectin domain (residues 29-157, E < 10
A more detailed analysis of the C-terminal, low complexity portion of
endosialin, covering amino acids 360-757, showed that the local amino
acid composition is dominated by the preferential occurrence or absence
of certain residue types. In accordance with analyses of known
three-dimensional protein structures (15), there is little reason to
expect such polypeptide segments to assume stable tertiary structures
with sterically predefined binding or catalytic activity. It is not
surprising that secondary structure tools (16, 17) predict almost
exclusively coil preferences for this region. Segment 363-380 is
highly negatively charged, with 6 glutamate and 7 aspartate residues,
and it may serve a nonspecific role in facilitating a binding contact
with a charged ligand molecule. Using the software tools DAS (18) and
TOPPRED2 (19), the existence of a single hydrophobic transmembrane
region (amino acids 686-706) can be detected with strong significance; thus, endosialin appears to be a type I membrane protein. The extracellular region spanning amino acids 391-660 is much richer than
the data base average (20) in prolines (21.9%), threonines (9.3%),
and serines (9.3%), but it contains surprisingly few charged residues
(DEKR total only 9.6%), glycines (2.2%), asparagines (1.1%), and
phenylalanines (1.5%). There is no truly sequentially similar protein
sequence in the data base. The small intracellular portion 707-757 is
compositionally rich in proline (11.8%) but depleted of phenylalanine
(none) and hydrophobic residues in general (LVIFM total only 17.6%).
This sequence region is also unique compared with other known protein sequences.
Although the prediction of O-glycosylation sites is not very
reliable even with the most advanced algorithms (21), we supplied the
endosialin sequence to the NetOGlyc 2.0 server offered by J. E. Hansen. This program predicts as probable O-glycosylation sites Thr-60 (in the C-type lectin domain) and a series of 34 serine
and threonine residues located in segment 400-669, which probably does
not have a globular structure.
Endosialin Is Related to Thrombomodulin and Complement Receptor
C1qRp--
The N-terminal endosialin segment between amino acids 1 and
360 shares 39% sequence identity with the corresponding region in the
precursor protein of human thrombomodulin (also referred to as
fetomodulin, CD141, or emb:CAA29045.1) and 33% sequence identity with
the human complement receptor C1qRp (also known as lymphocyte antigen
68, antigen AA4, or emb:CAC00597.1), respectively. The matching region
encompasses the C-type lectin domain, the putative Sushi domain, and
the three EGF-like repeats (Fig. 5B). Moreover, the
positions of cysteine residues critical for disulfide bridges are
highly conserved among the three proteins, consistent with a shared
three-dimensional structure in this segment. Finally, the three
proteins show a conserved WIGL consensus motif, a feature found in
several additional cell surface proteins that modulate endocytosis
(22).
Endosialin Is Conserved in Nonhuman Species and Maps to Human
Chromosome 11q13--
A number of mouse and rat ESTs, originating from
embryonic tissue sources and comprising fragments of 186-521 bp were
identified in public data base searches. These ESTs show 82-92%
identity to the corresponding human endosialin cDNA sequence, thus
suggesting the presence of a close homologue in nonhuman species.
Further data base searches identified a human genomic clone
(RP11-755F10, accession number AP000759) assigned to chromosome 11q13,
which contains more than 75% of the endosialin coding sequence
(positions +521 to +2533) in a putative single exon and in this portion
is 99% identical with the endosialin cDNA. This chromosomal
assignment for the endosialin confirms and refines the assignment to
11q13-qter made for the FB5 antigen using somatic cell hybrids (1).
Carbohydrate Analysis of Recombinant Endosialin--
In several
human cell types, endosialin shows a high degree of
O-glycosylation and sialic acid content (1), and we have examined the glycosylation pattern of recombinant endosialin in transiently transfected HeLa-S3 cells (Fig.
6A). When tested by immunoprecipitation with mAb FB5 immobilized to CNBr-Sepharose, a
165-kDa protein is detected in these cells, but not in mock-transfected HeLa-S3 cells. Pretreatment of matrix-bound antigen with
O-glycosidase or sialidase led to a marked reduction in the
size of the endosialin band on SDS gels, with a pattern identical to
endogenous endosialin in LA1-5s cells (Fig. 6A). Thus,
sialidase treatment alone yields a 120-kDa protein species, and
combined treatment with sialidase and O-glycanase generates
the putative 95-kDa core protein. No shift in the mobility of
endosialin on SDS gels was observed after PNGase F treatment,
consistent with the notion that minor, if any, N-linked
sugar moieties are present at the single Asn-Xaa-(Ser/Thr) consensus
site at position Asn-628. It is noteworthy that endogenous endosialin
in LA1-5s cells does not show the marked heterogeneity in
glycosylation, revealed by broad bands on SDS gels, that is typical for
a number of other glycoproteins with abundant
O-glycosylation. The appearance of two closely spaced
endosialin bands in the HeLa-S3 transfectants may be indicative of
limited heterogeneity of O-glycosylation in this particular
cell type, because the two bands merge into a single band after
deglycosylation.
Mucin-like Endopeptidase Susceptibility--
Because
O-sialoglycoprotein endopeptidase (OSGE) has been shown to
cleave specifically highly O-glycosylated mucin-like
molecules (23, 24), we examined whether endosialin is susceptible to this enzyme. To this end, detergent extracts of metabolically labeled
LA1-5s cells were purified with mAb FB5 and treated with OSGE for
1 h at 37 °C prior to SDS-PAGE. We found that OSGE completely degraded endosialin (Fig. 6B), whereas a control
glycoprotein, We have cloned the gene coding for the human endosialin core
protein based on several lines of evidence. First, transfection of
endosialin cDNA into HeLa-S3 cells leads to induction of cell surface reactivity with the cognate mAb FB5. Secondly, mAb FB5 detects
the expected 165-kDa sialoglycoprotein in the transfectants, with a
core protein migrating as a 95-kDa species on SDS gels. Third, the
endosialin gene maps to chromosome 11q13, refining the previous
assignment to 11q13-qter based on serologic analysis of somatic cell
hybrids with mAb FB5. Finally, the amino acid sequence provides the
requisite attachment sites for abundant O-glycosylation of endosialin.
The present study is not the only evidence linking endosialin induction
to vascular endothelial cells in human cancer. Rather, an independent
line of investigation, aimed at dissecting comprehensive gene
expression profiles in cancers with the serial analysis of gene
expression (SAGE) method, has implicated endosialin-specific ESTs in
tumor angiogenesis. St. Croix et al. (25) employed SAGE to
survey about 32,500 unique gene transcripts for differential expression
in purified tumor endothelium versus endothelium of nonmalignant tissue. The study identified 46 transcripts elevated at
least 10-fold in tumor endothelium, and the highest and most consistent
up-regulation was noted for a hitherto uncharacterized gene, deposited
as ESTs of unknown function in data bases and designated tumor
endothelial marker 1 (TEM1) by St. Croix et al. (25). We
show here that TEM1 is the endosialin gene.
In their study, St. Croix et al. (25) confirmed the SAGE
data and showed by in situ RNA hybridization that
TEM1/endosialin expression in vivo is selective for tumor
endothelium, with no detection in the malignant epithelial cells of
colorectal and other cancer, a range of normal organs, and a panel of
cultured tumor cell lines. Nevertheless, TEM1/endosialin mRNA was
not unique to tumor blood vessels because endothelial cells associated
with wound healing and corpus luteum formation also showed gene
expression by in situ RNA hybridization.
Taking together the endosialin expression data generated with mAb FB5
(1) and the RNA expression data derived from SAGE and in
situ RNA hybridization studies (25), a consistent picture of
endosialin induction in tumor endothelium emerges. Such a close agreement between epitope and protein/mRNA expression is
instructive for a molecule like endosialin, which falls into the group
of sialomucin-type glycoproteins. There is precedent for sialomucins and other glycoconjugates for which mAb-defined epitope expression and
protein/mRNA distribution sharply diverge. Several levels of
complexity have been described (26-29), including (i) epitopes on core
proteins that are masked by glycosylation or specific sialylation, (ii)
epitopes on carbohydrate moieties shared by several unrelated
glycoproteins or glycolipids, and (iii) epitopes on distinct splice
variants or processing variants of the same core protein. In the case
of endosialin, the observed cotyping for the FB5 epitope and
TEM1/endosialin mRNA suggests that mAb FB5 binds to an invariant
epitope on the 95-kDa core protein. Nevertheless, this conclusion needs
to be confirmed for each cell type under investigation, because epitope
masking and mimicry can show exquisite cell type selectivity.
A persistent question in the exploration of tumor angiogenesis is the
extent of phenotypic and functional heterogeneity that may distinguish
subsets of tumor endothelial cells in a macroscopic tumor lesion with
regard to their derivation from pre-existing blood vessels or new
vessel sprouting, and their function and state of activation in
response to varying milieus of tumor-derived, paracrine angiogenesis
inducers and inhibitors. Generally, the capillary bed of a locally
advanced or metastatic tumor mass will extend into areas of relatively
stable blood supply and tissue architecture and closely juxtaposed or
intermingled regions of hypoxia, necrosis, excessive proliferation,
tissue invasion, fibroblastic remodeling, or inflammatory cell
infiltration. Placed in this context of malignant tissue disruption,
markers of tumor angiogenesis might be expected to display a
heterogeneous pattern of expression as a rule rather than exception.
Considering the marked heterogeneity in FB5 staining in tumor vessels
(1), it may be surprising that the report on TEM1/endosialin mRNA
in situ hybridization (25) does not address this aspect. One
explanation would suggest that endosialin mRNA is uniformly
expressed in tumor endothelium and that only subsets of endothelial
cells accumulate the endosialin protein or protein with the FB5
epitope; however, as outlined above, there is currently no evidence
supporting such a mechanism. Alternatively, it may be argued that
mRNA in situ hybridization is the more sensitive
detection method for endosialin and that FB5 immunostaining marks tumor
endothelium with the highest levels of protein/epitope density rather
than a distinct tumor endothelial cell type. Finally, there may simply
be differences in the design of the two available studies on endosialin expression.
Our analysis shows that the N-terminal extracellular portion of
endosialin is likely to consist of five domains with globular structure, and in this region homology to thrombomodulin and the complement receptor C1qRp, with 39 and 33% amino acid sequence identity, respectively, is observed. Importantly, a pattern of cysteine
residues is highly conserved among the three proteins and provides a
scaffold of anchoring points for disulfide bridges, presumably imposing
similar three-dimensional structures on each protein. Thrombomodulin
exhibits a C-type lectin domain, six EGF-like repeats, and a
serine/threonine-rich region in the extracellular domain. Unlike
endosialin, thrombomodulin is expressed on a variety of normal cell
types and on normal vascular endothelium serves as a receptor for
thrombin, modulating the coagulation cascade and triggering the
thrombin-activable fibrinolysis inhibitor (30). The thrombin
interaction depends on the EGF repeats 5 and 6 of thrombomodulin (31,
32), which are not present in endosialin, making it unlikely that the
two molecules overlap in this particular function. The complement
receptor C1qRp is also expressed on a wider variety of cell types than
endosialin, including macrophages, neutrophilic granulocytes, and
normal vascular endothelium. The extracellular portion of C1qRp is
composed of a C-type lectin domain, a tandem of five EGF-like repeats,
and a mucin-like region. The molecule binds the complement factor C1q,
mannose-binding lectin, and pulmonary surfactant protein A (22). On
myeloid cells, C1qRp mediates phagocytosis of antibody-coated
particles, but the function of C1qRp on normal endothelial cells is
largely unknown (33).
Predictions about the membrane-proximal extracellular domain focus on
fewer structural features. Thus, the high content of proline,
threonine, and serine residues indicates likely attachment points for
more than 30 O-linked carbohydrate side chains consistent with the sialomucin-like features of endosialin (28, 34). Certain
sialomucins have been implicated in sequestering growth factors to the
plasma membrane and presenting these factors to endothelial cell
surface receptors (34, 35); endosialin may be a candidate for such a
function unique to tumor endothelium.
Apart from further biochemical investigations that will benefit from
the availability of mAb FB5 and recombinant endosialin protein, study
of defined alterations in endosialin gene expression and structure are
now possible, including genetic manipulations in mice and rats, which
harbor a close homologue of endosialin. In conclusion, key experiments
are now within reach to explore the endosialin system in human
physiology and disease and to extend these investigations into
experimentally more amenable rodent models. A better understanding of
endosialin may even enable new approaches to cancer detection and treatment.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-integrin chain (6).
2-macroglobulin
(Roche Molecular Biochemicals) at 107 cells/ml, filtered
through a 0.2 µm filter (Gelman/Pall, Ann Arbor, MI), and precleared
with bovine serum albumin-blocked CNBr-Sepharose for 30 min at 4 °C.
Endosialin was precipitated from lysates at a ratio of 1.5 µl of mAb
FB5 covalently coupled to CNBr-Sepharose (2.8 mg/ml of
gel)/107 lysed cells. The precipitate was washed three
times with RIPA buffer and eluted twice for 30 min at 55 °C, using 4 µl of elution buffer (1% SDS, 100 mM ammonium hydrogen
carbonate, pH 7.4)/1.5 µl of affinity matrix. The eluate was
concentrated in a bovine serum albumin-precoated Centricon 30 device
(Amicon, Beverly, MA), reduced with 50 mM dithiothreitol
(Roche Molecular Biochemicals) and carboxymethylated with 110 mM iodine acetamide (Fluka, Buchs, Switzerland). For Edman
sequencing, purified protein was separated via SDS-polyacrylamide gel
electrophoresis (PAGE) on a 6% gel and visualized by Coomassie
staining. The endosialin bands were excised from the gel, incubated
with trypsin solution (Promega, Madison, WI), and eluted peptides were
subjected, after HPLC (Waters, Milford, MA) separation, to a 494 cLC
ABI-PerkinElmer device. Peptides were desalted by ZipTip procedure and
subjected to mass spectroscopy analysis using a matrix-assisted
laser desorption ionization-time of flight instrument (Voyager DE-STR,
PerSeptive Biosystems/AB, Foster City, CA). Peptide samples were
prepared using dihydroxybenzoic acid as matrix (7).
1-chain
coupled to protein A-Sepharose (1 mg/ml of gel) via rabbit anti-rat IgG
(1 mg/ml of gel) for 2 h at 4 °C. The precipitates were washed,
split into equal aliquots, and treated either with 4 µl of
O-sialoglycoprotein endopeptidase from Pasteurella
hemolytica (activity: 1 µl cleaves 10 µg of glycophorin A/h at
37 °C; Cedarlane Laboratories, Hornby, Canada) or reaction buffer
only (50 mM Tris/HCl, pH 7.4), with incubation for 1 h
at 37 °C. Proteins were eluted and analyzed by SDS-PAGE as described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Affinity purification of endosialin.
Silver-stained gel showing proteins isolated with mAb
FB5-CNBr-Sepharose matrix (FB5) or negative control matrix
with mouse IgG (mIgG) from detergent extract of
108 LA1-5s cells. Matrix-bound material was eluted with
Laemmli sample buffer and electrophoresed under reducing conditions on
a 6% polyacrylamide gel. The expected 165-kDa endosialin protein
species is marked by the arrow; positions of molecular
mass markers (in kDa) are indicated to the
left.
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Fig. 2.
cDNA and deduced amino acid sequence of
human endosialin. The sequence of full-length cDNA was
assembled from ESTs, identified based on the five tryptic peptide
sequences derived from Edman analysis of purified endosialin
(double-underlined amino acids) and from 5'-RACE analyses.
The putative N-terminal signal sequence and the potential
polyadenylation signal are underlined. The predicted
transmembrane segment is boxed, and a single
N-linked glycosylation site is marked by a
circle.
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Fig. 3.
Northern blot analysis of endosialin.
RNA from two endosialin expressing cell lines (LA1-5s and MF-SH; 4 µg
of poly(A)+ RNA/lane) was probed with
32P-labeled endosialin cDNA or a probe for GAPDH.
Positions of size markers (in kilobases) are shown to the
left.
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Fig. 4.
Cell surface expression of endosialin on
HeLa-S3 cell transfectants. Photomicrograph of indirect
immunofluorescence staining of endosialin-transfected HeLa-S3 cells
(A; magnification, 200×) or vector control-transfected,
parental HeLa-S3 cells (B; magnification, 200×),
respectively, with mAb FB5. Binding was detected with Alexa
488-conjugated goat anti-mouse IgG.
5 with PFAM) and three EGF-like domains (residues
235-271, E < 10
5 with PFAM; residues
274-311, E < 10
2 with SMART; residues
316-350, E < 10
3 with PFAM). In
accordance with SMART assignments, the latter two EGF repeats may be
Ca2+ binding. The region 176-230 is probably a
Sushi/SCR/CCP domain. Although the E value in a PFAM search
is only 0.72, the typical motifs in the Sushi seed alignment with the
endosialin segment are well conserved, including all necessary
cysteines for disulfide bridges and a number of critical prolines,
glycines, and a tryptophan.
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Fig. 5.
Domain architecture of endosialin and
sequence comparison to human thrombomodulin and complement receptor
C1qRp. A, domain architecture of endosialin. The
diagram shows the arrangement of the N-terminal signal leader peptide
(triangle), the C-type lectin domain (C-LEC), the
domain similar to a Sushi/SCR/CCP domain (S), three EGF-like
repeats (EGF), and the transmembrane region (TM),
flanked by two segments with low complexity regions, namely a
sialomucin-like sequence (MUCIN) and a short, putative
cytoplasmic tail (CYT). B, protein sequence
alignment of endosialin with thrombomodulin (36) (SwissProt
P07204) and the complement receptor C1qRp (22) (TREMBL
U94333). The alignment shows the N-terminal 360 amino acids
of endosialin (end marked by black slash), which include the
C-type lectin domain, the Sushi-like domain, and the three EGF-like
repeats. In this region, endosialin shares 39% identity with the
matched region of thrombomodulin and 33% identity with C1qRp
(white letters on blue background), respectively.
Nearly all cysteine residues in the five domains are conserved among
the three proteins (red). The WIGL consensus motif is
depicted in green.
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Fig. 6.
Biochemical characterization of
endosialin. A, SDS-PAGE analysis of purified,
recombinant, and endogenous endosialin following pretreatment with
glycosidases. Detergent extracts of
[35S]methionine/[35S]cysteine-labeled
HeLa-S3 cells, transfected with the human endosialin cDNA
(HeLa transfectant), or LA1-5s cells (LA1-5s),
respectively, were precipitated with mAb FB5 conjugated to
CNBr-Sepharose or with control mouse IgG on CNBr-Sepharose.
Immunoprecipitated endosialin was either mock-treated (none)
or pretreated with PNGase F, sialidase, or with a combination of
O-glycosidase and sialidase. B, SDS-PAGE analysis
of endosialin following pretreatment with
O-sialoglycoprotein endopeptidase. To test the sensitivity
of endosialin to O-sialoglycoprotein endopeptidase,
detergent lysates of the labeled LA1-5s cells were precipitated with
mAb FB5 (FB5) or anti- 1 integrin mAb 9EG7
(anti-
1) as a control. Immunoprecipitated
antigens were treated with O-sialoglycoprotein endopeptidase
from P. hemolytica for 1 h at 37 °C (OSGE) or
mock-treated without enzyme (none). Note complete digestion
of endosialin by OSGE and only limited degradation, presumably
nonspecific, for
1-integrin. Molecular size markers (in
kDa) are indicated to the left.
1-integrin, was largely insensitive to
OSGE under these conditions.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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The expert technical assistance of Claudia Eiberle, Elfriede Müller, Werner Rust, and Kai Zuckschwerdt is gratefully acknowledged. Prof. Junichi Sakamoto kindly provided cell line MF-SH. Dr. Elisabeth Stockert kindly supplied purified mAb FB5. We thank Dr. Lloyd J. Old for valuable contributions and suggestions. Sven Christian's contribution was in partial fulfillment of his Ph.D. thesis; he acknowledges the support of Prof. Klaus Pfizenmaier throughout the work.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be 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 GenBankTM/EMBL Data Bank with accession number(s) AJ295846.
** To whom correspondence should be addressed: Boehringer Ingelheim Pharma KG, 88397 Biberach and der Riss, Germany. E-mail: martin.lenter@bc.boehringer-ingelheim.com.
Published, JBC Papers in Press, November 17, 2000, DOI 10.1074/jbc.M009604200
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ABBREVIATIONS |
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The abbreviations used are: mAb, monoclonal antibody; bp, base pair(s); EGF, epidermal growth factor; EST, expressed sequence tag; OSGE, O-sialoglycoprotein endopeptidase; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; MOPS, 4-morpholinepropanesulfonic acid; RACE, rapid amplification of cDNA ends; SAGE, serial analysis of gene expression; TEM1, tumor endothelial marker 1.
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