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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 1 (January 1), 1999:
pp. 165-175
Biochemical Characterization and Molecular Cloning of a Novel
Endothelial-Specific Sialomucin
By
Suzanne Marie Morgan,
Ulrike Samulowitz,
Liz Darley,
David L. Simmons, and
Dietmar Vestweber
From the Cell Adhesion Group, Institute of Molecular Medicine, John
Radcliffe Hospital, and the Sir William Dunn School of Pathology,
University of Oxford, Oxford, UK; and the Institute of Cell Biology,
University of Münster, Münster, Germany.
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ABSTRACT |
We have generated rat monoclonal antibodies (MoAbs) against cell
surface antigens of the mouse endothelioma cell line bEND.3. Three
antibodies (V.1A7, V.5C7, and V.7C7) were selected, all of which
recognize a 75-kD antigen on bEND.3 cells and bind selectively to
endothelial cells in cryostat sections of mouse tissues. A cDNA for the
antigen was isolated from a bEND.3 pCDM8 expression library by using
transient expression in COS-7 cells and immunoselection with the three
MoAbs. This cDNA coded for a novel, type I membrane protein of 248 amino acids with an extracellular domain rich in threonine and serine
residues (35%). The protein is sensitive to O-sialoglycoprotein
endopeptidase, indicating that it belongs to the class of
sialomucin-like proteins. Therefore, we suggest the name endomucin.
Treatment of isolated endomucin by sialidase and O-glycosidase reduced
the apparent molecular weight to 45 kD and abolished binding of all
three antibodies, indicating that carbohydrates are directly or
indirectly involved in the formation of the antibody epitopes.
Immunohistological analysis of all examined mouse tissues showed that
endomucin is an endothelial antigen found in venous endothelium as well
as in capillaries, but not on arterial endothelium. Interestingly, high
endothelial venules of peripheral and mesenteric lymph nodes as well as
of Peyers's patches were negative for staining with the three MoAbs.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
MUCIN-LIKE MEMBRANE glycoproteins contain
many serine and threonine residues that carry large amounts of O-linked
glycans, forcing the molecule into an extended
structure.1-4 By virtue of these features, cell
surface-associated mucins have a dual function in the regulation of
cell adhesion either by providing a repulsive barrier to prevent
cell-cell or cell-matrix interactions5-9 or by promoting
adhesion through facilitating lectin-type interactions with opposing
cells.10 Members of this family of proteins can be involved
in signal transduction,11-13 and recent evidence suggests that CD43, a leukocyte sialoglycoprotein,14,15 can function as a positive signaling molecule for  1 and
2 integrin-mediated cell adhesion.16
Growing evidence indicates that endothelial cell-associated mucins
function as cell adhesion molecules mediating adherence with
leukocytes. Leukocyte-endothelial cell interactions occur during
leukocyte trafficking from the blood into lymph nodes, peripheral
tissues, or sites of inflammation and are mediated at least in part by
interactions between mucin-like molecules and their receptors. A
well-described class of sialomucin-binding adhesion molecules are the
selectins.10,17-19 All selectins recognize sialylated and
fucosylated carbohydrates related to the tetrasaccharide sialyl Lewis
X, and several mucin-like glycoproteins have been shown to present such
structures to selectins. Glycosylation-dependent cell adhesion molecule
1 (GlyCAM-1) and CD34 are sialomucins that represent two of the
principle ligands for L-selectin in the high endothelial venules (HEV)
of peripheral lymph nodes.20,21 GlyCAM-1 is a secreted
protein and CD34 is a transmembrane protein of endothelial cells of
HEV.22 Increasing evidence indicates that sialylation, sulfation, and fucosylation of the O-linked side chains of GlyCAM-1 and
CD34 are requirements for the binding to the
selectins.23,24 Mucosal addressin cell adhesion molecule 1 (MAdCAM-1) is another cell adhesion molecule that contains a mucin-like
domain and is expressed on HEV in Peyer's patches and mesenteric lymph
nodes and on venules in intestinal lamina propria.25,26 In
the HEV of mesenteric lymph nodes, the mucin-like domain of a
subpopulation of MAdCAM-1 molecules contains sulfated carbohydrate side
chains that interact with L-selectin.27
The sialomucin CD43, which is found on all leukocytes except mature B
cells, was recently shown to have antiadhesive functions in T-cell
homing in vivo.28 T cells from CD43-deficient mice home
more frequently to secondary lymphoid organs and this effect is due to
CD43 interference with L-selectin-mediated interactions in vivo and in
vitro. The extended structure of CD43 (length, 45 nm)3 and
its strongly charged nature are likely to be responsible for the
antiadhesive function of CD43. A monoclonal antibody (MoAb) against
CD43 was found to block T-cell binding to lymph node HEV as well as
homing in vivo, suggesting yet another mechanism for the involvement of
CD43 in adhesive events.29 In summary, many sialomucins are
involved in cell contact formation either directly by supporting cell
adhesion or repelling the neighboring cell or indirectly by regulating
adhesion events via signaling to other adhesion molecules.
Raising MoAbs against endothelial cell surface molecules, we have
identified a novel endothelial-specific antigen. We have isolated a
full-length cDNA for this glycoprotein by immunoselection and describe
the sequence, the tissue distribution, and the biochemical properties
of this antigen. The predicted 248 amino acid polypeptide is a novel
type I integral membrane protein with similarities in structure to the
growing family of sialomucins.
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MATERIALS AND METHODS |
Cell culture.
The mouse endothelioma cell line bEND.3 (kindly provided by Dr Werner
Risau, Max-Planck-Institute for Physiological and Clinical Research,
Bad Nauheim, Germany), the SV-40-transformed African green monkey
kidney cell line COS-7, and the mouse hybridoma cell line SP2/0 were
cultured in Dulbecco's modified Eagles medium (DMEM) supplemented with
10% fetal calf serum (FCS), 100 IU/mL penicillin, 100 µg/mL
streptomycin, and 2 mmol/L L-glutamin (all GIBCO Life Technologies,
Karlsruhe, Germany). Serially passaged b.End.3 cells were grown to
confluence in tissue culture plates (Falcon, Heidelberg,
Germany) and stimulated for 24 hours at 200 U/mL with recombinant human
tumor necrosis factor  (TNF-; obtained by Knoll AG, Ludwigshafen,
Germany) to be used for immunization and in cell surface
enzyme-linked immunosorbent assays (ELISAs). Inducibility of endomucin
was examined by immunoblots of bEND.3 cells after stimulation for 12 hours with the cytokines interleukin-1 (IL-1; 10 pg/mL),
interferon- (IFN-; 0.4 ng/mL), and TNF- (0.05 ng/mL) according
to the manufacturer's guidelines (R&D, Abingdon, Oxon,
UK). Hybridoma cells were grown in DMEM as decribed above supplemented
with HAT (Boehringer Mannheim, Mannheim,
Germany) according to the manufacturer's instructions.
MoAbs.
MoAbs against endothelial cell surface antigens were produced as
described previously.30 Briefly, female Lewis rats were immunized with b.END.3 cells (passage 15 to 29) that had been stimulated with 200 U/mL TNF- for 24 hours before injection. Cells
mixed with Complete Freund's adjuvans for the first immunization and
incomplete for the other immunizations were injected six times (1 to
2 × 107 cells per injection) in 1-week
intervals, with 3 weeks between the fifth and sixth immunization. Three
days after the last boost, cells from peripheral lymph nodes and spleen
cells were fused with SP2/0 cells essentially as
described.31 Hybridoma supernatants were differentially
screened for indirect immunoperoxidase staining on TNF- stimulated
and nonstimulated endothelioma cells by cell surface ELISA, as
described.30 Hybridoma producing antibodies against cell
surface antigens were subcloned by limiting dilution. The isotype of
the three MoAbs against endomucin was determined to be IgG1 for V.1A7
and V.5C7 and IgG2a for V.7C7. MoAbs EA-3 (rat IgG1)32
against mouse CD31, 10E9 (rat IgG2a)33 against mouse
E-selectin, 9EG7 (rat IgG2a)34 against mouse 1-integrin chain, M1/70 (rat IgG2a; called TIB128 from the ATCC, Manassas, VA) against mouse Mac-1, MECA367 (rat IgG2a)25
against mouse MAdCAM-1, and polyclonal serum against human vWF (DAKO,
Hamburg, Germany) were used as controls in cell surface
ELISAs and for immunostaining of cryosections. Additionally, rat
anti-mouse MoAb OX40 (CD134; provided by M. Puklavec, MRC Cellular
Immunology Unit, Sir William Dunn School of Pathology, University of
Oxford, Oxford, UK), clone MRC OX86,35 rat anti-mouse
Sca-2, clone III.3A7 (U.S. and D.V., unpublished data),
rat anti-CD59, clone SM8 (S.M.M., unpublished data), and
rat anti-CD31, clone 390 (Serotec, Kidlington, Oxford,
UK), were used as controls in the staining of wax-embedded sections.
Immunohistochemistry.
Tissue sections (5 µm) of wax-embedded normal mouse organs were
washed with phosphate-buffered saline (PBS) containing 0.1% (vol/vol)
Triton X-100 and incubated in phosphate buffer containing 0.1 mol/L
glucose, 0.01 mol/L NaN3, and 40 U glucose oxidase (Sigma, St Louis, MO) for 15 minutes at 37°C to block
endogenous peroxidase activity. Sections were blocked with 5% normal
mouse serum for 30 minutes and incubated with the appropriate rat
antibodies (hybridoma supernatant). Binding was detected by incubation
of sections with biotinylated rabbit anti-rat IgG (Sigma) diluted in
blocking buffer, followed by avidin-biotin-peroxidase complex (ABC
elite; Vector Laboratories, Burlingame, CA) and 0.5 mg/mL
diaminobenzidine (Polysciences Inc, Warrington, PA) with
0.024% H2O2. Sections were counter-stained with crystal violet. The primary MoAb was replaced with MoAb MRC OX86
as a negative control.
For cryosections, organs and tissues from 10- to 14-week-old NMRI mice
were covered with tissue freezing medium (Jung, Nussloch, Germany),
snap-frozen in liquid nitrogen, and stored at  80°C. Sections
of 5 µm were made on a cryostat (Leica, Nussloch,
Germany), transferred to Poly-L-Lysin-covered slides,
dried overnight at room temperature, and fixed in 100%
ethanol, followed by acetone treatment. Primary antibodies as
supernatants or at 10 µg/µL in medium were added to the sections
after washing in PBS, followed by further washing steps and incubation
with peroxidase-coupled goat anti-rat or goat anti-rabbit IgG and IgM
(Dianova, Hamburg, Germany), respectively, diluted in DMEM at 1:1,000.
Signals were detected by incubation with 0.04% amino-ethyl-carbazole
in N,N-dimethylformamid with 0.006% H2O2. For
counterstaining, Mayers hematoxylin was used.
Flow cytometry.
Two-stage labeling of cell surface molecules was performed by
incubation of cells (1 × 106) with the appropriate
MoAb supernatant, followed after washing by the addition of purified
fluorescein isothiocyanate (FITC)-conjugated goat anti-rat Ig antibody
at 10 µg/mL (Serotec). Cell surface fluorescence was assayed by
FACScan analysis (Becton Dickinson, San Jose, CA). Cells
were gated on the scatter profiles to exclude dead cells.
Immunoprecipitations and immunoblots.
Cells were surface biotinylated with 0.5 mg/mL Sulfo-NHS-biotin
(Pierce, Rockford, IL) in PBS, lysed, and subjected to
immunoprecipitation as described.36,37 Immunoprecipitated
proteins were separated by electrophoresis on 8% or 10% sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and
transferred to nitrocellulose (Schleicher & Schüll, Dassel,
Germany). Filters were analyzed for biotinylated proteins with
peroxidase-conjugated streptavidin (Dianova) and the ECL system
(Amersham, Braunschweig, Germany). Alternatively, nonlabeled cells were
subjected to immunoprecipitations and precipitated antigens were
separated by electrophoresis and transferred to nitrocellulose filters.
Antigens were probed with hybridoma supernatant containing rat
anti-endomucin MoAbs, and first antibodies were detected with
peroxidase-conjugated rabbit anti-rat IgG (Dianova). Immunoreactive
proteins were detected by enhanced chemoluminescence (ECL kit; Amersham
Life Science, Braunschweig, Germany).
Enzyme treatments.
For treatments of purified endomucin with glycosidases, the antigen was
immunoprecipitated from 5 × 106 bEND.3 cells, divided
into five equal size aliquots, and treated with the appropriate enzyme
while still bound to protein A Sepharose beads. The following
treatments were performed on separate aliquots. (1) Beads were boiled
for 5 minutes in 10 µL 0.1% SDS and then 0.05 U of endoglycosidase F
(Boehringer Mannheim) in 30 µL of enzyme buffer (100 mmol/L
K2HPO4/KH2PO4, 20 mmol/L EDTA, 0.5% Triton X-100, 0.1% SDS) was added. (2) Beads were
incubated with 0.2 mU O-glycosidase (Boehringer Mannheim) in 40 µL
enzyme buffer. (3) Beads were treated with 0.5 mU neuraminidase (from
Arthrobacter ureafaciens; Boehringer Mannheim) in 40 µL
enzyme buffer. (4) The same treatment as in (3) followed by the
subsequent addition of 0.2 mU of O-glycosidase for 3 hours at 37°C.
All other digestions were performed at 37°C overnight. The antigen
was subsequently electrophoresed, transferred to nitrocellulose
membranes, and detected in immunoblots with the respective
anti-endomucin antibodies. Alternatively, the antigen was
immunoprecipitated from surface biotinylated cells, electrophoresed,
transferred to nitrocellulose, and detected by peroxidase-conjugated
streptavidin (Dianova) followed by ECL. For treatment of purified
endomucin or control antigens with O-sialoglycoprotein endopeptidase
(Cedarlane Laboratories, Hornby, Ontario, Canada) the
antigens were immunoprecipitated from (5 × 106)
surface biotinylated bEND.3 cells. The precipitated material was
divided in three aliquots and the different aliquots were either
mock-treated or treated with neuraminidase as described above or with
2.5 µL of O-sialoglycoprotein endopeptidase (activity, 1 µL cleaves
10 µg glycophorin A per hour at 37°C) in 30 µL 50 mmol/L
Tris/HCl, pH 7.4. The biotinylated antigens were subsequently electrophoresed, transferred to nitrocellulose, and analyzed as described above. For treatment of intact cells with O-sialoglycoprotein endopeptidase, 5 × 106 bEND.3 cells were washed,
resuspended in RPMI/HEPES (200 µL), and incubated with or without 10 µL of O-sialoglycoprotein endopeptidase at 37°C for 30 minutes.
Subsequently, cells were washed and analyzed by flow cytometry.
Expression cloning.
cDNA clones were isolated from a bEND.3 cDNA library using the COS-cell
expression cloning protocol by Aruffo and Seed.38 A bEND.3
cDNA library in the pCDM8 vector was introduced into COS-7 cells by
diethyl aminoethyl (DEAE)-dextran transfection. Forty-eight hours after
transfection, cells were detached with EDTA, washed, and immunoselected
with the rat MoAbs V.5C7, V.7C7, and V.1A7 and goat anti-rat Ig-coated
plates. Plasmid DNA was recovered from the adherent COS-7 cells,
amplified in E coli (strain MC1061/p3), and then reintroduced
into COS-7 cells by spheroblast fusion. Three rounds of enrichment were
performed to isolate positive clones.
DNA sequencing and sequence analysis.
Nucleotide sequencing was performed on both strands using
-32S-dATP with sequenase version 2.0 (USB, Little
Chalfont, Bucks, UK) and automated sequencing using the
dRodamine kit (Perkin-Elmer, Warrington, Cheshire, UK).
Forward and reverse primers in pCDM8 and sequence-specific 18-mer
oligonucleotide primers were used. Analysis of nucleic acid and protein
sequence data were performed using the Wisconsin-GCG
Package (Pittsburgh Supercomputing Center, Pittsburgh,
PA). Sequence comparison with the mouse and human EST databases
(GenBank)39 was performed using BLASTN 2.0.4 (NIH, Bethesda, MD).40
Northern blot analysis.
A mouse multiple tissue Northern blot prepared from mRNA (Origene
Technologies, Rockville, MD) was hybridized with a
32P-labeled cDNA probe for endomucin (Xho I
fragment containing the complete insert from pCDM8) or
32P-labeled -actin probe and washed in 0.25× SSC
in the presence of 0.1% SDS at 42°C.
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RESULTS |
Development of MoAbs against a cell surface antigen of mouse
endothelial cells.
To identify novel endothelial-specific cell surface antigens, we
immunized rats with intact mouse bEND.3 endothelioma cells and screened
MoAbs for binding to the surface of bEND.3 cells in a cell surface
ELISA, in a similar way as described before.30 Of 16 MoAbs
that gave strong signals in these assays, three MoAbs, designated
V.1A7, V.5C7, and V.7C7, were selected that all recognized a 75-kD cell
surface antigen in immuno-precipitations with cell surface biotinylated
bEND.3 cells (Fig 1A). In addition, a
second weak band was detected at approximately 66 kD that is possibly a
degradation product of the larger protein. Treatment of these cells
with a mixture of inflammatory cytokines (IL-1, IFN-, and
TNF-) for 12 hours did not alter the levels of expression of the
antigens recognized by these MoAbs (data not shown).
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| Fig 1.
Detection of the glycoprotein antigen for antibodies
V.1A7, V.5C7, and V.7C7 by immunoprecipitation. Mouse bEND.3 cells were
surface biotinylated and detergent extracts were incubated either with
protein A Sepharose loaded with rabbit antirat IgG and the MoAbs V.1A7,
V.5C7, or V.7C7 or a control antibody, as indicated. Specifically bound
proteins were eluted with SDS-PAGE loading buffer, electrophoresed on
an 8% polyacrylamide gel under reducing conditions, and transferred to
nitrocellulose. Filters were incubated with peroxidase-conjugated
streptavidin and analyzed by enhanced chemiluminescence.
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Endothelial cells are specifically stained by all three antibodies in
immunohistochemistry.
To determine the tissue distribution of the identified antigen, we
stained sections of wax-embedded mouse tissues and cryostat sections of
various mouse tissues with all three antibodies. An identical pattern
of staining was observed with all three antibodies. Best signals were
obtained with the MoAb V.5C7, and the results shown in
Figs 2, 3, and
4 were all obtained with this antibody. The
antigen was found in all tissues examined, and in all cases its
expression was specific for the vascular endothelium. Endothelial specificity was controlled by staining with antibodies against endothelial-specific antigens such as von Willebrand factor (vWF) or
platelet endothelial cell adhesion molecule (PECAM-1), as
is documented in Fig 2 for the uterus, brain, heart, small intestine, and tongue. The staining pattern of V.5C7 in the brain was the same as
that of an antibody against the integrin chain 1, which is known to be predominantly expressed on brain capillaries (Fig 2).
Intense staining was found in the glomerular capillaries and intertubular vessels in the kidney (Fig 3a). Venules and capillaries in
the skin (Fig 3b), liver (Fig 3c), adrenal tissue (Fig 3d), heart (Fig
3e), pancreas (Fig 3g), and thymus (Fig 3h) also stained strongly.
Endothelial cells lining the aorta did not stain for the antigen (Fig
3f), whereas the endocard in the heart was positive (Fig
2d). Staining of arterioles was absent, as shown in the kidney and
heart (Fig 3a and e).
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| Fig 2.
Staining of cryostat sections of mouse tissues with the
MoAb V.5C7. Cryostat sections of tongue (a, b, and c), heart (d, e, and
f), uterus (g, h, and i), brain (k, l, and m), and small intestine (n,
o, and p) were reacted with the MoAb V.5C7 (a, d, g, k, and n),
polyclonal rabbit antibodies against von Willebrand factor (e, h, and
o), the rat MoAb 9EG7 against integrin chain 1 (l), the
rat MoAb EA3 against mouse PECAM-1 (b), or no first antibody (c, f, i,
m, and p). First antibodies were detected by an immunoperoxidase
technique. The bar represents 50 µm.
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| Fig 3.
Staining of sections of wax-embedded mouse tissue with
the MoAb V.5C7. Sections of wax-embedded mouse kidney (a), skin (b),
liver (c), adrenal tissue (d), heart (e), aorta (f), pancreas (g), and
thymus (h) were incubated with MoAb V.5C7. First antibodies were
detected by an immunoperoxidase technique. The arrowheads point to
arterioles. The bar represents 100 µm.
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| Fig 4.
Endomucin is not expressed on HEV of lymphatic tissue.
Cryostat sections of mesenteric lymph nodes were incubated with MoAb
V.5C7 (a) (endomucin) or MoAb MECA 367 (b) against the vascular
addressin MAdCAM-1 (MAdCAM-1). Arrowheads point to HEV and arrows point
to capillaries. The bar represents 50 µm.
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Capillaries and small venules in lymph node tissue were positive for
the V.5C7 antigen, whereas HEV were clearly negative in peripheral
lymph nodes and Peyer's patches (data not shown) and in mesenteric
lymph nodes (Fig 4a). Clear staining of HEV was seen with MoAb MECA79
against the peripheral node addressin (PNAd; not shown) and MoAb
MECA367 against the mucosa-associated addressin MAdCAM-1, as found for
Peyer's patches (not shown) and documented on sections of mesenteric
lymph nodes (Fig 4b). HEV were the only type of venular endothelium
that we found to be negative for the staining with all three antibodies
V.1A7, V.5C7, and V. 7C7.
The cDNA for the endothelial antigen codes for a novel
sialomucin-like protein.
Using the three MoAbs V.1A7, V.5C7, and V.7C7 as panning reagents, we
isolated three cDNA clones from a bEND.3 pCDM8 expression library.
These clones were isolated after several rounds of panning and plasmid
rescue using the COS-cell expression cloning protocol described by
Aruffo and Seed.38 After the final round of enrichment using these three MoAbs, plasmid inserts from single colonies were
analyzed using restriction digests.
A representative clone from each group (DW-5C7.3, DW-7C7.17, and
DW-1A7.27) was transfected into COS-7 cells and found positive for
protein expression as determined by flow cytometry
(Fig 5A). Mock-transfected COS-7 cells were
unreactive for all three MoAbs (data not shown). In
immunoprecipitations of surface-biotinylated COS-7 cells transfected
with any of the three isolated cDNAs, each of the three MoAbs (V.1A7,
V.5C7, and V.7C7) recognized a glycoprotein of similar size as that
they recognize in bEND.3 cells. This is shown in Fig 5B for MoAb V.5C7
in an immunoprecipitation on COS-7 cells transfected with the cDNA
clone DW-7C7.17.
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| Fig 5.
Transfection of COS-7 cells with the novel cDNA confers
reactivity to MoAbs V.1A7, V.5C7, and V.7C7. (A) COS-7 cells were
transfected with the plasmid clones DW-5C7.3, DW-7C7.17, and DW-1A7.27
by the DEAE-dextran method. After 48 hours, the cells were detached and
incubated with saturating levels of the respective MoAbs (thick line)
or the control MoAb MRC OX86 (thin line), followed by a second
incubation with fluorescein-conjugated goat antirat Ig antibody.
Antibody binding was measured by flow cytometry. (B) Mock-transfected
COS-7 cells (lane 1) or COS-7 cells transfected with the cDNA clone
DW-7C7.17 (lane 2) were surface biotinylated and subjected to
immunoprecipitation with MoAb V.5C7. Specifically bound proteins were
analyzed and detected as described for Fig 1A. Molecular mass markers
(in kilodaltons) are indicated on the left.
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The three positive plasmid clones contained cDNA inserts of
approximately 1.2 kb and had identical restriction maps, suggesting that the same cDNA had been enriched by all three MoAbs, and each plasmid clone, when transfected into COS-7 cells, was found to express
protein recognized by each of the MoAbs (data not shown). One clone
(DW-7C7.17) was sequenced and the complete nucleotide sequence of 1,228 bp is shown in Fig 6 (76 to +1152
bp). A mouse EST clone (557127) overlapping the 5 region of the
cDNA clone DW-7C7.17 was sequenced and found to be identical across
this region of the cDNA insert (76 to +424 bp) and extended the
5 end of the cDNA clone by a further 21 bp (Fig 6; 97 to
77 bp). The DW-7C7.17 cDNA clone did not contain the complete
3 untranslated end. However, the sequence of the mouse EST
I.M.A.G.E. clone (749333) that overlapped with the 3 region of
the clone DW-7C7.17 showed complete homology in the region +962 to
+1159 bp and showed the polyadenylation signal (AATAAA, +1225 bp)
followed by the poly(A) tail. Highly homologous EST clones were also
found in the human EST database. Searching the GenEMBL database showed
an absence of homology to other known sequences, thus indicating that a
novel cDNA had been cloned. The first ATG of the complete cDNA sequence (position +1 bp), which has a strong translation initiation site in the
context of a Kozak41 consensus sequence
(GGCACCATGC), precedes an open reading frame of 744 nucleotides encoding a protein of 248 amino acids (26,332 Daltons; Fig 6) The coding sequence is started by a
putative 20 amino acid signal sequence containing a hydrophobic core,
with the predicted cleavage site assigned to Ser-20/Asp21 according to
von Heijne.42 Hydrophilicity (Kyte/Doolittle)43 analysis suggests a type I integral membrane protein with an
extracellular domain of 157 amino acids, a 23 amino acid transmembrane
domain, and a cytoplasmic tail of 48 amino acids (residues 201-248).
The putative extracellular domain contains two consensus sites
(Asn-X-Ser/Thr) for N-glycosylation (Asn-101 and Asn-119) and is rich
in serine and threonine residues (35%), suggesting that it is
abundantly O-glycosylated. Using the O-GLYCBASE database,44
the deduced amino acid sequence was predicted to have 25 O-linked
glycosylation sites. There are three potential protein kinase C
phosphorylation sites in the cytoplasmic tail (Ser-220, Ser-224, and
Thr-229). By virtue of the clusters of serine and threonine residues in the extracellular domain of endomucin, computer-generated alignments of
this sequence show homologies to mucins. Based on the predicted structure, it is likely that the processed protein is heavily glycosylated with O-linked glycans, which is in agreement with the fact
that the apparent molecular weight of the antigen as judged by SDS-PAGE
analysis is much larger than the molecular mass deduced from its amino
acid sequence. Members of the sialomucin family of adhesion molecules,
including CD34 and CD43, have similar features.3,45,46 We
assign the name endomucin to this novel molecule due to its prominent
localization on endothelial cells as well as the structural
similarities with mucin-like glycoproteins.
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| Fig 6.
Nucleotide and deduced amino acid sequence of endomucin.
Nucleotides and amino acids are numbered on the left. The putative
N-terminal signal sequence, the transmebrane region, and the
polyadenylation signal are underlined. The putative N-linked
glycosylation sites are in italics and double underlined. The putative
protein kinase C phosphorylation sites in the cytoplasmic tail are in
italics and marked by dashed lines. The sequence appears in the
GenBank/EMBL sequence databases under the GenBank accession no.
AF060883.
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Northern blot analysis.
Poly (A)+ RNA isolated from various mouse tissues was
examined by Northern blot analysis for the presence of endomucin
transcripts (Fig 7A). A prominent endomucin
transcript of approximately 1.7 kb was observed in all of the tissues
examined, including the brain, heart, kidney, spleen, thymus, and
liver. The transcript appeared to be highly abundant in the heart and
kidney. This difference in the level of expression was not due to
uneven loading of poly(A)+ RNA, because similar levels of
the -actin transcript were detected in all lanes (Fig 7B). However,
it may be attributed in part to the high density of blood vessels in
the heart and kidney. Overexposure of the blot showed weak
hybridization with an approximately 4-kb mRNA in the heart and kidney,
which may represent an incompletely spliced precursor. This broad
distribution of the endomucin transcript was anticipated, because
immunohistology showed constitutive expression of endomucin in all of
the tissues examined (Figs 2, 3, and 4). The difference in the size
between the 1.5-kb cDNA and the observed 1.7-kb mRNA transcript could
be accounted for by additional 5 untranslated sequence.
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| Fig 7.
Distribution of the endomucin RNA expression. Northern
blot analysis of endomucin. Northern blot containing RNA from various
mouse tissues (as indicated) with 2 µg Poly(A)+ RNA per
lane was hybridized with the 32P-labeled endomucin cDNA
probe (A) or a probe for -actin (B) as a control. Positions of size
markers (in kilobases) are shown on the left.
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Sensitivity of endomucin to glycosidases and O-sialoglycoprotein
endopeptidase.
Based on the high content of serine and threonine residues of
endomucin, we assumed that it would be highly O-glycosylated. O-sialoglycoprotein endopeptidase specifically cleaves highly O-glycosylated mucin-like molecules.47,48 To examine
whether endomucin is sensitive to this endopeptidase, bEND.3 cells were treated with the enzyme and, subsequently, endomucin expression was
analyzed by flow cytometry. As can be seen in
Fig 8A, treatment was effective in
producing a strong reduction of binding of the antiendomucin MoAb
V.5C7. Similar results were observed for the endomucin-specific MoAbs
V.7C7 and V.1A7 (data not shown). Untreated cells did not show any
change. To control for the lack of contaminating proteases in the
enzyme preparation, the cell surface expression of a number of
glycoproteins (CD31, Sca-2, and CD59) was also analyzed and was found
not to be altered on treatment with O-sialoglycoprotein endopeptidase.
To exclude that the removal of the endomucin epitope was due to a
contaminating neuraminidase in the O-sialoglycoprotein endopeptidase
preparation, we treated immunoprecipitated endomucin with the
endopeptidase. As shown in Fig 8B, endomucin was completely degraded by
O-sialoglcoprotein endopeptidase, whereas treatment with neuraminidase
did not alter the apparent molecular weight of endomucin. The control
antigen, PECAM-1, was not sensitive to the endopeptidase. These data
suggest that endomucin is modified by densely packed O-linked
carbohydrate side chains rich in sialic acid.
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| Fig 8.
Sensitivity of endomucin to O-sialoglycoprotein
endopeptidase. (A) bEND.3 cells were treated with O-sialoglycoprotein
endopeptidase from Pasteurella haemolytica for 30 minutes
(dotted line) or left untreated (thick line) and analyzed directly by
flow cytometry as described for Fig 5A. The profiles show the binding
of rat MoAb V.5C7 (endomucin), MoAb 390 (CD31), MoAb SM8 (CD59), and
MoAb III.3A7 (Sca-2). Negative control staining was analyzed with MoAb
MRC OX86 (thin line). (B) bEND.3 cells were surface biotinylated and
subjected to immunoprecipitation with the antiendomucin antibody MoAb
V.5C7 (lanes 1 through 3) or the anti-PECAM-1 antibody EA-3 (lanes 4 through 6). Immunoprecipitated antigens were either mock-treated (lanes
1 and 4), treated with O-sialoglycoprotein endopeptidase (lanes 2 and
5), or treated with neuraminidase (lanes 3 and 6). After the enzyme
treatment, proteins were electrophoresed on a 10% polyacrylamide gel,
transferred to nitrocellulose, and detected with peroxidase-conjugated
streptavidin followed by enhanced chemoluminescence. Molecular mass
markers (in kilodaltons) are indicated on the left.
|
|
To examine this further, we isolated endomucin from cell surface
biotinylated bEND.3 cells by immunoprecipitation. While still bound to
the affinity matrix, aliquots of the antigen were separately treated
with one of the enzymes (endoglycosidase F, O-glycosidase, or
sialidase) or with sialidase and subsequently with
O-glycosidase. The antigen was then electrophoresed,
transferred to a filter, and detected by peroxidase-conjugated
streptavidin and enhanced chemoluminescence. As shown in
Fig 9A endoglycosidase F reduced the
apparent molecular weight of endomucin to 60 kD. As expected, no effect
was seen if the molecule was treated with O-glycosidase alone, because
this enzyme is only active after the removal of sialic acid. After
pretreatment with sialidase, O-glycosidase digestion could reduce the
apparent molecular weight of endomucin to 45 kD. Removal of sialic acid
by sialidase did not affect the apparent molecular weight of endomucin.
We conclude that the antigen carries N-linked as well as O-linked
carbohydrate side chains.
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| Fig 9.
Sensitivity of the antibody epitopes on endomucin to
treatment with glycosidases. bEND.3 cells either surface biotinylated
(A) or unlabeled (B, C, and D) were subjected to immunoprecipitations
with the antiendomucin MoAb V.5C7 (A and C), V.1A7 (B), or V.7C7 (D).
Immunoprecipitated endomucin was either mock-treated (lane 1) or
treated with endoglycosidase F (lane 2), O-glycosidase (lane 3),
neuraminidase (lane 4), or neuraminidase and O-glycosidase
(lane 5). After the enzyme treatment, endomucin was electrophoresed on
a 10% polyacrylamide gel, transferred to nitrocellulose, and detected
either with peroxidase-conjugated streptavidin followed by enhanced
chemoluminescence (A) or by immunoblotting with the anti endomucin MoAb
V.1A7 (B), V.5C7 (C), and V.7C7 (D). Molecular mass markers (in
kilodaltons) are indicated on the left.
|
|
Similar experiments were performed with unlabeled bEND.3 cells and
immunoprecipitated and glycosidase-treated endomucin was subsequently
examined in immunoblots with the three MoAbs V.1A7, V.5C7, and V.7C7
(Fig 9B, C, and D). Whereas the epitopes of all three antibodies were
resistant to endoglycosidase F, they were highly sensitive to sialidase
treatment of the antigen. The weak detectable signal with V.1A7 after
sialidase treatment of the antigen is probably due to incomplete
digestion. Thus, it is possible that the three antibodies recognize
carbohydrate epitopes on endomucin. Alternatively, removal of sialic
acid might affect the overall folding of the mucin and thereby might
indirectly destroy the epitopes.
| |
DISCUSSION |
Searching for novel endothelial cell surface antigens, we have
generated three MoAb against a novel membrane glycoprotein. All three
antibodies specifically recognized endothelial cells in all mouse
tissues examined. Cloning showed a sequence with no significant
homology to any known glycoprotein. The high content of serine and
threonine residues suggested strong O-glycosylation of the protein
backbone, a characteristic feature of mucin-like glycoproteins. This
could be verified by the large decrease in molecular weight upon
O-glycosidase treatment as well as by the sensitivity of the
glycoprotein to O-sialoglycoprotein endopeptidase, a protease with
specificity for sialomucins. Thus, we conclude that the novel
endothelial-specific cell surface glycoprotein is a sialomucin, which
we suggest should be named endomucin.
The tissue distribution of endomucin suggests that it serves a specific
function important in endothelial cells. Although endomucin is
expressed on endothelial cells of all tissues examined, it is not
expressed on all types of endothelial cells. Most importantly, it is
not expressed on HEV of secondary lymphoid organs such as peripheral
lymph nodes, mesenteric lymph nodes, and Peyer's patches. These are
the sites where lymphocytes leave the blood stream and enter into
lymphatic tissue. Lymphocyte trafficking at these sites of the vascular
system is very efficient. The lack of endomucin at these sites might
lead us to speculate that endomucin is an antiadhesive molecule that
suppresses leukocyte endothelial interactions in other vascular beds
and whose absence in HEV might increase the probability of lymphocyte
endothelial interactions at these sites. This would be in line with the
antiadhesive effects of other sialomucins such as CD43,28
epiglycanin,8 and the sialomucin complex formed of the
mucins ASGP-1 and ASGP-2.9 Such a negative regulatory
effect of endomucin on cell adhesion would also be in line with the
absence of endomucin on arteriolar endothelium, because the higher
shear forces in these vessels are already sufficient to suppress
unwanted leukocyte endothelial interactions.
Although an antiadhesive effect of endomucin is still speculative, it
is intriguing that this novel antigen is found in all venules but is
absent from HEV. In contrast, several other antigens are known to be
specifically expressed in endothelium of HEV but not other endothelia.
Examples are the carbohydrate epitope MECA79 (also called
PNAd),49 the vascular addressin MAdCAM-1,25 and the selectin-binding glycoforms of GlyCAM-120 and
CD34.21,50 All of these molecules or epitopes
are thought to be involved in lymphocyte trafficking. The secreted
protein hevin, which is more prominent in HEV than in flat-walled
vessel endothelium,51,52 was shown to modulate the binding
of high endothelial cells to components of the basement membrane, a
process that could possibly influence features of HEV endothelium that
facilitate lymphocyte migration.
The sensitivity of endomucin to sialidase and O-glycosidase as well as
to O-sialoglycoprotein endopeptidase indicates the high content of this
antigen in sialic acid-carrying O-linked carbohydrate side chains, the
common feature of sialomucins. Surprisingly, sialidase treatment of
endomucin abolished the binding of each of the three MoAb against this
antigen. This argues for a direct or indirect role of the carbohydrates
in the generation of the antibody epitopes. Although it cannot be
excluded, it is unlikely that the epitopes of all three antibodies are
exclusively formed by carbohydrates, because each of the three
antibodies only recognizes endomucin and no other glycoprotein. The
effect of sialidase on the expression of the antibody epitopes could
well be due to indirect effects on the conformation or accessibility of
polypeptide epitopes. This was found for several antibody epitopes of
antibodies against the sialomucin CD43.3 The binding of
four antibodies against CD43 was abolished upon treatment of the
antigen with neuraminidase, although three of these antibodies
recognized bacterial, nonglycosylated fusion proteins of CD43 in
Western blots. Thus, these antibodies, possibly similar to the three
antiendomucin antibodies, recognize epitopes formed by the protein
backbone, which are dependent on the sialylation of the antigen, but
which are detectable in Western blots, ie, in the SDS-extended
conformation of the protein.
Major differences in O-linked glycosylation structures have been
described for leukocyte populations, with the complexity of the
O-linked structures altering according to the activation states.4 It has been shown, for example, that, whereas
simple O-linked oligosaccharide structures are found on CD43 on resting T cells, more complex structures are displayed on activated T cells.53,54 The larger forms of CD43 found on activated T
lymphocytes that resulted from the modification of O-linked
oligosaccharide could be resolved by SDS-PAGE.55,56 In
contrast to these data on CD43, we did not observe any changes in
molecular weight of endomucin upon activation of bEND.3 cells with the
alarm cytokines IL-1, IFN-, or TNF-. This might indicate that
substantial alterations in the O-glycan substitutions are not inducible
by the treatment of bEND.3 cells with these cytokines.
Endomucin is a novel endothelial-specific sialomucin. It is the first
constitutively expressed endothelial cell surface protein that is found
on all venules but that is absent from HEV, the specialized site for
most efficient lymphocyte trafficking. This could argue for an
antiadhesive function of endomucin, as it has been demonstrated for
other sialomucins. Alternatively, it is possible that endomucin is
differently glycosylated on HEV-endothelia. Indeed, the binding of each
of our antiendomucin antibodies is sensitive to changes in the
glycosylation of endomucin. A different HEV-specific glycosylation
might enable endomucin to bind to leukocyte lectins in a manner similar
to that shown for the selectin ligands GlyCAM-1 or CD34.
It is also possible that endomucin, like other sialomucins,11-13 might be involved in signal transduction.
The presence of three putative protein kinase C phosphorylation sites in the cytoplasmic tail indicates that endomucin might have the capacity to be a signaling molecule. Further studies are necessary in
the future to elucidate the physiological function of this novel
endothelial sialomucin.
| |
FOOTNOTES |
Submitted June 1, 1998;
accepted August 18, 1998.
S.M.M. and U.S. contributed equally to the work.
Supported in part by a grant of the Interdisziplinäres Klinisches
Forschungszentrum (IKF) Münster to D.V. and a grant from the
Wellcome Trust (UK) to D.L.S.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Dietmar Vestweber, PhD,
Institute of Cell Biology, ZMBE, University of Münster,
Von-Esmarch-Str. 56, D-48149 Münster, Germany; e-mail:
vestweb{at}uni-muenster.de.
| |
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Top
Abstract
Introduction