1 Laboratory of Molecular and Cellular Recognition, Osaka University Graduate School of Medicine, Suita 565-0871, Japan
2 Department of Molecular Oncology, Kyoto University Graduate School of Medicine, Kyoto, 606-8501, Japan
3 Program of Molecular Pathology, Aichi Cancer Center, Research Institute, Nagoya, 464-8681, Japan
4 Glycobiology Program, Cancer Research Center, The Burnham Institute, La Jolla, CA 92037, USA
5 Present address: Biochemistry and Cell Biology Unit, HMRO, Kyoto University Graduate School of Medicine, Kyoto, 606-8501, Japan
Correspondence to: M. Miyasaka; E-mail: mmiyasak{at}orgctl.med.osaka-u.ac.jp
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Abstract |
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Keywords: carbohydrate-modifying enzymes, glycosylation, L-selectin ligand, lymphocyte homing, MECA-79
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Introduction |
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In the mouse, at least five ligands for L-selectin have been identified in HEVs and all of them are decorated with mucin-like carbohydrates. These are sulfated glycoproteins (Sgp) of 50, 90, 170 and 200 kDa, which are collectively termed the peripheral node addressin (PNAd) and were first identified by immunoprecipitation with soluble L-selectin (4), and the MECA-79 mAb, which recognizes an HEV-specific carbohydrate epitope on L-selectin-reactive sugar chains (57). Subsequently, Sgp50 and Sgp90 were identified as glycosylation-dependent cell adhesion molecule-1 (GlyCAM-1) (8) and CD34 (9), respectively. Notably, the reactivity of the MECA-79 mAb with Sgp90 was detected even in CD34-deficient mice (10), suggesting that one or more glycoproteins besides CD34 that migrate to 90 kDa are also involved in lymphocyte homing. In addition, the mucosal addressin cell adhesion molecule-1 (MAdCAM-1), which is recognized by the MECA-367 mAb, has also been shown to carry the MECA-79 epitope, which mediates the rolling of L-selectin-expressing cells under flow conditions (11). In human tonsils, the MECA-79 mAb recognizes four distinct glycoproteins of 65, 105, 160 and 200 kDa (12). The 105 and 160 kDa glycoproteins were subsequently determined to be CD34 (13) and the podocalyxin-like protein (14), respectively. All these HEV-associated ligands for L-selectin have common structural features in their post-translational sugar modifications, i.e. sialylation, fucosylation and sulfation on O-linked oligosaccharides (2,3,15). The importance of the particular pattern of fucosylation and sulfation was demonstrated by the targeted disruption of the -1,3-fucosyltransferase VII (FucTVII) or the HEC N-acetylglucosamine-6-sulfotransferase (HEC-GlcNAc6ST) gene, which led to a drastic reduction in lymphocyte homing to peripheral LNs (16,17). Recently, another sialomucin expressed in endothelial cells, endoglycan, has been added to the list of candidate L-selectin ligands (18).
Endomucin was identified by Vestweber and his colleagues by expression cloning (19). Endomucin is apparently highly O-glycosylated, as indicated by its sensitivity to O-sialoglycoprotein endopeptidase. Endomucin is specifically expressed in the vascular endothelial cells of adult mice and also in putative hematopoietic cell clusters and the endothelium of the dorsal aorta of mouse embryos (20,21). Endomucin mRNA is most abundantly expressed in the heart and kidney, where four different splicing isoforms are found (21). In separate studies, endomucin has been shown to have anti-adhesive activity when expressed in endothelial or non-endothelial cells (21,22). Interestingly, although endomucin is expressed in human HEVs in a form that is decorated with the MECA-79 epitope (23), it remains to be verified whether endomucin indeed presents L-selectin-reactive oligosaccharides to serve as a ligand for L-selectin in HEVs.
During the course of a gene expression analysis of mouse HEV cells (24,25), we found that endomucin is also expressed in LN HEVs. Because endomucin is a heavily O-glycosylated molecule, we attempted to characterize it, particularly from the viewpoint of whether the endomucin on HEVs could serve as a ligand for L-selectin. Here, we show that the endomucin expressed in HEVs binds L-selectin and that when expressed in combination with a specific set of carbohydrate-modifying enzymes, it can efficiently mediate L-selectin-dependent rolling.
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Methods |
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Reagents
The G72 mAb [anti-sialyl 6-sulfo LacNAc (26)] was used as a hybridoma culture supernatant. The CSLEX-1 mAb (anti-sialyl Lewis X) was obtained from Becton Dickinson (San Jose, CA). The RAM34 (anti-CD34) and MEL-14 (anti-L-selectin/CD62L) mAbs were purchased from BD PharMingen (San Diego, CA). The MECA-79 and MECA-367 hybridoma cell lines were kindly provided by Dr E. C. Butcher (Stanford University, Stanford, CA). Mouse L-selectinIgG chimeric protein (LEC/IgG) was produced in COS-7 cells and purified by protein ASepharose from culture supernatants as previously described (27). The plasmid encoding the mouse L-selectinIgM chimera (LEC/IgM) was kindly provided by Dr J. B. Lowe (University of Michigan, Ann Arbor, MI) and the chimeric protein was produced as previously described (28).
RTPCR
Total RNA samples were extracted using the Trizol reagent (Invitrogen, Carlsbad, CA) from purified MECA-79+ HEV cells and MECA-367+ HEV cells, which were from pooled inguinal and mesenteric LNs (24,25), and total RNA was also extracted from cell lines including the bEnd.3, F-2 and L lines. They were then used for the synthesis of the first strand cDNA with a Ready-To-Go kit (Amersham, Piscataway, NJ), according to the manufacturer's instructions. PCR was carried out using the following primers: for endomucin, 5'-CACCATGCGGCTGCTTCAAGCGACTGTTCT-3' (forward) and 5'-GTTCTTGGTTTTCCCCTGTGCAGAGTGTTC-3' (reverse); for GlyCAM-1, 5'-CACCATGAAATTCTTCACTGTCCTGCTATT-3' (forward) and 5'-TGACTTCGTGATACGACTGGCACCAGAGAT-3' (reverse); for CD31, 5'-AAAGATGCTCCTGGCTCTGGGACTCACGCT-3' (forward) and 5'-AGTTCCATTAAGGGAGCCTTCCGTTCT-3' (reverse); for GAPDH, 5'-GCATCGAAGGTGGAAGAGTGGGAGTTGCTG-3' (forward) and 5'-ATGGTGAAGGTCGGTGTGAACGGATTTGGC-3' (reverse). cDNA fragments were amplified by ThermalAce Taq polymerase (Invitrogen) under the following conditions: 95°C for 5 min; 95°C for 30 s, 65°C for 30 s, 72°C for 2 min, 30 cycles; 72°C for 5 min. The sizes of PCR products amplified by the endomucin primer were as follows: endomucin isoform a (792 bp), isoform b (753 bp), isoform c (678 bp) and isoform d (639 bp). The PCR products were analyzed by polyacrylamide gel electrophoresis.
Generation of rabbit polyclonal antibodies against the mouse endomucin extracellular domain
A cDNA fragment encoding the extracellular domain of mouse endomucin, excluding the signal sequence, was amplified by PCR using the MAdCAM-1+ HEV cDNA preparation and subsequently inserted between the EcoRI and BamHI sites of the pGEX-6P-1 expression vector (Amersham). To produce a GST fusion protein, the expression construct was introduced into Escherichia coli strain BL21 (Invitrogen). The recombinant endomucin fusion protein was purified using glutathioneSepharose 4B (Amersham), and the GST tail was removed by PreScission protease (Amersham). Rabbits were subcutaneously immunized with the purified recombinant endomucin protein (200 µg/rabbit) emulsified in complete Freund's adjuvant (for the initial injection) or in incomplete Freund's adjuvant (for the four booster injections). The polyclonal anti-endomucin antibodies from immunized rabbits were purified using HiTrap Protein G (Amersham).
Plasmid construction and CHO transfectants
CHO cells stably expressing both human core 2 ß-1,6-N-acetylglucosaminyltransferase (C2GnT) and human -1,3-fucosyltransferase VII (FucTVII) (CD7II cells) (29) were kindly provided by Dr R. P. McEver (Oklahoma Medical Research Foundation, Oklahoma City, OK). To prepare an expression plasmid for human L-selectin ligand sulfotransferase (30) [LSST, also known as HEC GlcNAc-6-sulfotransferase (31)], the open reading frame of LSST, amplified by PCR, was inserted between the KpnI and BamHI sites of pZeoSV2 (Invitrogen). After transfection with the resultant plasmid, the CD7II cells expressing LSST were selected with zeocin (300 µg/ml). The LSST-expressing CD7II cells were purified by autoMACS (Miltenyi Biotec, Gladbach, Germany) using the G72 mAb, cloned by limiting dilution, and designated as A5 cells. To construct expression plasmids for endomucin and CD34, their open reading frames were amplified by PCR using mouse MAdCAM-1+ HEV cDNA as a template and inserted into EcoRI- and XhoI-digested pcDNA6 (Invitrogen). After transfection, the A5 cells stably expressing endomucin or CD34 were selected with blasticidine (10 µg/ml), purified by autoMACS using the anti-endomucin antibodies or anti-CD34 mAb, and cloned by limiting dilution. These cell lines were designated as A5-EM and A5-CD34, respectively. All cDNA fragments generated by PCR were sequenced from both strands to exclude the influence of mutations.
Immunohistochemistry
Frozen sections (10 µm) of mouse mesenteric LNs were fixed in acetone for 10 min at 4°C and blocked with 500 µg/ml goat IgG and 3% BSA in PBS for 1 h. For the detection of endomucin, the sections were first incubated with anti-endomucin antibodies, followed by biotin-conjugated goat anti-rabbit IgG(H+L) (American Qualex, San Clemente, CA). For assessment of L-selectin binding, sections were incubated with LEC/IgM, followed by biotin-conjugated goat anti-human IgM (American Qualex). For MECA-79 and MECA-367 staining, biotin-conjugated goat anti-rat IgG+M (Southern Biotechnology, Birmingham, AL) was used as a secondary antibody. The sections were further incubated with Alexa Fluor 594-conjugated streptavidin (Molecular Probes, Eugene, OR), counterstained with Hoechst 33258 and examined by fluorescence microscopy (BX-50; Olympus, Tokyo, Japan).
Immunoprecipitation
Mesenteric and inguinal LNs obtained from 21 mice were homogenized in 4 ml of lysis buffer (PBS containing 2% Triton X-100, 1 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM PMSF and 1 mM EDTA) in a glass homogenizer. The homogenate was further solubilized by incubation on ice for 30 min, followed by centrifugation at 14 000 g for 45 min at 4°C. After pre-clearing with protein GSepharose, aliquots of the lysate (0.3 ml) were mixed with lysis buffer containing CaCl2 (0.7 ml), to restore the calcium ion concentration (final 2 mM). For immunoprecipitation, anti-endomucin antibody (2.5 µg) or LEC/IgG (30 µg) that was pre-mixed with protein GSepharose (10 µl) was added and the mixture was incubated for 8 h at 4°C. Human IgG and normal rabbit IgG were used as controls. Materials precipitated by LEC/IgG or anti-endomucin antibody were eluted with 10 mM EDTA or 1x SDS sample buffer, respectively. The samples were then separated by SDSPAGE under reducing conditions and transferred to PVDF filters that were then blocked with PBS containing 3% BSA. The filter was incubated with a combination of MECA-79 mAb and horseradish peroxidase (HRP)-conjugated anti-rat IgG+M (Southern Biotechnology) or anti-endomucin antibodies and HRP-conjugated anti-rabbit IgG (American Qualex), followed by ECL reagents (Amersham). For reprecipitation experiments, LN lysates were first precipitated with LEC/IgG or control human IgG. The LEC/IgG-reactive materials were then eluted with 5 mM EDTA and subjected to reprecipitation with rabbit anti-endomucin antibody or control rabbit IgG. The precipitated proteins were analyzed by western blotting with MECA-79 mAb as described above. For the precipitation of endomucin expressed in CHO transfectants, the A5 cells (3 x 106 cells) were transiently transfected with expression plasmids for endomucin (10 µg) by electroporation. The transfected cells were then cultured for 72 h in SO4-free minimum essential medium (Sigma) with 10% dialyzed FCS containing Na235SO4 (20 µCi/ml; 1.4 Ci/mmol) (ICN Biomedicals Inc., Irvine, CA). 35SO4-labeled cell lysates were subjected to precipitation experiments with LEC/IgG followed by SDSPAGE as described above. The gels were exposed to an image plate for 48 h and analyzed by a Typhoon 9200 imager (Amersham).
Flow cytometry
CHO transfectants (1 x 105) were incubated with anti-endomucin antibodies, CSLEX-1, G72, or RAM34 on ice for 30 min. After being washed in PBS containing 0.1% BSA, the cells were incubated with the appropriate biotinylated secondary antibody, followed by streptavidinPE. The cells were then analyzed on a FACScan (Becton Dickinson) using CELLQuest software (Becton Dickinson) and WinMDI software (The Scripps Research Institute, La Jolla, CA).
Cell binding assay
The cell binding assay was performed as previously described (27). In brief, 96-well microtiter plates (Sumilon H; Sumitomo Bakelite, Tokyo, Japan) were coated with 40 µg/ml of LEC/IgG overnight at 4°C and blocked with 1% BSA in PBS for 3 h at room temperature. Cells (1 x 105) suspended in Hanks' balanced salt solution (HBSS) containing 5 mM CaCl2 and 1 mM MgCl2 (HBSS/Ca2+/Mg2+) were added to each well and incubated for 20 min at 4°C with rotation (140 r.p.m.). The plates were gently washed with HBSS/Ca2+/Mg2+ twice, and adherent cells were photographed and counted.
Sandwich ELISA
Anti-endomucin antibody, RAM34 or control IgG (100 ng/well) were immobilized onto 96-well assay plates (Immulon 2HB; Thermo Labsystems, Franklin, MA) at 4°C. After blocking with 3% BSA/PBS, cell lysates (A5, A5-EM and A5-CD34) were added and incubated for 1 h at room temperature with rocking. After gentle washing, L-selectin reactivity was determined as previously described (32) using a preformed complex of LEC/IgG (final concentration at 1 µg/ml), biotinylated F(ab')2 fragment of rabbit anti-human IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) and streptavidin-conjugated alkaline phosphatase (Caltag Laboratories, Burlingame, CA). P-nitrophenyl phosphate was used as a substrate. The optical density at 405 nm was measured in a microplate reader. Each experiment was done in triplicate and background signals obtained with control capture antibody (normal rabbit IgG and rat IgG for anti-endomucin and RAM34, respectively) were subtracted. Results are shown as mean ± SD.
Rolling assay
The inner surface of the wall of 0.69 mm-diameter glass capillary tubes (Drummond, Broomall, PA) was coated with LEC/IgG or human IgG (40 µg/ml) overnight at 4°C. After the unbound materials were removed, glass tubes were blocked with 2% BSA/PBS at room temperature for 3 h. For antibody inhibition experiments, glass tubes were further treated with a neutralizing anti-L-selectin mAb (MEL-14; 30 µg/ml) or isotype matched-control antibody at room temperature for 2 h. The flow rate was controlled with a Harvard PHD 2000 syringe pump (Harvard Apparatus, South Natick, MA). Wall shear stress was calculated from Poiseuille's law for Newtonian fluids with a viscosity of 0.015 poise as follows (33): wall shear stress (dyn/cm2) = mean flow velocity (mm/s) x [8/tube diameter (mm)] x viscosity (poise). The cells suspended in HBSS/Ca2+/Mg2+ (15 x 105 cells/ml) were injected into the glass capillary tube at room temperature and the behavior of the cells was recorded on videotape. The cells stably rolled along the wall of the glass capillary tube for at least 3 s were considered as the rolling cells in this assay. The velocity of the rolling cells was measured by tracing at least 100 cells frame by frame for 3 s, and the mean of the distance traveled in 1 s was calculated as the velocity of the cells. For quantitation of the A5-EM cell rolling, the numbers of A5-EM cells with rolling velocity <30 µm/s at 0.75 dyn/cm2 and <50 µm/s at 1.5 dyn/cm2 were counted, and results are expressed as the number of rolling cells in a 6.25 mm2 field per min (mean ± SD). The cells with faster velocities showed neither typical rolling behavior nor stable interactions with the glass capillary wall.
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Results |
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To examine the expression of endomucin in HEVs in detail, we generated polyclonal antibodies specific to the extracellular domain of mouse endomucin and used them in an immunohistochemical analysis. As shown in Fig. 1(A), immunofluorescence staining with the polyclonal antibodies in combination with HEV-specific reagents revealed the expression of the endomucin protein in HEVs that were positive for LEC/IgM binding as well as those that were positive for PNAd or MAdCAM-1, and the staining was evident at the luminal aspects of these HEVs as well. The anti-endomucin antibody also reacted with small non-HEV-type blood vessels (Fig. 1A, arrow), confirming the previous observation that endomucin is expressed in various blood vessels (19,23).
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Endomucin in LN HEVs displays the MECA-79 epitope and binds L-selectin
To examine whether the endomucin expressed in HEVs is decorated with L-selectin-reactive sugars, endomucin was affinity-purified from pooled LNs or control endothelial cells (F-2) and subjected to western blot analysis with the MECA-79 mAb, which recognizes a subset of L-selectin-reactive oligosaccharides (35). As shown in Fig. 2(A), the endomucin that was immunoprecipitated from LNs, but not from F-2 endothelial cells, was recognized by the MECA-79 mAb, as has been reported for the endomucin expressed in human tonsils (23). In addition, when materials affinity-purified from LNs by LEC/IgG were subjected to immunoblotting with the anti-endomucin antibody and the MECA-79 mAb, the LEC/IgG-purified preparation showed a strong reactivity with both antibodies (Fig. 2B). A close examination of the gel indicated that a higher molecular weight species (90100 kDa) but not a lower molecular weight species (80 kDa) of endomucin appeared to be selectively modified with L-selectin-reactive oligosaccharides (Fig. 2B, upper panels). No signal was observed in material obtained from F-2 cells or when the control IgG was used. To test whether L-selectin-reactive endomucin carries MECA-79 epitopes, LEC/IgG-purified materials were subjected to reprecipitation with anti-endomucin antibody and immunoblotting with the MECA-79 mAb. As shown in Fig. 2(C) (right panel), the endomucin reprecipitated from LEC/IgG-purified materials was recognized by MECA-79 mAb, demonstrating that at least a portion of the endomucin expressed in HEVs carries specific oligosaccharides that interact with L-selectin and bear the MECA-79 epitope.
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Discussion |
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In the present study, we confirmed that endomucin is expressed in HEVs, including their luminal aspect, in LNs and found that the endomucin isoform predominantly expressed in PNAd+ HEVs and MAdCAM-1+ HEVs is isoform a, as has been seen with other tissues. Because the primers used to detect isoform a cover the whole open reading frame of endomucin, they would have detected any HEV-specific forms that were expressed. Thus, no tissue-specific isoform appears to be expressed in HEVs. We also demonstrated that the endomucin expressed in LNs binds both recombinant L-selectin and the anti-PNAd mAb, MECA-79. Because MECA-79 and recombinant L-selectin are selectively reactive with HEVs, the present observation is in accordance with the hypothesis that the endomucin expressed in HEVs binds L-selectin and bears the MECA-79 epitope. Indeed, reconstitution experiments showed that, in the presence of a specific set of carbohydrate-modifying enzymes, endomucin becomes decorated with sulfated sugars and can interact with L-selectin to mediate cell adhesion and rolling.
We detected two apparent molecular species of endomucin in LNs. One was a predominant component of 80 kDa, the other was a relatively minor component of 90100 kDa. Using affinity purification with soluble L-selectin, we found that the 90100 kDa endomucin species was preferentially decorated with L-selectin-reactive sugar chains. Based on the observation that both HEVs and non-HEV-type blood vessels expressed endomucin, whereas only HEVs express L-selectin-reactive sugar chains, it seemed likely that the 90100 kDa species of endomucin represented one or more HEV-specific forms, whereas the 80 kDa species represented one or more of the conventional forms that are expressed ubiquitously in non-HEV-type vascular endothelial cells. Based on the RTPCR data, we speculate that both of these species represent mainly isoform a, but are differentially glycosylated. Previous studies identified a PNAd component with a similar molecular size (90100 kDa) to the L-selectin-reactive glycoform of endomucin in CD34-deficient mouse LNs (10) and CD34-depleted PNAd fractions isolated from human tonsils (13). It is tempting to speculate that the 90100 kDa glycoform of endomucin actually represents the non-CD34-type PNAd; this, however, requires further investigation.
Although it has been reported that human lymphatic endothelium of the secondary lymphoid tissues expresses endomucin (23) and ligands for L-selectin (37), endomucin expression and L-selectin binding in lymphatic endothelial cells in mouse LNs are very weak in our hands, if they exist at all, and thus the significance of these reports remains unclear from our study.
When endomucin was expressed in CHO cells together with an array of carbohydrate-modifying enzymes, C2GnT, FucTVII and LSST, that can elaborate L-selectin-reactive 6-sulfo sialyl Lewis X in core 2-branched O-glycans attached to sialomucin core proteins (30,31,36), the cells showed L-selectin-dependent rolling under physiological flow conditions, as did CHO cells transfected with CD34 and the carbohydrate-modifying enzymes. These observations support the hypothesis that when endomucin is appropriately glycosylated, it can mediate cell rolling by interacting with L-selectin. Unexpectedly, however, the A5 cells, which are negative for endomucin expression but stably express C2GnT, FucTVII and LSST, showed rolling along immobilized L-selectin. Their rolling behavior was quite distinct and their velocity was considerably higher than that seen with A5 cells expressing endomucin or CD34. In addition, rather paradoxically, the A5 cells failed to show binding to immobilized L-selectin in the adhesion assay that was performed under shear stress. One possibility that may account for this paradox is that the highly expressed carbohydrate-modifying enzymes generated a threshold level of mucin-type carbohydrate modification on some non-endomucin protein(s) in A5 cells, which allowed only weak interactions of the cells with L-selectin, yielding only high-velocity rolling but no cell adhesion under conditions of shear stress that exceeded a certain threshold. In fact, the A5 cells might have adhered weakly to L-selectin but been removed by the washing processes that were used for removing non-adherent or only weakly associated cells in the adhesion assay. In contrast, A5 cells that expressed properly glycosylated and sulfated endomucin seemed to interact with L-selectin efficiently and exhibited cell adhesion and rolling at readily detectable levels. Interestingly, flow cytometric analysis showed that A5 and A5-EM cells expressed comparable levels of G72 epitope on their surface (see Supplementary figure S1), indicating that these cells express a comparable amount of carbohydrate ligands but the way that they express these ligands may be qualitatively different (e.g. the extent of clustering of the carbohydrate chains). It could be that endomucin presents mucin-like carbohydrates to L-selectin more efficiently than non-endomucin protein(s) in A5 cells.
While we used C2GnT in conjunction with FucTVII and LSST in the reconstitution experiments, it was previously shown that L-selectin-reactive sulfated sialyl Lewis X capping groups are generated in extended core-1 O-glycans, core-2 branched O-glycans, or biantennary O-glycans containing both core-2 branch and core-1 extension in HEVs (35). Although extended core-1-based structures, including the MECA-79 mAb-reactive oligosaccharide structures, have been shown to account for a large portion of the L-selectin ligands in vivo, sulfated sialyl Lewis X on core-2 branched O-glycans are among the L-selectin ligands expressed in HEVs (30,31,36) and hence we think that the use of C2GnT is justified for the reconstruction of L-selectin-reactive carbohydrates on the endomucin core protein. When core1-ß3-N-acetylglucosaminyltransferase (C1GnT), which is required for synthesis of extended core-1 O-glycans including MECA-79 epitope (35), was expressed in A5-EM cells, endomucin can be modified with MECA-79+ oligosaccharides (see Supplementary figure S2), confirming the importance of this enzyme in the synthesis of L-selectin-reactive carbohydrates. We are now examining the ability of endomucin decorated with core 1 O-glycans to interact with L-selectin.
Previous studies by others demonstrated that endomucin functions as an anti-adhesive molecule (21,22). Ueno et al. reported that over-expressed endomucin strongly inhibits the adhesion and aggregation of aortic endothelial cells (21). Kinoshita et al. also showed that endomucin negatively regulated the cell adhesion of HEK293, HeLa and NIH3T3 cells (22). Other sialomucins, such as podocalyxin, which is expressed in the kidney, have also been suggested to be anti-adhesive (38) and the anti-adhesive properties are, at least in part, ascribed to their strong negative charge (38). However, rather than assigning a dual role to a single endomucin molecule, it might be more reasonable to assume that endomucin can serve different functions depending on the cell type that expresses it and also on the type of glycosylation it receives. Given that properly glycosylated endomucin can bind L-selectin, we speculate that endomucin expressed in HEVs is in fact pro-adhesive, serving as a potential ligand for L-selectin, whereas endomucin expressed in other tissues may be anti-adhesive, particularly when it is highly expressed. Further study is clearly warranted.
Other than functioning as a scaffold protein for L-selectin-reactive carbohydrates, endomucin may play a distinct role in vascular endothelial cells. However, its extracellular domain currently affords no functional information, because, unlike CD34 and podocalyxin, the endomucin's extracellular region shows no significant structural similarity to that of any other sialomucin members (19,22). On the other hand, its cytoplasmic region carries three putative protein kinase C phosphorylation sites and is highly homologous between human and mouse, suggesting that endomucin may participate in intracellular signaling. To gain further insights into the physiological roles of endomucin, it is now necessary to inactivate the gene in vivo. Such experiments should allow us to test endomucin's function directly, and, together with previous as well as on-going investigations on the gene ablation of sialomucins, the results will tell us whether multiple sialomucins expressed in HEVs perform overlapping functions or play unique individual roles.
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Supplementary data |
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Acknowledgements |
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Abbreviations |
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C2GnT | core-2 ß-1,6-N-acetylglucosaminyltransferase I |
FucTVII | ![]() |
GlyCAM-1 | glycosylation-dependent cell adhesion molecule-1 |
HBSS | Hanks' balanced salt solution |
HEV | high endothelial venule |
HRP | horseradish peroxidase |
LEC/IgG | L-selectin fused to human immunoglobulin G1 Fc region |
LEC/IgM | L-selectin fused to human immunoglubulin M Fc region |
LN | lymph node |
LSST | L-selectin ligand sulfotransferase |
MAdCAM-1 | mucosal addressin cell adhesion molecule-1 |
PNAd | peripheral node addressin |
PP | Peyer's patch |
Sgp | sulfated glycoprotein |
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Notes |
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Transmitting editor: H. Karasuyama
Received 25 September 2003, accepted 17 June 2004.
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References |
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