JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M001703200 on August 10, 2000

J. Biol. Chem., Vol. 275, Issue 42, 32642-32648, October 20, 2000
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
275/42/32642    most recent
M001703200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bruehl, R. E.
Right arrow Articles by Rosen, S. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bruehl, R. E.
Right arrow Articles by Rosen, S. D.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Minimal Sulfated Carbohydrates for Recognition by L-selectin and the MECA-79 Antibody*

Richard E. BruehlDagger , Carolyn R. Bertozzi§, and Steven D. RosenDagger

From the Dagger  Department of Anatomy, Programs in Immunology and Biomedical Sciences, and the Cardiovascular Research Institute, University of California, San Francisco, California 94143 and the § Departments of Chemistry and Molecular and Cell Biology, University of California, Berkeley, California 94720

Received for publication, March 2, 2000, and in revised form, August 8, 2000


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Sulfated forms of sialyl-LeX containing Gal-6-SO4 or GlcNAc-6-SO4 have been implicated as potential recognition determinants on high endothelial venule ligands for L-selectin. The optimal configuration of sulfate esters on the N-acetyllactosamine (Galbeta 1right-arrow4GlcNAc) core of sulfosialyl-LeX, however, remains unsettled. Using a panel of sulfated lactose (Galbeta 1right-arrow4Glc) neoglycolipids as substrates in direct binding assays, we found that 6',6-disulfolactose was the preferred structure for L-selectin, although significant binding to 6'- and 6-sulfolactose was observed as well. Binding was EDTA-sensitive and blocked by L-selectin-specific monoclonal antibodies. Surprisingly, 6',6-disulfolactose was poorly recognized by MECA-79, a carbohydrate- and sulfate-dependent monoclonal antibody that binds competitively to L-selectin ligands. Instead, MECA-79 bound preferentially to 6-sulfolactose. The difference in preferred substrates between L-selectin and MECA-79 may explain the variable activity of MECA-79 as an inhibitor of lymphocyte adhesion to high endothelial venules in lymphoid organs. Our results suggest that both Gal-6-SO4 and GlcNAc-6-SO4 may contribute to L-selectin recognition, either as components of sulfosialyl-LeX capping groups or in internal structures. By contrast, only GlcNAc-6-SO4 appears to contribute to MECA-79 binding.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

L-selectin, the "leukocyte selectin," mediates the tethering and rolling of lymphocytes along high endothelial venules (HEVs)1 in peripheral lymph nodes, a prerequisite for extravasation of the lymphocytes (1-3). By virtue of a calcium-type lectin domain at its amino terminus, L-selectin functions as a calcium-dependent, carbohydrate-binding receptor that recognizes a set of discrete counter-receptors (generally termed ligands) displayed on the luminal aspect of HEVs (reviewed in Refs. 4 and 5). Several HEV-expressed, L-selectin-reactive ligands (all of which possess mucin-like domains) have been identified as potential physiological ligands for L-selectin (reviewed in Ref. 6). These include GlyCAM-1 (7), CD34 (8, 9), and podocalyxin (10).

A large body of evidence has established that the optimal interaction between L-selectin and these HEV-expressed ligands requires sialylation, fucosylation, and carbohydrate sulfation (11-17). In an attempt to rationalize these requirements in terms of carbohydrate structures, a detailed analysis of the O-linked oligosaccharide side chains of GlyCAM-1 was conducted (18-20). Sulfation analysis of acid-hydrolyzed glycans revealed monosulfated monosaccharides and disaccharides (N-acetyllactosamine) with equivalent levels of Gal-6-SO4 and GlcNAc-6-SO4. Analysis of the simplest oligosaccharide side chains identified equivalent levels of two novel sulfated isomers of sialyl-LeX (sLeX), 6'-sulfo-sLeX, containing Gal-6-SO4, and 6-sulfo-sLeX, containing GlcNAc-6-SO4 (see Fig. 1), as capping groups of core-2 branched oligosaccharides. The finding of a sLeX motif was significant because this tetrasaccharide binds to all three selectins (reviewed in Refs. 21-25), although weakly.

Following the identification of the 6'- and 6-sulfo forms of sLeX within GlyCAM-1, a number of studies have been directed at examining the binding of these and analogous structures to L-selectin (reviewed in Ref. 26). There is general agreement that sulfation at C-6 of GlcNAc, in the context of 6-sulfo-sLeX, enhances L-selectin binding relative to sLeX (27-29). Similar findings have been obtained using 3'-sulfo-LeX and 3'-sulfo-Lea as mimetics of sLeX (28, 30-32). Furthermore, Kannagi and coworkers (33, 34) have described 6-sulfo-sLeX-reactive antibodies that stain HEVs and inhibit L-selectin binding to HEVs. With respect to the contribution of Gal-6-SO4, several groups have reported that this modification of sLeX or of sLeX mimetics either enhances or does not affect L-selectin binding (29, 30, 35-37). In marked contrast, Feizi and co-workers found that this modification eliminates binding to L-selectin (28, 38).

Previously, in an attempt to understand the contribution of the two sulfate esters in question to L-selectin binding, we synthesized a limited series of sulfated lactose derivatives (39). As inferred from the ability of these compounds to compete the binding of an L-selectin/IgG chimera to GlyCAM-1, 6',6-disulfolactose (containing sulfate esters in positions analogous to those in 6',6-disulfo-sLeX) exhibited significant affinity for L-selectin. In the present study, we have synthesized a more extensive series of mono- and disulfated lactose derivatives with various combinations of sulfate esters on Gal and Glc. These compounds were derivatized with lipid tails, creating neoglycolipids that could be coated onto wells of microtiter plates. Thus, direct binding studies could be done with L-selectin chimeras and L-selectin bearing lymphocytes, allowing specificity controls to verify that the observed binding was functionally relevant. These studies have led to the definition of the minimal sulfated structures that support recognition by L-selectin. In addition, we employed these compounds to investigate the epitope corresponding to MECA-79, a widely used monoclonal antibody (mAb) that recognizes HEV-expressed ligands for L-selectin in human and other species (6, 40). The MECA-79 epitope has been shown to be sulfation-dependent, but the structural context for this modification has heretofore not been defined (14-16). In the present study, we find that L-selectin favors the disulfated lactose derivative with modifications at both C-6 of Gal and C-6 of Glc, whereas MECA-79 favors the monosulfated lactose derivative bearing Glc-6-SO4.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

General-- Ninety-six-well polyvinyl chloride microtiter plates (Falcon 3912) were from Becton Dickinson, and 96-well polystyrene plates (Immulon II) were from Dynatech Laboratories, Inc. (Alexandria, VA). The sialyl-LeX analog neoglycolipid (aLeX), N-[O-(3-O-(2-acetic acid)-beta -D-galactopyranosyl)-(1right-arrow4)-O-[(alpha -l-fucopyranosyl)-(1right-arrow3)]-O-(beta -D-glucopyranosyl)]-1-acetamido-6-(10,12-pentacosadiynamide)-3-thiohexane, was a gift of Dr. Jon Nagy. Sulfated lactose neoglycolipids were synthesized and verified as described.2 Cholesterol, phosphatidylcholine, bovine serum albumin (BSA), EDTA, and p-nitrophenyl phosphate (phosphatase substrate) were from Sigma. BCECF/AM was from Calbiochem. Allylamine, diisopropylethylamine, dimethylformamide, and steroyl chloride were from Aldrich.

Sialyllactose Neoglycolipid-- The sialyllactose neoglycolipid was synthesized from 1-beta -O-allyllactose by first forming an N-allyl glycoside (42) and then coupling the glycosylamine to steroyl chloride. A solution of 0.63 g (1.0 mmol) of sialyllactose (NeuAcalpha 2right-arrow3Galbeta 1right-arrow4Glc) (Neose, Horsham, PA) in 10 ml of allylamine was stirred at room temperature for 72 h. The solvent was removed by rotary evaporation, and the resulting residue was suspended in hexanes and evaporated five times. The resulting N-allyl glycoside was suspended in 2.5 ml of dimethylformamide; 0.675 ml of steroyl chloride (0.61 g, 2.0 mmol) in 1 ml of dimethylformamide containing 0.697 ml of diisopropylethylamine (0.52 g, 4.0 mmol) was added dropwise over 10 min; and the solution was stirred vigorously for 60 min. The reaction was quenched by the addition of 1 ml of methanol and extracted in a solution of 5 ml of hexanes and 5 ml of distilled water. The solution was centrifuged at high speed in a bench-top microcentrifuge. The unreacted steroyl chloride and diisopropylethylamine partitioned into the organic layer; the unreacted N-allylsialyllactose and diisopropylethylamine salts partitioned into the aqueous layer; and the neoglycolipid was trapped in a densely packed foam layer at the interface between the two solvents. This extraction was repeated five times, and the recovered foam layer was suspended in 40% aqueous acetonitrile, frozen, and lyophilized to yield 0.242 g (26%) of sialyllactose neoglycolipid. The calculated mass for C44H78N2O19 was 938.52, and the measured mass was 961.6 [M + Na]+ (liquid secondary ionization mass spectrometry, positive mode).

Preparation of GlyCAM-1-- GlyCAM-1 was prepared as described previously (43). Briefly, mouse serum from Pel-Freez Biologicals (Rogers, AR) was extracted with 4 volumes of 2:1 chloroform/methanol, and the layers were separated by centrifugation at 2000 × g. The upper aqueous layer was separated from the organic and precipitated protein layers, concentrated to one-half of the original serum volume by boiling, and dialyzed against 20 volumes of Dulbecco's phosphate-buffered saline (PBS) with two changes. This preparation was then diluted with PBS back to the original serum volume and used as an enriched source of GlyCAM-1. GlyCAM-1 was captured onto Immulon II microtiter plate wells using a purified rabbit anti-GlyCAM-1 peptide polyclonal antibody, CAM02 (44).

Selectin/IgM Chimeras-- The recombinant murine L-selectin/human IgM chimera cDNA and the tissue culture supernatant containing murine E-selectin/human IgM were gifts from Dr. Lloyd Stoolman and have been described previously (45). The recombinant human L-selectin/human IgM chimera was prepared by subcloning human L-selectin cDNA, a gift of Dr. Thomas Tedder, into the human IgM vector used to make the murine L-selectin/human IgM chimera. Recombinant protein was produced in transiently transfected COS-7 cells grown in Opti-MEM serum-free medium (Life Technologies, Inc.). The level of expression was determined by enzyme-linked immunosorbent assay using immobilized goat anti-human IgM polyclonal antibody to capture the chimera from the supernatant and a goat anti-human IgM/alkaline phosphatase conjugate to detect the immobilized chimera. Human IgM was used as a standard. The chimeras were used as crude tissue culture supernatant.

Antibodies-- Purified MECA-79 (rat IgM) (40) and the control antibody OZ-42 (46) were kindly provided by Dr. Stefan Hemmerich. Human IgM and the goat anti-human IgM/alkaline phosphatase conjugate were from Sigma. Rabbit anti-rat IgM/alkaline phosphatase was from Zymed Laboratories Inc. (South San Francisco, CA). The anti-murine L-selectin monoclonal antibody MEL-14 (rat IgG2a) (1) was purified by ammonium sulfate precipitation from hybridoma cell culture supernatant. The control for MEL-14 was an irrelevant rat IgG2a (Zymed Laboratories Inc.). The anti-human L-selectin mAb DREG-56 (murine IgG1) and control murine IgG1 were from Caltag Laboratories (Burlingame, CA). The purified rabbit anti-GlyCAM-1 peptide polyclonal antibody CAM02 was generated as described previously (44).

Selectin Direct Binding Assay-- Neoglycolipids were resuspended in 85% aqueous ethanol containing 5 µg/ml cholesterol and 2.5 µg/ml phosphatidylcholine. Serial dilutions were coated onto polyvinyl chloride microtiter plates at 40 µl/well, and the solvent was evaporated at 37 °C. It is assumed in enzyme-linked immunosorbent assays of this type that the input concentration of lipid accurately predicts the coating concentration. The finding that the rank orders of reactivity of the neoglycolipids for L-selectin, E-selectin, and MECA-79 are very distinct (see "Results") indicates that the differences are not just due to variations in the amount of lipid coated. The coated wells were washed once with distilled water and then blocked with 3% BSA in PBS for 2 h. Tissue culture supernatant containing the selectin/IgM chimera was diluted 1:1 in 2% BSA in PBS and incubated at 100 µl/well for 2 h. After two washes in PBS, goat anti-human IgM/alkaline phosphatase conjugate diluted 1:1000 in 1% BSA in PBS was added at 100 µl/well for 45 min. After five washes in PBS, bound selectin/IgM chimera and human IgM were detected by the addition of phosphatase substrate (p-nitrophenyl phosphate (1 mg/ml) in 10% diethanolamine and 0.1 mM MgCl2) at 100 µl/well. Absorbances were recorded at 405 nm on a Bio-Rad microplate reader. All incubations and wash steps were performed at ambient temperature.

MECA-79 Direct Binding Assay-- Microtiter wells were coated with neoglycolipids and blocked with 3% BSA in PBS as described for the L-selectin direct binding assay. MECA-79 and the OZ-42 control antibody were diluted to 10 µg/ml in 1% BSA in PBS and incubated at 100 µl/well for 2 h. The wells were washed twice with PBS, and rabbit anti-rat IgM/alkaline phosphatase diluted 1:1000 in 1% BSA in PBS was added at 100 µl/well for 45 min. After five washes in PBS, bound antibody was detected by the addition of p-nitrophenyl phosphate at 100 µl/well. Absorbances were recorded at 405 nm. All incubations and wash steps were performed at ambient temperature.

Antibody Inhibition Assay-- Microtiter plates were coated with 200 µM solutions of neoglycolipid in 85% ethanol, 5 µg/ml cholesterol, and 2.5 µg/ml phosphatidylcholine at 40 µl/well (8 nmol/well). The solvent was evaporated at 37 °C, and the wells were blocked with 3% BSA in PBS. Anti-L-selectin or control mAb was diluted to 2 µg/ml in 2% BSA in PBS and added in equal volumes to tissue culture supernatants containing the L-selectin/IgM chimera. The solutions were incubated for 30 min and then transferred to the neoglycolipid-coated plates at 100 µl/well. After a 2-h incubation, bound chimera was probed and detected as described for the direct binding assay. All incubations and wash steps were performed at ambient temperature.

EDTA Inhibition Assay-- Microtiter plates were coated with neoglycolipid and blocked as described for the antibody inhibition assay. Serial dilutions of EDTA in 2% BSA in PBS were combined in equal volumes with undiluted L-selectin/IgM-containing tissue culture supernatant and added to the neoglycolipid-coated wells at 100 µl/well. After a 2-h incubation, bound chimera was probed and detected as described for the direct binding assay. All incubations and wash steps were at ambient temperature. The EqCal software package (Biosoft, Cambridge, United Kingdom) was used to calculate the free calcium and magnesium levels as a function of the amount of EDTA added to the Dulbecco's PBS/Opti-MEM sample buffer. The calculations were based on the following parameters: an initial concentration of 0.90 mM for calcium, an initial concentration of 0.75 mM for magnesium, pH 7.4, 25 °C, and an ionic strength of 0.1 M.

Cell Adhesion Assay-- L-selectin-expressing Jurkat T-cells were obtained from the laboratory of Dr. Arthur Weiss and maintained in RPMI 1640 medium supplemented with 100 units/ml penicillin, 100 mg/ml streptomycin, 2 mM glutamine, and 5% heat-inactivated fetal calf serum (Hyclone Laboratories, Logan, UT). Neoglycolipids were diluted to 200 µM in 85% ethanol, 5 µg/ml cholesterol, and 2.5 µg/ml phosphatidylcholine and coated onto microtiter plates at 40 µl/well (8 nmol/well). After drying, the wells were blocked with 3% BSA in PBS for 2 h. Jurkat cells were centrifuged and resuspended to 5 × 106/ml in 0.1% BSA in PBS. The fluorescent dye BCECF/AM was added at a dilution of 1:1000 from a 2 mM stock solution in dimethyl sulfoxide and incubated with the cells for 20 min in the dark. The labeled cells were centrifuged, resuspended to 2 × 106/ml in 0.1% BSA in PBS, and transferred to the neoglycolipid-coated plate at 100 µl/well. After 30 min, the plate was washed twice with PBS, and 0.1% BSA in PBS was added at 100 µl/well. Arbitrary fluorescence intensity units were recorded at 485 nm excitation and 530 nm emission on a CytoFluor II fluorescence multiwell plate reader (PerSeptive Biosystems, Foster City, CA). To demonstrate L- selectin-dependent binding, some cells were incubated with 10 mM EDTA or with DREG-56 or a class-matched control antibody at 10 µg/ml for 20 min prior to incubation with the immobilized neoglycolipids. All reactions, washes, and centrifugation steps were performed at ambient temperature.

Statistics-- Significant differences among means were determined by one-way analysis of variance. When significant differences were detected, multiple pairwise comparisons were performed using a Tukey test (SigmaStat statistical software, SPSS Inc., Chicago, IL). Unless stated otherwise, the data shown are from representative experiments and are the average of duplicate wells after subtraction of background signal (carrier lipids only), with error bars denoting the range in signal.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Binding of Recombinant L-selectin to Sulfated Lactose Neoglycolipids-- To investigate the contribution of sulfate esters in defined positions to L-selectin binding, serial dilutions of a panel of sulfated lactose neoglycolipids with 25-atom single chain hydrocarbon tails (Fig. 1) were immobilized on microtiter plate wells and assayed for their ability to support the binding of L-selectin/IgM chimeras. For both the human and murine L-selectin chimeras, the best substrate for binding was 6',6-disulfolactose ((SO4-6)Galbeta 1right-arrow4(SO4-6)Glc), but significant binding was also observed with the 6'-sulfolactose ((SO4-6)Galbeta 1right-arrow4Glc) and 6-sulfolactose (Galbeta 1right-arrow4(SO4-6)Glc) derivatives (Fig. 2, A and B). At a coating concentration of 5 nmol/well, 6',6-disulfolactose generated a signal 2.4-fold greater than 6-sulfolactose (p < 0.001) and 2.8-3.6-fold greater than 6'- and 3'-sulfolactose ((SO4-3)Galbeta 1right-arrow4Glc) (p < 0.001). Differences in reactivity between 6',6-disulfolactose and the other sulfated derivatives and between all of the sulfated derivatives and lactose or carrier lipids only (data not shown) were statistically significant (p < 0.001). These results are consistent with previous data in which similar compounds were used as soluble inhibitors of L-selectin binding to GlyCAM-1 (39). There were no significant differences in the reactivity profiles between the human and murine chimeras; however, the murine chimera consistently generated higher signals than the human chimera at comparable concentrations. There was no detectable binding of either chimera to non-sulfated lactose or sialyllactose (data not shown).


View larger version (16K):
[in this window]
[in a new window]
  		Fig. 1.   Sulfated lactose derivatives modeled after the sulfated N-acetyllactosamine core of sulfo-sLeX. A, sulfo-sLeX. The N-acetyllactosamine core on which the sulfated lactose neoglycolipids are based in B is shown boxed. Sulfation at C-6 of Gal (R1) defines 6'-sulfo-sLeX. Sulfation at C-6 of GlcNAc (R2) defines 6-sulfo-sLeX. Sulfation at C-6 of Gal and GlcNAc defines 6',6-disulfo-sLeX. B, sulfated lactose derivatives and sialyllactose. C, aLeX. D, lipid tail of sulfated lactose neoglycolipids.


View larger version (13K):
[in this window]
[in a new window]
  		Fig. 2.   Binding of L-selectin/IgM to sulfated lactose neoglycolipids. Serial dilutions of sulfated lactose neoglycolipids were dried onto wells of microtiter plates and assayed for their ability to support binding of the human (A) and murine (B) L-selectin/IgM chimeras. The enhanced binding to 6',6-disulfolactose relative to the monosulfated compounds was observed in 12 independent experiments using three different preparations of 6',6-disulfolactose and two different preparations each of 6'- and 6-sulfolactose at different times. black-square, 6',6-disulfolactose; open circle , 6'-sulfolactose; , 6-sulfolactose; triangle , 3'-sulfolactose.

Gal-3-SO4 has not been detected in acid hydrolysates of GlyCAM-1 (18). However, a number of studies have established binding of L-selectin to Gal-3-SO4-containing structures, including sulfatides, 3'-sulfo-Lea ((SO4-3)Galbeta 1right-arrow3(Fucalpha 1right-arrow4)GlcNAc), and 3'-sulfo-LeX ((SO4-3)Galbeta 1right-arrow4(Fucalpha 1right-arrow3)GlcNAc) (28, 30-32, 47, 48). Structures of this kind could be important determinants in the L-selectin-mediated association of leukocytes with tumor cells (49-51). As shown in Fig. 2 (A and B), L-selectin binding to 3'-sulfolactose was relatively weak compared with that to 6'- and 6-sulfolactose.

To address the possibility that the greater reactivity of L-selectin for 6',6-disulfolactose relative to the monosulfated derivatives was due to an increase in negative charge rather than to a specific configuration of sulfate esters, L-selectin binding to 3',6'-disulfolactose ((SO4-3)(SO4-6)Galbeta 1right-arrow4Glc) and 3',6-disulfolactose ((SO4-3)Galbeta 1right-arrow4(SO4-6)Glc) was determined (Fig. 3). The addition of a sulfate ester to C-3' of either 6'- or 6-sulfolactose did not yield binding greater than that of the parent monosulfated lactose derivative.


View larger version (13K):
[in this window]
[in a new window]
  		Fig. 3.   Binding of L-selectin/IgM to disulfated lactose neoglycolipids. Serial dilutions of neoglycolipids were dried onto microtiter plate wells, and human L-selectin/IgM was assayed for binding. The enhanced binding of 6',6-disulfolactose relative to the other disulfated derivatives was observed in four independent experiments. The data shown for 6',6-disulfolactose are the same as those shown in Fig. 2A; all compounds were tested in the same experiment. At a coating concentration of 5 nmol of neoglycolipid/well, differences in reactivity between 6',6-disulfolactose and the other sulfated derivatives (3',6'- and 3',6-disulfolactose) were statistically significant (p < 0.001). black-square, 6',6-disulfolactose; open circle , 3',6'-disulfolactose; , 3',6-disulfolactose.

To further characterize the binding of L-selectin to the sulfated lactose neoglycolipids, we determined the effects of a function-blocking mAb (DREG-56) (52) and divalent cation chelation on the binding of the human L-selectin chimera. As shown in Fig. 4, the binding of L-selectin to all of the sulfated lactose derivatives was strongly inhibited by DREG-56 or by 10 mM EDTA. Similarly, mouse L-selectin binding to these sulfated compounds was inhibited by a function-blocking mAb (MEL-14) or by EDTA (data not shown). The binding of wheat germ agglutinin and ricin toxin agglutinin to lactose or 6',6-disulfolactose, respectively, was not inhibited by 20 mM EDTA, indicating that EDTA does not strip the neoglycolipid substrate from the microtiter well (data not shown).


View larger version (15K):
[in this window]
[in a new window]
  		Fig. 4.   Antibody and EDTA inhibition of L-selectin/IgM binding. Neoglycolipids were dried onto microtiter wells at a concentration of 8 nmol/well. Prior to incubation with the dried lipids, the L-selectin chimera was incubated with antibody or EDTA for 30 min at room temperature. A, effects of DREG-56 (black bars) or a class-matched control antibody (gray bars); B, effects of 10 mM EDTA. These results were obtained in three independent experiments. 6',6, 3',6', and 3',6, 6',6-, 3',6'-, and 3',6-disulfolactose, respectively; 6', 6, and 3', 6'-, 6-, and 3'-sulfolactose, respectively.

We investigated the divalent cation dependence of L-selectin binding to the most active sulfated lactose neoglycolipids in detail by measuring chimera binding as a function of EDTA concentration. As shown in Fig. 5, L-selectin binding to 6'-sulfolactose, 6-sulfolactose, and 6',6-disulfolactose exhibited the identical divalent cation dependence as observed for its binding to GlyCAM-1. 50% inhibition of binding was achieved at 0.9 mM EDTA, which corresponds to a calcium concentration of 80 µM. This value is in accord with previous estimates of the amount of calcium needed for L-selectin function (66).


View larger version (14K):
[in this window]
[in a new window]
  		Fig. 5.   Effect of EDTA on L-selectin binding to sulfated lactose neoglycolipids. GlyCAM-1 or sulfated lactose neoglycolipids were coated onto microtiter wells, and human L-selectin/IgM binding was measured in the presence of serial dilutions of EDTA. The concentrations of free Ca2+ and Mg2+ were calculated to be as follows: 593 and 745 µM, 296 and 729 µM, 13 and 388 µM, 0.01 and 0.81 µM, 3.4 × 10-3 and 0.21 µM, and 1.4 × 10-3 and 0.08 µM for EDTA concentrations of 0.31, 0.63, 1.3, 2.5, 5.0, and 10 mM, respectively. black-triangle, GlyCAM-1; black-square, 6',6-disulfolactose; open circle , 6'-sulfolactose; , 6-sulfolactose.

Binding of Jurkat T-cells-- To investigate the binding of L-selectin in a cellular context, we examined the adhesion of an L-selectin-expressing T-cell line (Jurkat) to the same panel of sulfated lactose neoglycolipids. As was the case for the L-selectin chimeras, the Jurkat cells bound best to 6',6-disulfolactose (Fig. 6), and differences in reactivity between 6',6-disulfolactose and the other sulfated derivatives were statistically significant (p < 0.001). Binding to the other sulfated derivatives was clearly above background levels, (p < 0.001), but without notable differences in potency among them. Jurkat cell binding to 6',6-disulfolactose was effectively inhibited by EDTA and the DREG-56 mAb (Fig. 6, inset), verifying that the interaction was L-selectin-dependent and exhibited the same characteristics as the chimera binding.


View larger version (19K):
[in this window]
[in a new window]
  		Fig. 6.   Binding of Jurkat cells to sulfated lactose neoglycolipids. Jurkat T-cells were labeled with the fluorescent dye BCECF/AM and assayed for adhesion to the sulfated lactose neoglycolipids. The superior binding to 6',6-disulfolactose relative to the other derivatives was observed in four independent experiments. Inset, effect of 10 mM EDTA or the DREG-56 mAb on binding of Jurkat cells to 6',6-disulfolactose. Wells contained buffer (control), 10 mM EDTA, the DREG-56 mAb, or a class-matched IgG1. All data shown are the average of triplicate wells after subtraction of background signal (carrier lipids only), and the error bars indicate the S.D. in signal. 6',6, 3',6', and 3',6, 6',6-, 3',6'-, and 3',6-disulfolactose, respectively; 6', 6, and 3', 6'-, 6-, and 3'-sulfolactose, respectively.

Binding of MECA-79-- To determine whether there was overlap between the MECA-79 epitope and the L-selectin recognition determinant, we assayed MECA-79 binding to the sulfated lactose neoglycolipids (Fig. 7). The strongest binding was observed with 6-sulfolactose, whereas much less binding was observed with 6'- and 3'-sulfolactose. Surprisingly, the weakest signal among the sulfated compounds was seen with 6',6-disulfolactose. MECA-79 binding, however, was much weaker relative to the L-selectin/IgM chimeras, as evidenced by a significantly longer substrate conversion time. The control rat IgM was not reactive with any of the compounds tested (data not shown).


View larger version (14K):
[in this window]
[in a new window]
  		Fig. 7.   Binding of MECA-79 to sulfated lactose neoglycolipids. Serial dilutions of neoglycolipids were dried onto microtiter wells and assayed for MECA-79 binding. Enhanced binding of MECA-79 to 6-sulfolactose over the other compounds was observed in three independent experiments and was statistically significant (p < 0.001) at a coating concentration of 8 nmol of neoglycolipid/well. , 6-sulfolactose; open circle , 6'-sulfolactose; triangle , 3'-sulfolactose; black-square, 6',6-disulfolactose; down-triangle, lactose.

Binding of E-selectin-- E-selectin has previously been shown to bind to sulfated derivatives of LeX (Galbeta 1right-arrow4) (Fucalpha 1right-arrow3)GlcNAc) or Lea (Galbeta 1right-arrow3 (Fucalpha 1right-arrow4) GlcNAc) (30, 38, 53, 54). In view of the binding of L-selectin to sulfated lactose derivatives, we wanted to examine the interaction of E-selectin with the same compounds in parallel assays. The preferred structure for the E-selectin/IgM chimera was the non-sulfated sLeX analog, aLeX (Fig. 8A). This compound consists of a LeX-like trisaccharide with Glc substituting for GlcNAc and with a 3'-O-acetic acid substituting for the 3'-sialic acid of sLeX (Fig. 1C). The L-selectin chimera did not show detectable binding to this sLeX analog at a concentration of 8 nmol/well, although (as above) L-selectin bound markedly to various sulfated lactose derivatives at this concentration. A markedly different pattern of E-selectin/IgM binding to the sulfated compounds was observed compared with L-selectin/IgM (Fig. 8B). The strongest binding was to 6-sulfolactose, followed by 6',6-disulfolactose. In contrast to the results with L-selectin, E-selectin binding was seen only at the highest concentration of neoglycolipid coated (8 nmol/well). 6'-Sulfolactose failed to support E-selectin binding at any concentration (Fig. 8A). Despite the ability of E-selectin to bind to 3'-sulfo-LeX and 3'-sulfo-Lea (31, 32, 55), E-selectin did not bind to 3'-sulfolactose or 3',6-disulfolactose and bound only weakly to 3',6'-disulfolactose (Fig. 8A). Like L-selectin binding, E-selectin binding was strongly inhibited by divalent cation chelation using EDTA (Fig. 8A). These comparisons add to the existing evidence for a fundamental difference in the specificities of these two selectins (56-58).


View larger version (10K):
[in this window]
[in a new window]
  		Fig. 8.   Comparison of E- and L-selectin binding to sulfated lactose and sLeX analog neoglycolipids. Neoglycolipids were coated onto polyvinyl chloride plates at 8 nmol/well, and binding was assayed as described under "Experimental Procedures." A, E-selectin binding in buffer (black bars) or 10 mM EDTA (gray bars); B, L-selectin binding. The enhanced binding of E-selectin to aLeX relative to the sulfated compounds was observed in three independent experiments. 6',6, 3',6', and 3',6, 6',6-, 3',6'-, and 3',6-disulfolactose, respectively; 6', 6, and 3', 6'-, 6-, and 3'-sulfolactose, respectively.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Interest in the Gal-6-SO4 and GlcNAc-6-SO4 modifications within L-selectin ligands was originally prompted by our analysis of the acid hydrolysis products of GlyCAM-1 (18). Reconstitution experiments performed with recently cloned Gal- and GlcNAc-6-O-sulfotransferases have justified this interest by confirming that both of these modifications (i.e. Gal-6-SO4 and GlcNAc-6-SO4) can contribute to L-selectin ligand activity (59-61). Interestingly, transfection of Chinese hamster ovary cells with a combination of GlcNAc- and Gal-6-O-sulfotransferase cDNAs imparted much greater L-selectin ligand activity than either sulfotransferase alone, indicating a synergistic contribution from the two kinds of sulfation. The present study was directed at examining the contribution of Gal-6-SO4 and GlcNAc-6-SO4 to L-selectin ligand activity in isolation from the influence of sialylation and fucosylation. Guided by the analysis of the simplest chains of GlyCAM-1 (18-20), the minimal structures that we synthesized were based on the N-acetyllactosamine core (Galbeta 1right-arrow4GlcNAc) of sulfo-sLeX. Although the C-2 N-acetate of GlcNAc in N-acetyllactosamine is missing in the sulfated lactose (Galbeta 1right-arrow4Glc) derivatives studied here, this alteration has been shown to have a minimal effect on L-selectin binding (62).

The best substrate for the recombinant L-selectin/IgM chimeras was 6',6-disulfolactose, which supported binding significantly better than the next most adhesive substrate, 6-sulfolactose (containing Glc-6-SO4), over a range of neoglycolipid concentrations. Binding to other disulfated structures containing Gal-3-SO4 (3',6'- and 3',6-disulfolactose) was distinctly less than that to 6',6-disulfolactose, demonstrating that the position of sulfate esters, rather than the overall charge, is responsible for efficacy of this compound. A similar pattern of binding was seen with L-selectin-expressing Jurkat T-cells, with the 6',6-disulfolactose neoglycolipid clearly superior to the other compounds. These parallels strongly validate the use of the L-selectin/IgM chimera as a probe for L-selectin binding.

A significant advantage of the direct binding studies over competition studies is that important specificity controls could be performed. Thus, we were able to establish that the binding of the L-selectin/IgM chimeras to the sulfated neoglycolipids (as well as Jurkat cell binding to 6',6-disulfolactose) could be effectively inhibited by function-blocking antibodies or by divalent cation chelation. EDTA titration demonstrated that the divalent cation requirement for L-selectin binding to the preferred sulfated lactose derivatives (6',6-disulfolactose, 6'-sulfolactose, and 6-sulfolactose) was identical to that of native GlyCAM-1. These results strongly argue that these compounds are engaged by a site in the L-selectin calcium-type lectin domain that is critical for recognition of physiological ligands. Further supporting evidence for this conclusion derives from our previous observation that soluble sulfated lactose derivatives (with 6',6-disulfolactose as the best inhibitor) can compete the binding of L-selectin to GlyCAM-1 (39). The fact that specific sulfated lactose derivatives, lacking fucose and sialic acid, can bind to relevant sites in L-selectin again emphasizes the importance of sulfate esters with the appropriate spatial orientation as recognition elements. The observation that L-selectin bound better to the sulfated lactose neoglycolipids than to the non-sulfated sLeX analog is consistent with our previous report that 6',6-disulfolactose is a superior inhibitor of L-selectin binding to GlyCAM-1 than sLeX (39).

Further work will be necessary to understand the relationship between the minimal binding structures defined above and actual recognition determinants of HEV-expressed ligands for L-selectin. As reviewed above, a variety of studies have established that 6-sulfo-sLeX (NeuAcalpha 2right-arrow3Galbeta 1right-arrow4(Fucalpha 1right-arrow3)(SO4-6)GlcNAc) is an important recognition determinant (27, 34, 38, 59-61, 63). Contained within this structure is 6-sulfo-N-acetyllactosamine, which, from the data presented herein, can contribute a significant degree of L-selectin binding by itself. Given that L-selectin functions in the dynamic processes of tethering and rolling of lymphocytes, a complete analysis of 6-sulfo-sLeX must examine the contribution of each modification (including sulfation) to kinetic constants as well as to the overall equilibrium constant. A previous study has shown that sulfated LeX derivatives can exhibit very different inhibitory activities against L-selectin depending upon whether equilibrium or flow chamber assays are used (37).

The structural context for the contribution of Gal-6-SO4 is less certain at the present time. As reviewed above, there is significant controversy as to whether sulfation at C-6 of Gal augments the affinity of sLeX or 6-sulfo-sLeX for L-selectin. Furthermore, antibody staining studies by Kannagi and co-workers (34) have failed to detect the presence of 6'-sulfo-sLeX (NeuAcalpha 2right-arrow3(SO4-6)Galbeta 1right-arrow4(Fucalpha 1right-arrow3)GlcNAc) or 6',6-disulfo-sLeX (NeuAcalpha 2right-arrow3(SO4-6)Galbeta 1right-arrow4(Fucalpha 1right-arrow3)(SO4-6)GlcNAc) determinants on HEVs in human lymphoid organs. The present study demonstrates that 6'-sulfolactose and especially 6',6-disulfolactose can support L-selectin binding without sialylation or fucosylation. One possibility is that Gal-6-SO4 may contribute to L-selectin binding in the context of 6'-sulfo- or 6',6-disulfo-N-acetyllactosamine, lacking fucose and/or sialic acid. Such structures might exist as glycan capping groups or be present internally in extended oligosaccharide side chains. In this context, it should be stressed that native GlyCAM-1 possesses extended and multisulfated chains, the structures of which have not been solved (20).

The finding that L-selectin binding to the sulfated lactose derivatives was highly sensitive to divalent cation chelation by EDTA is somewhat surprising. Structural analysis of an engineered form of E-selectin complexed with sLeX revealed a role for calcium in coordinating the C-2 and C-3 hydroxyls of fucose, in addition to four amino acids in the lectin domain (64). Furthermore, calcium-independent L-selectin binding has been demonstrated for a number of non-fucosylated anionic molecules, e.g. sulfatide, lipid A, lipopolysaccharide, and cardiolipin (48, 65, 67).3 Thus, it was expected that binding to the sulfated lactose neoglycolipids, which lack fucose, would be calcium-independent. It is possible that in addition to coordinating vicinal hydroxyls on fucose, calcium is important for maintaining a properly folded lectin domain for optimal engagement of carbohydrate or specific sulfate esters. Indeed, this is the case with other calcium-type lectins (68, 69). Further study of this issue is warranted.

The MECA-79 antibody has been an invaluable probe for L-selectin ligands on HEVs of normal lymphoid organs and on activated endothelium at sites of chronic inflammation (6). Sulfation, but not fucosylation or sialylation, is required for this epitope, but detailed structural information has been lacking (15, 17). Recent experiments using a cloned sulfotransferase have revealed an essential contribution of GlcNAc-6-SO4 for MECA-79 binding, but again did not illuminate a structural context for this modification (60). The experiments conducted here showed that 6-sulfolactose supported MECA-79 binding considerably better than the other sulfated derivatives. This result is consistent with the demonstrated requirement for GlcNAc-6-SO4, but the inactivity of the 6',6-disulfated derivative was unexpected. In light of our results, one possibility is that the function-blocking activity of this antibody is achieved by neutralizing recognition determinants such as 6-sulfo-sLeX and 6-sulfo-N-acetyllactosamine. The reduced affinity of MECA-79 for 6'-sulfolactose and 6',6-disulfolactose may signify that this antibody cannot directly neutralize the contribution of Gal-6-SO4 to ligand activity. These considerations may explain the variable ability of MECA-79 to block lymphocyte attachment to HEVs at different anatomical sites (40, 70, 71).

The studies reported here advance our understanding of the minimal sulfated structures that may be involved in L-selectin binding. A key question for future work will be to define the full recognition determinants of its endothelial ligands. There is considerable indirect evidence that the structures of these determinants differ as a function of the anatomical location of the secondary lymphoid organ (34, 72). Varying contributions from Gal-6-SO4 and GlcNAc-6-SO4 may underlie this apparent diversity of recognition elements. The need for further information also applies to the inducible L-selectin ligands on non-lymphoid endothelium, which are implicated in the inflammatory trafficking of leukocytes (41, 73).

    ACKNOWLEDGEMENT

We thank Mark Singer for assistance with the cell binding assay and for providing the human L-selectin/human IgM chimera.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants MERIT R37GM23547 and R01GM5741 (to S. D. R.) and Grant R01GM59907-01 (to C. R. B.).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.

To whom correspondence should be addressed. Tel.: 415-476-1759; Fax: 415-476-4845; E-mail: sdr@itsa.ucsf.edu.

Published, JBC Papers in Press, August 10, 2000, DOI 10.1074/jbc.M001703200

2 R. E. Bruehl, S. D. Rosen, and C. R. Bertozzi, submitted for publication.

3 R. E. Bruehl and S. D. Rosen, unpublished data.

    ABBREVIATIONS

The abbreviations used are: HEVs, high endothelial venules; sLeX, sialyl-LeX (NeuAcalpha 2right-arrow3Galbeta 1right-arrow4(Fucalpha 1right-arrow3)GlcNAc); aLeX, sialyl-LeX analog ((-O2CCH2O-3)Galbeta 1right-arrow4(Fucalpha 1right-arrow3)Glc); mAb, monoclonal antibody; BSA, bovine serum albumin; BCECF/AM, 2',7'-bis(2-carboxyethyl)-5(6')-carboxyfluorescein acetoxymethyl ester; PBS, phosphate-buffered saline. With the exception of fucose, which is in the L- configuration, all sugars are in the D-configuration.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Gallatin, W. M., Weissman, I. L., and Butcher, E. C. (1983) Nature 304, 30-34
2. Arbonés, M. L., Ord, D. C., Ley, K., Ratech, H., Matnard-Curry, C., Otten, G., Capon, D. J., and Tedder, T. F. (1994) Immunity 1, 247-260
3. Geoffroy, J. S., and Rosen, S. D. (1989) J. Cell Biol. 109, 2463-2469
4. Bertozzi, C. R. (1995) Curr. Biol. 2, 703-708
5. Kansas, G. S. (1996) Blood 88, 3259-3287
6. Rosen, S. D. (1999) Am. J. Pathol. 155, 1013-1020
7. Lasky, L. A., Singer, M. S., Dowbenko, D., Imai, Y., Henzel, W., Fennie, C., Watson, S., and Rosen, S. D. (1992) Cold Spring Harbor Symp. Quant. Biol. 57, 259-269
8. Baumhueter, S., Singer, M. S., Henzel, W., Hemmerich, S., Renz, M., Rosen, S. D., and Lasky, L. A. (1993) Science 262, 436-438
9. Puri, K. D., Finger, E. B., Gaudernack, G., and Springer, T. A. (1995) J. Cell Biol. 131, 261-270
10. Sassetti, C., Tangemann, K., Singer, M. D., Kershaw, D. B., and Rosen, S. D. (1998) J. Exp. Med. 187, 1965-1975
11. Rosen, S. D., Singer, M. S., Yednock, T. A., and Stoolman, L. M. (1985) Science 228, 1005-1007
12. Malý, P., Thall, A. D., Petryniak, B., Rogers, C. E., Mith, P. L., Marks, R. M., Kelly, R. J., Gersten, K. M., Cheng, G., Saunders, T. L., Camper, S. A., Camphausen, R. T., Sullivan, F. X., Isogai, Y., Hinndsgaul, O., Von Andrian, U. H., and Lowe, J. B. (1996) Cell 86, 643-653
13. Imai, Y., Singer, M. S., Fennie, C., Lasky, L. A., and Rosen, S. D. (1991) J. Cell Biol. 113, 1213-1221
14. Imai, Y., Lasky, L. A., and Rosen, S. D. (1993) Nature 361, 555-557
15. Hemmerich, S., Butcher, E. C., and Rosen, S. D. (1994) J. Exp. Med. 180, 2219-2226
16. Crommie, D., and Rosen, S. D. (1995) J. Biol. Chem. 270, 22614-22624
17. Shailubhai, K., Streeter, P. R., Smith, C. E., and Jacob, G. S. (1997) Glycobiology 7, 305-314
18. Hemmerich, S., Bertozzi, C. R., Leffler, H., and Rosen, S. D. (1994) Biochemistry 33, 4820-4829
19. Hemmerich, S., and Rosen, S. D. (1994) Biochemistry 33, 4830-4835
20. Hemmerich, S., Leffler, H., and Rosen, S. D. (1995) J. Biol. Chem. 270, 12035-12047
21. Rosen, S. D., and Bertozzi, C. R. (1994) Curr. Opin. Biol. 6, 663-673
22. Varki, A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7390-7397
23. Fukuda, M., Hiraoka, N., and Yeh, J. C. (1999) J. Cell Biol. 147, 467-470
24. Foxall, C., Watson, S. R., Dowbenko, D., Fennie, C., Lasky, L. A., Kiso, M., Hasegawa, A., Asa, D., and Brandley, B. K. (1992) J. Cell Biol. 117, 895-902
25. McEver, R. P., Moore, K. L., and Cummings, R. D. (1995) J. Biol. Chem. 270, 11025-11028
26. Simanek, E. E., McGarvey, G. J., Jablonowski, J. A., and Wong, C.-H. (1998) Chem. Rev. 98, 833-862
27. Scudder, P. R., Shailubhai, K., Duffin, K. L., Streeter, P. R., and Jacob, G. S. (1994) Glycobiology 4, 929-933
28. Galustian, C., Lubineau, A., le Narvor, C., Kiso, M., Brown, G., and Feizi, T. (1999) J. Biol. Chem. 274, 18213-18217
29. Yoshino, K., Ohmoto, H., Kondo, N., Tsujishita, H., Hiramatsu, Y., Inoue, Y., and Kondo, H. (1997) J. Med. Chem. 40, 455-462
30. Sanders, W. J., Katsumoto, T. R., Bertozzi, C. R., Rosen, S. D., and Kiessling, L. L. (1996) Biochemistry 35, 14862-14867
31. Green, P. J., Tamatani, T., Watanabe, T., Miyasaka, M., Hasegawa, A., Kiso, M., Yuen, C.-T., Stoll, M. S., and Feizi, T. (1992) Biochem. Cell Biol. 188, 244-251
32. Green, P. J., Yuen, C.-T., Childs, R. A., Chai, W., Miyasaka, M., Lemoine, R., Lubineau, A., Smith, B., Ueno, H., Nicolaou, K. C., and Feizi, T. (1995) Glycobiology 5, 29-38
33. Mitsuoka, C., Kawakami-Kimura, N., Kasugai-Sawada, M., Hiraiwa, N., Toda, K., Ishida, H., Kiso, M., Hasegawa, A., and Kannagi, R. (1997) Biochem. Cell Biol. 230, 546-551
34. Mitsuoka, C., Sawada-Kasugai, M., Ando-Furui, K., Izawa, M., Nakanishi, H., Nakamura, S., Ishida, H., Kiso, M., and Kannagi, R. (1998) J. Biol. Chem. 273, 11225-11233
35. Tsuboi, S., Isogai, Y., Hada, N., King, J. K., Hindsgaul, O., and Fukuda, M. (1996) J. Biol. Chem. 271, 27213-27216
36. Koenig, A., Jain, R., Vig, R., Norgard-Sumnicht, K. E., Matta, K. L., and Varki, A. (1997) Glycobiology 7, 79-93
37. Sanders, W. J., Gordon, E. J., Dwir, O., Beck, P. J., Alon, R., and Kiessling, L. L. (1999) J. Biol. Chem. 274, 5271-5278
38. Galustian, C., Lawson, A. M., Komba, S., Ishida, H., Kiso, M., and Feizi, T. (1997) Biochem. Cell Biol. 240, 748-751
39. Bertozzi, C. R., Fukuda, S., and Rosen, S. D. (1995) Biochemistry 34, 14271-14278
40. Streeter, P. R., Rouse, B. T. N., and Butcher, E. C. (1988) J. Cell Biol. 107, 1853-1862
41. Tu, L., Delahunty, M. D., Ding, H., Luscinskas, F. W., and Tedder, T. F. (1999) J. Exp. Med. 189, 241-252
42. Spevak, W., Dasgupta, F., Hobbs, C. J., and Nagy, J. O. (1996) J. Org. Chem. 61, 3417-3244
43. Singer, M., and Rosen, S. D. (1996) J. Immunol. Methods 196, 153-161
44. Lasky, L. A., Singer, M. S., Dowbenko, D., Imai, Y., Henzel, W. J., Grimley, C., Fennie, C., Gillett, N., Watson, S. R., and Rosen, S. D. (1992) Cell 69, 927-938
45. Knibbs, R. N., Craig, R. A., Natsuka, S., Chang, A., Cameron, M., Lowe, J. B., and Stoolman, L. M. (1996) J. Cell Biol. 133, 911-920
46. Pickford, L. B., Mayer, D. N., Bolin, L. M., and Rouse, R. V. (1989) J. Neurocytol. 18, 465-478
47. Imai, Y., True, D. D., Singer, M. S., and Rosen, S. D. (1990) J. Cell Biol. 111, 1225-1232
48. Brandley, B. K., Kiso, M., Abbas, S., Nikrad, P., Srivastava, O., Foxall, C., Oda, Y., and Hasegawa, A. (1993) Glycobiology 3, 633-641
49. Kim, Y. J., Borsig, L., Han, H. L., Varki, N. M., and Varki, A. (1999) Am. J. Pathol. 155, 461-472
50. Capon, C., Wieruszeski, J. M., Lemoine, J., Byrd, J. C., Leffler, H., and Kim, Y. S. (1997) J. Biol. Chem. 272, 31957-31968
51. Mannori, G., Crottet, P., Cecconi, O., Hanasaki, K., Aruffo, A., Nelson, R. M., Varki, A., and Bevilacqua, M. P. (1995) Cancer Res. 55, 4425-4431
52. Kishimoto, T. K., Jutila, M. A., and Butcher, E. C. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2244-2248
53. Yuen, C.-T., Lawson, A. M., Chai, W., Larkin, M., Stoll, M. S., Stuart, A. C., Sullivan, F. X., Ahern, T. J., and Feizi, T. (1992) Biochemistry 31, 9126-9131
54. Larkin, M. T., Ahern, T. J., Stoll, M. S., Shafer, M., Sako, D., O'Brien, J., Yuen, C.-T., Lawson, A. M., Childs, R. A., Barone, K. M., Langer-Safer, P. R., Hasegawa, A., Kiso, M., Larsen, G. R., and Feizi, T. (1992) J. Biol. Chem. 267, 13661-13668
55. Yuen, C.-T., Bezouska, K., O'Brien, J., Stoll, M., Lemoine, R., Lubineau, A., Kiso, M., Hasegawa, A., Bockovich, N. J., Nicolaou, K. C., and Feizi, T. (1994) J. Biol. Chem. 269, 1595-1598
56. Nelson, R. M., Cecconi, O., Roberts, W. G., Aruffo, A., Linhardt, R. J., and Bevilacqua, M. P. (1993) Blood 82, 3253-3258
57. Pouyani, T., and Seed, B. (1995) Cell 83, 333-343
58. Tangemann, K., Bistrup, A., Hemmerich, S., and Rosen, S. D. (1999) J. Exp. Med. 190, 935-942
59. Bistrup, A., Bhakta, S., Lee, J. K., Belov, Y. Y., Gunn, M. D., Zuo, F. R., Huang, C. C., Kannagi, R., Rosen, S. D., and Hemmerich, S. (1999) J. Cell Biol. 145, 899-910
60. Kimura, N., Mitsuoka, C., Kanamori, A., Hiraiwa, N., Uchimura, K., Muramatsu, T., Tamatani, T., Kansas, G. S., and Kannagi, R. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 4530-4535
61. Hiraoka, N., Petryniak, B., Nakayama, J., Tsuboi, S., Suzuki, M., Yeh, J. C., Izawa, D., Tanaka, T., Miyasaka, M., Lowe, J. B., and Fukuda, M. (1999) Immunity 11, 79-89
62. Dasgupta, F., and Rao, B. N. N. (1994) Exp. Opin. Invest. Drugs 3, 709-724
63. Uchimura, K., Muramatsu, H., Kaname, T., Ogawa, H., Yamakawa, T., Fan, Q. W., Mitsuoka, C., Kannagi, R., Habuchi, O., Yokoyama, I., Yamamura, K., Ozaki, T., Nakagawara, A., Kadomatsu, K., and Muramatsu, T. (1998) J. Biochem. (Tokyo) 124, 670-678
64. Ng, K. K., and Weis, W. I. (1997) Biochemistry 36, 979-988
65. Malhotra, R., Priest, R., and Bird, M. I. (1996) Biochem. J. 320, 589-593
66. Koenig, A., Norgard-Sumnicht, K., Linhardt, R., and Varki, A. (1998) J. Clin. Invest. 101, 877-889
67. Malhotra, R., Taylor, N., and Bird, M. I. (1996) Biochem. J. 314, 297-303
68. Ng, K. K., Park-Snyder, S., and Weis, W. I. (1998) Biochemistry 37, 17965-17976
69. Ng, K. K., and Weis, W. I. (1998) Biochemistry 37, 17977-17989
70. Clark, R. A., Fuhlbrigge, R. C., and Springer, T. A. (1998) J. Cell Biol. 140, 721-731
71. Michie, S. A., Streeter, P. R., Bolt, P. A., Butcher, E. C., and Picker, L. J. (1993) Am. J. Pathol. 143, 1688-1698
72. Berg, E. L., Mullowney, A. T., Andrew, D. P., Goldberg, J. E., and Butcher, E. C. (1998) Am. J. Pathol. 152, 469-477
73. Zakrzewic, A., Gräfe, M., Terbeek, D., Bongrazio, M., Auch-Schwelk, W., Walzog, B., Graf, K., Fleck, E., Ley, K., and Gaehtgens, P. (1997) Blood 89, 3228-3235

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 GlycobiologyHome page
N. Hiraoka, B. Petryniak, H. Kawashima, J. Mitoma, T. O Akama, M. N Fukuda, J. B Lowe, and M. Fukuda
Significant decrease in {alpha}1,3-linked fucose in association with increase in 6-sulfated N-acetylglucosamine in peripheral lymph node addressin of FucT-VII-deficient mice exhibiting diminished lymphocyte homing
Glycobiology, March 1, 2007; 17(3): 277 - 293.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
K. Uchimura, K. Kadomatsu, F. M. El-Fasakhany, M. S. Singer, M. Izawa, R. Kannagi, N. Takeda, S. D. Rosen, and T. Muramatsu
N-Acetylglucosamine 6-O-Sulfotransferase-1 Regulates Expression of L-Selectin Ligands and Lymphocyte Homing
J. Biol. Chem., August 13, 2004; 279(33): 35001 - 35008.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
N. Hiraoka, H. Kawashima, B. Petryniak, J. Nakayama, J. Mitoma, J. D. Marth, J. B. Lowe, and M. Fukuda
Core 2 Branching {beta}1,6-N-Acetylglucosaminyltransferase and High Endothelial Venule-restricted Sulfotransferase Collaboratively Control Lymphocyte Homing
J. Biol. Chem., January 23, 2004; 279(4): 3058 - 3067.
[Abstract] [Full Text] [PDF]

Home page
 GlycobiologyHome page
M. Delcommenne, R. Kannagi, and P. Johnson
TNF-{alpha} increases the carbohydrate sulfation of CD44: induction of 6-sulfo N-acetyl lactosamine on N- and O-linked glycans
Glycobiology, October 1, 2002; 12(10): 613 - 622.
[Abstract] [Full Text] [PDF]