A carbohydrate domain common to both sialyl Le (a) and sialyl Le (X ...

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Olle Mansson11 , Eugene C. Butcher$, and. John L. Magnani**$$ ..... Johnson, G. I., Cook, R. G., and McEver, R. P. (1989) Cell 56,1033-1044. 2 4 3 , 1160-1165.
T H EJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 266, No. 23, Issue of August 15, pp. 14869-14872.1991 Printed in U.S.A.

Communication

recently by the identification of several key inflamA Carbohydrate DomainCommon forced matory cell adhesion receptors, the LEC-CAMS or selectins, to Both Sialyl Le" and Sialyl LeX (1,2). These molecules comprise as calcium-dependent lectins a gene family whosethree members, ELAM-1, LECAM-1 (the Is Recognized bythe Endothelial lymph node homingreceptor),andGMP-140 Cell Leukocyte Adhesion Molecule peripheral (PADGEM or CD62),display similar domain structures conELAM- l* sisting of anN-terminaldomain homologous to calciumdependent lectins, an epidermalgrowth factor-like domain, a variable numberof repeat sequences similar to those found in Ellen L. Berg$$, Martyn K.Robinson$ll, complement regulatory proteins, a transmembrane domain, Olle Mansson11, Eugene C. Butcher$, and andshortcytoplasmictail (3-7). One of the LEC-CAMS, John L. Magnani**$$ endothelial cell leukocyte adhesion molecule-1 (ELAM-l),' is From the $Department of Pathology, Stanford University, a n inducible endothelial cell antigen believed to be involved Stanford, California 94305, Veterans Administration in the initial adhesion of neutrophils to the endothelium at Medical Center, Palo Alto, California 94304,11 BioCarb sites of acuteinflammation (8, 9). Recently,a neutrophil Technology AB, S-223 70 Lund, Sweden, and **BioCarb Inc., Gaithersburg, Maryland 20879 carbohydrate ligand for ELAM-1 has beenidentified by a number of investigators as sialylated lacto-N-fucopentaose I11 The specificityof endothelial cell leukocyte adhesion (NeuAca2,3-Gal~l-4(Fuc~l,3)-GlcNAc), the sialylatedLewis molecule- 1,ELAM- 1, for binding to a panel of carbo- X antigen (Sialyl Le") (10-12). hydrate structures was determinedby a sensitive cell ELAM-1 does not appear tobe an adhesionmolecule exclubinding assay with immobilized synthetic glycoconju- sively for neutrophils, however, or for Sialyl Le". Our laboragates. ELAM- 1cDNA transfectants were found to bind tory haspreviously identified a subpopulation of lymphocytes Sialyl Le" (sialylated lacto-N-fucopentaose 11) or sial- that bind avidly to ELAM-1 transfectants(13) although lymylated Lewis a antigen (NeuAccu2-3Gal@1-3(Fuc~l- phocytes do not express significant levels of Sialyl Le" (14). 4)GlcNAc), as well as or slightly better than Sialyl Le" These lymphocytes, which are found selectively in sites of (sialylated lacto-N-fucopentaose 111) or sialylated chronic inflammation in the skin, are characterized by the Lewis X antigen (NeuAca2-3Gal@l-4(Fuccu1-3)- expression of anovel carbohydrate antigen, the cutaneous GlcNAc). A monoclonal antibody, HECA-452, which has been identified recently as recognizing ELAM-1 lymphocyte-associatedantigen, CLA, identifiedbymonoligands in addition to those containing Sialyl Le", was clonal antibody HECA-452 (15). HECA-452 recognizes neurto also found to bind both Sialyl Le" and SialylLe". Hard aminidase-sensitive carbohydrate structures that appear compriseELAM-1 ligands inadditionto Sialyl Le". For sphere exo-anomeric (HSEA) calculations were performed on these two hexasaccharides. The conforma- example, the HECA-452 antigen isolated from tonsil orfrom tions indicate that Sialyl Le" and Sialyl Le" show a myeloid cells binds ELAM-1 cDNA transfectants, and this high degree of similarity in both the nonreducing andbinding is inhibitedby mAb HECA-452.Furthermore HECAreducing termini. As Le" and Le" show much weaker 452 inhibits adhesionof CLA' lymphocytes as well as neutrophils to ELAM-1 (data not shown). In this paper, ELAM-1 reactivity, the determinants recognized by ELAM-1 and HECA-452 probably involve neuraminic acid and and HECA-452 were found to share a similar specificity for fucose residues which on one face of both Sialyl Le" oligosaccharide structure.Mostinterestingly,bothHECAand SialylLe" can be similarly positioned. The finding 452 and cells transfected with ELAM-1 cDNAwere found to that SialylLe" is a potent ligand forELAM-1 is impor- bind Sialyl Le', a n oligosaccharide structure not normally tant, as circulating SialylLe" and SialylLe" containing found on either neutrophils or lymphocytes (16), as well as mucins whichare elevated in the serum of many cancer Sialyl Le". This finding has implicationsfor the components patients mayblock leukocyte interactions with ELAM- and conflguration of these carbohydrates that are likely to 1 and maycontributetothepathological immuno- directly interact with ELAM-1 andHECA-452 binding sites. depression observed in these patients. Furthermore, Sialyl Le" and Sialyl Le" are important tumor antigens found in high levels in the serum of patients with gastrointestinal, pancreatic, and breast cancer(16-18). Intercellular interactions have long been postulated to be EXPERIMENTALPROCEDURES influenced by the recognition of carbohydrates by membrane receptors. The importanceof cell surface carbohydrate strucSynthetic Glycoproteins (Neoglycoproteins-The neoglycoproteins tures to the targeting and positioningof cells has been rein- used in this paperwere produced a t BioCarb AB (Lund, Sweden) by (Received for publication, April 23, 1991)

* This work was supported in partby National Institutes of Health Grant GM37734. The costs of publication of this articlewere defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" inaccordancewith 18 U.S.C. Section 1734 solely to indicate this fact. ยง Senior Fellow of the Leukemia Society. ll Present address: CellTech Ltd., 216 Bath Rd., Slough, SL1 4EN, Berkshire, Great Britain. $$ TOwhom correspondence should be addressed: BioCarb Inc., 300 Professional Dr., Gaithersburg, MD 20879.

The abbreviations used are: ELAM-1, endothelial cell leukocyte adhesion molecule-1; Sialyl Le", sialylated lacto-N-fucopentaose 11; Sialyl Le", sialylated lacto-N-fucopentaose 111; CLA, cutaneous lymphocyte-associated antigen;mAb, monoclonal antibody: HSA, human serum albumin: PAP,p-aminophenyl; APE, aminophenylethyl; APD, acetyl phenylenediamine;ELISA,enzyme-linkedimmunoassays; PBS,phosphate-bufferedsaline:HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; DMEM, Dulbecco'smodifiedEagle's medium; HSEA, hard sphere exo-anomeric;Fuc, fucose.

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Sialyl Le" and Sialyl Le" Bind ELAM-1

chemically coupling 10-20mol of a oligosaccharide to 1 molof nonglycosylated albumin (bovine or human). The hapten densities of the Sialyl Le"-HSA and Sialyl Le"-HSA neoglycoproteins are 15 and 12, respectively. The resulting synthetic glycoprotein (neoglycoprotein) contains multiple copies of the identical carbohydrate sequence, thereby producing a well characterized multivalent glycoconjugate that is extremely effective for studying carbohydrate-protein interactions. Depending on the size of the oligosaccharide, three different chemical spacer arms were used to couple the oligosaccharides to proteins: 1)p-aminophenyl (PAP), 2) aminophenylethyl (APE), and 3) acetyl phenylenediamine (APD) (19-21). PAP andAPE were used to couple the shorter oligosaccharides to albumin since they will retain the anomeric configuration of the reducing sugars that may be involved in a potential binding site. ADP was used to couple the larger sugars to protein by reductive amination, which converts the reducing sugar to an aminoalditol. The abbreviation of the chemical spacer arm coupled to each oligosaccharide in a conjugate is presented in the legend for Fig. 1. Purity was verified by NMR analysis. Monoclonal Antihodies-The monoclonal antibodies employed in these studies include the following: HECA-452 (anti-CLA), arat IgM (15, 22); MECA-79 (anti-peripheral lymph node addressin), rat IgM (23); PA3-2C2, rat IgM control (24); CL2 (anti-ELAM-1), a mouse IgG, kindly supplied by C. Wayne Smith (Baylor College of Medicine, Houston, TX) (13); Dreg-56 (anti-human LECAM-1) mouse IgGl (25); CSLEX-1, (anti-Sialyl Le") (26), a mouse IgM kindly supplied by P. Terasaki (UCLA); and lHlO (anti-Sialyl Le"), a mouse IgG,. Direct Binding of Antibodies to Synthetic Glycoproteins (Neoglycoproteins)-Synthetic glycoproteins were coated onto microtiter plates by filling each well with 100 ng of the neoglycoprotein in 100 pl of phosphate-buffered saline (PBS), pH 7.4, 0.1% azide, overnight at 4 "C. Enzyme-linked immunoassays (ELISA) were then performed onthe solid phase carbohydratestructures using the appropriate antibody diluted to 10 pg/ml. Production of ELAM-1 cDNA-tranfected Cell Lines-L1-2/ pMRB107 cells (L1-2ELAM")were prepared by transfecting the ELAM-1 gene into the murine pre-B cell line L1-2 (27, 28). A cDNA clone encoding ELAM-1 was obtained from a cDNA library made from activated human umbilical vein endothelial cell cultures by polymerase chain reaction amplification. The ELAM-1 genewas inserted downstream of the hCMV promoter in pMRBlOl (a derivative of EE6 which contains the Escherichia coli gpt gene (29, 30)). DNA was introduced into L1-2 cells by electroporation and the cells selected for resistance to mycophenolic acid. The identity of the cDNA was confirmed by restriction analyses and by comparison of the expressed product with ELAM-1 expressed by an independent ELAM. 1 cDNA clone provided by Dr. B. Seed, Harvard University (5). COS cells transfected with both ELAM-1 cDNAs in pCDM8 reacted with anti-ELAM-1 and gave indistinguishable results in assays of neutrophil and T cell binding (13). A population of cells staining brightly for ELAM-1 was selected by fluorescence-activated cell sorting and cloned by limiting dilution. These cells are ELAMIh'LFA-lmnd CD45h' CDUneg LECAM-lneg, differing from the parent cell line or control vector transfectants only in their expression of ELAM-1. L1-2/pMRB101 (Ll-2""'"') cells area similarly transformed derivative ofL1-2 transfected with pMBRlOl and lack ELAM-1 expression. Cell Binding Assays-One hundred-microliter samples of each synthetic glycoconjugate in PBS, pH 7.2, were absorbed onto glass wells of eight-chamber slides (LabTek) for 2 h at room temperature. For some experiments glass slides were pre-coated with rabbit anti-human serum albumin (Sigma) at 200 pg/ml overnight at 4 "C and washed with PBS prior to the addition of the glycoconjugate. After blocking with 5% newborn bovine serum, 10 mM HEPES in Dulbecco's modified Eagle's medium (DMEM),pH 7.0 (CM), L1-ZELAM" or L1-2"'t0r cells were applied to each well (1.5 X 106/0.15 ml in CM). After a 25min incubation at room temperature on a rotating shaker at 50 rpm, the tops of the wells were removedand theslides washed three times in DMEM andthen fixed by incubation in 1.5% glutaraldehyde (Kodak) in DMEM. Three to six 1 0 0 ~ fields were counted for each data point, and the average and standard error are reported. Data reported are from representative experiments which were performed two to five times with similar results. Hard SphereExo-anomeric (HSEA) Calculations-Conformational models of the oligosaccharides in solution were obtained by HSEA calculations. Hydroxyl groups are represented by the oxygen atoms. A fixed bond angle of 117" was used for the glycosidic linkages. The energy calculated by an HSEA potential (31) was minimized using simultaneous variation of dihedral angles (multidimensional binary

chop). This algorithm shows a slow convergence near a local minimum, when compared with other methods utilizing the firstand second derivative, but has the advantage of allowing a large initial search area of the conformational space, whereby the chances of finding the lowest local minima increases. Other applications of this program are described in Refs. 32 and 33. RESULTSANDDISCUSSION

Carbohydrate Epitope of Antibody HECA-452"Antibody HECA-452 was tested for direct binding to a wide variety of carbohydrate structures by assaying a panel of neoglycoproteins that were produced by chemically coupling specific oligosaccharides to serum albumin. As shown in Fig. l A , HECA452 binds both Sialyl Le" and Sialyl Le" hexasaccharides but not a large variety of other similar but not identical carbohydrate sequences. In comparison, antibody CSLEX-1, which has been reported to bind Sialyl Le" (26), recognizes an epitope specific to Sialyl Le" and not present in Sialyl Le" (Fig. 1B). Likewise, antibody 1H10, developed at BioCarb, binds to Sialyl Le" but not Sialyl Le". lHlO also weakly crossreacts with LSTa which is defucosylated Sialyl Le" (Fig. IC).

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11.5

0.5

0 Neoglycoconjugate

10

20

(Ccdml)

FIG. 1. Direct binding of antibodies to neoglycoproteins. The average value of duplicate wells coated with a neoglycoprotein and assayed by ELISA is depicted by histograms. The structures of the carbohydrates and chemical linker arm on the neoglycoproteins in A-C, presented from left to right are: lacto-N-fucopentaose I-APD (H-type l), lacto-N-fucopentaose 11-APD (Lea), lacto-N-fucopentaose 111-APD (Lex), lacto-N-difuconeohexaoseI-APE (Ley), lactoN-difucohexaose I-APD (Leb), maltose-PAP, lactose-PAP, 1acto-Ntetraose-APD, lacto-N-neotetraose-APD, lacto-N-hexaose-APD, lacto-N-neohexaose-APD, melibiose-PAP, cellobiose-PAP, A-trisaccharide-APE, B-trisaccharide-APE, A-tetrasaccharide-APD, A-heptasaccharide-APD, 2 fucosyllactosamine-APE (H-type 2), gangliotetraose-APD, T-antigen-APE, 3'-sialylactose-APD, 6'-sialylactoseAPD, sialyllacto-N-tetraose a-APD(LSTa), sialyllacto-N-tetraose bAPD (LSTb), sialyllacto-N-tetraose c-APD (LSTc), sialylated lactoN-fucopentaose 11-APD (SLe"), sialylated lacto-N-fucopentaose IIIAPD (SL"),disialylated lacto-N-tetraose-APD, chitotriose-APD, ManaGlcNAc-APD,Man2GlcNAc-APD,biantennery-octasaccharideAPD,globotriose-APD,globotetraose-APD,GlcNAcT-APE, difucosyl Le"/Le"-APE, and difucosyl Le"-APE. The last sample represents the background binding to nonglycosylated bovine serum albumin. The two samples followingthe line of separation are internal controls representing an anti-Leaantibody (BioCarb Product 90/40) binding to theLea-active neoglycoprotein and bovine serum albumin, respectively. A-C represent the screening of neoglycoproteins by antibodies HECA-452, CSLEX-1, and 1H10, respectively. Neoglycoproteins that bound antibodies (Sialyl Lea,B, Sialyl Le", 0 , LSTa, 0, bovine serum albumin, A) were used to assay the binding of varying concentrations of antigen to a constant concentration of antibodies (10 pg/ml). D, E, and F represent the binding curves for antibodies HECA-452, CSLEX-1, and 1H10, respectively.

Sialyl Le" and Sialyl Le" Bind ELAM-1

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FIG. 2. Direct binding of ELAM-1-transfected cells to neoglycoproteins. L1-2"',AM" cells were incubated with glass slides previously coated with neoglycoproteins as described under "Experimental Procedures." Alter washing and fixation the number of cells bound per X 100 field was determined microscopically. The number of L1-2""'"' (control) cells bound per X 100 field was 23.

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Titration of the antigens (Fig. 1, D-F) further demonstrates that HECA-452 binds both Sialyl Le" and Sialyl Le" with similar relative affinities, whereas CSLEX-1 and lHlO maintain specificity to either Sialyl Le" or Sialyl Lea, respectively. Carbohydrate Structure Recognized by ELAM-1-Studies fromourlaboratories have indicatedthatELAM-1binds structures other thanSialyl Le". A population of skin-homing memory T lymphocytes, characterized by their expression of a carbohydrate antigen, the cutaneous lymphocyte-associated 10 100 1000 antigen (CLA) defined by antibody HECA-452, was found to [ugW interact specifically with ELAM-1 even though lymphocytes FIG. 3. Comparison of the relative affinities of neoglycodo not express significant levels of Sialyl Le" (14-15). Fur- proteins with ELAM-1-transfected L1-2 cells. The number of L I - ~ E L A M - I cells bound to glass slides coated with various neoglycothermore, when isolated from various sources, glycoproteins recognized by the anti-CLA, mAb, and HECA-452 are adhe- proteins was determined microscopically. A, L1-ZELA"' cells bound sive for ELAM-1 cDNA transfectants (data not shown). In- most strongly to Sialyl Le" followed by Sialyl Le" and weakly to Le". deed, the association between expression of the HECA-452 B, weak binding of L1-2ELAM"cells to nonsialylated oligosaccharides is observed at higher concentrations of neoglycoproteins. An exepitope and the ability to bind ELAM-1 appeared to be quite panded y axis reveals weak binding to Le" and very little binding to close and suggested that ELAM-1 and HECA-452 recognize Le". In these experiments slides were preincubated with polyclonal very similar carbohydrate structures. Therefore the reactivity anti-HSA antibodies prior to coating with HSA-glycoconjugates(neoof HECA-452 with Sialyl Le" raised the possibility that this glycoproteins). Similar results are obtained without this step. carbohydrate, in addition, might be recognized by ELAM-1. We developed a sensitive binding assay usingcells perma- stereochemically in that the galactose and thefucose residues nently transfected with ELAM-1 cDNA. The mouse pre-B are attached toGlcNAc in the4 and 3 positions, respectively, cell line L1-2, transfected with ELAM-1 cDNA(L1-2ELAM"), in Sialyl Le" (type 2 chain) and in the reverse positions in but not vector control cDNA, L1-2""ct0', expresses very high Sialyl Le" (type 1 chain): levels of ELAM-1 (data not shown). The ELAM-1 expressed Neu5Aca2-3Gal~l-x-GlcNAcpl-3Galpl-4(Glc)-ADP by these cells is functional as L1-2ELAM"cells are adhesive 3' for neutrophils, and this adhesion is blocked by anti-ELAMI 1 monoclonal antibodies.' When added to glass slides coated Fuca 1 with various synthetic glycoconjugates, L1-2ELAM"cells bound selectively to Sialyl Le" and Sialyl Le" neoglycoproteins but where x and y are 3 and 4 for SialylLe" and x and y are 4 and Lex, respectively. The low level of binding of not to a number of other glycoconjugates (Fig. 2). L1-2ELAM" 3forSialyl cells also bound, albeit more weakly, to Le" neoglycoprotein. ELAM-1 transfectants to Le" is consistent with an essential T h e binding to Le" is significant as L1-2ELAM"cells bound role of fucose in recognition (10-12) and argues that neurapoorly to Le" and not at all to the glycoconjugates prepared minic acid also plays a key role. Furthermore, as the binding with the structural analogs such as LNFI (Fig. 3). That L1- to eitherSialyl Le"-HSA orSialyl Le"-HSA is totally inhibited 2ELAM.l cells did not bind other monosialylated carbohydratesby SialylLe"-HSA (data not shown), ELAM-1 contains a binding site that recognizes a carbohydrate domain common suchas3'SL,6'SL,LSTaorLSTc (Fig. 2 and data not shown) demonstrates that the bindingSialyl to Le" and Sialyl to both the Sialyl Le" and Sialyl Le" antigens. Graphic Representation of the Carbohydrate Domain RecLe" is not due tononspecific charge effects but rather reflects specific structural features of these oligosaccharides. Our re- ognized by ELAM-I-The calculations show high similarity sults confirm that ELAM-1 andHECA-452 recognize a very in the corresponding dihedral angles for the two structures, linkage between the N-acetylsimilar carbohydrate domain, presented by both the Sialyl also at the bonds with different Le" and Sialyl Le" antigens. As depictedin Fig. 4, these glucosamine andthe fucose and Neu5Aca2-3Ga1, respechexasaccharides have the same sugar composition but differ tively. The only major discrepancy is a difference in 118"for 6 position of the glucose the hydroxymethyl group in the T. Yoshino and R. A. Warnock, personal observations. residue at the reducing terminal which is a result of the very

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Sialyl Le" and Sialyl Le" Bind ELAM-1 tified this epitope as atumor-associated antigen (16).Mucins containing both of these structures areelevated in the sera of cancer patients, including gastrointestinal, pancreatic, and breast cancer patients (16-18). Preliminary experiments indicate that some Sialyl Le"- and Sialyl Lex-containingmucins and the Sialyl Le" glycolipid (16) are recognized by ELAM-1 transfectants (data notshown). By interacting with ELAM-1 on venules in acute and chronically inflammed tissues and interfering with the recruitment of leukocytes to these locations, these mucins secreted by tumors may contribute to the immunodepressed state of cancer patients. Acknowledgments-We are grateful to Dr. David Zopf for helpful discussions, to Cynthia Andrews for technical assistance, and toCarol Culwell and Cheryl Lambert for expert secretarial assistance. REFERENCES 1. Brandley, B. K, Swidler,S. J., and Robbins, P. W. (1990) Cell 6 3 , 861-863 2. Springer, T. A,, and Lasky, L. (1990) Nature 3 4 9 , 196-197 M., and Weissman, I. L. (1989) Science 3. Siegelman, M. H., van de Rijn, 2 4 3 , 1165-1172 4. Lasky, L. A,, Singer, M. S., Yednock, T. A,, Dowbenko, D., Fennie, C., Rodriguez, N., Nguyen, T., Stachel, S., and Rosen, S. D. (1989) Cell 5 6 , 1045-1055 5. Bevilacqua, M., Stenglein, S., Gimbrone, M., and Seed, B. (1989) Science 2 4 3 , 1160-1165 6. Johnson, G. I., Cook, R. G., and McEver, R. P. (1989) Cell 56,1033-1044

FIG. 4. Computer-generated graphic representation of Sialyl Le" and Sialyl Lex hexasaccharides as determined by HSEA calculations. A stereopair of Sialyl Le" and Sialyl Le" hexasacacharides, computer-generated by HSEA calculations, are presented in A and B, respectively. Atoms are displayed as spheres with radii proportional to the van der Waals radii. Hydroxyl groups are represented by the oxygen atoms. Carbons are coded d a r k g r a y , oxygens are light gray, and nitrogens and hydrogens (substantially smaller than nitrogens) are white. The models are oriented so as to keep the sialic acid-substituted galactose residues in the same position in both case A and B. The reducing terminals of both carbohydrate images are located on the right (2 o'clock position), the fucose residues at theupper left (10o'clock position) and the sialic acids in the lower p a r t (6 o'clock position) of the figures. Three-dimensional images of the oligosaccharide surfaces are revealed when the figure is observed through a stereo viewer.

nearly equal energies for this molecular group after a rotation of 120". As this group is far away from the linkages differing between Sialyl Le" and Sialyl Lex, its direction is of no importance for the conformational structure in this region. The resulting computer-generated stereo images of the structures arerepresented graphically in Fig. 4. The conformations indicate that the structures show a high degree of similarity inboth the nonreducing and reducing terminalparts. In particular, the structures of the terminal carbohydrate sequence up to, but not including, the N-acetyl group on the internal GlcNAc residue show a high degree of homology and may represent the domain recognized by both ELAM-1 and the monoclonal antibody HECA-452. As the conformations are based solely on HSEA calculations, this area of structural homology requires empirical evidence as itmay beparticularly useful for the design of potential anti-inflammatory drugs. Examples of other carbohydrate-binding proteinsthat recognize type 1 and type 2 chain isomers are the antibodies E123-48 and E166-18which bind the blood group B antigen (34) andthe lectin, Griffoniasirnplicifolia IV, which recognizes both Leh and LeYantigens (35). The recognition of the Sialyl Le" antigen andthe Sialyl Le" antigen by ELAM-1 may be of pathologic importance. The first description of the Sialyl Le" carbohydrate structureiden-

7. Tedder, T. F., Isaacs, C. M., Ernst, T. J., Demetri, G. D., Adler, D.A., and Disteche, C. M. (1989) J. Exp. Med. 1 7 0 , 123-133 8. Bevilacqua, M. P.,Pober, J. S., Mendrick, D. L., Cotran, R. S., and Gimbrone, M. A. (1987) Proc. Natl. Acad. Sci. U. S. A. 84,9238-9242 9. Cotran, R. S., Gimbrone, M. A., Bevlicqua, M. P., Mendrick, D. L., and Pober, J. S. (1986) J. Exp. Med. 164,661-666 10. Lowe, J. B., Stoolman, L. M., Nair, R. P., Larsen, R. D., Berbend, T. L., and Marks, R. M. (1990) Cell 63,475-484 11. Phillips, M. L., Nudelman, E., Gaeta, F. C. A,, Perez, M., Singhal, A. K., Hakomori, %I., and Paulson, J. C. (1990) Science 2 5 0 , 1130-1132 12. Walz, G., Aruffo, A,, Kolanus, W., Bevlicqua, M., and Seed, B. (1990) Science 2 5 0 , 1132-1135 13. Picker, L.J., Kishimoto, T. K., Smith, C. W., Warnock, R.A,, and Butcher, E. C. (1991) Nature 349,796-799 14. Ohmori, K., Yoneda, R., Ishihara, G., Shigata, K., Hiroshima, K., Kanai, M., Itai, S., Sasaoki, T., Arii, S., Arita, H., and Kannagi, R. (1989) Blood 74.255-261 - -,--15. Picker, L. J., Michie, S. A,, Rott, L. S., and Butcher, E. C. (1990) Am. J. Pathol. 136,1053-1068 16. Magnani, J. L., Nilsson, B., Brockhaus, M., Zopf, D.,Steplewski, Z., Koprowski, H., andGinsburg, V. (1982) J. Biol. Chem. 2 5 7 , 1436514369 17. Magnani, J. L., Steplewski, Z., Koprowski, H., and Ginsburg, V. (1983) Cancer Res. 43,5489-5492 18. Kannagi, R., Fukushi, Y., Tachikawa, T., Noda, A., Shin, S., Shigeta, K., Hirauia, N., Fukiida, Y., Inamoto, T., Hakomori, S., and Imura, H.(1986) Cancer Res. 46,2619-2626 19. Kallin, E., Lonn, H., and Norberg, T. (1986) Glycoconjugate J. 3,311-319 20. Svenson, S. B., and Linberg, A.A. (1979) J. Immunol. Methods 2 5 , 323335 21. Zopf, D. A,, Smith, D. F., Drzeniek, Z., Tsai, C. M., and Ginsburg,V. (1978) Methods Enzymol. 5 0 , 171-175 22. Picker, L. J., Terstappen, L. W. M. M., Rott, L. S., Streeter, P. R., Stein, H., and Butcher, E. C. (1990) J. Zmmunol. 145,3247-3255 23. Streeter. P. R.. Rouse. B. T. N.. and Butcher. E.C. (1988) J. Cell Biol. 107, 1853-1862 ' 24. Coffman, R. L., and Weissman, I. L. (1981) J . Exp. Med. 153,269-279 25. Kishimoto, T. K., Jutila, M. A,, and Butcher, E.C. (1990) Proc. Natl. Acud. Sci. U. S. A . 8 7 , 2244-2248 26. Fukushima, K., Hirota, M., Terosaki, P. I., Wakisaka, A,, Tagoshi, H., ~~~

Chia,D.,Suyama, N., Fukushi, Y., Nudelman, E., and Hakomori, S. (1984) Cancer Res. 44,5279-5286 27. Butcher, E. C., Scollary, R.G., and Weissman,I. L. (1980) Eur. J. Immunol.

10,556-561 28. Gallatin, W., Weissman, I. L., and Butcher, E. C. (1983) Nature 304, 3034 29. Mulligan, R. C., and Berg, P. (1981) Proc. Natl. Acad. Sci. U. S. A . 7 8 , 2072-2076 30. Stephens, P. E., and Cockett, M. I. (1989) Nucleic Acids Res. 17,7110 31. Bock, K (1983) Pure Appl. Chem. 55,605-622 32. Kumlien,J.,Gronberg, G., Nilsson, B., Minsson, O., Zopf, D. A., and Lundblad, A. (1989) Arch. Eiochem. Biophys. 269,678-689 33. Wieslander, J., Minsson, O., Kallin, E., Gabrielu, A,, Nowack, H., and Timpl, R. (1990) Glycoconjugate J. 7,85-100 34. Hansson, G. C., Karisson, K.-A,, Larson, G., McKibbin, J. M., Blaszczyk, M., Herlyn, M., Steplewski, 2., and Koprowski, H. (1983) J. Biol. Chem. 258,4901-4097 35. Spohr, U., Hindsgaul, O., and Lemieux, R. V. (1985) Can. J. Chem. 6 3 , 2644-2652