Identification of Lewis x structures of the cell adhesion molecule CEACAM1 from human granulocytes

Lothar Lucka1,3, Malkanthi Fernando1,4, Detlef Grunow3, Christoph Kannicht5, Andrea K. Horst4, Peter Nollau4 and Christoph Wagener2,4

3 Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, Institut für Biochemie und Molekularbiologie, Arnimallee 22, D-14195 Berlin-Dahlem, Germany; 4 Universitätsklinkum Hamburg-Eppendorf, Institut für Klinische Chemie, Martinistraße 52, D-20251 Hamburg, Germany; 5 Octapharma, Molecular Biochemistry Berlin, Arnimallee 22, D-14195 Berlin, Germany


2 To whom correspondence should be addressed; e-mail: wagener{at}uke.uni-hamburg.de

Received on June 30, 2004; revised on August 11, 2004; accepted on August 13, 2004


    Abstract
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 Abstract
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 Results
 Discussion
 Materials and methods
 References
 
Carcinoembryonic antigen–related cell adhesion molecule 1 (CEACAM1) is expressed on epithelia, blood vessel endothelia, and leukocytes. A variety of physiological functions have been assigned to CEACAM1. It is involved in the formation of glands and blood vessels, in immune reactions, and in the regulation of tumor growth. As a homophilic and heterophilic adhesion receptor, it signals through different cellular pathways. The existence of special oligosaccharide structures such as Lewis x or sialyl-Lewis x glycans within this highly glycosylated protein has been postulated, but chemical proof is missing so far. Because such structures are known to be essential for different cell–cell recognition and adhesion processes, characterizing the CEACAM1 glycan structure is of pivotal importance in revealing the biological function of CEACAM1. We examine the terminal glycosylation pattern of CEACAM1 from human granulocytes, focusing on Lewis x epitopes. Lewis x–specific antibodies react with immunoaffinity-purified native CEACAM1. Antibody binding was completely abolished by treatment with fucosidase III, confirming a terminal {alpha}(1-3,4) fucose linkage to the N-acetylglucosamine of lactosamine residues, a key feature of Lewis epitopes. To verify these data, MALDI-TOF MS analysis after stepwise exoglycosidase digestion of the CEACAM1 N-glycan mixture was performed. A complex mixture of CEACAM1-bound oligosaccharides could be characterized with an unusually high amount of fucose. The sequential digestions clearly identified several different Lewis x glycan epitopes, which may modulate the cell adhesive functions of CEACAM1.

Key words: carcinoembryonic antigen / cell adhesion / Lewis x glycan epitope / mass spectrometry / N-glycan analysis


    Introduction
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Carcinoembryonic antigen (CEA)–related cell adhesion molecule 1 (CEACAM1) has been implicated in a number of physiological processes, such as cell–cell adhesion, microbial receptor activity, and regulation of tumor growth (for review see Beauchemin et al., 1999Go; Horst and Wagener, 2004Go; Öbrink, 1997Go). In the majority of carcinomas, CEACAM1 is down-regulated or dysregulated (Becker et al., 1986Go; Huang et al., 1998Go; Nollau et al., 1997Go). However, in epithelial malignancies, such as lung cancer and in melanomas, an increased expression of CEACAM1 has been observed, and CEACAM1 expression has been found to be an independent prognostic parameter (Laack et al., 2003; Sienel et al., 2003Go; Thies et al., 2002Go). CEACAM1 is a signaling molecule that regulates cell proliferation, differentiation, apoptosis, and angiogenesis (Ergün et al., 2000Go; Kirshner et al., 2003Go; Singer et al., 2000Go). In addition, it is involved in the activation of leukocytes (Kammerer et al., 2002; Singer et al., 2002Go). Initially, CEACAM1 has been identified as a cell–cell adhesion molecule mediating Ca2+-independent homophilic binding between the N-terminal Ig domains presented by the surfaces of adjacent cells (Ocklind and Öbrink, 1982Go). In addition, it has also been reported that CEACAM1 mediates heterophilic binding to other CEA family members (Oikawa et al., 1992Go), to Opa proteins of Neisseria meningitidis and Neisseria gonorrhoeae and to virulent strains of Haemophilus influenzae (Virji et al., 1996Go, 2000Go). Furthermore, mouse CEACAM1 is the receptor for the mouse hepatitis virus (Williams et al., 1991Go).

The CEACAM1 gene encodes four Ig-like domains followed by a transmembrane and a cytoplasmic domain. Like other CEA family members, CEACAM1 is highly glycosylated, but so far a detailed chemical analysis of the N-glycan structure is available only for CEACAM1 from rat liver (Kannicht et al., 1999Go). The N-linked oligosaccharide side chains of rat liver CEACAM1 are highly sialylated and mostly of the complex type and, to a much lesser extent, of the high-mannose type. A high-mannose oligosaccharide structure has also been described in a glycopeptide of CEACAM1 isolated from granulocytes (Mahrenholz et al., 1993Go). Biological functions of CEACAM1 glycans have been shown for high-mannose structures through the specific binding to type 1 fimbriae (Leusch et al., 1990Go; Sauter et al., 1993Go) and for lactosamine glycan structures through the binding to galectin-1 and galectin-3 (Feuk-Lagerstedt et al., 1999Go; Ohannesian et al., 1995Go). CEACAM1 has been reported to be the major membrane glycoprotein of human granulocytes that binds monoclonal Lewis x (Le x) antibodies (Stocks and Kerr, 1993Go). It has been postulated that the glycan structure of CEACAM1 mediates the binding of granulocytes to endothelial cells (Stocks et al., 1995Go). Le x structures, also known as SSEA-1 or CD15, are defined as terminal epitopes of oligosaccharide chains with the common structure Galß(1-4)Fuc{alpha}(1-3)GlcNAc-R (Gooi et al., 1981Go; Hakomori et al., 1981Go; Kobata and Ginsburg, 1969Go). These structures are expressed on glycoproteins as well as on glycolipids (Spooncer et al., 1984Go). It has been suggested that they are involved in cellular recognition during embryogenesis, neural development, or fertilization (Eggens et al., 1989Go; Johnston et al., 1998Go; Streit et al., 1996Go). For example, Le x antibodies inhibit the compactation of mouse blastomere cells (Fenderson et al., 1984Go; Solter and Knowles, 1978Go). It has been postulated that opposing Le x glycans bind to each other (Geyer et al., 2000Go). Recently, renewed interest in the adhesive function of Le x glycans is being noted because the C-type lectin DC-SIGN (dendritic cell–specific ICAM [intercellular adhesion molecule]-3-grabbing nonintegrin) and related lectins of dendritic cells specifically bind to Le x glycans on pathogens and possibly adhesion molecules (Appelmelk et al., 2003Go; Geijtenbeek et al., 2004Go; Liu et al., 2004Go; van Die et al., 2003Go).

CEACAM1 is expressed on granulocytes, lymphocytes, and endothelial cells and in a variety of tumors, such as carcinomas and malignant melanoma. At least in granulocytes, CEACAM1 is the major membrane glycoprotein that binds Le x antibodies, and, in addition, carries high-mannose residues. Thus CEACAM1 may be an endogenous ligand for C-type lectins, such as DC-SIGN. To verify this hypothesis, it appears mandatory to confirm the presence of Le x and high-mannose groups by structural methods. Therefore, in the present study we analyze the fucose-containing glycan structures of CEACAM1 purified from human granulocytes in a systematic manner. To characterize the type of terminal carbohydrate structures on CEACAM1, we employed immunoblot methods using sialyl-Lewis x (sLe x)– and Le x–antibodies in combination with sialidase and fucosidase treatment. To specify these data on a structural level, we developed a sensitive strategy based on the final determination of released oligosaccharides by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS). CEACAM1 was sequentially treated with different linkage-specific exoglycosidases, and fragmentation products were analyzed. From the observed mass values we were able to determine the carbohydrate composition of CEACAM1-bound N-glycan side chains. We found an unusually high amount of fucose-containing oligosaccharides and succeeded to structurally define a number of Le x groups that may function as specific binding structures for other proteins, such as C-type lectins.


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 Results
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Purification of CEACAM1 and reactivity with Le x and sialyl-Le x antibodies
CEACAM1 was purified from crude membrane extracts of human granulocytes by immunoaffinity chromatography using the monoclonal antibody (mAb) T84.1, which binds to a common epitope on CEACAM1, CEACAM5, and CEACAM6, respectively. Resulting protein fractions were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) to check the purity of the preparation. As shown in Figure 1A, in addition to CEACAM1, which migrates as a broad 160 kDa band, several other proteins were detectable in the lower molecular weight range (lane b). To achieve a higher purity, this protein fraction was treated by size-exclusion chromatography. The resulting CEACAM1 preparation contains one major band of about 160 kDa (lane c) and only minor contaminants of other proteins. To detect Le x structures, extracts from granulocytes as well as the CEACAM1 preparation after size-exclusion chromatography were subjected to western blotting with the specific antibody L5 (Figure 1B). This results in a major ~160-kDa band in both lanes, confirming that in our preparation CEACAM1 is a major carrier of the Le x glycotope, as published previously (Stocks and Kerr, 1993Go). The same blot was stripped and treated with the CEACAM1-specific mAb 4D1C2 to demonstrate that the Mr of CEACAM1 is in the same range as the Mr of the L5-reactive band. To confirm and further characterize Le x structures in crude granulocyte extracts and in purified CEACAM1, the antibodies MMA and 80H5 were also used (Table I). Both showed the same reactivity to proteins from granulocytes and to purified CEACAM1 as the mAb L5. The fact that mAb L5 binds to {alpha}1-acid glycoprotein ({alpha}1-GP) only after enzymatic removal of sialic acids indicates that sialic acid masks the Le x structure recognized by this antibody.



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Fig. 1. SDS–PAGE of CEACAM1 purified from human granulocytes. (A) CEACAM1 was purified from crude extract of granulocytes by immunaffinitiy chromatography followed by size exclusion filtration as described in Materials and methods. Obtained protein fractions were separated by SDS–PAGE and stained with silver dye. (a) Granulocyte extract; (b) immunoaffinity-purified CEACAM1; (c) immunoaffinity-purified CEACAM1 fraction after size-exclusion chromatography. The arrow indicates purified CEACAM1. (B) Protein extracts from human granulocytes (a) and CEACAM1 after the final purification step (c) were used in western blot analysis with the Lewis x–specific mAb L5. The same blot was stripped and incubated with the CEACAM1-specific mAb 4D1C2.

 

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Table I. Reactivity of different Lewis x antibodies with extract from human granulocytes, purified CEACAM1 and different control proteins

 
To further analyze Lewis glycan epitopes on CEACAM1 and other members of the CEACAM family, the Le x–specific antibody L5 and sLe x–specific antibody KM93 were used in western blot analysis (Figure 2). L5 reacted with CEACAM1 from granulocytes but failed to react with recombinant CEACAM1 expressed in HEK293 cells, indicating that fucosyltransferases necessary for the synthesis of Le x structures are not expressed in this cell line. CEACAM5 (CEA) and CEACAM6 (NCA) were also recognized by the L5 antibody. These results reveal that native CEACAM1 and the other CEA family members contain epitopes immunoreactive for Le x antibodies. In contrast, the sLe x antibody KM93 did not react with either CEACAM1 from granulocytes or with recombinant CEACAM1, indicating the absence of sLe x epitopes on both preparations. The same blots were stripped and treated with mAb T84.1. A series of other antibodies against sLe x, such as HECA493, 2H5, or CSLEX1, also failed to recognize CEACAM1 (data not shown).



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Fig. 2. Reactivity of CEACAM1 and other family members with Lewis x and sialyl-Lewis x antibodies. Human CEACAM1, CEACAM5, and CEACAM6 proteins were used in western Blot analysis. Detection was done with the Lewis x–specific antibody L5 or the sialyl-Lewis x–specific antibody KM93. Lewis x–BSA and sialyl-Lewis x–BSA were used as positive controls and recombinant CEACAM1 as a negative control. The same blots were stripped and incubated with mAb T84.1 cross-reactive with different CEACAM antigens.

 
Western blot analysis of exoglycosidase-treated CEACAM1
To confirm the presence of Lewis epitopes, purified CEACAM1 from granulocytes was treated with exoglycosidases prior to immunoblotting. The data presented in Figure 3 show that Le x epitopes were detectable in the native and the sialidase-treated CEACAM1 preparation. The N4-(N-acetyl-beta-glucosaminyl)asparagine amidase (PNGase F) cleavage product shifted to the estimated molecular weight ranges of the CEACAM1 polypeptide backbones, probably representing the long and short isoforms (Öbrink, 1997Go). Consequently, the peptides could not be detected by the Le x–specific mAb L5. Fucosidase III treatment, which led to the release of {alpha}(1-3,4)-linked terminal fucose but not fucose linked to the core structure of N-glycans, completely abolished binding of L5, confirming the presence of fucose linked to the N-acetylglucosamine of lactosamine residues, which is a key feature of the Lewis glycan epitope. Presence of CEACAM1 in all lanes was demonstrated by mAb T84.1.



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Fig. 3. Rectivity of the Le x antibody L5 with CEACAM1 after glycosidase treatment. Human CEACAM1 was treated with different enzymes as indicated. Western blot analysis was performed with the Lewis x–specific antibody L5. Lewis x–BSA was taken as positive control. The same blot was stripped and treated with the CEACAM mAb T84.1 to confirm the presence of CEACAM1.

 
CEACAM1 monosaccharide analysis
Typical constituents of complex-type sugars were detected by carbohydrate composition analysis. For this purpose, N-glycans were released from CEACAM1 tryptic peptides by PNGase F treatment and hydrolyzed, and monosaccharides were detected and quantified via high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Fucose, glucosamine, galactose, and mannose were found at a ratio of 8:34:40:17 (264 pmol:1102 pmol:1296 pmol:543 pmol). Thus CEACAM1 has to be considered a highly fucosylated glycoprotein. The overall 2:1 proportion of glucosamine or galactose to mannose indicates the presence of complex-type oligosaccharides. The monosaccharide composition analysis revealed fucose as the only deoxyhexose sugar in N-glycans released from CEACAM1.

CEACAM1 N-glycan structures
The mixture of CEACAM1 N-glycans was characterized after sialidase digestion with subsequent MALDI-TOF MS. We obtained an extremely complex pattern of putative N-glycan structures, which is summarized in Table II (the corresponding positive-ion mass spectrogram is shown later). The mixture of neutral N-glycans after sialidase cleavage is much more complex than for {alpha}1-GP (Figure 4A). Besides the relative masses corresponding to the Na+ adducts of bi-, tri-, and tetraantennary complex N-glycans (1663, 2028, and 2394, respectively), the relative masses corresponding to high-mannose type oligosaccharides and a series of complex oligosaccharides with repeating units have been found. Also, the existence of hybrid-type oligosaccharide structures can be assumed. In total, we identified 35 different oligosaccharide compositions. Twenty of these contain at least one fucose residue and eight contain two or three fucose molecules, indicating that fucose is linked not only to the core GlcNAc but also to the antennae, a characteristic feature of fucose within the Lewis epitope.


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Table II. MS data of desialylated N-glycans released from CEACAM1 and putative corresponding oligosaccharide composition

 


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Fig. 4. MALDI-TOF MS of N-glycans released from {alpha}1-GP and treated with different glycosidases. MS analysis was performed by MALDI-TOF MS. Detected ions could be interpreted as Na adducts (M+Na)+ of the glycans. Mass values are specified in Table III. Mass peaks corresponding to the K+ adduct are not indicated. N-glycans were stepwise digested by sialidase (sia), a mixture of ß-N-acetylhexosaminidase and ß-galactosidase (hex/gal), {alpha}(1-3,4)-specific fucosidase (fucIII), and a ß(1-4)-specific galactosidase (gal). Some not indicated mass peaks are probably due to incomplete enzymatic cleavage.

 


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Fig. 5. MALDI-TOF MS of N-glycans released from CEACAM1 and treated with different glycosidases. MS analysis was performed by MALDI-TOF MS. Detected ions could be interpreted as Na+ adducts (M+Na)+ of the glycans. Desialylated N-glycans in A correspond to the data presented in Table II. Indicated mass values in the other parts of the figure correspond to the data presented in Table IV. Mass peaks corresponding to the K+ adduct are not indicated. N-glycans were stepwise digested by sialidase (sia), a mixture of ß-N-acetylhexosaminidase and ß-galactosidase (hex/gal), {alpha}(1-3,4)-specific fucosidase (fucIII), and a ß(1-4)-specific galactosidase (gal).

 

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Table III. Procedure for the identification of Lewis structures, demonstrated for an {alpha}1-GP-linked N-glycan

 
Identification of Le x oligosaccharides by enzymatic sequencing
Lewis structures were identified by stepwise exoglycosidases treatment. This was first demonstrated for desialylated {alpha}1-GP, which is known to contain Lewis structures (Fournier et al., 2000Go). The strategy for identification of Le x structures on N-linked oligosaccharides, specificity of the applied enzymes, and an example showing sequential digestion of a Le x–type N-glycan of one {alpha}1-GP N-glycan is summarized in Table III. The corresponding positive ion mass spectrograms are shown in Figure 4. For enzyme specificities see Kobata (1979), Dwek et al. (1993), and Prime et al. (1996). Because sLe x groups were absent in the CEACAM1 preparation, PNGase F–released N-glycans were desialylated by sialidase to avoid the difficulties associated with the MS analysis of charged oligosaccharides. Sialidase cleaves off {alpha}(2-3,6,8,9)-linked neuraminic acid.


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Table IV. MS data of desialylated N-glycans released from CEACAM1 after treatment with different glycosidases and putative corresponding oligosaccharide composition

 
In a next step, a mixture of ß-galactosidase and ß-N-acetylhexosaminidase was applied. ß-Galactosidase cleaves ß(1-3,4,6)-linked galactose on condition that no fucose is bound to the subterminal GlcNAc. ß-N-acetylhexosaminidase removes ß(1-2,3,4,6)-bound GlcNAc residues. Consequently, Lewis structures within N-glycan antennae cannot be cleaved. In turn, unsubstituted type I or type II antennae will be removed completely, resulting in the common N-glycan core structure with or without fucose bound to the reducing GlcNAc. The use of {alpha}(1-3,4)-specific fucosidase (FucIII) then leads to the release of fucose that is bound to the antennae, not of fucose that is bound to the core structure.

Finally, we used the ß-galactosidase with the cleavage specificity ß(1-4) to demonstrate that the released fucose residues were part of Le x and not Le a structures. Incubation of the neutral {alpha}1-GP oligosaccharide mixture with ß-galactosidase/ß-N-acetylhexosaminidase resulted in a major peak with a relative mass of 1444.43 (Figure 4B). This can be assigned to the theoretical mass (Mcalc+Na)+ 1444.507 of the oligosaccharide (Hex)1(HexNAc)1(Deoxyhexose)1(Man)3(GlcNAc)2. This mass peak shifted to 1298.56 after treatment with FucIII (Figure 4C). Finally, mass shifted to 1136.39 corresponding to the loss of one hexose residue after digestion with the ß(1-4)-specific galactosidase that specifically cleaves the ß(1-4) linkage (Figure 4D). The loss of one galactose of the defucosylated oligosaccharides confirms the presence of the Le x epitope.

The same strategy was performed for the analysis of CEACAM1-released N-glycans. The data are summarized in Table IV and the corresponding positive-ion mass spectrograms are shown in Figure 5B–D. From these results it is concluded that at least seven structures were sensitive to FucIII treatment. These could be followed through the spectra and corresponding mass peaks shifted after digestion with the ß(1-4)-specific galactosidase to masses that correspond to the loss of one or two galactose residues after the cleavage of one or two fucose residues linked {alpha}(1-3) to subterminal GlcNAc residues. For example, we detected the relative mass of 1444.69 corresponding to the deduced Na+ adduct of a complex-type structure with one terminal linked fucose residue with the composition (Hex)1(HexNAc)1(Deoxyhexose)1(Man)3(GlcNAc)2. It is the same structure that was demonstrated to be part of the {alpha}1-GP N-glycan (for comparison, see Table III and Figure 4). The structures with masses of 1590.80 and 1956.05, respectively, both contain two fucose residues. The former with the likely carbohydrate composition (Hex)1(HexNAc)1(Deoxyhexose)2(Man)3(GlcNAc)2 shifted to a mass of 1282.26 after fucosidase III and ß-galactosidase digestion, indicating the presence of one terminal fucose residue, the other fucose is core-linked. The mass peak of 1956.05 corresponds to the Na+ adduct of a complex-type structure with one antenna of two lactosamine units. One fucose residue is presumably linked to the core as still present in the deduced oligosaccharide composition after fucosidase III and ß-galactosidase digestion. The appropriate mass shift corresponding to the loss of one fucose and one galactose residue could be detected.

Also, structures with three fucose residues, sensitive to FucIII treatment, were detected and mass shifts could be followed through the digestions. One of these with a mass of 2467.34 corresponds to the Na+-adduct of a complex type structure with three antennae. After ß(1–4)-specific galactosidase digestion one structure still contains two fucose residues, indicating incomplete cleavage or that one of the remaining fucose residues is bound in another linkage than {alpha}(1-3,4). It should be noted that all listed oligosaccharide compositions represent putative cleavage products. Because of the complexity of the structures, we could not follow exactly all mass shifts. However, we treated the final N-glycan mixture with the galactosidase from bovine testes which cleaves ß1-3,4,6 glycosidic linkages (data not shown). None of the mass peaks that were assumed to correlate with the product of a digestion of a Lewis-containing N-glycan disappeared. This indicates that these oligosaccharides did not contain Le a structures.


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In the present study, we provide the conclusive immunochemical and structural proof that CEACAM1 is a major carrier of Le x structures among membrane glycoproteins of human granulocytes. In crude membrane extracts from granulocytes, CEACAM1 is the most prominent antigen binding monoclonal Le x antibodies (Stocks and Kerr, 1993Go) (see Figures 1B and 2). Among the Le x antibodies used here, the specificity of mAb L5 is well established. The antibody has been shown to recognize Le x structures within neutral N-glycans (Streit et al., 1996Go). This is consistent with our finding that the antibody does not recognize sialylated {alpha}1-GP. Previously, it has been postulated that in addition to Le x structures, CEACAM1 may also carry sLe x groups (Stocks and Kerr, 1993Go). However, in our hands, sLe x antibodies such as KM93, HECA452, 2H5, or CSLEX1 did not bind to native granulocyte CEACAM1. These findings indicate that in the CEACAM1 preparation used, Le x epitopes were not masked by sialic acid. The presence of Le x structures is supported further by treatment of CEACAM1 with fucosidase III, which completely abolished the binding of the Le x antibody L5 (see Figure 3). The final proof that the CEACAM1 glycans contain Le x structures is being provided by MS. N-linked-glycans of CEACAM1 isolated from human granulocytes carry an unusually high amount of fucose and within the highly complex N-glycan structures several Le x glycan epitopes.

Our experimental strategy aimed to unequivocally demonstrate the presence of Le x structures in CEACAM1 (see Table IV). For this purpose, sialidase-treated PNGase-released N-glycans were further subjected to digestion by a mixture of ß-galactosidase and ß-N-acetylhexosaminidase. By this treatment, glycan chains in which fucose is bound to the subterminal GlcNAc remain undigested. By further treatment with an {alpha}(1-3,4)-specific fucosidase (FucIII), fucose residues bound to the antennae but not to the core structure are released and could thus be identified. By the use of a ß-galactosidase with the ß(1-4) cleavage specificity, Le x groups could be differentiated from Le a groups.

{alpha}1-GP is known to contain sialylated Le x structures (for review see Fournier et al., 2000Go). Therefore, the usefulness of the strategy was first demonstrated for N-glycans released from desialylated {alpha}1-GP. The same strategy was applied to the analysis of CEACAM1-bound N-glycans. The analysis of desialylated CEACAM1 N-glycans revealed the presence of at least 20 fucosylated structures. This is consistent with the high fucose content found in the monosaccharide analysis. In general, membrane glycoproteins contain fucose residues of less than 1%. The mixture of neutral N-glycans after neuraminidase cleavage is much more complex than, for example, for {alpha}1-GP. In addition to the bi-, tri-, and tetraantennary complex N-glycans, high-mannose type oligosaccharides and a series of complex oligosaccharides with repeating units could be identified. This is consistent with the heterogeneous molecular weight of CEACAM1 in SDS–PAGE and with the data obtained from the analysis of N-glycans released from rat liver CEACAM1 (Kannicht et al., 1999Go). For CEACAM1 from human granulocytes, the apparent Mr ranges from 140 to 170 kDa and shifted to a doublet band in the range of ~55–58 kDa after treatment with PNGase. The doublet band most probably represents the long and short isoforms of CEACAM1 (Öbrink, 1997Go). Out of the 20 fucose-containing structures of desialylated N-glycans, at least 7 structures could be identified to be insensitive to ß-galactosidase and ß-N-acetylhexosaminidase but sensitive to FucIII, thus containing {alpha}(1-3,4) bound fucose at terminal positions. This linkage is characteristic for fucose within the Lewis epitope (Kobata and Ginsburg, 1969Go). Further digestion with a ß-galactosidase with the cleavage specificity ß(1-4) excluded Le a structures.

In human granulocytes, CEACAM1 is the major glycoprotein bound by Le x antibodies. In FutIX transfection studies, we showed that CEACAMs but not other members of the immunoglobulin superfamily (such as the FGF receptor 1), are decorated with Le x groups (unpublished data). These findings suggest that the presence of Le x groups on CEACAM1 is rather specific. It has been argued that defined glycotopes present on a limited number of glycoproteins most probably serve a biological function (Varki, 1993Go). It has been reported that Le x glycan epitopes associate with each other, although with very low affinity (Geyer et al., 2000Go). The association of Le x structures may initiate the homophilic binding of CEACAM1 residues in cis or trans. However, according to our preliminary data, the homotypic binding of CEACAM1-transfected Chinese hamster ovary cells appears not to be affected by FutIX cotransfection.

Alternatively, the Le x groups of CEACAM1 may bind to lectins, such as C-type lectins. Only very recently Le x–binding C-type lectins were described: DC-SIGN was originally detected as a ligand of the cell adhesion molecules ICAM-3 (Geijtenbeek et al., 2000Go). Because this lectin binds to Le x and high-mannose residues (Appelmelk et al., 2003Go), which are both present on CEACAM1, CEACAM1 could be a binding partner of DC-SIGN and other related C-type lectins, such as DC-SIGN-related lectins and liver and lymph node sinusoidal endothelial cell C-type lectin (Liu et al., 2004Go). There are several scenarios in which these lectins could interact with glycans of CEACAM1. It has been reported that DC-SIGN binds strongly to granulocytes (Appelmelk et al., 2003Go). CEACAM1 as the major Le x–positive membrane glycoprotein would be one of the putative binding partners. In addition, because CEACAM1 is expressed on certain subpopulations of T cells, it could mediate their interaction with dendritic cells and in this way regulate self–nonself recognition (Geijtenbeek et al., 2004Go).

A related function in immunosurveillance could be envisaged for CEACAM1 expressed on melanoma cells and other malignancies in which CEACAM1 is overexpressed (Laack et al., 2002Go; Sienel et al., 2003Go; Thies et al., 2002Go). Interestingly, DC-SIGN behaves as a dendritic cell–specific rolling receptor for ICAM-2 and is thus similar to the selectins (Geijtenbeek et al., 2004Go). If CEACAM1 interacts with DC-SIGN and related lectins expressed in endothelia, the extravasation of leukocytes to specific sites would be an important potential function assigned to the Le x residues of CEACAM1. The extensive characterization of the glycan structures of CEACAM1 reported here provides a solid basis to address pertinent questions regarding the function of the glycans of CEACAM1 in cell–cell adhesion, immune recognition, angiogenesis, and tumorigenesis.


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 Abstract
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Antibodies, control proteins, and enzymes
mAb T84.1 recognizing several members of the CEACAM family was used for immunopurification of CEACAM1. For immundetection on western blots, either mAb T84.1 or the CEACAM1-specific mAb 4D1C2 were applied (Stoffel et al., 1993Go) and detected by a horseradish peroxidase (HRP)–conjugated goat anti-mouse IgG secondary antibody (Dianova, Hamburg, Germany). The Le x–specific antibody L5 was a kind gift from Prof. M. Schachner, Zentrum für Molekulare Neurobiologie, Universtät Hamburg, Germany (Streit et al., 1996Go); detection was performed with a HRP-coupled rat anti-IgM antibody (Dianova). The anti-sialyl-Le x antibody KM-93 was obtained from Calbiochem (Schwalbach, Germany) and the anti-Le x antibodies MMA and 80H5 were purchased from BD Bioscience (Heidelberg, Germany) and Beckman & Coulter (Krefeld, Germany), respectively; for immunodetection an HRP-conjugated goat anti-mouse IgM secondary antibody was applied (Dianova).

Le x–bovine serum albumin (BSA), sLe x–BSA, and {alpha}1-GP purchased from Calbiochem (Schwalbach, Germany) as well as lacto-N-fucopentaose III-BSA (Dextra Lab, Göttingen, Germany) served as controls for binding and specificity of anti-Le x and anti-sialyl-Le x antibodies, respectively. CEACAM5 purified from liver metastasis of colon cancer patients was purchased from Dianova. The following enzymes were used for the specific release of oligo- or monosaccharides. Recombinant PNGaseF asparagine amidase from Flavobacterium meningosepticum (EC 3.5.1.52) was purchased from Roche (Mannheim, Germany). Recombinant sialidase from Arthrobacter ureafaciens (exo-alpha-sialidase, EC 3.2.1.18), ß-N-acetylhexosaminidase from jack bean (EC 3.2.1.52), ß-galactosidase from bovine testes (EC 3.2.1.23), {alpha}(1-4)-specific galactosidase from jack bean (EC 3.2.1.23), and {alpha}(1-3,4)-fucosidase from Xanthomonas manihotis ({alpha}-fucosidase III, EC 3.2.1.51) were obtained from PROzyme/Glyko (Novaton CA) and Calbiochem.

Cell lines
Human embryonic kidney cells (HEK293) cells were obtained from the American Type Culture Collection and cultured in Dulbeco's modified Eagle's medium containing 10% fetal calf serum, 100 U/ml penicillin, and 10 mg/ml streptomycin. For expression of soluble recombinant CEACAM1, the extracellular part of CEACAM1 was amplified by polymerase chain reaction and cloned into the pcDNA3.1 expression vector. DNA was transfected into HEK293 cells from and stable transfectants were selected in the presence of zeocine by limited dilution. After selection, stable transfectants were adapted to serum-free growth conditions, and soluble CEACAM1 was harvested from the tissue culture supernatant.

Isolation of granulocytes from whole blood
Isolation of human granulocytes was performed according to Stoffel et al. (1993)Go. Granulocytes were isolated from fresh pooled buffy coats of normal human blood donors. Blood was layered on Ficoll-Paque (d = 1.077 g/cm3; Pharmacia, Freiburg, Germany) and spun at 2000 rpm at 4°C for 20 min; the supernatant was carefully removed, and granulocytes were harvested. To remove contaminating erythrocytes, cells were resuspended in erythrocyte lysis buffer (155 mM ammonium chloride, 10 mM potassium bicarbonate, 0.1 mM ethylenediamine tetra-acetic acid [EDTA], pH 8.0). After centrifugation (1500 rpm, 20 min, 4°C), the supernatant was removed and granulocytes were washed three times with cold phosphate buffered saline (PBS). For the extraction of membrane proteins, granulocytes were lysed in PBS (pH 7.4) containing 1% Triton X-100, 5 mM benzamidine, 10 mM EDTA, 100 mM 6-aminohexane acid, and 2 mM phenylmethylsulfonyl fluoride under rotation for 16 h at 4°C. Lysates were cleared by centrifugation (13,000 x g, 1 h, 4°C), the supernatant containing membrane proteins of granulocytes was removed and stored at –20°C.

Antibody coupling and purification of CEACAM1
Immunoaffinity purification was applied for the purification of native CEACAM1. To remove additives from CNBr-activated Sepharose 4B beads (Pharmacia), beads were soaked in 1 mM HCl (pH 2.8–3.0) at room temperature for 15 min. Subsequently, beads were transferred to a G2 glass filter, washed thoroughly with ice-cold 1 mM HCl, and dried under reduced pressure. Coupling of CEACAM mAb T84.1 to the column matrix (2.5 mg/ml mAb) was performed at 4°C for 16 h on a shaker in coupling buffer (0.1 M NaHC03, pH 8.0). After packing of the column (XK 16, column volume 25 ml, Pharmacia), CEACAM purification was performed by FPLC (Biologic DuoFlow, BioRad, Hercules, CA). To remove unbound antibodies, the matrix was washed with 10 column volumes of coupling buffer. Unoccupied functional groups were blocked by ethanolamine (1 M, pH 8.0) for 6 h at 4°C at a flow rate of 1 ml/min; the column was washed with 3 column volumes of ethanolamine and 1 column volume of dimethylformamide to prevent antibody aggregation. Subsequently, the column was washed 5 times with 5 column volumes of coupling buffer, followed by alternating washing steps (5 times with 5 column volumes each) with sodium acetate/sodium chloride buffer (pH 4.0) and Tris–HCl/sodium acetate buffer (pH 8.0) at a flow rate of 2 ml/min. Finally, the column was equilibrated with PBS (pH 7.4), and 50 ml of granulocyte lysate corresponding to 2000 ml of buffy coat was loaded onto the T84.1-affinity column with a flow rate of 1 ml/min. The column was washed with 3 column volumes of PBS (pH 7.4), and bound CEACAM was eluted with 40 ml 0.2 M glycine (pH 3.0) followed by elution with 0.2 M glycine (pH 1.8) at a flow rate of 2 ml/min. Elution was performed in fractions of 2.5 ml each; CEACAM-containing fractions were pooled and immediately neutralized with 1 M Na2HPO4 (pH 8.0) to pH 7.0. Eluates were ~100-fold concentrated by ultrafiltration (Centricon, cutoff 10 kDa) at 3000 rpm at 4°C.

To separate CEACAM1 from other coeluted CEACAM family members, size-exclusion chromatography was performed using Superdex 200 prep grade (Pharmacia). The column was equilibrated with 900 ml 0.05 M Tris–HCl (pH 7.5) containing 0.35 M sodium chloride and 0.1% Triton X-100 at a flow rate of 1 ml/min. Subsequently, 2 ml of the immunopurified, concentrated sample was loaded and eluted at a flow rate of 0.25 ml/min with the same buffer applied for equilibration. CEACAM1-positive fractions were pooled, and Triton X-100 was removed using Oasis HLB columns (3.9 x 20 mm, Waters, Germany). Prior to use, columns were washed with 100% methanol and subsequently with water. Samples were 10-fold diluted in PBS and passed over the column (flow rate 0.5 ml/min). Finally, eluates were concentrated ~50-fold by ultrafiltration (Centricon, cutoff 100 kDa) and stored at –20°C. The purity of preparations was controlled by silver staining and immunodetection with CEACAM1-specific antibodies after SDS–PAGE. For CEACAM6, appropriate fractions of size-exclusion chromatography were pooled and further processed as described for CEACAM1.

Recombinant CEACAM1 was purified from cell culture supernatant of transfected HEK293 cells by Concanavalin A affinity chromatography. Bound protein was eluted with 1 M {alpha}-methylmannopyranosid dissolved in 20 mM Tris, 1 mM MgCl2, 1 mM CaCl2, 500 mM NaCl (pH 7.5). Elution buffer was exchanged to PBS by PD10 columns (Pharmacia) and purity of preparations were controlled by SDS–PAGE and Coomassie staining.

SDS-PAGE and western blot analysis
After determination of the total protein amount by Bradford assay, protein preparations were separated by SDS–PAGE and transferred to polyvinylidene difluoride (Immunobilon Millipore, Bedford, MA) or nitrocellulose membranes (Schleicher & Schüll, Germany). After blocking of membranes overnight at 4°C in 1% blocking reagent (Roche), membranes were incubated with primary antibodies in Tris-buffered saline with Tween (150 mM NaCl, 10 mM Tris (pH 8.0), 0.05% Tween 20) overnight at 4°C, washed three times with the same solution, and incubated with the appropriate secondary antibody for 1 h at room temperature. After washing for 3 h at room temperature in Tris-buffered saline with Tween, signals were detected by chemiluminescence (ECL reagent, Amersham, Germany).

Glycosidase treatment of purified CEACAM1 prior to western blot analysis
For PNGase F digestion, ~5 µg of native CEACAM1 were denatured in 50 mM sodium phosphate (pH 7.5) containing 0.1% SDS and 50 mM ß-mercaptoethanol by heating at 100°C for 5 min. After cooling to room temperature, ~200 U/ml of PNGase F and Triton X-100 was added to a final concentration of 0.75% and incubated overnight at 37°C. Sialidase treatment of native CEACAM1 was performed overnight at 37°C in 50 mM sodium phosphate (pH 6.0) in the presence of ~0.5 U/ml sialidase A. Defucosylation was carried out overnight at 37°C with ~25 mU/ml of {alpha}(1-3,4)-specific fucosidase III in 50 mM sodium phosphate buffer (pH 5.0). Complete cleavage was confirmed by SDS–PAGE and subsequent western blotting.

Trypsin digestion and release of N-linked glycans from purified CEACAM1
Trypsin (sequencing grade) was purchased from Serva (Heidelberg, Germany) and trifluoroacetic acid was obtained from Sigma (Taufkirchen, Germany). One hundred micrograms of native CEACAM1 was digested with trypsin according to standard procedures (Nuck et al., 2002). The protein:enzyme ratio was 1:50 (w/w). Digestion at 37°C was carried out overnight in a buffer containing 50 mM N-methyl-2,2-iminodiethanol (pH 8.0) and stopped by heat inactivation. For release of N-glycans, tryptic peptides derived from 100 µg CEACAM1 were digested with 5 mU PNGase F in 500 µl of the described N-methyl-2,2-iminodiethanol buffer for 18 h at 37°C. Peptides were separated by cation-exchange chromatography (AG-50WX12; BioRad, Munich, Germany). Released oligosaccharides were further purified on a reversed phase cartridge (RP18).

Carbohydrate composition analysis
Monosaccharide analysis was performed by hydrolysis of the oligosaccharides with 2 M trifluoroacetic acid for 3.5 h at 100°C, followed by HPAEC-PAD as described previously (Gohlke et al., 1996Go).

Sequential exoglycosidase digestions
Digestion of purified CEACAM1 derived N-glycan mixture led to specific fragments, which could be determined with subsequent MS. All digestions were carried out with oligosaccharides derived from 100 µg CEACAM1 peptides according to the manufacturer's protocol within the provided incubation buffer at 37°C. After each enzymatic treatment, appropriate aliquots were taken and analyzed by MALDI-TOF MS as described later. Prior to the next digestion, oligosaccharides were desalted by anion/cation exchange chromatography. For specificities of the used enzymes see Kobata (1979), Dwek et et al. (1993) and Prime et al. (1996).

Sialidase with the specificity for {alpha}(2-3,6,8,9)-linked sialic acid was used to remove all bound sialic acid residues. Oligosaccharides were incubated with 100 mU in a final volume of 100 µl for 48 h, with a fresh enzyme aliquot added after 24 h. A mixture of ß-galactosidase with ß(1-3,4,6) cleavage specificity from bovine testes and ß-N-acetylhexosamidase with the cleavage specificity ß(1-2,3,4,6) was used to remove galactose and GlcNAc residues from antennae that do not carry a fucose residue. Desialylated CEACAM1 oligosaccharides were incubated with 0.15 U ß-N-acetylhexosaminidase and 1.5 U ß-galactosidase in a final volume of 100 µl for 18 h. {alpha}-Fucosidase III with cleavage specificity for {alpha}(1-3,4)-linked fucose was used to remove terminal linked fucose residues; 1.5 mU was incubated in a final volume of 30 µl for 3 h. ß-Galactosidase led to the release of ß(1,4)-linked galactose residues, which carried an {alpha}(1-3)-linked fucose residue prior to the fucosidase treatment. Eight milliunits were used in 100 µl of provided incubation buffer for 16 h.

MS
MALDI-TOF MS was carried out on a Bruker Biflex instrument equipped with a 337 nm nitrogen laser (Bruker, Bremen, Germany) as described previously (Gohlke et al., 1996Go; Kannicht and Flechner, 2002Go). Measurement was done using the positive ion mode. One-half microliter of each digestion mixture was used in 50 µl arabinosazon matrix solution.

The GlycoMod tool (available online at www.expasy.org/tools/glycomod) of the Expasy Molecular Biology Server was used to verify all calculation of oligosaccharide compositions. Experimental masses were calculated as monoisotopic Na+ adducts of underivatized N-glycans from PNGase F release.


    Acknowledgements
 
We thank Melitta Schachner for the L5 antibody. C. Frenz, K. Scheike, and S. Wuttke are acknowledged for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 470, Universität Hamburg and SFB 366, Freie Universität Berlin) and the Sonnenfeld-Stiftung.


    Footnotes
 
1 These authors contributed equally to this work. Back


    Abbreviations
 
{alpha}1-GP, {alpha}1-acid glycoprotein; BSA, bovine serum albumin; CEA, carcinoembryonic antigen; CEACAM, CEA-related cell adhesion molecule; DC-SIGN, dendritic cell–specific ICAM-3 grabbing nonintegrin; EDTA, ethylenediamine tetra-acetic acid; HPAEC-PAD, high-performance anion-exchange chromatography with pulsed amperometric detection; HRP, horseradish peroxidase; ICAM, intercellular adhesion molecule; Le x, Lewis x; LNFIII, lacto-N-fucopentaose III; mAb, monoclonal antibody; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy; PBS, phosphate buffered saline; PNGase, N4-(N-acetyl-beta-glucosaminyl)asparagine amidase; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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