Journal of Leukocyte Biology eBioscience full spectrum cell analysis
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
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 Kuroki, M.
Right arrow Articles by Kuroki, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kuroki, M.
Right arrow Articles by Kuroki, M.
(Journal of Leukocyte Biology. 2001;70:543-550.)
© 2001 by Society for Leukocyte Biology

Identification and comparison of residues critical for cell-adhesion activities of two neutrophil CD66 antigens, CEACAM6 and CEACAM8

Motomu Kuroki*, Hironori Abe*, Takayuki Imakiirei*, Shaoxi Liao*, Hiroko Uchida*, Yasushi Yamauchi*, Shinzo Oikawa2 and Masahide Kuroki*

* Department of Biochemistry, School of Medicine, Fukuoka University, Fukuoka, Japan; and
{dagger} Suntory Biomedical Research Limited, Osaka, Japan

Correspondence: Dr. Motomu Kuroki, Department of Biochemistry, School of Medicine, Fukuoka University, 7-45-1 Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan. E-mail: mokuroki{at}fukuoka-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CEACAM6 (CD66c) and CEACAM8 (CD66b) are cell-adhesion proteins on neutrophils that belong to the human carcinoembryonic antigen (CEA) family. CEACAM6 reveals homophilic adhesion and heterophilic adhesion to other CEACAM family antigens including CEACAM8, CEACAM1, and CEA, whereas CEACAM8 exhibits only heterophilic adhesion to CEACAM6. Here, we investigated and compared structural requirements for the homophilic adhesion of CEACAM6 and heterophilic adhesion between CEACAM6 and CEACAM8 at the amino acid level by using CHO transfectants expressing their mutant and chimeric proteins. The NH2-terminal domain (N-domain) of CEACAM6 expressed on a CHO cell was suggested to bind the N-domain of CEACAM6 or CEACAM8 on the opposing cell. By homologue-scanning mutagenesis, we found that the locations of the sequences critical for the adhesion of CEACAM6 to itself and to CEACAM8 are overlapped and that they are highly similar but not identical to the locations of the residues previously shown to be essential for the binding of CEACAM antigens to Opa proteins of pathogenic Neisseriae. Our findings imply that subtle differences in the N-domain sequences determine the specificity of the CEACAM antigens on neutrophils for interaction with the same or different CEACAM antigens and the bacterial proteins.

Key Words: CEA family • CD66b • CD66c • homologue-scanning mutagenesis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The human CD66 antigens, including CD66a, CD66b, CD66c, CD66d, and CD66e, are a group of glycoproteins encoded by the carcinoembryonic antigen (CEA) family genes, which are clustered on chromosome 19 as BGP, CGM6, NCA, CGM1, and CEA, respectively [1 , 2 ]. These antigens were recently renamed CEACAMs [3 ]. CEA, known as a tumor-associated protein encoded by the CEACAM5 gene, is chiefly expressed on epithelial cells of the gastrointestinal mucosa, whereas the other four antigens, CEACAM1 (CD66a, BGP1), CEACAM3 (CD66d), CEACAM6 (CD66c, NCA-50/90), and CEACAM8 (CD66b, NCA-95), are expressed on neutrophils [2 ]. CEACAM3 and CEACAM8 have been identified only on neutrophils, and CEACAM6 exists on the epithelial cells of the gastrointestinal tract as well as neutrophils. CEACAM1 is also expressed on lymphocytes and epithelial cells of a variety of tissues [4 ]. Except for CEACAM3, these CEA family antigens exhibit homophilic and/or heterophilic intercellular adhesion activities in vitro [5 6 7 8 ]. Cell-surface expression of the CEACAM proteins on neutrophils is up-regulated during cell activation [9 ]. Ligation of CEACAMs with antibodies induces neutrophil activation, including tyrosine phosphorylation and up-regulation of adhesion activity of CD11/CD18 [10 , 11 ]. CEACAM6 seems to play a role in one type of antibody-dependent cellular cytotoxicity (ADCC) by neutrophils [12 ]. In addition, CEACAM1, CEACAM3, CEA, and CEACAM6 bind a variety of bacteria including Escherichia coli, Salmonella [13 ], Neisseria gonorrhoeae [14 ], and Neisseria meningitides [15 ] through interactions with the carbohydrate or protein structures [13 , 16 ]. These observations imply that the CEACAM antigens contribute to self-defense mechanisms in which neutrophils and mucosal epithelial cells are involved.

Each CEACAM protein consists of several domains similar to the immunoglobulin (Ig) V or C domains: one NH2-terminal IgV-like domain (N-domain) of 108 amino acids and zero to six IgC2-like domains (A- and B-domains) followed by a glycolipid membrane anchor or a transmembrane-spanning region with a cytoplasmic tail [1 , 3 ]. The N-domains of CEACAM family proteins reveal sequence similarity with 70–90% identity to each other. CEA [5 ], CEACAM1 [7 , 17 ], and CEACAM6 [6 ] exhibit homophilic adhesion and heterophilic adhesion amongst them. Conversely, CEACAM8 binds only CEACAM6 relatively strongly compared with the binding activities of the other CEACAM members without showing homophilic-adhesion activity [8 ]. In contrast to these CEACAM antigens, CEACAM3 exhibits neither homophilic adhesion nor heterophilic adhesion to the other CEACAM members [18 ]. However, CEACAM3 binds pathogenic bacteria belonging to Neisseria through recognition of the cell-surface Opa proteins as do CEA, CEACAM1, and CEACAM6 [14 , 15 ]. No adhesion activity has been demonstrated for CEACAM4 or CEACAM7. It has been demonstrated that the homophilic adhesion of human [19 ] and mouse CEACAM1 [20 ] is mediated by direct interaction between their N-domains. Conversely, double-reciprocal interaction between the N-domain of one molecule and the A3-domain of the other was proposed for the homophilic adhesion of CEA [21 ]. In our previous studies [8 , 22 ], we have demonstrated that the N-domains of CEACAM6 and CEACAM8 are important for their heterophilic adhesion by using blocking monoclonal antibodies (mAbs) and chimeric proteins between CEA and CEACAM6. Recently, several amino acid residues in the N-domains of CEA [23 ] and CEACAM1 [24 ] have been identified as critical residues for binding to the neisserial Opa proteins. However, the structural basis is still unclear for the homophilic and heterophilic interactions between the CEACAM family molecules as well as their binding to the bacterial proteins.

In the present study, to elucidate the structure/function relationships in the homophilic adhesion of CEACAM6 and in the heterophilic adhesion between CEACAM6 and CEACAM8, we first analyzed the importance of their N-domains and then located and compared the critical amino acid residues for their adhesion activities by introducing single and multiple homologue substitutions into their sequences with particular emphasis on the residues that are unique to CEACAM6 or CEACAM8.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies and reagents
CD66 mAbs (F34-187, F106-88, TET-2, 4/3/17, 80H3, and B13.9) with different specificities against CEACAM antigens were described previously [2 ]. Fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgG was purchased from DAKO (Glostrup, Denmark). MOPC21 IgG1 was from Sigma Chemical Co. (St. Louis, MO). Alpha modified Eagle’s medium (MEM), fetal bovine serum (FBS), and geneticin were obtained from Life Technologies (Rockville, MD). Restriction enzymes and oligo-DNA primers were from Nippon Gene (Tokyo, Japan) and Hokkaido System Science (Sapporo, Japan), respectively. Fluorochrome bisbenzimide H33342 was purchased from Calbiochem (La Jolla, CA).

Preparation of Chinese hamster ovary (CHO) cells expressing recombinant CEACAM proteins
N-domains of the CEACAM6 and CEACAM8 were exchanged to each other as follows: cDNA fragments encoding CEACAM6 [25 ] and CEACAM8 [26 ] were inserted into the EcoRI restriction enzyme site of the plasmid pUC118, yielding pUC118-CEACAM6 and pUC118-CEACAM8, respectively. The 313-bp fragment that covers the leader sequence and the residues 1–71 in the N-domain of each CEACAM antigen was obtained by digestion of the plasmids with NcoI and NsiI restriction enzymes. Each fragment of CEACAM6 and CEACAM8 was inserted into the NcoI and NsiI-digested pUC118-CEACAM8 and pUC118-CEACAM6, respectively. The resultant plasmid pUC118-CEACAM6/8 encodes a chimeric protein comprised of the residues 1–71 of the CEACAM6 N-domain followed by the residues 72–108 of the CEACAM8 N-domain and the AB-domains of CEACAM8 (see Fig. 1 ). Similarly, the plasmid pUC118-CEACAM8/6 encodes a chimeric protein consisting of the residues 1–71 of the CEACAM8 N-domain followed by the residues 72–108 of the CEACAM6 N-domain and the AB-domains of CEACAM6. For preparation of CEACAM3/6, a chimeric protein consisting of the residues 1–93 of the CEACAM3 N-domain, followed by the residues 94–108 of the CEACAM6 N-domain and the AB-domains of CEACAM6, the NcoI and BgIII-digested fragment of pUC118-CEACAM3 [25 ], was replaced with the NcoI and BgIII-digested fragment of pUC118-CEACAM6. The EcoRI-digested insert of each plasmid was ligated into the unique EcoRI site of the mammalian expression vector pdKCR-neo [27 ]. CHO cells were transfected with the expression vectors by the calcium-phosphate method using a kit (Cell-Phect Kit; Amersham Pharmacia Biotech, Uppsala, Sweden) and selected by culture in alpha-MEM containing 10% FBS and 500 µg/ml geneticin. Expression of the recombinant proteins was analyzed by flow cytometry on a FACSCaliver (Becton Dickinson, San Jose, CA) after successive treatments with CD66 mAbs and FITC-labeled anti-mouse IgG and by immunoblotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described [16 ].



View larger version (54K):
[in this window]
[in a new window]
  		Figure 1. Involvement of N-domains in the cell-adhesion activities of CEACAM6 and CEACAM8. (A) Portion (residues 1–71) of each N-domain of CEACAM6 (indicated by 6) and CEACAM8 (8) was replaced by recombination of their cDNAs to yield chimeric proteins CEACAM8/6 (8/6) and CEACAM6/8 (6/8), respectively. The domain structures of these recombinant proteins are schematically shown on the right-hand side (N, N-terminal IgV-like domain; A and B, IgC2-like domains; GPI, glycosylphosphatidyl inositol). CHO cells expressing the native and chimeric proteins were mixed for 30 min, and cell aggregation was determined by calculating single cells, which were expressed as percentage of total cells. Data are means ± SD of one representative experiment performed in triplicate out of three similar ones. (B) Expression levels of the recombinant proteins on CHO cells shown by flow cytometry with F34-187, reactive with N-domain of CEACAM6; 80H3, reactive with N-domain of CEACAM8; and MOPC21, negative control.

 
Site-directed mutagenesis
To introduce mutations into the sequences of CEACAM6, CEACAM8, CEACAM6/8, and CEACAM8/6, we performed site-directed mutagenesis by polymerase chain reaction (PCR) using the long and accurate (LA) PCR in vitro Mutagenesis Kit (Takara, Tokyo, Japan), which is based on the method of Ito et al. [28 ], according to the manufacturer’s instructions. Primers used for introducing mutations were: CEACAM6-V21, 5'-GGT TGT GGA CGA GTA GAA GA-3'; CEACAM6-H27, 5'-TGC TGT AAC CAA TAC GAT GCT G-3'; CEACAM6-L28, 5'-TGC TGT AAC CAA TAA GA TTC TG-3'; CEACAM6-F29, 5'-TGC TGT AAC CAA AAC GAT TCT G-3'; CEACAM6-Q44, 5'-CCT ATA ATT TGA CTG TTG CCA T-3'; CEACAM6-N32, 5'-CTT TGT ACC AGT TGT AAC CAA-3'; CEACAM8-STFV, 5'-ACC AGC TGT AGC CAC GAG G-3'; CEACAM8-NRI, 5'-AGT TGT AGC CAA TAC GGT TCT GG-3'; CEACAM8-S32, 5'-TGT ACC AGC TGT AGC CAA TA-3'; CEACAM8-RG, 5'-CGG TTG CCA TCC ACT CTT TCC-3'; CEACAM8-Q44, 5'-TCC TAT AAT TTG ACG GTT GGC-3'; CEACAM8-GTA, 5'-TAA GCT GTT GAG TTC CTA TTA CAT-3'; CEACAM8-G63, 5'-TCG ACG GTT GCC ATC CAC TCT-3'; CEACAM3(6)/6, 5'-AGC TGT AGC CAA TAA GAT TCT G-3'; and CEACAM3(8)/6, 5'-TGT ACC AGC TGT AGC CAA TA-3'/5'-CTA CAA TTC GAC TGT TGC CA-3'. The sequences and mutations were confirmed by sequencing with a DNA sequencer (PE Applied Biosystem, Foster City, CA). An EcoRI fragment that encodes each mutant was ligated into the EcoRI site of the pdKCR-neo vector for transfection of CHO cells as described above.

Cell-adhesion assays
We used two different methods, a cell-aggregation assay and a cell-attachment assay, to evaluate cell-adhesion activities. For the cell-aggregation assay, CHO transfectants expressing CEACAM proteins were dissociated by incubation in phosphate-buffered saline (PBS) containing 0.1% trypsin and 5 mM ethylenediaminetetraacetate (EDTA) and were resuspended in alpha-MEM at 1 x 106 cells/ml. Cell aggregation was performed in aliquots of 200 µl in wells of round-bottom 96-well plates (Nunc, Roskilde, Denmark), which were rotated at 240 rpm on a platform shaker at room temperature. After 30 min, the number of nonaggregated cells was microscopically counted with a hemacytometer, and the percentage of cell aggregation was calculated. Alternatively, cell-adhesion activity was assessed by attachment of CHO transfectants to CEACAM protein-coated plates. Recombinant CEACAM6 and CEACAM8 proteins or bovine serum albumin (BSA; 1 µg/well) were dried up onto the wells of 96-well flat-bottom plates (Greiner, Frickenhausen, Germany) as described previously [22 ]. CHO transfectants were labeled with H33342 (50 µg/ml in serum-free medium) for 30 min at 37°C, plated into the CEACAM-coated wells (5x104 cells/100 µl/well), and allowed to attach to the bottom for 20 min at room temperature. Wells were gently washed twice with 100 µl PBS, and cells adhered to the bottom were solubilized in 100 µl of 0.2% (w/v) Nonidet P-40. Fluorescence of the cell lysates was determined by using a Fluoroskan II reader (Labsystems Oy, Helsinki, Finland). Percent-adherence was calculated by the following formula: (fluorescence of adherent cells - fluorescence of solubilizing buffer)/fluorescence of total cells added - fluorescence of solubilizing buffer) x 100.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Importance of N-domains for cell-adhesion activities of CEACAM6 and CEACAM8
CEACAM6 exhibits homophilic adhesion and heterophilic adhesion to CEA, CEACAM1, and CEACAM8, whereas CEACAM8 shows only heterophilic adhesion to CEACAM6 [6 , 8 ]. We have demonstrated recently that contact between the N-domains of CEACAM6 and CEACAM8 is sufficient for their heterophilic adhesion, by using CHO transfectants that express chimeric proteins comprised of the N-domain of CEACAM6 or CEACAM8 and the transmembrane and cytoplasmic regions of CEACAM3 [18 ]. However, the chimeric protein possessing the N-domain of CEACAM6 did not show homophilic adhesion, possibly because of the lack of the membrane-proximal A- and B-domains. Here, to clarify whether the homophilic adhesion of CEACAM6 is mediated by direct interaction between two N-domains, we verified cell-adhesion activity of a chimeric protein, CEACAM6/8, which consisted of the residues 1–71 of the CEACAM6 N-domain and the residues 72–108 of the CEACAM8 N-domain and the AB-domains of CEACAM8. Figure 1A shows that CEACAM6/8 revealed a homophilic-adhesion activity similar to that of CEACAM6, suggesting that the N-domain of a CEACAM6 molecule interacts with the N-domain of the opposite CEACAM6 molecule. In our previous study [8 ], we showed that the first 93 residues, but not the last 15 residues, of the N-domain of CEACAM6 are important for its heterophilic adhesion to CEACAM8. As shown in Figure 1A , CEACAM6/8 bound CEACAM8/6, another chimeric protein comprised of the residues 1–71 of the CEACAM8 N-domain, the residues 72–108 of the CEACAM6 N-domain, and the AB-domains of CEACAM6. Figure 1B demonstrates that the expression levels of the recombinant CEACAMs were similar to each other. These findings suggest that the sequences of the NH2-terminal 71 residues of CEACAM6 and CEACAM8 are essential for their adhesion activities and also confirm our previous finding that the heterophilic adhesion between CEACAM6 and CEACAM8 is mediated by their N–N interaction [18 ].

N-domain residues critical for the homophilic adhesion of CEACAM6 and its heterophilic adhesion to CEACAM8
To locate the amino acid residues critical for the homophilic adhesion of CEACAM6, we carried out homologue mutagenesis screening of the N-domain. The CEACAM family antigens show similar amino acid sequences of their N-domains to each other with about 70–90% identities (Fig. 2 ). Each residue unique to the N-domain (the residues 1–71) of CEACAM6 was replaced with the respective residue of CEA by site-directed mutagenesis (Fig. 3A ). As shown in Figure 4A , CHO cells expressing CEACAM6-H27, a CEACAM6 mutant whose 27th residue of the N-domain is histidine, were reacted with F34-187 and F106-88, mAbs for epitopes on the N- and the AB-domains of CEACAM6, respectively [2 ]. Further, 4/3/17, which recognizes CEA and CEACAM1 but hardly CEACAM6 [2 ], bound to CEACAM6-H27, indicating that the epitope for this mAb was newly generated on CEACAM6 by substitution of N27 with histidine. Similar reactivities with F34-187 and F106-88 were observed for CEACAM6-N32 (Fig. 4B) and the other CEACAM6 mutants (unpublished results). Moreover, these mutants showed similar molecular masses on SDS-PAGE to that of the CEACAM6 (about 80 kDa; unpublished results). These findings imply that the extracellular domains of the CEACAM6 mutants were expressed properly on the cell surfaces and that their expression levels are similar enough to compare their cell-adhesion activities.



View larger version (33K):
[in this window]
[in a new window]
  		Figure 2. Amino acid sequences of the N-domains of CEACAM antigens deduced from their cDNA sequences. The residues identical to those of CEA are indicated by dashes.

 


View larger version (24K):
[in this window]
[in a new window]
  		Figure 3. Amino acid sequences of CEACAM mutants and their adhesion activities. Residues 1–64 of N-domains are shown. (A) CEACAM8 mutants; (B) CEACAM6 mutants; (C) CEACAM3/6 mutants. Dashes indicate the residues identical to those of each parental protein. ++, Strong binding; +, weak binding; -, no binding.

 


View larger version (37K):
[in this window]
[in a new window]
  		Figure 4. Flow cytometric analysis of CHO cells expressing CEACAM mutants. CHO transfectants expressing CEACAM mutants—(A) CEACAM6-H27; (B) CEACAM6-N32; (C) CEACAM8-S32; (D) CEACAM8-Q44—were reacted with mAbs for the N-domain (F34-187, 4/3/17) or AB-domains (F106-88) of CEACAM6 (A and B) and for the N-domain (80H3 and B13.9) or AB-domains (TET-2) of CEACAM8 (C and D). See Figure 3A and 3B , for the locations of mutation. MOPC21 IgG was used for negative control. Binding of mAbs was analyzed by flow cytometry after incubation with FITC-conjugated anti-mouse IgG.

 
When homophilic-adhesion activity was examined by the cell-aggregation assay, CEACAM6-H27 showed no binding activity to itself (Fig. 5 ). In contrast, the exchange of alanine at position 21 with valine (CEACAM6-V21) did not result in the loss of the homophilic adhesion. CEACAM6-L28 and CEACAM6-F29 also retained homophilic-adhesion activity. Although neither serine at position 32 nor leucine at position 44 of CEACAM6 is unique to CEACAM6 (see Fig. 2 ), we examined the importance of these two residues, because as shown below, the 32nd and the 44th residues of CEACAM8 were essential for its heterophilic adhesion to CEACAM6. Replacement of S32 with asparagine (CEACAM6-N32) or replacement of L44 with glutamine (CEACAM6-Q44) resulted in the loss of the homophilic-adhesion activity of CEACAM6.



View larger version (70K):
[in this window]
[in a new window]
  		Figure 5. Effects of mutations in the N-domain amino acid sequence on the homophilic-adhesion activity of CEACAM6. CHO cells expressing CEACAM6 (indicated by 6) or CEACAM6 mutants, CEACAM6-V21 (6-V21), CEACAM6-H27 (6-H27), CEACAM6-L28 (6-L28), CEACAM6-F29 (6-F29), CEACAM6-N32 (6-N32), or CEACAM6-Q44 (6-Q44), were mixed for 30 min. Cell aggregation was determined by calculating single cells, which were expressed as percentage of total cells. See Figure 3A for the locations of mutation. Data are means ± SD of one representative experiment performed in triplicate out of three similar ones.

 
These mutants were also used for analyzing the heterophilic adhesion of CEACAM6 to CEACAM8. CEACAM6-H27, which did not show homophilic adhesion (Fig. 5) , exhibited no heterophilic adhesion to CEACAM8 either (Fig. 6 ). Although CEACAM6-F29 retained homophilic-adhesion activity (Fig. 5) , it did not show heterophilic adhesion to CEACAM8. A moderate inhibition was observed in the heterophilic adhesion of CEACAM6-L28 and CEACAM6-N32 to CEACAM8, whereas no inhibition was seen for CEACAM6-V21 or CEACAM6-Q44. These results are summarized in Figure 3A .



View larger version (65K):
[in this window]
[in a new window]
  		Figure 6. Effects of mutations in the N-domain amino acid sequence on the heterophilic-adhesion activity of CEACAM6 to CEACAM8. CHO cells expressing CEACAM6 mutants, CEACAM6-V21 (indicated by 6-V21) CEACAM6-H27 (6-H27), CEACAM6-L28 (6-L28), CEACAM6-F29 (6-F29), CEACAM6-N32 (6-N32), or CEACAM6-Q44 (6-Q44), were mixed with CHO cells expressing CEACAM8 (8) for 30 min. Cell aggregation was determined by calculating single cells, which were expressed as percentage of total cells. See Figure 3A for the locations of mutation. Data are means ± SD of one representative experiment performed in triplicate out of three similar ones.

 
N-domain residues of CEACAM8 essential for the heterophilic adhesion to CEACAM6
Similar analyses were carried out for identification of critical residues in the N-domain of CEACAM8 for its heterophilic adhesion to CEACAM6. When N32 of CEACAM8 was replaced with serine (CEACAM8-S32, see Fig. 3B ), which is common to the 32nd residues of CEACAM6, CEACAM1, and CEA, the heterophilic-adhesion activity to CEACAM6 was abrogated (Fig. 7 ). The substitution of R44 of CEACAM8 with glutamine (CEACAM8-Q44) also resulted in loss of the adhesion activity to CEACAM6. The molecular integrity of these two mutants lacking heterophilic-adhesion activity was suggested by the following findings: Both mutants were recognized by mAbs 80H3 and TET.2, which are reactive with the N- and the AB-domains of CEACAM8, respectively [2 ] (Fig. 4C and 4D) , and they showed similar molecular masses on SDS-PAGE to that of CEACAM8 (about 90 kDa; unpublished results). A modification in the secondary structure of CEACAM8-Q44 was indicated by the result that it was not reactive with B13.9 (Fig. 4D) , which recognizes an epitope different from that for 80H3 on the N-domain of CEACAM8 [2 ]. No significant change in the heterophilic-adhesion activity was observed by replacement of the other 13 residues unique to CEACAM8, including the 27th–29th residues, and none of these changes resulted in acquisition of homophilic-adhesion activity (summarized in Fig. 3B ).



View larger version (48K):
[in this window]
[in a new window]
  		Figure 7. Effects of mutations in the N-domain amino acid sequence on the heterophilic-adhesion activity of CEACAM8 to CEACAM6. CHO cells expressing CEACAM8 mutants, CEACAM8-N27, R28, I29 (indicated by 8-NRI), CEACAM8-S32 (8-S32), or CEACAM8-Q44 (8-Q44), were mixed with CHO cells expressing CEACAM6 (6) for 30 min. Cell aggregation was determined by calculating single cells, which were expressed as percentage of total cells. Homophilic adhesion of CEACAM8 (8; negative) and CEACAM6 (positive) is also shown. See Figure 3B for the locations of mutation. Data are means ± SD of one representative experiment performed in triplicate out of three similar ones.

 
Acquisition of adhesion activity by CEACAM3 after introduction of the residues important for the adhesion activities of CEACAM6 and CEACAM8
To confirm the importance of the N-domain residues demonstrated above, we prepared a chimeric molecule between CEACAM3 and CEACAM6 (designated CEACAM3/6), whose CEACAM3-derived N-domain (the residues 1–93) was partly altered by introducing the residues critical for the adhesion activity of CEACAM6 or CEACAM8. Just as CEACAM3 reveals no binding activity to any of the CEACAM family antigens [18 ], CEACAM3/6 exhibited no adhesion activity to itself (Fig. 8A ), CEACAM6, or CEACAM8 (unpublished results). N27 and I29, the residues critical for the adhesion activities of CEACAM6, were introduced into CEACAM3/6, yielding the chimeric mutant CEACAM3(6)/6 (see Fig. 3C ). The other two residues, S32 and L44, important for the adhesion activity of CEACAM6, are shared by CEACAM3/6. Conversely, N32 and R44, the residues essential for the heterophilic-adhesion activity of CEACAM8, were introduced into CEACAM3/6, which was designated CEACAM3(8)/6. Surprisingly, as shown in Figure 8A , CEACAM3(8)/6 as well as CEACAM3(6)/6 exhibited homophilic-adhesion activity. In this cell-aggregation assay, detection of the heterophilic-adhesion activity of CEACAM3(6)/6 to CEACAM8 and that of CEACAM3(8)/6 to CEACAM6 was difficult because of their own homophilic-adhesion activities. Therefore, we used a different experimental system in which attachment of the CHO transfectants to CEACAM protein-coated plates was evaluated. Figure 8B shows that the CHO cells expressing CEACAM3(6)/6 adhered to the CEACAM8-coated plate, and the cells expressing CEACAM3(8)/6 adhered to the CEACAM6-coated plates. It was thus demonstrated that N27, S32, and L44 are crucial for the homophilic adhesion of CEACAM6 and that N27 and I29 of CEACAM6 and N32 and R44 of CEACAM8 are essential for their heterophilic adhesion.



View larger version (42K):
[in this window]
[in a new window]
  		Figure 8. Acquisition of adhesion activity by CEACAM3/6 after introduction of the residues critical for the binding between CEACAM6 and CEACAM8. N27 and I29 of CEACAM6 were introduced into CEACAM3/6, which consists of the N-domain (residues 1–93) of CEACAM3 followed by residues 94–108 of the CEACAM6 N-domain and the AB-domains of CEACAM6, yielding CEACAM3(6)/6. N32 and Q44 of CEACAM8 were also introduced into CEACAM3/6, yielding CEACAM3(8)/6. See Figure 3C for the locations of mutation. (A) Homophilic-adhesion activity of CHO cells expressing CEACAM3(6)/6 and CEACAM3(8)/6 was examined by cell aggregation. Single cells were expressed as percentage of total cells. (B) CHO cells expressing CEACAM3(6)/6 and CEACAM3(8)/6 were labeled with H33342, their attachment to the plates was coated with BSA, and CEACAM6 and CEACAM8 proteins were assessed as described in Materials and Methods. Data are means ± SD of one representative experiment performed in triplicate out of two similar ones. *P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
N-terminal domains are generally important for the interactions of cell-adhesion molecules that belong to the immunoglobulin supergene family, including CD80 [29 ], CD31 [30 ], and intercellular adhesion molecule-3 (ICAM-3) [31 ]. Among the CEACAM family members, CEACAM1, which exhibits about 90% homology in the N-domain sequence with CEA and CEACAM6, has been shown to exhibit homophilic binding by direct contact between two N-domains [19 ]. The heterophilic adhesion between CEACAM6 and CEACAM8 was also shown to be mediated by the same head-to-head interaction [18 ]. It was demonstrated, however, that the homophilic binding of CEA is mediated by contact of the N-domain of one molecule with the B3-domain of the other [21 ]. We have shown in the present study that the homophilic adhesion of CEACAM6 is mediated by the N–N interaction. In our previous study [18 ], the chimeric molecule consisting of the N-domain of CEACAM6 and the membrane-spanning and cytoplasmic domains of CEACAM3 did not exhibit homophilic adhesion with the parental CEACAM6, although it bound CEACAM8. Because the two membrane-binding modes of proteins, the transmembrane form and the glycolipid-linked form, do not affect the cell-adhesion activity of CEACAM6 (unpublished data), it is likely that the length of the outer membrane portion is critical for the relatively weak homophilic-adhesion activity of CEACAM6.

We next located the amino acid residues in the N-domains crucial for the homophilic adhesion of CEACAM6 and the heterophilic adhesion between CEACAM6 and CEACAM8 by homologue-scanning mutagenesis. It was shown that N27, S32, and L44 are key residues for the homophilic adhesion of CEACAM6, whereas N27 and I29, and to a lesser degree, L28 and S32, also are important for the heterophilic adhesion of CEACAM6 to CEACAM8. Conversely, in CEACAM8, N32 and R44 were essential for its heterophilic adhesion to CEACAM6. However, in contrast to the 27th–29th residues of CEACAM6, those of CEACAM8 did not appear important for its heterophilic-adhesion activity to CEACAM6. The region containing these 27th–29th residues in CEACAM8 is of a strong hydrophilic nature in contrast to the hydrophobic or weak hydrophilic nature of this region in the other CEACAM antigens. This might be partly associated with the lack of homophilic-adhesion activity for CEACAM8.

As demonstrated in this paper, substitution of one residue in CEACAM6 induced strong inhibition of its homophilic-adhesion activity. Introduction of each critical residue alone into CEACAM8 did not result in acquisition of homophilic-adhesion activity. However, as shown in Figure 8 , CEACAM3(6)/6, which had the four key residues N27, I29, S32, and L44 of CEACAM6, acquired homophilic- and heterophilic-adhesion activities to CEACAM6. The key residues located in different positions in the N-domains of CEACAM6 and CEACAM8 are thus equally prerequisites for their full adhesion activities.

The residues critical for the homophilic and heterophilic adhesion of these CEACAM antigens may be located on the potential GFCC'C'' face of their N-domains predicted from the three-dimensional model of CEA based on the structures of CD2, CD4, and REI [23 , 24 , 32 , 33 ]. The possible secondary structure predicted from the sequence, however, suggested that the N-domain of CEACAM6 lacks the C strand that is predicted to exist in CEA and CEACAM1, both of which display no adhesion activity to CEACAM8 [8 ]. The absence of the C strand might be related to the specific affinity of CEACAM6 to CEACAM8. As we have suggested in a previous paper, that the carbohydrate portion is not required for the adhesion between CEACAM6 and CEACAM8 [22 ], the N-linked carbohydrate chains possibly existing in the N-domains of CEACAM6 and CEACAM8 [22 , 26 ] are not located in the regions analyzed in the present study.

In addition to the adhesion among the family members, most of the CEACAM antigens bind Opa proteins of N. meningitides [14 ] and N. gonorrhoeae [15 , 16 ]. This is likely to be related to infectious activity of the bacteria or recognition of the bacteria by phagocytes that express the CEACAM antigens. The recent studies using site-directed mutagenesis demonstrated several amino acid residues critical for the binding activity of the CEACAM family antigens to the Opa proteins. Virji et al. [24 ] showed by alanine-scanning mutagenesis that the substitution of Y34 of CEACAM1 resulted in a complete loss of the binding of CEACAM1 to OpaA, OpaB, and OpaC and that differential effects against these three Opa variants were seen by alanine substitution of S32, V39, or Q44. The positions of two of these residues, S32 and Q44, are the same as those of the residues prerequisite for the homophilic adhesion of CEACAM6 (S32 and L44). In a similar study, Bos et al. [23 ] showed that crucial residues in the N-domain of CEA for the adhesion of OpaB, OpaC, and OpaI include F29 and S32, by using homologue-scanning mutagenesis between CEA and CEACAM8, which shows no affinity for any Opa proteins. It is interesting that the 29th and 32nd residues in CEACAM6 were essential for its heterophilic adhesion to CEACAM8, which lacks any adhesion activity except to CEACAM6. It is intriguing to speculate that the three-dimensional structures of the Opa proteins required for the binding to CEACAM6 may resemble that of CEACAM8.

The recent study on the mechanism of homophilic binding of CEA [33 ] demonstrated that three regions in the N-domain, the residues 30–35, 42–46, and 80–84, are important for the binding, similar to our present findings on the adhesion activity of CEACAM6 and CEACAM8. It remains to be elucidated, however, whether the residues critical for the adhesion activity of CEACAM6 to self or to CEACAM8 are also important for its heterophilic adhesion to CEA and CEACAM1. Our present study thus suggests that subtle variations on several restricted regions in the N-domains alter the specificity of the adhesion activity of the CEACAM antigens against self, the same family antigens, and the bacterial proteins.


   ACKNOWLEDGEMENTS
 
This work was supported in part by Grant-in-Aids for High-Technology Research Center and for Scientific Research (C) from the Ministry of Education, Science, Sports and Culture, Japan. We thank Yoshino Matsuo for technical assistance.

Received March 7, 2001; revised June 4, 2001; accepted June 4, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Thompson, J. A., Grunert, F., Zimmermann, W. (1991) Carcinoembryonic antigen gene family: molecular biology and clinical perspectives J. Clin. Lab. Anal. 5,344-366[Medline]
  2. Skubitz, K. M., Grunert, F., Jantscheff, P., Kuroki, M., Skubitz, P. N. (1996) CD66 family workshop panel report Kishimoto, T. Kikutani, H. von dem Borne, A. E. G. K. Goyert, S. M. Mason, D. Y. Miyasaka, M. Moretta, L. Okumura, K. Shaw, S. Springer, T. A. Sugamura, S. Zola, H. eds. Leukocyte Typing VI New York London.
  3. Beauchemin, N., Draber, P., Dveksler, G., Gold, P., Gray-Owen, S., Grunert, F., Hammarström, S., Holmes, K. V., Karlsson, A., Kuroki, M., Lin, S. H., Lucka, L., Najjar, S. M., Neumaier, M., Öbrink, B., Shively, J. E., Skubitz, K. M., Stanners, C. P., Thomas, P., Thompson, J. A., Virji, M., von Kleist, S., Wagener, C., Watt, S., Zimmermann, W. (1999) Redefined nomenclature for members of the carcinoembryonic antigen family Exp. Cell Res. 252,243-249[Medline]
  4. Prall, F., Nollau, P., Neumaier, M., Haubeck, H. D., Drzeniek, Z., Helmchen, U., Loning, T., Wagener, C. (1996) CD66a (BGP), an adhesion molecule of the carcinoembryonic antigen family, is expressed in epithelium, endothelium, and myeloid cells in a wide range of normal human tissues J. Histochem. Cytochem. 44,35-41[Abstract]
  5. Benchimol, S., Fuks, A., Jothy, S., Beauchemin, N., Shirota, K., Stanners, C. P. (1989) Carcinoembryonic antigen, a human tumor marker, functions as an intercellular adhesion molecule Cell 57,327-334[Medline]
  6. Oikawa, S., Inuzuka, C., Kuroki, M., Matsuoka, Y., Kosaki, G., Nakazato, H. (1989) Cell adhesion activity of non-specific cross-reacting antigen (NCA) and carcinoembryonic antigen (CEA) expressed on CHO cell surface: homophilic and heterophilic adhesion Biochem. Biophys. Res. Commun. 164,39-45[Medline]
  7. Rojas, M., Fuks, A., Stanners, C. P. (1990) Biliary glycoprotein, a member of the immunoglobulin supergene family, functions in vitro as a Ca2(+)-dependent intercellular adhesion molecule Cell Growth Differ 1,527-533[Abstract]
  8. Oikawa, S., Inuzuka, C., Kuroki, M., Arakawa, F., Matsuoka, Y., Kosaki, G., Nakazato, H. (1991) A specific heterotypic cell adhesion activity between members of carcinoembryonic antigen family, W272 and NCA, is mediated by N-domains J. Biol. Chem. 266,7995-8001[Abstract/Free Full Text]
  9. Kuroki, M., Matsuo, Y., Kinugasa, T., Matsuoka, Y. (1992) Augmented expression and release of nonspecific cross-reacting antigens (NCAs), members of the CEA family, by human neutrophils during cell activation J. Leukoc. Biol. 52,551-557[Abstract]
  10. Skubitz, K. M., Campbell, K. D., Skubitz, A. P. N. (1996) CD66a, CD66b, CD66c, and CD66d each independently stimulate neutrophils J. Leukoc. Biol. 60,106-117[Abstract]
  11. Stocks, S. C., Ruchaud-Sparagano, M. H., Kerr, M. A., Grunert, F., Haslett, C., Dransfield, I. (1996) CD66: role in the regulation of neutrophil effector function Eur. J. Immunol. 26,2924-2932[Medline]
  12. Ottonello, L., Epstein, A. L., Dapino, P., Barbara, P., Morone, P., Dallegri, F. (1999) Monoclonal Lym-1 antibody-dependent cytolysis by neutrophils exposed to granulocyte-macrophage colony-stimulating factor: intervention of Fc gamma R-II(CD32), CD11b-CD18 integrins, and CD66b glycoproteins Blood 93,3505-3511[Abstract/Free Full Text]
  13. Leusch, H. G., Drzeniek, Z., Markos-Pusztai, Z., Wagener, C. (1991) Binding of Escherichia coli and Salmonella strains to members of the carcinoembryonic antigen family: differential binding inhibition by aromatic alpha-glycosides of mannose Infect. Immun. 59,2051-2057[Abstract/Free Full Text]
  14. Chen, T., Grunert, F., Medina-Marino, A., Gotschlich, E. C. (1997) Several carcinoembryonic antigens (CD66) serve as receptors for gonococcal opacity proteins J. Exp. Med. 185,1557-1564[Abstract/Free Full Text]
  15. Virji, M., Makepeace, K., Ferguson, D. J. P., Watt, S. M. (1996) Carcinoembryonic antigens (CD66) on epithelial cells and neutrophils are receptors for Opa proteins of pathogenic neisseriae Mol. Microbiol. 22,941-950[Medline]
  16. Bos, M. P., Kuroki, M., Krop-Watorek, A., Hogan, D., Belland, R. J. (1998) CD66 receptor specificity exhibited by neisserial Opa variants controlled by protein determinants in CD66 N-domains Proc. Natl. Acad. Sci. USA 95,9584-9589[Abstract/Free Full Text]
  17. Oikawa, S., Kuroki, M., Matsuoka, Y., Kosaki, G., Nakazato, H. (1992) Homotypic and heterotypic Ca(++)-independent cell adhesion activities of biliary glycoprotein, a member of carcinoembryonic antigen family, expressed on CHO cell surface Biochem. Biophys. Res. Commun. 186,881-887[Medline]
  18. Oikawa, S., Sugiyama, M., Kuroki, M., Kuroki, M., Nakazato, H. (2000) Extracellular N-domain alone can mediate specific heterophilic adhesion between members of the carcinoembryonic antigen family, CEACAM6 and CEACAM8 Biochem. Biophys. Res. Commun. 278,564-568[Medline]
  19. Teixeira, A. M., Fawcett, J., Simmons, D. L., Watt, S. M. (1994) The N-domain of the biliary glycoprotein (BGP) adhesion molecule mediates homotypic binding: domain interactions and epitope analysis of BGPc Blood 84,211-219[Abstract/Free Full Text]
  20. Wikstrom, K., Kjellstrom, G., Obrink, B. (1996) Homophilic intercellular adhesion mediated by C-CAM is due to a domain 1-domain 1 reciprocal binding Exp. Cell Res. 227,360-366[Medline]
  21. Zhou, H., Fuks, A., Alcaraz, G., Bolling, T. J., Stanners, C. P. (1993) Homophilic adhesion between Ig superfamily carcinoembryonic antigen molecules involves double reciprocal bonds J. Cell Biol. 122,951-960[Abstract/Free Full Text]
  22. Yamanaka, T., Kuroki, M., Matsuo, Y., Matsuoka, Y. (1996) Analysis of heterophilic cell adhesion mediated by CD66b and CD66c using their soluble recombinant proteins Biochem. Biophys. Res. Commun. 219,842-847[Medline]
  23. Bos, M. P., Hogan, D., Belland, R. J. (1999) Homologue scanning mutagenesis reveals CD66 receptor residues required for neisserial Opa protein binding J. Exp. Med. 190,331-340[Abstract/Free Full Text]
  24. Virji, M., Evans, D., Hadfield, A., Grunert, F., Teixeira, A. M., Watt, S. M. (1999) Critical determinants of host receptor targeting by Neisseria meningitides and Neisseria gonorrhoeae: identification of Opa adhesiotopes on the N-domain of CD66 molecules Mol. Microbiol. 34,538-551[Medline]
  25. Kuroki, M., Arakawa, F., Matsuo, Y., Oikawa, S., Misumi, Y., Nakazato, H., Matsuoka, Y. (1991) Molecular cloning of nonspecific cross-reacting antigens in human granulocytes J. Biol. Chem. 266,11810-11817[Abstract/Free Full Text]
  26. Arakawa, F., Kuroki, M., Misumi, Y., Oikawa, S., Nakazato, H., Matsuoka, Y. (1990) Characterization of a cDNA clone encoding a new species of the nonspecific cross-reacting antigen (NCA), a member of the CEA gene family Biochem. Biophys. Res. Commun. 166,1063-1071[Medline]
  27. Hashino, J., Fukuda, Y., Oikawa, S., Nakazato, H., Nakanishi, T. (1994) Metastatic potential of human colorectal carcinoma SW1222 cells transfected with cDNA encoding carcinoembryonic antigen Clin. Exp. Metastasis 12,324-328[Medline]
  28. Ito, W., Ishiguro, H., Kurosawa, Y. (1991) A general method for introducing a series of mutations into cloned DNA using the polymerase chain reaction Gene 102,67-70[Medline]
  29. Fargeas, C. A., Truneh, A., Reddy, M., Hurle, M., Sweet, R., Sekaly, R. P. (1995) Identification of residues in the V domain of CD80 (B7-1) implicated in functional interactions with CD28 and CTLA4 J. Exp. Med. 182,667-675[Abstract/Free Full Text]
  30. Newton, J. P., Buckley, C. D., Jones, E. Y., Simmons, D. L. (1997) Residues on both faces of the first immunoglobulin fold contribute to hemophilic binding sites of PECAM-1/CD31 J. Biol. Chem. 272,20555-20563[Abstract/Free Full Text]
  31. Klickstein, L. B., York, M. R., Fougerolles, A. R., Springer, T. A. (1996) Localization of the binding site on intercellular adhesion molecule-3 (ICAM-3) for lymphocyte function-associated antigen 1 (LFA-1) J. Biol. Chem. 271,23920-23927[Abstract/Free Full Text]
  32. Bates, P. A., Luo, J., Sternberg, M. J. (1992) A predicted three-dimensional structure for the carcinoembryonic antigen (CEA) FEBS Lett 301,207-214[Medline]
  33. Taheri, M., Saragovi, U., Fuks, A., Makkerh, J., Mort, J., Stanners, C. P. (2000) Self recognition in the Ig superfamily. Identification of precise subdomains in carcinoembryonic antigen required for intercellular adhesion J. Biol. Chem. 275,26935-26943[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Leukoc. Biol.Home page
M. Ozeki and J. E. Shively
Differential cell fates induced by all-trans retinoic acid-treated HL-60 human leukemia cells
J. Leukoc. Biol., September 1, 2008; 84(3): 769 - 779.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
T. Tanaka, J. Huang, S. Hirai, M. Kuroki, M. Kuroki, N. Watanabe, K. Tomihara, K. Kato, and H. Hamada
Carcinoembryonic Antigen-Targeted Selective Gene Therapy for Gastric Cancer through FZ33 Fiber-Modified Adenovirus Vectors.
Clin. Cancer Res., June 15, 2006; 12(12): 3803 - 3813.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
N. Stern, G. Markel, T. I. Arnon, R. Gruda, H. Wong, S. D. Gray-Owen, and O. Mandelboim
Carcinoembryonic Antigen (CEA) Inhibits NK Killing via Interaction with CEA-Related Cell Adhesion Molecule 1
J. Immunol., June 1, 2005; 174(11): 6692 - 6701.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
G. Markel, R. Gruda, H. Achdout, G. Katz, M. Nechama, R. S. Blumberg, R. Kammerer, W. Zimmermann, and O. Mandelboim
The Critical Role of Residues 43R and 44Q of Carcinoembryonic Antigen Cell Adhesion Molecules-1 in the Protection from Killing by Human NK Cells
J. Immunol., September 15, 2004; 173(6): 3732 - 3739.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. M. Comegys, S.-H. Lin, D. Rand, D. Britt, D. Flanagan, H. Callanan, K. Brilliant, and D. C. Hixson
Two Variable Regions in Carcinoembryonic Antigen-related Cell Adhesion Molecule1 N-terminal Domains Located in or Next to Monoclonal Antibody and Adhesion Epitopes Show Evidence of Recombination in Rat but Not in Human
J. Biol. Chem., August 13, 2004; 279(33): 35063 - 35078.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
D. Finkenzeller, B. Fischer, S. Lutz, H. Schrewe, T. Shimizu, and W. Zimmermann
Carcinoembryonic Antigen-Related Cell Adhesion Molecule 10 Expressed Specifically Early in Pregnancy in the Decidua Is Dispensable for Normal Murine Development
Mol. Cell. Biol., January 1, 2003; 23(1): 272 - 279.
[Abstract] [Full Text]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
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 Kuroki, M.
Right arrow Articles by Kuroki, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kuroki, M.
Right arrow Articles by Kuroki, M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS