Homophilic adhesion of human CEACAM1 involves N-terminal domain interactions: structural analysis of the binding site.

CEACAM1 on leukocytic, endothelial, and epithelial cells functions in homophilic adhesion, tumor suppression, regulating cell adhesion and proliferation, and in heterophilic adhesion as a receptor for E-selectin and Neisseria meningiditis, Neisseria gonorrhoeae, Haemophilus influenzae, and murine coronaviruses. The 8 transmembrane isoforms of human CEACAM1 possess an extracellular N-terminal IgV domain, followed by variable numbers of IgC2 domains. To establish which key amino acids contribute specifically to CEACAM1 homophilic adhesion, exposed amino acids in the N-terminal domain of a soluble form of CEACAM1 were subjected to mutagenesis. Analyses of mutant proteins with conformationally dependent antibodies indicated that most mutations did not substantially affect the structural integrity of CEACAM1. Nevertheless, decreased adhesion was observed for the single mutants V39A or D40A (single-letter amino acid codes) in the CC' loop and for the triple mutants located in the GFCC'C" face of the N-terminal domain. Interestingly, whereas single mutations in R64 or D82 that are predicted to form a salt bridge between the base of the D and F beta strands close to the critical V39 and D40 residues also abolish adhesion, an amino acid swap (R64D and D82R), which maintains the salt bridge was without significant effect. These studies indicate that the CC' loop plays a crucial role in the homophilic adhesion of CEACAM1. They further predict that specific hydrophobic amino acid residues on the nonglycosylated GFCC'C" face of CEACAM1 N-terminal domain are not only involved in heterophilic interactions with Opa proteins and H influenzae, but are also critical for protein-protein interactions between 2 CEACAM1 molecules on opposing cells.

The CGM1a (CEACAM3/CD66d)-mediated phagocytic pathway of Neisseria gonorrhoeae expressing opacity proteins is also the pathway to cell death.

Phagocytosis of Opa+ Neisseria gonorrhoeae (gonococcus, GC) by neutrophils is in part dependent on the interaction of Opa proteins with CGM1a (CEACAM3/CD66d) antigens, a neutrophil-specific receptor. However, the signaling pathways leading to phagocytosis have not been characterized. Here we show that interaction of OpaI bacteria with neutrophils or CGM1a-transfected DT40 cells induces calcium flux, which correlates with phagocytosis of bacteria. We identified an immunoreceptor tyrosine-based activation motif (ITAM) in CGM1a, and showed that the ability of CGM1a to transduce signals and mediate phagocytosis was abolished by mutation of the ITAM tyrosines. We also demonstrated that CGM1a-ITAM-mediated bacterial phagocytosis is dependent on Syk and phospholipase C activity in DT40 cells. Unexpectedly, the activation of the CGM1a-ITAM phagocytic pathway by Opa+ GC results in induction of cell death.

Locally inducible CD66a (CEACAM1) as an amplifier of the human intestinal T cell response.

CD66a is an adhesion molecule member of the carcinoembryonic antigen immunoglobulin-like family present on the surface of epithelial cells, granulocytes and IL-2 activated T cells. We studied whether CD66a is expressed in vivo by T lymphocytes and whether it affects TCR-mediated activation. CD66a was detected by histochemistry, flow cytometry analysis, reverse transcription PCR and Western blot on fresh colon biopsies and T cell clones. A fraction of T cells in the lamina propria express CD66a, which is induced by IL-7 and IL-15 cytokines. T cells express four different CD66a splice variants and at least two forms of the protein are glycosylated in a cell type-specific manner. Triggering of CD66a on T cells with physiological ligands or with specific mAb increases TCR-mediated lymphokine release, in an antigen dose-independent manner. This effect requires the presence of the CD66a intracytoplasmic domain, which contains two immunoglobulin receptor family tyrosine-based inhibitory motif-like domains, as shown by stimulation of Jurkat cells transfected with different CD66a isoforms and is associated with increased induction of AP1 and NFkappaB transcription factors. These data indicate that CD66a amplifies T cell activation and thus could facilitate crosstalk between epithelial cells and T lymphocytes in intestinal immune response.

Carcinoembryonic antigen family members CEACAM6 and CEACAM7 are differentially expressed in normal tissues and oppositely deregulated in hyperplastic colorectal polyps and early adenomas.

Four members of the carcinoembryonic antigen (CEA) family, CEA, CEACAM1 (BGP), CEACAM6 (NCA-50/90), and CEACAM7 (CGM2), are coexpressed in normal colorectal epithelia but are deregulated in colorectal cancers, where they could play a role in tumorigenesis. As a basis for functional studies, their expression patterns in normal tissues first need to be clarified. This is well documented for CEACAM1 and CEA but not for CEACAM6 or CEACAM7. We have now carried out immunohistochemical expression studies on 35 different organs, using CEACAM6-specific (9A6) and CEACAM7-specific (BAC2) monoclonal antibodies. CEACAM7 was only found on the apical surface of highly differentiated epithelial cells in the colorectal mucosa and on isolated ductal epithelial cells within the pancreas. CEACAM6 was expressed in granulocytes and epithelia from various organs. CEACAM6 and CEACAM7 expression correlated with apoptosis at the table region of the normal colon, and both were absent from highly proliferating cells at the base of colonic crypts. CEACAM6 revealed a broader expression zone in proliferating cells in hyperplastic polyps and adenomas compared with normal mucosa, whereas CEACAM7 was completely absent. Down-regulation of CEACAM7 and up-regulation of CEACAM6 expression in hyperplastic polyps and early adenomas represent some of the earliest observable molecular events leading to colorectal tumors.

Critical determinants of host receptor targeting by Neisseria meningitidis and Neisseria gonorrhoeae: identification of Opa adhesiotopes on the N-domain of CD66 molecules.

The human pathogens Neisseria meningitidis and Neisseria gonorrhoeae express a family of variable outer membrane opacity-associated (Opa) proteins that recognize multiple human cell surface receptors. Most Opa proteins target the highly conserved N-terminal domain of the CD66 family of adhesion molecules, although a few also interact with heparan sulphate proteoglycans. In this study, we observed that at least two Opa proteins of a N. meningitidis strain C751 have the dual capacity to interact with both receptors. In addition, all three Opa proteins of C751 bind equally well to HeLa cells transfected with cDNA encoding the carcinoembryonic antigen [CEA (CD66e)] subgroup of the CD66 family, but show distinct tropism for CGM1- (CD66d) and NCA (CD66c)-expressing cells. Because the C751 Opa proteins make up distinct structures via the surface-exposed hypervariable domains (HV-1 and HV-2), these combinations appear to be involved in tropism for the distinct CD66 subgroups. To define the determinants of receptor recognition, we used mutant proteins of biliary glycoprotein [BGP (CD66a)] carrying substitutions at several predicted exposed sites in the N-domain and compared their interactions with several Opa proteins of both N. meningitidis and N. gonorrhoeae. The observations applied to the molecular model of the BGP N-domain that we constructed show that the binding of all Opa proteins tested occurs at the non-glycosylated (CFG) face of the molecule and, in general, appears to require Tyr-34 and Ile-91. Further, efficient interaction of distinct Opa proteins depends on different non-adjacent amino acids. In the three-dimensional model, these residues lie in close proximity to Tyr-34 and Ile-91 at the CFG face, making continuous binding domains (adhesiotopes). The epitope of the monoclonal antibody YTH71.3 that inhibits Opa/CD66 interactions was also identified within the Opa adhesiotopes on the N-domain. These studies define the molecular basis that directs the Opa specificity for the CD66 family and the rationale for tropism of the Opa proteins for the CD66 subgroups.

Synovial PMN show a coordinated up-regulation of CD66 molecules.

Changes in the expression of various activation-dependent surface markers have been reported for polymorphonuclear neutrophils (PMN) isolated from synovial fluid of patients with inflammatory joint diseases. We extend these findings to the expression of CD66 molecules and several other surface markers. Three members of the CD66 family, namely CD66a, CD66b, and CD66c, showed an up to fourfold up-regulation on synovial fluid PMN compared with peripheral blood PMN (PBG) of the same patients; CD59 was increased twofold, the expression of CD16 did not change, whereas CD62L was reduced by more than 50% on synovial fluid PMN. It is interesting that CD66a, CD66b, and CD66c showed a coordinated expression on PBG of patients and controls and a coordinated up-regulation on synovial neutrophils. In contrast, after in vitro stimulation of peripheral blood PMN with phorbol myristate acetate, CD66c was much less up-regulated compared with CD66a and CD66b. All samples of synovial fluid PMN exhibited an additional increase in the expression of CD66a, CD66b, and CD66c when stimulated with phorbol myristate acetate in vitro. Prostaglandins are known to inhibit various responses of neutrophils to inflammatory stimuli. We could show that prostaglandins inhibit N-formyl-methionyl-leucyl-phenylalanine-induced up-regulation of CD66 on peripheral blood PMN in a concentration-dependent manner.

CEA-related proteins on human urothelial cell lines of different transformation grades.

CEA family proteins from human urothelial cell lines of different transformation grades were characterized by flow cytometry and Western blotting using monoclonal antibodies: 26/3/13, D14HD11, 9A6 and 4/3/17. The following observations were made: (i) the urothelial cell lines, representing transformation grade III (TGr III, tumorigenic, invasive cells), were characterized by the presence of a component with molecular mass 110-135 kDa, most probably representing biliary glycoprotein (BGP); (ii) BGP was absent in non-tumorigenic and non-invasive TGr II urothelial cell lines; (iii) a protein band with apparent molecular mass 180 kDa, and migrating as a CEA standard was detected in only one of seven urothelial cell lines analyzed; (iv) a broad band of apparent molecular mass migrating at 65-90 kDa, probably representing NCA-50/90, was found in two tumorigenic and invasive cell lines, HCV 29T and Hu 1703He.

Molecular analysis of neisserial Opa protein interactions with the CEA family of receptors: identification of determinants contributing to the differential specificities of binding.

The carcinoembryonic antigen (CEA) gene family members, CEACAM1, CEACAM3, CEACAM5 and CEACAM6, are bound by the Opa outer membrane proteins of pathogenic Neisseria spp., whereas CEACAM8 is not. In this study, we demonstrate that the closely related CEACAM4 and CEACAM7, which are also members of the CEA family, are not Opa receptors. We exploited the high conservation between CEACAM6 and CEACAM8 to generate an extensive set of chimeric receptors in order to delineate the sequences necessary for Opa binding. Using a transfection-based infection system, we showed that binding of Opa52 involves residues 27-42, which are predicted to form beta-strand C and short loops adjacent to it, and residues lying between amino acids 60 and 108 in the amino-terminal domain. The replacement of residues 27-29 in CEACAM6 with the CEACAM1 or CEACAM5 sequences generated recombinant CEACAM6 receptors that are bound by CEACAM1/CEACAM5-specific Opa variants. Together, our data demonstrate that Opa proteins bind to residues exposed on the GFCC' face of the N-terminal domain of CEACAM receptors, and identify an amino acid triplet sequence that is responsible for the differential binding of Opa proteins to CEACAM1, CEACAM5 and CEACAM6.

Highly specific monoclonal antibody demonstrates that pregnancy-specific glycoprotein (PSG) is limited to syncytiotrophoblast in human early and term placenta.

Pregnancy specific glycoproteins (PSG) in humans constitute a family of 11 closely related glycoproteins (PSG1-8, PSG11-13) of unknown function(s), which are produced in large amounts by the placenta. As a step toward understanding the biology of PSG, specific monoclonal antibodies (mAbs) against PSG were developed and used to investigate the ultrastructural localization of PSG in the early and term placenta and in first trimester decidua. One mAb, BAP-3, was found to react with all six individually expressed PSGs representing five alternatively spliced forms, but not with any of the seven expressed members of the carcinoembryonic antigen (CEA) subfamily. The BAP-3 epitope is located in the PSG B2 domain. Using the BAP-3 mAb, PSGs were found to be expressed exclusively by the syncytiotrophoblast of first trimester and term villi. The intensity of the staining was much higher in early than in term placenta. All three main cellular compartments involved in the biosynthesis pathway of secreted proteins, i.e. rough endoplasmic reticulum, the Golgi complex and secretory vesicles, were stained for PSG. A second PSG-reactive mAb, BAP-1, also stained the apical plasma membrane of some glandular epithelial cells in first trimester decidua in addition to syncytiotrophoblast. This staining was most likely due to cross-reactivity with biliary glycoprotein (BGP).

Differential recognition of members of the carcinoembryonic antigen family by Opa variants of Neisseria gonorrhoeae.

Opacity (Opa) protein variation in Neisseria gonorrhoeae is implicated in the pathogenesis of gonorrhea, possibly by mediating adherence and entry of the bacteria into human tissues. One particular Opa protein mediates adherence to epithelial cells through cell surface proteoglycans. Recently, two other eukaryotic cell receptors for Opa proteins have been reported. These receptors are members of a subgroup of the carcinoembryonic (CEA) gene family that express CD66 antigens. CEA family members vary in their distribution in human tissues. In order to understand whether interactions between Opa and CEA-like molecules play any role in pathogenesis, we must investigate which CEA family members are able to serve as Opa receptors and which Opa proteins recognize CEA-like molecules. We therefore studied HeLa cells that were stably transfected with five different members of the CEA family, i.e., CEA, CEA gene family member 1a (CGM1a), CGM6, nonspecific cross-reacting antigen (NCA), and biliary glycoprotein a (BGPa). We infected these transfectants with all possible 11 Opa variants of gonococcal strain MS11 and determined the numbers of bacteria that were bound and internalized. To account for proteoglycan-mediated adherence, infection assays were also performed in the presence of heparin. Our results show that of the 11 Opa variants of MS11, the same 4 recognized CGM1a and NCA. CGM6, however, was not recognized by any Opa variant of MS11. CEA was recognized by at least 9 of 11 Opa variants, and the BGP transfectants specifically bound and internalized 10 of 11 Opa variants and also bound Opa-negative gonococci. Immunofluorescence experiments showed that clustering of CEA-like molecules occurred upon infection of HeLa transfectants with those Opa variants that interacted specifically with the CEA family member. Together these data show that CEA family members are differentially recognized by gonococcal Opa variants, suggesting that this phenomenon may contribute to cell tropism displayed by gonococci.

CD66 carcinoembryonic antigens mediate interactions between Opa-expressing Neisseria gonorrhoeae and human polymorphonuclear phagocytes.

Colonization of urogenital tissues by the human pathogen Neisseria gonorrhoeae is characteristically associated with purulent exudates of polymorphonuclear phagocytes (PMNs) containing apparently viable bacteria. Distinct variant forms of the phase-variable opacity-associated (Opa) outer membrane proteins mediate the non-opsonized binding and internalization of N. gonorrhoeae by human PMNs. Using overlay assays and an affinity isolation technique, we demonstrate the direct interaction between Opa52-expressing gonococci and members of the human carcinoembryonic antigen (CEA) family which express the CD66 epitope. Gonococci and recombinant Escherichia coli strains synthesizing Opa52 showed specific binding and internalization by transfected HeLa cell lines expressing the CD66 family members BGP (CD66a), NCA (CD66c), CGM1 (CD66d) and CEA (CD66e), but not that expressing CGM6 (CD66b). Bacterial strains expressing either no opacity protein or the epithelial cell invasion-associated Opa50 do not bind these CEA family members. Consistent with their different receptor specificities, Opa52-mediated interactions could be inhibited by polyclonal anti-CEA sera, while Opa50 binding was instead inhibited by heparin. Using confocal laser scanning microscopy, we observed a marked recruitment of CD66 antigen by Opa52-expressing gonococci on both the transfected cell lines and infected PMNs. These data indicate that members of the CEA family constitute the cellular receptors for the interaction with, and internalization of, N. gonorrhoeae.

Several carcinoembryonic antigens (CD66) serve as receptors for gonococcal opacity proteins.

Neisseria gonorrhoeae (GC) is a human pathogen that adheres to and invades genital surfaces. Although pili are required for the initial adherence, the interaction of GC with epithelial cells is also promoted by a family of outer membrane proteins, the opacity (Opa) proteins such as OpaA protein from strain MS11. Studies have demonstrated that the interaction of the OpaA GC with epithelial cells involves binding to heparan sulfate attached to syndecan receptors. However, other Opa proteins interact with CEA gene family member 1 (CGM1) or biliary glycoprotein (BGP), members of the CD66 antigen family. In this study, we demonstrate that, in addition, the 180-kD carcinoembryonic antigen (CEA) is a receptor for Opa proteins. This conclusion was based on the following observations. First, transfected HeLa cells expressing CEA (HeLa-CEA) and the CEA-expressing colon cancer cell line (LS 174T) bound and subsequently engulfed the Opa+ bacteria. These interactions were inhibited by anti-CEA antibody, but could not be inhibited by addition of heparin. Furthermore, OpaI E. coli directly bound purified CEA. We also compared the adherence and invasion by Opa+ bacteria of CD66 transfected HeLa cells: HeLa-BGPa, HeLa-CGM6, HeLa-NCA, HeLa-CGM1a, HeLa-CEA, and HeLa-Neo serving as negative control. Using OpaI as the prototype, the relative ability of the transfected HeLa cell lines to support adherence was (CEA = BGPa >CGM1a >NCA >>CGM6 = Neo). The ability to mediate invasion of the transfectant cells was (CGM1a >CEA >BGPa >NCA >CGM6 = Neo). Among the Opa proteins tested, OpaC proved to be bifunctional, able to mediate adherence to both syndecan receptors and to CD66 antigens.

CD66: role in the regulation of neutrophil effector function.

Neutrophils express several heavily glycosylated carcinoembryonic antigen (CEA)-related glycoproteins (CD66 antigens) which have been implicated in adhesion to E-selectin and as receptors for the lectins galectin 3 and bacterial type-1 fimbriae. The role of the CD66 antigens in neutrophil effector function was examined using non-cross-reacting and cross-reacting domain-mapped CD66 monoclonal antibody (mAb), which recognize epitopes on biliary glycoprotein (BGP; CD66a), CEA gene family member 6 (CGM6; CD66b), nonspecific cross-reacting antigen 90 (NCA90; CD66c) or CGM1 (CD66d). We show that BGP-specific mAb which recognize an AB-domain epitope strongly augment adhesion to fibrinogen by an Fc receptor- and beta2 integrin-dependent mechanism. Co-ligation of BGP with the glycophosphatidylinositol (GPI)-anchored CGM6 and NCA90 also caused increased beta2 integrin-mediated adhesion, receptor clustering and priming of formyl-Met-Leu-Phe (fMLP)-induced oxidant production by neutrophils, but only a small change in expression of L-selectin and CR3 compared to the chemotactic peptide fMLP. Ligation of CGM6 or NCA90 alone did not cause activation of the neutrophil in any of the assays used and did not cause priming of fMLP-induced oxidant production even when a secondary cross-linking reagent was used. We propose that specific cross-linking of neutrophil BGP with CGM6 and NCA90 contributes significantly to the regulation of neutrophil function during neutrophil recruitment.