Extracellular N-domain alone can mediate specific heterophilic adhesion between members of the carcinoembryonic antigen family, CEACAM6 and CEACAM8

The domain(s) responsible for the specific heterophilic adhesion between two members of the carcinoembryonic antigen (CEA) family, CEACAM6 and CEACAM8, both of which with three extracellular domains, were investigated using Chinese hamster ovary (CHO) transfectants expressing chimeric antigens. Using a chimeric antigen in which the N-domain, a sole extracellular domain, of CEACAM3 was substituted with that of CEACAM6, it was shown that the N-domain of CEACAM6 alone was able to mediate specific adhesion to CEACAM8. Furthermore, the chimeric antigen was shown to bind significantly to chimeric CEA whose N-domain was substituted with that of CEACAM8, but not to unsubstituted CEA. These results demonstrate that the N-domain alone is sufficient and other domains of CEACAM6 or CEACAM8 are not required for this specific binding. We therefore propose a model of heterophilic interaction between the N-domains, which is distinct from that of CEA-CEA homophilic binding.     

Carcinoembryonic antigen (CEA) inhibits NK killing via interaction with CEA-related cell adhesion molecule 1

The NK killing activity is regulated by activating and inhibitory NK receptors. All of the activating ligands identified so far are either viral or stress-induced proteins. The class I MHC proteins are the ligands for most of the inhibitory NK receptors. However, in the past few years, several receptors have been identified that are able to inhibit NK killing independently of class I MHC recognition. We have previously demonstrated the existence of a novel inhibitory mechanism of NK cell cytotoxicity mediated by the homophilic carcinoembryonic Ag (CEA)-related cell adhesion molecule 1 (CEACAM1) interactions. In this study, we demonstrate that CEACAM1 also interacts heterophilically with the CEA protein. Importantly, we show that these heterophilic interactions of CEA and CEACAM1 inhibit the killing by NK cells. Because CEA is expressed on a wide range of carcinomas and commonly used as tumor marker, these results represent a novel role for the CEA protein enabling the escape of tumor cells from NK-mediated killing. We further characterize, for the first time, the CEACAM1-CEA interactions. Using functional and binding assays, we demonstrate that the N domains of CEACAM1 and CEA are crucial but not sufficient for both the CEACAM1-CEACAM1 homophilic and CEACAM1-CEA heterophilic interactions. Finally, we suggest that the involvement of additional domains beside the N domain in the heterophilic and homophilic interactions is important for regulating the balance between cis and trans interactions.     

The adhesion and differentiation-inhibitory activities of the immunoglobulin superfamily member,carcinoembryonic antigen,can be independently blocked

The external domains of Ig superfamily members are involved in multiple binding interactions, both homophilic and heterophilic, that initiate molecular events leading to the execution of diverse cell functions. Human carcinoembryonic antigen (CEA), an Ig superfamily cell surface glycoprotein used widely as a clinical tumor marker, undergoes homophilic interactions that mediate intercellular adhesion. Recent evidence supports the view that deregulated overexpression of CEA has an instrumental role in tumorigenesis through the inhibition of cell differentiation and the disruption of tissue architecture. The CEA-mediated block of the myogenic differentiation of rat L6 myoblasts depends on homophilic binding of its external domains. We show here that L6 transfectant cells expressing CEA can "trans-block" the myogenesis of juxtaposed differentiation-competent L6 transfectant cells expressing a deletion mutant of CEA (DeltaNCEA). This result implies the efficacy of antiparallel CEA-CEA interactions between cells in the differentiation block. In addition, DeltaNCEA can acquire differentiation blocking activity by cross-linking with specific anti-CEA antibodies, thus implying the efficacy of parallel CEA-CEA interactions on the same cell surface. The myogenic differentiation blocking activity of CEA was demonstrated by site-directed mutations to involve three subdomains of the amino-terminal domain, shown previously to be critical for its intercellular adhesion function. Monovalent Fab fragments of monoclonal antibodies binding to the region bridging subdomains 1 and 2 could both inhibit intercellular adhesion and release the myogenic differentiation block. Amino acid substitutions Q80A, Q80R, and D82N in subdomain 3, QNDTG, however, were found to completely ablate the differentiation blocking activity of CEA but had no effect on intercellular adhesion activity. A cyclized peptide representing this subdomain was the most effective at releasing the differentiation block.     

Homotypic and heterotypic Ca(++)-independent cell adhesion activities of biliary glycoprotein, a member of carcinoembryonic antigen family, expressed on CHO cell surface.

Homotypic and heterotypic cell adhesion activities of a carcinoembryonic antigen (CEA) family member, biliary glycoprotein a (BGPa), have been examined. CHO cells transfected with the cDNA for BGPa, CEA, non-specific cross-reacting antigen (NCA) and CGM6 have been used. The BGPa producers showed both homotypic and heterotypic adhesion to CEA and NCA producers. However, they hardly adhered to CGM6 producers. Calcium ion was not required for BGPa-mediated homotypic and heterotypic cell adhesion as well as for the adhesions of other members of CEA family. The results strongly suggested that BGPa may play some important roles through Ca(++)-independent cell adhesion activities.     

A specific heterotypic cell adhesion activity between members of carcinoembryonic antigen family, W272 and NCA, is mediated by N-domains

The Ca(2+)-independent homotypic and heterotypic cell adhesion activities of a carcinoembryonic antigen (CEA) family member, W272 (CGM6), whose cDNA has recently been isolated from libraries of human peripheral leukocytes of apparently normal subjects (Arakawa, F., Kuroki, Mo., Misumi, Y., Oikawa, S., Nakazato, H., and Matsuoka, Y. (1990) Biochem. Biophys. Res. Commun. 166, 1063-1071) and spleen of chronic myelogenous leukemia patients (Berling, B., Kolbinger, F., Grunert, F., Thompson, J. A., Brombacher, F., Buchegger, F., von Kleist, S., and Zimmermann, W. (1990) Cancer Res. 50, 6534-6539) has been examined. Chinese hamster ovary cells transfected with the cDNA for W272, CEA, nonspecific cross-reacting antigen (NCA), and various antigens containing chimeric N-domain have been used. The W272 producers did not show homotypic binding at all but bound only to the cells expressing NCA and a chimeric CEA whose N-domain is substituted by that of NCA, indicating the major contribution of N-domain of NCA in the specific binding. The importance of the N-terminal region of NCA N-domain for the W272-NCA binding has been shown by detailed analysis using COS-1 cells producing various NCA whose N-domain are chimera of that of NCA and CEA. The strict heterotypic nature of the W272-NCA adhesion strongly suggests that the cell adhesion activities exhibited by CEA family members are not the fortuitous activity but the specific one which have some important physiological roles.     

Expression of the CD15 differentiation antigen (3-fucosyl-N-acetyl-lactosamine, LeX) on putative neutrophil adhesion molecules CR3 and NCA-160.

The expression of the carbohydrate antigen 3-fucosyl-N-acetyl-lactosamine (CD15, LeX) on human neutrophil glycoproteins has been studied by immunoprecipitation and immunoblotting by using monoclonal antibody MC2. The antigen is expressed on membrane glycoproteins of approximate molecular mass 165 and 105 kDa. These glycoproteins include the complement receptor and adhesion molecule, CR3, in which the beta-chain (CD18, 105 kDa) shows much greater expression than the alpha-chain (CD11b, 165 kDa). Most of the 165 kDa CD15 antigen is accounted for by expression on the carcinoembryonic antigen (CEA)-related molecule NCA160. Other members of this family, NCA95, NCA90 and NCA55, which are also found in neutrophils, do not express the CD15 antigen. There is a marked increase in the surface expression of CD15, CR3 and the antigen recognized by anti-CEA antibodies upon activation of neutrophils by the chemotactic peptide N-formylmethionyl-leucylphenylalanine.     

C-CAM (cell-CAM 105)--a member of the growing immunoglobulin superfamily of cell adhesion proteins

Cell recognition and adhesion, being of prime importance for the formation and integrity of tissues, are mediated by cell adhesion molecules, which can be divided into several distinct protein superfamilies. The cell adhesion molecule C-CAM (cell-CAM 105) belongs to the immunoglobulin superfamily, and more specifically is a member of the carcinoembryonic antigen (CEA) gene family. C-CAM can mediate adhesion between hepatocytes in vitro in a homophilic, calcium-independent binding reaction. The molecule, which occurs in various isoforms, is expressed in liver, several epithelia, vessel endothelia, platelets and granulocytes and its expression is dynamically regulated under various physiological and pathological conditions. It is proposed that C-CAM in different cells and tissues plays different functional roles, where the common denominator is membrane-membrane binding.     

Specificity of intercellular adhesion mediated by various members of the immunoglobulin supergene family

The immunoglobulin supergene family members have been shown to be involved in cell-cell recognition and interaction during cell growth and differentiation. Neural cell adhesion molecule, myelin-associated glycoprotein, and carcinoembryonic antigen (CEA) are immunoglobulin supergene family members which can mediate cell adhesion. We show here that nonspecific cross-reacting antigen (NCA), a closely related CEA family member, is found on the surface of rodent cells transfected with functional NCA complementary DNA in different glycosylated forms, all of which can be deglycosylated to an Mr 35,000 core protein. Furthermore, NCA can mediate Ca2(+)-independent, homotypic aggregation of these NCA-producing transfectant cells. Since CEA has three internal repeated C2-set, immunoglobulin-like domains, whereas NCA has one, only one such domain is required for the intercellular adhesive function. We also demonstrate that NCA- and CEA-producing transfectants can form heterotypic aggregates, whereas mixtures of CEA or NCA transfectants and neural cell adhesion molecule or long form-myelin-associated glycoprotein transfectants sort themselves out into homotypic aggregates. The results suggest that subsets of the immunoglobulin superfamily, such as the CEA family, can be used in both homotypic and heterotypic cellular interactions, whereas less closely related members of the family can be used to separate different cell types by strictly homotypic interactions.     

Critical role of the fifth domain of E-cadherin for heterophilic adhesion with alpha E beta 7, but not for homophilic adhesion

The integrin alpha(E)beta(7) is expressed on intestinal intraepithelial T lymphocytes and CD8(+) T lymphocytes in inflammatory lesions near epithelial cells. Adhesion between alpha(E)beta(7)(+) T and epithelial cells is mediated by the adhesive interaction of alpha(E)beta(7) and E-cadherin; this interaction plays a key role in the damage of target epithelia. To explore the structure-function relationship of the heterophilic adhesive interaction between E-cadherin and alpha(E)beta(7), we performed cell aggregation assays using L cells transfected with an extracellular domain-deletion mutant of E-cadherin. In homophilic adhesion assays, L cells transfected with wild-type or a domain 5-deficient mutant formed aggregates, whereas transfectants with domain 1-, 2-, 3-, or 4-deficient mutants did not. These results indicate that not only domain 1, but domains 2, 3, and 4 are involved in homophilic adhesion. When alpha(E)beta(7)(+) K562 cells were incubated with L cells expressing the wild type, 23% of the resulting cell aggregates consisted of alpha(E)beta(7)(+) K562 cells. In contrast, the binding of alpha(E)beta(7)(+) K562 cells to L cells expressing a domain 5-deficient mutant was significantly decreased, with alpha(E)beta(7)(+) K562 cells accounting for only 4% of the cell aggregates, while homophilic adhesion was completely preserved. These results suggest that domain 5 is involved in heterophilic adhesion with alpha(E)beta(7), but not in homophilic adhesion, leading to the hypothesis that the fifth domain of E-cadherin may play a critical role in the regulation of heterophilic adhesion to alpha(E)beta(7) and may be a potential target for treatments altering the adhesion of alpha(E)beta(7)(+) T cells to epithelial cells in inflammatory epithelial diseases.     

The homophilic binding of junctional adhesion molecule-C mediates tumor cell-endothelial cell interactions

The junctional adhesion molecule C (JAM-C) was recently shown to undergo a heterophilic interaction with the leukocyte beta2 integrin Mac-1, thereby mediating interactions between vascular cells in inflammatory cell recruitment. Here, the homophilic interaction of JAM-C is presented and functionally characterized to mediate tumor cell-endothelial cell interactions. Recombinant soluble JAM-C in fluid phase bound to immobilized JAM-C as assessed in a purified system; moreover, JAM-C-transfected Chinese hamster ovary (CHO) cells adhered to immobilized JAM-C. The homophilic interaction of JAM-C was mediated by the isolated amino-terminal Ig domain (D1), but not the carboxyl-terminal Ig domain (D2), of the molecule. Dimerization of JAM-A is dependent on the sequence RVE in the amino-terminal Ig domain. This motif is conserved in JAM-C (Arg64-Ile65-Glu66), and a single amino acid mutation in this motif (E66R) abolished the homophilic interaction of JAM-C. The lung carcinoma cell line NCI-H522 was found to express JAM-C. NCI-H522 cells adhered to immobilized JAM-C, as well as to JAM-C-transfected CHO cells, but not to mock-transfected CHO cells or to CHO cells transfected with the JAM-C mutant (E66R). Adhesion of NCI-H522 cells to JAM-C protein or JAM-C-transfected CHO cells was abolished in the presence of soluble JAM-C or the isolated D1. Furthermore, the adhesion of NCI-H522 cells to endothelial cells was significantly blocked by soluble JAM-C or the isolated D1. Thus, JAM-C undergoes a homophilic interaction via the Arg64-Ile65-Glu66 motif on the membrane-distal Ig domain of the molecule. The homophilic interaction of JAM-C can mediate tumor cell-endothelial cell interactions and may thereby be involved in the process of tumor cell metastasis.     

Molecular basis for the homophilic activated leukocyte cell adhesion molecule (ALCAM)-ALCAM interaction

Activated leukocyte cell adhesion molecule (ALCAM/CD166), a member of the immunoglobulin superfamily with five extracellular immunoglobulin-like domains, facilitates heterophilic (ALCAM-CD6) and homophilic (ALCAM-ALCAM) cell-cell interactions. While expressed in a wide variety of tissues and cells, ALCAM is restricted to subsets of cells usually involved in dynamic growth and/or migration processes. A structure-function analysis, using two monoclonal anti-ALCAM antibodies and a series of amino-terminally deleted ALCAM constructs, revealed that homophilic cell adhesion depended on ligand binding mediated by the membrane-distal amino-terminal immunoglobulin domain and on avidity controlled by ALCAM clustering at the cell surface involving membrane-proximal immunoglobulin domains. Co-expression of a transmembrane ALCAM deletion mutant, which lacks the ligand binding domain, and endogenous wild-type ALCAM inhibited homophilic cell-cell interactions by interference with ALCAM avidity, while homophilic, soluble ligand binding remained unaltered. The extracellular structures of ALCAM thus provide two structurally and functionally distinguishable modules, one involved in ligand binding and the other in avidity. Functionality of both modules is required for stable homophilic ALCAM-ALCAM cell-cell adhesion.     

Escherichia coli of human origin binds to carcinoembryonic antigen (CEA) and non-specific crossreacting antigen (NCA)

Immobilized carcinoembryonic antigen (CEA) and non-specific crossreacting antigen (NCA) bound 3 strains of E. coli of human origin. The binding was dose dependent, saturable, and of high avidity. Binding of the bacteria to CEA and NCA was completely abolished in the presence of 10 mM alpha-methyl D-mannopyranoside. Bacteria did not bind to concanavalin A. In addition, binding to deglycosylated CEA was either absent or significantly reduced. These findings indicate that the E. coli strains bind to D-mannosyl residues in CEA and NCA. Considering the tissue distribution of CEA (brush border of colonic epithelium) and NCA (granulocytes), these glycoproteins may be involved in the recognition of bacteria.     

Primary structure of nonspecific crossreacting antigen (NCA), a member of carcinoembryonic antigen (CEA) gene family, deduced from cDNA sequence

A cDNA containing the entire coding region for a member of carcinoembryonic antigen (CEA) gene family has been cloned from cDNA library of HLC-1 cells by immunochemical screening with the antibody specific to nonspecific crossreacting antigen (NCA). The cDNA encodes a precursor form of a polypeptide consisting of a 34-residue signal sequence, a 108-residue N-terminal (N-) domain, a 178-residue domain (NCA-I domain) and a 24-residue domain rich in hydrophobic amino acids (M-domain). Each domain has a distinct but homologous amino acid sequence to that of the corresponding domain of CEA. Unlike the coding sequences, the 3'-untranslated sequences differ markedly in the NCA and CEA cDNAs facilitating the preparation of probes that will discriminate between nucleotide sequences for CEA and NCA.     

Self recognition in the Ig superfamily. Identification of precise subdomains in carcinoembryonic antigen required for intercellular adhesion

The homophilic binding of extracellular domains of membrane-bound immunoglobulin superfamily (IgSF) molecules is often required for intercellular adhesion and signaling. Carcinoembryonic antigen (CEA), a member of the IgSF, is a widely used tumor marker that functions in vitro as a homotypic intercellular adhesion molecule. CEA has also been shown to contribute to tumorigenicity by inhibiting cellular differentiation, an effect that requires the homophilic binding of its extracellular domains. It was of interest, therefore, to identify small subdomain sequences in CEA that could serve as a focus in the design of peptides that disrupt CEA-mediated intercellular adhesion. Three subdomains in the N-terminal domain of CEA, identified by site-directed deletions and point mutations, were shown to be required for intercellular adhesion. Cyclized peptides representing two of these subdomains, (42)NRQII and (80)QNDTG, were found to be effective in blocking CEA-mediated cellular aggregation when added to CEA-expressing transfectants in suspension. Intermolecular binding involving each of these subdomains is therefore essential for intercellular adhesion and cannot be compensated for by known binding contributions of other regions in the CEA molecule. In further support of this assumption, the binding epitope of an anti-CEA monoclonal antibody (monoclonal antibody A20) known to block CEA-mediated adhesion, was shown to bridge two of the three required subdomains: (42)NRQII and (30)GYSWYK.     

Intercellular adhesion molecule-5 induces dendritic outgrowth by homophilic adhesion

Intercellular adhesion molecule-5 (ICAM-5) is a dendritically polarized membrane glycoprotein in telencephalic neurons, which shows heterophilic binding to leukocyte beta(2)-integrins. Here, we show that the human ICAM-5 protein interacts in a homophilic manner through the binding of the immunoglobulin domain 1 to domains 4-5. Surface coated ICAM-5-Fc promoted dendritic outgrowth and arborization of ICAM- 5-expressing hippocampal neurons. During dendritogenesis in developing rat brain, ICAM-5 was in monomer form, whereas in mature neurons it migrated as a high molecular weight complex. The findings indicate that its homophilic binding activity was regulated by nonmonomer/monomer transition. Thus, ICAM-5 displays two types of adhesion activity, homophilic binding between neurons and heterophilic binding between neurons and leukocytes.     

The receptor tyrosine kinase ARK mediates cell aggregation by homophilic binding.

The ARK (AXL, UFO) receptor is a member of a new family of receptor tyrosine kinases whose extracellular domain contains a combination of fibronectin type III and immunoglobulin motifs similar to those found in many cell adhesion molecules. ARK mRNA is expressed at high levels in the mouse brain, prevalently in the hippocampus and cerebellum, and this pattern of expression resembles that of adhesion molecules that are capable of promoting cell aggregation through homophilic or heterophilic binding. We report here the ability of the murine ARK receptor to mediate homophilic binding. Expression of the ARK protein in Drosophila S2 cells induces formation of cell aggregates consisting of ARK-expressing cells, and aggregation leads to receptor activation, with an increase in receptor phosphorylation. Homophilic binding does not require ARK tyrosine kinase activity, since S2 cells expressing a receptor in which the intracellular domain was deleted were able to undergo aggregation as well as cells expressing the wild-type ARK receptor. Similar results were obtained with NIH 3T3 and CHO cells expressing high levels of ARK, although in this case ARK expression appeared to be accompanied by constitutive activation. The purified recombinant extracellular domain of ARK can induce homotypic aggregation of coated fluorescent beads (Covaspheres), and this protein can also function as a substrate for adhesion by S2 and NIH 3T3 cells expressing ARK. These results suggest that ARK represents a new cell adhesion molecule that through its homophilic interaction may regulate cellular functions during cell recognition.     

Tyrosine Residue in Exon 14 of the Cytoplasmic Domain of Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1/CD31) Regulates Ligand Binding Specificity

Platelet/endothelial cell adhesion molecule (PECAM-1) is a cell adhesion molecule of the immunoglobulin superfamily that plays a role in a number of vascular processes including leukocyte transmigration through endothelium. The presence of a specific 19– amino acid exon within the cytoplasmic domain of PECAM-1 regulates the binding specificity of the molecule; specifically, isoforms containing exon 14 mediate heterophilic cell–cell aggregation while those variants missing exon 14 mediate homophilic cell–cell aggregation. To more precisely identify the region of exon 14 responsible for ligand specificity, a series of deletion mutants were created in which smaller regions of exon 14 were removed. After transfection into L cells, they were tested for their ability to mediate aggregation. For heterophilic aggregation to occur, a conserved 5–amino acid region (VYSEI in the murine sequence or VYSEV in the human sequence) in the mid-portion of the exon was required. A final construct, in which this tyrosine was mutated into a phenylalanine, aggregated in a homophilic manner when transfected into L cells. Inhibition of phosphatase activity by exposure of cells expressing wild type or mutant forms of PECAM-1 to sodium orthovanadate resulted in high levels of cytoplasmic tyrosine phosphorylation and led to a switch from heterophilic to homophilic aggregation. Our data thus indicate either loss of this tyrosine from exon 14 or its phosphorylation results in a change in ligand specificity from heterophilic to homophilic binding. Vascular cells could thus determine whether PECAM-1 functions as a heterophilic or homophilic adhesion molecule by processes such as alternative splicing or by regulation of the balance between tyrosine phosphorylation or dephosphorylation. Defining the conditions under which these changes occur will be important in understanding the biology of PECAM-1 in transmigration, angiogenesis, development, and other processes in which this molecule plays a role.     

Complementary expression and heterophilic interactions between IgLON family members neurotrimin and LAMP

Neurotrimin (Ntm) and the limbic system-associated membrane protein (LAMP) are members of the IgLON (LAMP, OBCAM, Ntm) family of glycorylphosphatidylinositol anchored neural cell adhesion molecules. We previously reported that LAMP and Ntm promote adhesion and neurite outgrowth via a homophilic mechanism, suggesting that these proteins promote the formation of specific neuronal circuits by homophilic interactions. In this report, we have further characterized the expression and binding specificity of Ntm. Using a newly generated monoclonal antibody to Ntm, we demonstrated that this protein is largely expressed in a complementary pattern to that of LAMP in the nervous system, with co-expression at a few sites. Ntm is expressed at high levels in sensory-motor cortex and, of particular note, is transiently expressed in neurons of cortical barrel fields and corresponding thalamic "barreloids." Binding of a recombinant, soluble form of Ntm to CHO cells expressing either Ntm or LAMP demonstrates that Ntm and LAMP interact both homophilically and heterophilically. In contrast to conventional growth-promoting activity of Ig superfamily members, LAMP strongly inhibits the outgrowth of Ntm-expressing dorsal root ganglion (DRG) neurons in a heterophilic manner. These anatomical and functional data support the concept that homophilic and heterophilic interactions between IgLON family members are likely to play a role in the specification of neuronal projections via growth promoting and inhibiting effects, respectively.     

Antigenic variants of the nonspecific cross-reacting antigen (NCA)

Monoclonal antibodies (Mab) were prepared against nonspecific cross-reacting antigen (NCA) and were selected on the basis of their absence of reactivity with carcinoembryonic antigen (CEA). Four Mab were found which allowed the characterization on CEA of three epitopes, defined A, B, and C. These epitopes were all located on the peptidic moiety of this highly glycosylated antigen and were present on NCA molecules of heterogeneous m.w. (greater than 100,000, 80,000, and 48,000 m.w., the latter being the most abundant). The amount of NCA was estimated in 251 human sera both by a conventional RIA, using a rabbit antiserum, and by EIA, using different Mab: Mab 4, 18, and 33, which reacted, respectively, with epitopes A, B, and C. Each assay gave a different value of the absolute concentration of NCA in the serum. On the whole, Mab 4 gave lower values, whereas Mab 18 and 33 gave higher values as compared to RIA. Furthermore, whereas all of the human sera contained NCA which was measurable by RIA, 67 sera typed negative in EIA when using Mab 4 or 18. Eight additional sera were negative in more than one EIA. Negativity when using Mab 33 was observed in only one serum, which was also negative with Mab 4 and 18. Twenty-five of 30 sera which were negative with Mab 4 came from cancer patients, and 32 of 37 sera negative with Mab 18 came from normal subjects and noncancer patients, giving a statistically highly significant difference between the two groups of sera (p less than 0.001). Analysis of tissue perchloric extracts and NCA samples purified from these extracts gave similar results. Three extracts (one from lung, two from cancer tissue) and the corresponding NCA samples were negative with Mab 18. The discrepancies observed in these assays are best explained by assuming the existence of antigenic variants of NCA which have not been described previously. These variants appear to exist in various proportions in the different sera. The variants may represent antigenically complete and incomplete molecules. Alternatively, most of the NCA molecules may be incomplete, lacking one or another of the several NCA-specific epitopes. Sequential immunoprecipitation experiments were in favor of the second hypothesis, showing that most of the NCA molecules were incomplete, lacking either epitope A or B.