Kupffer Cell Receptor Research Articles

Kupffer cell receptor CLEC4F is important for the destruction of desialylated platelets in mice

The liver has recently been identified as a major organ for destruction of desialylated platelets. However, the underlying mechanism remains unclear. Kupffer cells, which are professional phagocytic cells in the liver, comprise the largest population of resident tissue macrophages in the body. Kupffer cells express a C-type lectin receptor, CLEC4F, that recognizes desialylated glycans with an unclear in vivo role in mediating platelet destruction. In this study, we generated a CLEC4F-deficient mouse model (Clec4f−/−) and found that CLEC4F was specifically expressed by Kupffer cells. Using the Clec4f−/− mice and a newly generated platelet-specific reporter mouse line, we revealed a critical role for CLEC4F on Kupffer cells in mediating destruction of desialylated platelets in the liver in vivo. Platelet clearance experiments and ultrastructural analysis revealed that desialylated platelets were phagocytized predominantly by Kupffer cells in a CLEC4F-dependent manner in mice. Collectively, these findings identify CLEC4F as a Kupffer cell receptor important for the destruction of desialylated platelets induced by bacteria-derived neuraminidases, which provide new insights into the pathogenesis of thrombocytopenia in disease conditions such as sepsis.

Development and Characterization of Nanobodies Targeting the Kupffer Cell.

Nanobodies that are derived from single-chain antibodies of camelids have served as powerful tools in diagnostics, therapeutics and investigation of membrane receptors' structure and function. In this study, we developed a series of nanobodies by a phage display screening building from lymphocytes isolated from an alpaca immunized with recombinant mouse Kupffer cell receptor Clec4F, which is involved in pathogen recognition by binding to galactose and N-acetylgalactosamine. Bio-panning selections retrieved 14 different nanobodies against Clec4F with an affinity ranging from 0.2 to 2 nM as determined by SPR. Those nanobodies mainly recognize 4 different epitopes as analyzed via competitive epitope binning. By analysis of the radioactivity in each organ after injection of 99mTc labeled Clec4F nanobodies in naïve mice, we found that these nanobodies are targeting the liver. Furthermore, we performed a structural characterization at atomic resolution of two of the Clec4F nanobodies from different epitope groups, which revealed distinct features within the CDR2 and CDR3 regions. Taken together, we developed a series of nanobodies targeting multiple distinct recognition epitopes of the Kupffer cell-specific receptor Clec4F which may be useful for its structural and functional investigation as well as for use as molecular imaging and therapeutic agents.

Absence of a human ortholog of rodent Kupffer cell galactose-binding receptor encoded by the CLEC4f gene.

The murine CLEC4f gene encodes the Kupffer cell receptor, a galactose-binding receptor containing a C-type carbohydrate-recognition domain. Orthologs have been identified in nearly 100 species. The receptors from rat and mouse have previously been characterized and data presented here show that functional CLEC4f protein is expressed in domestic cattle (Bos taurus). However, the human CLEC4f gene does not encode a functional receptor because a mutation in the splice acceptor site of the final exon prevents appropriate splicing and a missense mutation disrupts the sugar-binding site. Transcriptomic and PCR analysis of transcripts confirms the absence of a spliced transcript containing the final exon and only background levels of transcripts are detected in human tissues. These mutations are also present in the CLEC4f gene in Neanderthals. In contrast to humans, closely related species, including chimpanzees, do have CLEC4f genes that encode full-length receptors. Affinity chromatography and glycan array results demonstrate that the chimpanzee, bovine and murine proteins all bind to galactose, but they show preferences for different subsets of galactose-containing glycans. In non-human primates, the receptor is expressed in spleen rather than in liver. The results indicate that the CLEC4f protein probably has distinct functions in different species. Absence of the receptor precludes using it for targeting of glycoconjugates to cells in human liver. The fact that CLEC4f protein is expressed in spleen in non-human primates and the close evolutionary relationship of the CLEC4f protein to langerin (CD207) suggest that it may function in the immune system, possibly as a pathogen receptor.

The Roles of Carcinoembryonic Antigen in Liver Metastasis and Therapeutic Approaches.

Metastasis is a highly complicated and sequential process in which primary cancer spreads to secondary organic sites. Liver is a well-known metastatic organ from colorectal cancer. Carcinoembryonic antigen (CEA) is expressed in most gastrointestinal, breast, and lung cancer cells. Overexpression of CEA is closely associated with liver metastasis, which is the main cause of death from colorectal cancer. CEA is widely used as a diagnostic and prognostic tumor marker in cancer patients. It affects many steps of liver metastasis from colorectal cancer cells. CEA inhibits circulating cancer cell death. CEA also binds to heterogeneous nuclear RNA binding protein M4 (hnRNP M4), a Kupffer cell receptor protein, and activates Kupffer cells to secrete various cytokines that change the microenvironments for the survival of colorectal cancer cells in the liver. CEA also activates cell adhesion-related molecules. The close correlation between CEA and cancer has spurred the exploration of many CEA-targeted approaches as anticancer therapeutics. Understanding the detailed functions and mechanisms of CEA in liver metastasis will provide great opportunities for the improvement of anticancer approaches against colorectal cancers. In this report, the roles of CEA in liver metastasis and CEA-targeting anticancer modalities are reviewed.

The surface carbohydrates of the Echinococcus granulosus larva interact selectively with the rodent Kupffer cell receptor

The larvae of the cestodes belonging to the genus Echinococcus dwell primarily in mammalian liver. They are protected by the laminated layer (LL), an acellular mucin-based structure. The glycans decorating these mucins constitute the overwhelming majority of molecules exposed by these larvae to their hosts. However, their decoding by host innate immunity has not been studied. Out of 36 mammalian innate receptors with carbohydrate-binding domains, expressed as Fc fusions, only the mouse Kupffer cell receptor (KCR; CLEC4F) bound significantly to the Echinococcus granulosus LL mucins. The receptor also bound the Echinococcus multilocularis LL. Out of several synthetic glycans representing Echinococcus LL structures, the KCR bound strongly in particular to those ending in Galα1-4Galβ1-3 or Galα1-4Galβ1-4GlcNAc, both characteristic LL carbohydrate motifs. LL carbohydrates may be optimized to interact with the KCR, expressed only in liver macrophages, cells known to contribute to the tolerogenic antigen presentation that is characteristic of this organ.

Liver Membrane Proteome Glycosylation Changes in Mice Bearing an Extra-hepatic Tumor

Cancer is well known to be associated with alterations in membrane protein glycosylation (Bird, N. C., Mangnall, D., and Majeed, A. W. (2006) Biology of colorectal liver metastases: A review. J. Surg. Oncol. 94, 68-80; Dimitroff, C. J., Pera, P., Dall'Olio, F., Matta, K. L., Chandrasekaran, E. V., Lau, J. T., and Bernacki, R. J. (1999) Cell surface n-acetylneuraminic acid alpha2,3-galactoside-dependent intercellular adhesion of human colon cancer cells. Biochem. Biophys. Res. Commun. 256, 631-636; and Arcinas, A., Yen, T. Y., Kebebew, E., and Macher, B. A. (2009) Cell surface and secreted protein profiles of human thyroid cancer cell lines reveal distinct glycoprotein patterns. J. Proteome Res. 8, 3958-3968). Equally, it has been well established that tumor-associated inflammation through the release of pro-inflammatory cytokines is a common cause of reduced hepatic drug metabolism and increased toxicity in advanced cancer patients being treated with cytotoxic chemotherapies. However, little is known about the impact of bearing a tumor (and downstream effects like inflammation) on liver membrane protein glycosylation. In this study, proteomic and glycomic analyses were used in combination to determine whether liver membrane protein glycosylation was affected in mice bearing the Engelbreth-Holm Swarm sarcoma. Peptide IPG-IEF and label-free quantitation determined that many enzymes involved in the protein glycosylation pathway specifically; mannosidases (Man1a-I, Man1b-I and Man2a-I), mannoside N-acetylglucosaminyltransferases (Mgat-I and Mgat-II), galactosyltransferases (B3GalT-VII, B4GalT-I, B4GalT-III, C1GalT-I, C1GalT-II, and GalNT-I), and sialyltransferases (ST3Gal-I, ST6Gal-I, and ST6GalNAc-VI) were up-regulated in all livers of tumor-bearing mice (n = 3) compared with nontumor bearing controls (n = 3). In addition, many cell surface lectins: Sialoadhesin-1 (Siglec-1), C-type lectin family 4f (Kupffer cell receptor), and Galactose-binding lectin 9 (Galectin-9) were determined to be up-regulated in the liver of tumor-bearing compared with control mice. Global glycan analysis identified seven N-glycans and two O-glycans that had changed on the liver membrane proteins derived from tumor-bearing mice. Interestingly, α (2,3) sialic acid was found to be up-regulated on the liver membrane of tumor-bearing mice, which reflected the increased expression of its associated sialyltransferase and lectin receptor (siglec-1). The overall increased sialylation on the liver membrane of Engelbreth-Holm Swarm bearing mice correlates with the increased expression of their associated glycosyltransferases and suggests that glycosylation of proteins in the liver plays a role in tumor-induced liver inflammation.

Profiling Carbohydrate-Receptor Interaction with Recombinant Innate Immunity Receptor-Fc Fusion Proteins

The recognition of bacteria, viruses, fungi, and other microbes is controlled by host immune cells, which are equipped with many innate immunity receptors, such as Toll-like receptors, C-type lectin receptors, and immunoglobulin-like receptors. Our studies indicate that the immune modulating properties of many herbal drugs, for instance, the medicinal fungus Reishi (Ganoderma lucidum) and Cordyceps sinensis, could be attributed to their polysaccharide components. These polysaccharides specifically interact with and activate surface receptors involved in innate immunity. However, due to the complexity of polysaccharides and their various sources from medicinal fungi, quantitative analysis of medicinal polysaccharide extracts with regard to their functions represents a major challenge. To profile carbohydrate-immune receptor interactions, the extracellular domains of 17 receptors were cloned as Fc-fusion proteins, such that their interactions with immobilized polysaccharides could be probed in an enzyme-linked immunosorbent assay. The results show that several innate immune receptors, including Dectin-1, DC-SIGN, Langerin, Kupffer cell receptor, macrophage mannose receptor, TLR2, and TLR4, interact with the polysaccharide extracts from G. lucidum (GLPS). This analysis revealed distinct polysaccharide profiles from different sources of medicinal fungi, and the innate immune receptor-based enzyme-linked immunosorbent assay described here can serve as a high-throughput profiling method for the characterization and quality control of medicinal polysaccharides. It also provides a means to dissect the molecular mechanism of medicinal polysaccharide-induced immunomodulation events.

Laser capture microdissection and genetic analysis of carbon-labeled Kupffer cells

To develop a method of labeling and micro-dissecting mouse Kupffer cells within an extraordinarily short period of time using laser capture microdissection (LCM). Tissues are complex structures comprised of a heterogeneous population of interconnected cells. LCM offers a method of isolating a single cell type from specific regions of a tissue section. LCM is an essential approach used in conjunction with molecular analysis to study the functional interaction of cells in their native tissue environment. The process of labeling and acquiring cells by LCM prior to mRNA isolation can be elaborate, thereby subjecting the RNA to considerable degradation. Kupffer cell labeling is achieved by injecting India ink intravenously, thus circumventing the need for in vitro staining. The significance of this novel approach was validated using a cholestatic liver injury model. mRNA extracted from the microdissected cell population displayed marked increases in colony-stimulating factor-1 receptor and Kupffer cell receptor message expression, which demonstrated Kupffer cell enrichment. Gene expression by Kupffer cells derived from bile-duct-ligated, versus sham-operated, mice was compared. Microarray analysis revealed a significant (2.5-fold, q value < 10) change in 493 genes. Based on this fold-change and a standardized PubMed search, 10 genes were identified that were relevant to the ability of Kupffer cells to suppress liver injury. The methodology outlined herein provides an approach to isolating high quality RNA from Kupffer cells, without altering the tissue integrity.

Two categories of mammalian galactose-binding receptors distinguished by glycan array profiling

Profiling of the four known galactose-binding receptors in the C-type lectin family has been undertaken in parallel on a glycan array. The results are generally consistent with those of previous assays using various different formats, but they provide a direct comparison of the properties of the four receptors, revealing that they fall into two distinct groups. The major subunit of the rat asialoglycoprotein receptor and the rat Kupffer cell receptor show similar broad preferences for GalNAc-terminated glycans, while the rat macrophage galactose lectin and the human scavenger receptor C-type lectin (SRCL) bind more restricted sets of glycans. Both of these receptors bind to Lewis x-type structures, but the macrophage galactose lectin also interacts strongly with biantennary galactose- and GalNAc-terminated glycans. Although the similar glycan-binding profiles for the asialoglycoprotein receptor and the Kupffer cell receptor might suggest that these receptors are functionally redundant, analysis of fibroblasts transfected with full-length Kupffer cell receptor reveals that they fail to endocytose glycosylated ligand.

Use of a cDNA microarray to study immunity against viral hemorrhagic septicemia (VHS) in Japanese flounder ( Paralichthys olivaceus) following DNA vaccination

Japanese flounder, Paralichthys olivaceus juveniles were vaccinated against viral hemorrhagic septicemia (VHS) by intramuscular injection of 10 μg of a plasmid DNA vector which encodes the viral hemorrhagic septicemia virus (VHSV) glycoprotein (G) gene under the control of the cytomegalovirus promoter. Experimental challenge of two viral doses (1 × 10 2 TCID 50 and 1 × 10 3 TCID 50) one month post-vaccination revealed that the G gene was able to induce protective immunity against VHS and this lasted until 21 days after the challenge. The VHSV G-protein gene DNA vaccine had a high protective efficiency, giving relative percentage survival (RPS) values of at least 93%. The defense mechanisms activated by the DNA vaccine were further elucidated by microarray analysis. Non-specific immune response genes such as NK, Kupffer cell receptor, MIP1-α and Mx1 protein gene were observed to be up-regulated by the VHSV G-protein DNA vaccine at 1 and 3 days post-immunization. Also, specific immune-related genes including the CD20 receptor, CD8 alpha chain, CD40 and B lymphocyte cell adhesion molecule were also up-regulated during that time. We observed significant up-regulation of some immune-related genes that are necessary for antiviral defense. Significant up- and/or down-regulation of unknown genes was also observed upon DNA vaccination. Our results confirm previous reports that the VHSV G gene elicits strong humoral and cellular immune responses which may play a pivotal role in protecting the fish during virus infections.

Gene expression changes in rat liver following exposure to liver growth agents: role of Kupffer cells in xenobiotic-mediated liver growth

Many xenobiotics are known to cause liver enlargement and hepatocarcinogenesis in rats, although the molecular mechanisms that underlie this effect remain largely undefined. Human exposure to several of these compounds, including glucocorticoids and peroxisome proliferators may be significant, due to their use in both pharmaceutical and industrial processes. It is therefore important to elucidate the molecular mechanisms underlying this abnormal liver enlargement in rats, as this will enable more accurate extrapolation of the possible outcomes of human exposure. Male Sprague–Dawley rats were dosed with the peroxisome proliferator Wy-14,643 and changes in liver gene expression examined using subtractive suppression hybridisation examined either 12 of 24 hr later. Twenty-five transcripts were identified which showed differential gene expression in liver following exposure toWy-14,643. Biochemical indices of liver growth (DNA synthesis, apoptosis) showed that these changes correlated with the initiation of liver enlargement. Rats were next treated with either Wy-14,643, cyproterone acetate and dexamethasone, chemically and mechanistically-distinct hepatomegalic compounds. Carboxylesterase and Kupffer cell receptor mRNA levels were seen to alter in a qualitatively similar fashion for all three compounds, and in a liver specific fashion. In addition, these changes correlated with a decrease in the density of Kupffer cells within the liver, which are known to release mitogenic cytokines, and have been linked to Wy-14,643-induced cell proliferation. We therefore propose that Kupffer cells play a role in a general mechanism of xenobiotic-mediated liver enlargement.

Molecular characterization of the rat Kupffer cell glycoprotein receptor.

The Kupffer cell receptor for glycoproteins has been reported to have a role in clearance of galactose- and fucose-terminated glycoproteins from circulation. Although the gene and a cDNA encoding the receptor have been described, there has been little study of the receptor protein. To address some questions about possible ligands and functions for this receptor, fragments representing portions of the extracellular domain have been expressed and characterized. The extracellular domain consists of a trimer stabilized by an extended coiled-coil of alpha-helices. The receptor displays monosaccharide-binding characteristics similar to the hepatic asialoglycoprotein receptor, but with somewhat less selectivity. The two best monosaccharide ligands are GalNAc and galactose. alpha-Methyl fucoside is a particularly poor ligand. Analysis of Kupffer cell receptor binding to glycoproteins and oligosaccharides released from them reveals highest affinity for desialylated, complex N-linked glycans. The best glycoprotein ligands contain multiple highly branched oligosaccharides. A human ortholog of the rat receptor gene does not encode a full-length protein and is not expressed in liver. These characteristics suggest that the receptor may have functions parallel to those of the hepatocyte asialoglycoprotein receptor in some (but not all) mammalian species.

Adhesion of colorectal carcinoma cells to the endothelium is mediated by cytokines from CEA stimulated Kupffer cells.

We hypothesize that a major factor regulating hepatic metastasis is the ability of CEA (carcinoembryonic antigen) producing colorectal carcinomas to activate Kupffer cells. CEA and NCA (nonspecific cross-reacting antigen) bind to an 80 kDa Kupffer cell receptor by the peptide sequence PELPK and stimulate cytokine production. Cytokines induce sinusoidal endothelial cells to express intercellular adhesion molecules and increase adhesion of the tumor cells and retention in the liver. In this study human Kupffer cells were activated in vitro with CEA, NCA, and the peptide PELPK. This resulted in release of IL-1beta, TNF-alpha and IL-6. CEA non-producing MIP-101 colon carcinoma cells labeled with 51Cr were incubated on monolayers of ECV-304 human umbilical vein endothelial cells treated with these Kupffer cell derived cytokines or with comparable recombinant human (rH) cytokines. Specific antibodies to the adhesion molecules ICAM-1, VCAM-1, E-selectin and beta2integrin were used to block their functions. A significant enhancement in the adhesion of colorectal carcinoma cells occurred when endothelial cells were stimulated with a very low concentration of Kupffer-cell derived cytokines. Activated endothelium demonstrated significant up-regulation primarily of ICAM-1. The adhesion was blocked by an antibody to ICAM-1. A combination of Kupffer-cell derived cytokines was more effective than IL-1beta or TNF-alpha alone. IL-6 alone did not influence adhesion under our conditions. Our results suggest a mechanism for CEA in the modulation of colorectal carcinoma adhesion to the hepatic endothelium and its enhancement of metastatic potential.

Gadolinium chloride blocks alcohol-dependent liver toxicity in rats treated chronically with intragastric alcohol despite the induction of CYP2E1.

Hepatic CYP2E1 is induced in several models of alcohol administration, but clinically relevant pathology is only observed in rats in a model involving the continuous intragastric administration of an ethanol-containing, corn oil-based, high-fat diet. The level of CYP2E1 correlates with the degree of liver pathology in the intragastric feeding model, which leads to the hypothesis that radical production by CYP2E1 is responsible for the pathology. Destruction of the Kupffer cells with gadolinium chloride (GdCl3) prevented the development of ethanol-dependent pathology and decreased the production of radicals that appeared in the bile of intragastrically alcohol-fed rats. If the induction of CYP2E1 and subsequent formation of oxidant species by the enzyme is causative in the ethanol-dependent hepatic pathology, then protection by GdCl3 could be due an inhibition of CYP2E1 induction. In the current study, ethanol-administration for 4 wk produced marked steatosis, necrosis, and inflammation not seen in control rats. Immunochemically, CYP2E1 was induced 5- to 6-fold in microsomes from the ethanol-treated animals. Rates of p-nitrophenol and chlorzoxazone hydroxylation were elevated approximately 3-fold, consistent with CYP2E1 induction. When GdCl3 was administered with ethanol, there was a decrease of approximately 80% in Kupffer cell receptor expression, and there was a significant decrease in hepatic pathology, which confirms previous studies. However, in the ethanol and GdCl3-treated animals, there was no significant decrease in the induction of CYP2E1. CYP2E1 was elevated approximately 5-fold, as estimated by immunoblot analysis, and rates of p-nitrophenol and chlorzoxazone hydroxylation were elevated 3- to 4-fold in ethanol + GdCl3-treated rats. Thus, these results clearly dissociate the induction of CYP2E1 by intragastric infusion of ethanol from the generation of early alcohol-induced liver disease. It is concluded that Kupffer cells rather than CYP2E1 play the major role in the initiation of hepatocyte damage caused by alcohol.

Alpha-helical coiled-coil stalks in the low-affinity receptor for IgE (Fc epsilon RII/CD23) and related C-type lectins.

The low-affinity receptor for IgE (Fc epsilon RII/CD23) is a cell surface glycoprotein that plays a role in both cellular immunity and allergic inflammation. Its extracellular IgE-binding domain bears homology to C-type animal lectins, and the protein is, therefore, classified as a member of this superfamily. We predict that this lectin-like domain is separated from the cell membrane by an extensive region of alpha-helical coiled-coil structure, based upon sequence comparisons with tropomyosin, the archetypal alpha-helical coiled-coil structure, and detection of characteristic heptad repeats. Analysis of other receptor protein sequences identified a similar structural motif in other membrane-bound members of the C-type lectin superfamily, including the asialoglycoprotein receptor, the Kupffer cell receptor, and the B-cell differentiation antigen Lyb-2 (CD72). It appears that within the C-type lectin superfamily, there is a subfamily of structurally related membrane-bound receptor proteins that contain alpha-helical coiled-coil stalks of various lengths.

Structure of the gene for a carbohydrate-binding receptor unique to rat kupffer cells

The gene encoding the rat Kupffer cell receptor for fucose and galactose (Lehrman, M. A., and Hill, R. L. (1986) J. Biol. Chem. 261, 7419-7425 and Lehrman, M. A., Haltiwanger, R. S., and Hill, R. L. (1986) J. Biol. Chem. 261, 7426-7432) has been isolated and characterized. The nucleotide sequence of the 9.7-kilobase gene and of 1 kilobase of 5'-flanking sequence was determined. The 5' end of the receptor coding sequence was determined by primer extension and S1 mapping of the receptor transcript. Knowledge of the transcription start site and the 5'-flanking sequence allowed potential promoter elements to be identified. The Kupffer cell receptor coding sequence is encoded by seven exons that are split by six introns. The divisions among domains in the receptor can be correlated with divisions among exons in the gene. The carbohydrate recognition domain is encoded by three exons similar to those in the rat hepatic lectin, human Fc receptor for IgE, and rat cartilage proteoglycan core protein genes. A model for the evolution of the vertebrate lectin genes that have been analyzed is presented.

Molecular cloning and sequencing of a cDNA for a carbohydrate binding receptor unique to rat Kupffer cells.

cDNA comprising the entire length of the rat Kupffer cell receptor (Mr = 88,000 and 77,000) for carbohydrates with an affinity for fucose and galactose was isolated, and its nucleotide sequence was determined. Receptor cDNA encoded a protein containing 550 amino acid residues with a Mr = 61,104. This Mr was consistent with the size of the deglycosylated receptor which was found to contain two polypeptides by gel electrophoresis with Mr = 58,000 and 52,000, respectively. Edman degradation of the receptor yielded a sequence which corresponded to amino acid residues 83-104 of the sequence derived from the cDNA. This confirmed that the cDNA which had been isolated corresponded to mRNA for the receptor and suggested that the smaller polypeptide in receptor preparations arises by proteolysis of the intact receptor. Amino acid composition of the receptor was nearly identical to that predicted by the cDNA. The Kupffer cell receptor was found to be homologous to other carbohydrate binding proteins including the hepatic receptors with different binding specificities. The Kupffer cell receptor also contained a series of 18 contiguous, homologous sequences with an average length of 14 residues.

A new Kupffer cell receptor mediating plasma clearance of carcinoembryonic antigen by the rat.

Native human carcinoembryonic antigen is rapidly removed from the circulation by the rat liver Kupffer cell after intravenous injection. The molecule is subsequently transferred to the hepatocyte in an immunologically identifiable form. Carcinoembryonic antigen has a circulatory half-life of 3.7 (+/- 0.8) min, and cellular entry is by receptor-mediated endocytosis. Non-specific fluid pinocytosis and phagocytosis can be excluded as possible mechanisms by the kinetics of clearance and failure of colloidal carbon to inhibit uptake. Substances with known affinity for the hepatic receptors for mannose, N-acetylglucosamine, fucose and galactose all fail to inhibit carcinoembryonic antigen clearance. After two cycles of the Smith degradation, carcinoembryonic antigen is still able to inhibit clearance of the native molecule. Receptor specificity is apparently not dependent on those non-reducing terminal sugars of the native molecule. Performic acid-oxidized carcinoembryonic antigen also inhibits clearance of carcinoembryonic antigen in vivo. Receptor binding is not dependent on tertiary protein conformation. Non-specific cross-reacting antigen, a glycoprotein structurally similar to carcinoembryonic antigen, is cleared by the same mechanism.