A carcinoembryonic antigen (CEA) binding protein from ascites influences CEA uptake by macrophages

A variant of CEA which is less readily endocytosed by macrophages has been isolated from malignant ascites. In vivo, CEA is cleared more slowly by the liver (t1/2 = 15.1 minutes) than CEAs isolated from hepatic metastases (t1/2 = 3.1 minutes). In vitro, rat and human Kupffer cells and rat alveolar macrophages endocytose this CEA less effectively. This slow clearing form of CEA is associated with a smaller (45kD) acidic glycoprotein (CORA) with which it forms a stable complex. CORA can be visualized on reducing gels but not on non reducing gels or by HPLC run under non reducing conditions. This suggests a non-covalent complex between the two glycoproteins. Analysis of protein conformation by circular dichroism revealed changes in the ascites CEA consistent with binding of CORA to the molecule. Western blot showed that CORA crossreacts with antisera to alpha 1-acid glycoprotein and double immunodiffusion demonstrated cross-reactivity but not identify. Sequencing of CNBr peptides showed sequence homology with alpha 1-acid glycoprotein but areas of unique sequence were also found. It is suggested that binding of CORA to CEA blocks the macrophage receptor binding of CEA.     

Carcinoembryonic antigen binding to Kupffer cells is via a peptide located at the junction of the N-terminal and first loop domains

A 11kD glycopeptide has been isolated by pepsin digestion of carcinoembryonic antigen (CEA) that is rapidly endocytosed by isolated rat Kupffer cells and lung alveolar macrophages. Uptake of this glycopeptide by the isolated cells can be inhibited by excess unmodified CEA. Removal of the N-linked oligosaccharide chains by N-glycanase did not alter cellular uptake but reduced the MW to approximately 5500. A seventeen amino acid N-terminal sequence locates this peptide at the junction of the N-terminal and first loop domain of CEA. It is suggested that the recognition of a peptide sequence in this area of CEA is responsible for its clearance from the circulation.     

Hexapeptide fragment of carcinoembryonic antigen which acts as an agonist of heterogeneous ribonucleoprotein M

Colorectal cancers with metastatic potential secrete the glycoprotein carcinoembryonic antigen (CEA). CEA has been implicated in colorectal cancer metastasis by inducing Kupffer cells to produce inflammatory cytokines which, in turn, make the hepatic micro‐environment ideal for tumor cell implantation. CEA binds to the heterogeneous ribonucleoprotein M (hnRNP M) which acts as a cell surface receptor in Kupffer cells. The amino acid sequence in CEA, which binds the hnRNP M receptor, is Tyr‐Pro‐Glu‐Leu‐Pro‐Lys. In this study, the structure of Ac‐Tyr‐Pro‐Glu‐Leu‐Pro‐Lys‐NH₂ (YPELPK) was investigated using electronic circular dichroism, vibrational circular dichroism, and molecular dynamics simulations. The binding of the peptide to hnRNP M was also investigated using molecular docking calculations. The biological activity of YPELPK was studied using differentiated human THP‐1 cells, which express hnRNP M on their surface and secrete IL‐6 when stimulated by CEA. YPELPK forms a stable polyproline‐II helix and stimulates IL‐6 production of THP‐1 cells at micromolar concentrations. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Surface expression of heterogeneous nuclear RNA binding protein M4 on Kupffer cell relates to its function as a carcinoembryonic antigen receptor

Elevated concentrations of carcinoembryonic antigen (CEA) in the blood are associated with the development of hepatic metastases from colorectal cancers. Clearance of circulating CEA occurs through endocytosis by liver macrophages, Kupffer cells. Previously we identified heterogeneous nuclear ribonucleoproteins M4 (hnRNP M4) as a receptor (CEAR) for CEA. HnRNP M4 has two isoform proteins (p80, p76), the full-length hnRNP M4 (CEARL) and a truncated form (CEARS) with a deletion of 39 amino acids between RNA binding domains 1 and 2, generated by alternative splicing. The present study was undertaken to clarify any isoform-specific differences in terms of their function as CEA receptor and localization. We develop anti-CEAR isoform-specific antibodies and show that both CEAR splicing isoforms are expressed on the surface of Kupffer cells and can function as CEA receptor. Alternatively, in P388D1 macrophages CEARS protein has nuclear and CEARL has cytoplasmic localization. In MIP101 colon cancer and HeLa cells the CEARS protein is localized to the nucleus and CEARL to the cytoplasm. These findings imply that different functions are assigned to CEAR isoforms depending on the cell type. The search of 39 amino acids deleted region against the Prosite data base revealed the presence of N-myristylation signal PGGPGMITIP that may be involved in protein targeting to the plasma membrane. Overall, this report demonstrates that the cellular distribution, level of expression, and relative amount of CEARL and CEARS isoforms determine specificity for CEA binding and the expression of alternative spliced forms of CEAR is regulated in a tissue-specific manner.     

Identification of a carcinoembryonic antigen binding protein on monocytes

Tumor-associated monocyte/macrophage cells are important stromal components involved in tumor development. A protein on human monocyte is identified that binds to carcinoembryonic antigen (CEA), a glycoprotein overexpressed in colon tumors. This implicates a role for this protein in CEA processing and establishes a link between monocytes and colon tumor cells. In vitro uptake of 125I-labeled CEA with isolated monocytes showed time and temperature dependence. The binding of 125I-CEA was specific and saturable as it could be inhibited by an excess of unlabeled CEA. To identify the binding protein on monocyte, we used a radiolabeled photoactivable heterobifunctional crosslinking agent and demonstrated that CEA reacts with a 115kDa protein as determined by SDS-polyacrylamide gel electrophoresis and autoradiography. Treatment of human monocytes in vitro with CEA resulted in a several fold increase in the production of proinflammatory cytokines TNF-alpha, IL-1 beta, and IL-6 compared to untreated controls. Binding of CEA to the monocyte protein may have implications in colon tumorigenesis.     

Pro-inflammatory cytokines from Kupffer cells downregulate hepatocyte expression of adrenomedullin binding protein-1

Polymicrobial sepsis is characterized by an early, hyperdynamic phase followed by a late hypodynamic phase. Adrenomedullin (AM), a vasodilatory peptide, inhibits this transition from the early phase to the late phase. Adrenomedullin binding protein-1 (AMBP-1) enhances AM-mediated activities. The decrease of AMBP-1 levels in late sepsis reduces the vascular response to AM and produces the hypodynamic phase. Studies have indicated that the administration of LPS downregulates AMBP-1 production in the liver. Since hepatocytes are the primary source of AMBP-1 biosynthesis in the liver, we employed a co-culture strategy using hepatocyte and Kupffer cells to determine whether LPS directly or by increasing pro-inflammatory cytokines from Kupffer cells downregulates AMBP-1 production. Hepatocytes and Kupffer cells isolated from rats were co-cultured and treated with LPS for 24 h. LPS significantly attenuated AMBP-1 protein expression in a dose-dependent manner. Since AMBP-1 is basically a secretory protein, cell supernatants from co-culture cells treated with LPS were examined for AMBP-1 protein levels. LPS treatment caused a dose related decrease in AMBP-1 protein secretion. Similarly, LPS treatment produced a significant decrease in AMBP-1 protein expression in hepatocytes and Kupffer cells cultured using transwell inserts. LPS had no direct effect on AMBP-1 levels in cultured hepatocytes or Kupffer cells alone. To confirm that the observed effects in co-culture were due to the cytokines released from Kupffer cells, hepatocytes were treated with IL-1beta or TNF-alpha for 24 h and AMBP-1 expression was examined. The results indicated that both cytokines significantly inhibited AMBP-1 protein levels. Thus, pro-inflammatory cytokines released from Kupffer cells are responsible for downregulation of AMBP-1.     

Lipopolysaccharides in liver injury: molecular mechanisms of Kupffer cell activation

Endogenous gut-derived bacterial lipopolysaccharides have been implicated as important cofactors in the pathogenesis of liver injury. However, the molecular mechanisms by which lipopolysaccharides exert their effect are not entirely clear. Recent studies have pointed to proinflammatory cytokines such as tumor necrosis factor-alpha as mediators of hepatocyte injury. Within the liver, Kupffer cells are major sources of proinflammatory cytokines that are produced in response to lipopolysaccharides. This review will focus on three important molecular components of the pathway by which lipopolysaccharides activate Kupffer cells: CD14, Toll-like receptor 4, and lipopolysaccharide binding protein. Within the liver, lipopolysaccharides bind to lipopolysaccharide binding protein, which then facilitates its transfer to membrane CD14 on the surface of Kupffer cells. Signaling of lipopolysaccharide through CD14 is mediated by the downstream receptor Toll-like receptor 4 and results in activation of Kupffer cells. The role played by these molecules in liver injury will be examined.     

A Peptidic Unconjugated GRP78/BiP Ligand Modulates the Unfolded Protein Response and Induces Prostate Cancer Cell Death

The molecular chaperone GRP78/BiP is a key regulator of protein folding in the endoplasmic reticulum, and it plays a pivotal role in cancer cell survival and chemoresistance. Inhibition of its function has therefore been an important strategy for inhibiting tumor cell growth in cancer therapy. Previous efforts to achieve this goal have used peptides that bind to GRP78/BiP conjugated to pro-drugs or cell-death-inducing sequences. Here, we describe a peptide that induces prostate tumor cell death without the need of any conjugating sequences. This peptide is a sequence derived from the cochaperone Bag-1. We have shown that this sequence interacts with and inhibits the refolding activity of GRP78/BiP. Furthermore, we have demonstrated that it modulates the unfolded protein response in ER stress resulting in PARP and caspase-4 cleavage. Prostate cancer cells stably expressing this peptide showed reduced growth and increased apoptosis in in vivo xenograft tumor models. Amino acid substitutions that destroyed binding of the Bag-1 peptide to GRP78/BiP or downregulation of the expression of GRP78 compromised the inhibitory effect of this peptide. This sequence therefore represents a candidate lead peptide for anti-tumor therapy.     

Interaction of DDSDEEN peptide with N-CAM protein. Possible mechanism enhancing neuronal differentiation

DDSDEEN chromatin peptide, after dansylation, was studied for its ability to bind N-CAM protein. The binding causes a quenching of the Dns-peptide fluorescence emission. Dose- and time-dependent binding of Dns-peptide with N-CAM has been shown. Fluorescence quenching is completely lost if the Dns-peptide is subjected to carboxypeptidase digestion. Moreover the undansylated peptide pEDDSDEEN competes with the DnsDDSDEEN peptide for the binding with the N-CAM protein. The Dns-peptide-N-CAM bond has been related to the peptide biological activity probably involved in the promotion of neuronal differentiation. An attempt to recognize a possible N-CAM binding site for Dns-peptide was performed by alignment of N-CAM from various sources with some sequences that have been previously reported as binding sites for the pEDDSDEEN and DDSDEEN peptides. Interestingly, the alignment of N-CAM from various sources with the peptides WHPREGWAL and WFPRWAGQA recognizes on rat and human N-CAM a unique sequence that could be the specific binding site for chromatin peptide: WHSKWYDAK. This sequence is present in fibronectin type-III domain of N-CAM. In addition molecular modeling studies indicate the N-CAM sequence WHSKWYDAK as, probably, the main active site for DnsDDSDEEN (or pEDDSDEEN) peptide ligand. Accordingly the binding experiments show a high affinity between WHSKWYDAK and DnsDDSDEEN peptides.     

Molecular evidence for a glycine-gated chloride channel in macrophages and leukocytes

Recent studies have demonstrated that glycine blunts the response of Kupffer cells to endotoxin. Based on pharmacological evidence, it was hypothesized that Kupffer cells and other macrophages contain a glycine-gated chloride channel similar to the glycine receptor expressed in neuronal tissues. Moreover, glycine stimulates influx of radiolabeled chloride in Kupffer cells in a dose-dependent manner. RT-PCR was used to identify mRNA of both alpha- and beta-subunits of the glycine receptor in rat Kupffer cells, peritoneal neutrophils, and splenic and alveolar macrophages, similar to the sequence generated from rat spinal cord. Importantly, the sequence of the cloned Kupffer cell glycine receptor fragment for the beta-subunit was >95% homologous with the receptor from the spinal cord. Membranes of these cells also contain a protein that is immunoreactive with antibodies against the glycine-gated chloride channel. These data demonstrate that Kupffer cells, as well as other macrophages and leukocytes, express mRNA and protein for a glycine-gated chloride channel with both molecular and pharmacological properties similar to the channel expressed in the central nervous system.     

Arg9-peptide facilitates the internalization of an anti-CEA immunotoxin and potentiates its specific cytotoxicity to target cells

Arginines-containing membrane translocational signals (MTS), such as Tat(47-57) and VP22(267-300), and synthetic arginine-rich peptides have been reported to be efficient carriers for transporting various types of biomolecules into cytoplasmic and nuclear compartments of living cells. Among those arginines-containing MTS, a 9-mer arginine peptide (Arg9) was proved the most economical and efficient. We fused Arg9-peptide to an anti-CEA (Carcinoembryonic antigen) immunotoxin, PE35/CEA(Fv)/KDEL, at the position between the toxin moiety and the binding moiety. Strong binding and internalization of this fusion protein was observed in all detected cell lines, but little cytotoxicity to the cells that lack the CEA molecules on the cell surface was detected. However, the cytotoxicity besides the binding activity of the fusion protein to specific tumor cells expressing large amount of CEA molecules on the cell surface was improved markedly, indicating that the Arg9-peptide is capable of facilitating the receptor-mediated endocytosis of this immunotoxin, which leads to the increase of the specific cytotoxicity of this immunotoxin.     

Proteoglycans mediate malaria sporozoite targeting to the liver

Malaria sporozoites are rapidly targeted to the liver where they pass through Kupffer cells and infect hepatocytes, their initial site of replication in the mammalian host. We show that sporozoites, as well as their major surface proteins, the CS protein and TRAP, recognize distinct cell type-specific surface proteoglycans from primary Kupffer cells, hepatocytes and stellate cells, but not from sinusoidal endothelia. Recombinant Plasmodium falciparum CS protein and TRAP bind to heparan sulphate on hepatocytes and both heparan and chondroitin sulphate proteoglycans on stellate cells. On Kupffer cells, CS protein predominantly recognizes chondroitin sulphate, whereas TRAP binding is glycosaminoglycan independent. Plasmodium berghei sporozoites attach to heparan sulphate on hepatocytes and stellate cells, whereas Kupffer cell recognition involves both chondroitin sulphate and heparan sulphate proteoglycans. CS protein also interacts with secreted proteoglycans from stellate cells, the major producers of extracellular matrix in the liver. In situ binding studies using frozen liver sections indicate that the majority of the CS protein binding sites are associated with these matrix proteoglycans. Our data suggest that sporozoites are first arrested in the sinusoid by binding to extracellular matrix proteoglycans and then recognize proteoglycans on the surface of Kupffer cells, which they use to traverse the sinusoidal cell barrier.     

The aggregated form of an AAMP derived peptide behaves as a heparin sensitive cell binding agent

A peptide termed P189, derived from the sequence of a newly discovered protein, AAMP (angio-associated migratory cell protein), contains a motif that is predicted to a bind heparin. It occurs near the amino terminal end of AAMP. Previous studies have shown that in its solubilized form P189 (RRLRRMESESES) binds heparin and melanoma cells. The peptide is bipolar in that it contains positive charges at its amino end and negative charges at its carboxyl end. It forms strongly aggregated particles that require exposure to 50% DMSO and 100 degrees C for solubilization to occur. Now heparin and cell binding (heparin sensitive) are also demonstrated for the peptide in its particular form. Cell binding/clustering to the peptide particles is strong and resists exposure to various reagents (sugars and inhibitors of glycolysis and protein synthesis) except heparin. Tumor cell migration is partially inhibited by the presence of the peptide. On electron photomicrographs the peptide is seen in close apposition to cell membranes. Heparin sensitivity of the cell binding indicates that cell surface glycosaminoglycans are involved. The aggregated peptide binds heparin in a saturable manner with a dissociation constant, K(d), of 306 pmol. Cell binding/clustering studies using peptide variants of P189, which have substitutions in either the charged and/or nonpolar residues, show that the specific sequence of P189 optimizes heparin-sensitive cell aggregation. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 365-372, 1997.     

Characterization of the chylomicron-remnant-recognition sites on parenchymal and Kupffer cells of rat liver. Selective inhibition of parenchymal cell recognition by lactoferrin.

Upon injection of chylomicrons into rats, chylomicron remnants are predominantly taken up by parenchymal cells, with a limited contribution (8.6% of the injected dose) by Kupffer cells. In vitro storage of partially processed chylomicron remnants for only 24 h leads, after in vivo injection, to an avid recognition by Kupffer cells (uptake up to 80% of the total liver-associated radioactivity). Lactoferrin greatly reduces the liver uptake of chylomicron remnants, which appears to be the consequence of a specific inhibition of the uptake by parenchymal cells. Kupffer-cell uptake is not influenced by lactoferrin. In vitro studies with isolated parenchymal and Kupffer cells show that both contain a specific recognition site for chylomicron remnants. The Kupffer-cell recognition site differs in several ways from the recognition site on parenchymal cells as follows. (a) The maximum level of binding is 3.7-fold higher/mg cell protein than with parenchymal cells. (b) Binding of chylomicron remnants is partially dependent on the presence of calcium, while binding to parenchymal cells is not. (c) beta-Migrating very-low-density lipoprotein is a less effective competitor for chylomicron-remnant binding to Kupffer cells compared to parenchymal cells. (d) Lactoferrin leaves Kupffer-cell binding uninfluenced, while it greatly reduces binding of chylomicron remnants to parenchymal cells. The properties of chylomicron-remnant recognition by parenchymal cells are consistent with apolipoprotein E being the determinant for recognition. It can be concluded that the chylomicron-remnant recognition site on Kupffer cells possesses properties which are distinct from the recognition site on parenchymal cells. It might be suggested that partially processed chylomicron remnants are specifically sensitive to a modification, which induces an avid interaction with the Kupffer cells. The recognition site for (modified) chylomicron remnants on Kupffer cells might function as a protection system against the occurrence of these potential atherogenic chylomicron-remnant particles in the blood.     

A monoclonal anti-peptide antibody recognizes the adrenocorticotropic receptor

We have produced a monoclonal antibody that specifically recognizes the adrenocorticotropic receptor on rat adrenal cells. The immunogen was designed from an RNA sequence complementary to the mRNA coding for ACTH1-24. This complementary peptide, termed HTCA, has been shown to specifically bind ACTH and was proposed to mimic the ACTH binding site of the hormone receptor. The monoclonal anti-HTCA antibody recognized a restricted domain of the HTCA peptide, bound to Y-1 adrenal cells with a KD of 1.8 nM, and blocked the binding of 125I-ACTH to rat adrenal cells. These findings show that anti-HTCA competes with ACTH for binding to the ACTH receptor.     

Development of a functional backbone cyclic mimetic of the HIV-1 Tat arginine-rich motif

We have used the backbone cyclic proteinomimetics approach to develop peptides that functionally mimic the arginine-rich motif (ARM) of the HIV-1 Tat protein. This consensus sequence serves both as a nuclear localization signal (NLS) and as an RNA binding domain. Based on the NMR structure of Tat, we have designed and synthesized a backbone cyclic ARM mimetic peptide library. The peptides were screened for their ability to mediate nuclear import of the corresponding BSA conjugates in permeabilized cells. One peptide, designated "Tat11," displayed active NLS properties. Nuclear import of Tat11-BSA was found to proceed by the same distinct pathway used by the Tat-NLS and not by the common importin alpha pathway, which is used by the SV40-NLS. Most of the Tat-derived backbone cyclic peptides display selective inhibitory activity as demonstrated by the inhibition of the nuclear import mediated by the Tat-NLS and not by the SV40-NLS. The Tat-ARM-derived peptides, including Tat-11, also inhibited binding of the HIV-1 Rev-ARM to its corresponding RNA element (Rev response element) with inhibition constants of 5 nm. Here we have shown for the first time (a) a functional mimetic of a protein sequence, which activates a nuclear import receptor and (b) a mimetic of a protein sequence with a dual functionality. Tat11 is a lead compound which can potentially inhibit the HIV-1 life cycle by a dual mechanism: inhibition of nuclear import and of RNA binding.     

乙型肝炎病毒表面抗原preS1在大肠杆菌中的表达与鉴定

本文报告利用pWR590质粒为载体,构建了含1ac启动子、β-半乳糖苷酶(1—590)基因、Xa因子的四肽识别位点和HBV preS1、preS2编码序列的表达质粒,并成功地在大肠杆菌中获得稳定表达。融合蛋白经Xa因子消化和高效液相层析,得到了preS1(1—91)纯肽。此肽特异性地与人肝细胞质膜结合,从而为肝细胞上存在preS1受体提供了直接的实验依据,也为分离和鉴定肝细胞上preS1受体打下了良好的基础。     

Adiponectin normalizes LPS-stimulated TNF-alpha production by rat Kupffer cells after chronic ethanol feeding

Chronic ethanol feeding sensitizes Kupffer cells to activation by lipopolysaccharide (LPS), leading to increased production of tumor necrosis factor-alpha (TNF-alpha). Adiponectin treatment protects mice from ethanol-induced liver injury. Because adiponectin has anti-inflammatory effects on macrophages, we hypothesized that adiponectin would normalize chronic ethanol-induced sensitization of Kupffer cells to LPS-mediated signals. Serum adiponectin concentrations were decreased by 45% in rats fed an ethanol-containing diet for 4 wk compared with pair-fed rats. Adiponectin dose dependently inhibited LPS-stimulated accumulation of TNF-alpha mRNA and peptide in Kupffer cells from both pair- and ethanol-fed rats. Kupffer cells from ethanol-fed rats were more sensitive to both globular (gAcrp) and full-length adiponectin (flAcrp) than Kupffer cells from pair-fed controls with suppression at 10 ng/ml adiponectin after chronic ethanol feeding. Kupffer cells expressed both adiponectin receptors 1 and 2; chronic ethanol feeding did not change the expression of adiponectin receptor mRNA or protein. gAcrp suppressed LPS-stimulated ERK1/2 and p38 phosphorylation as well as IkappaB degradation at 100-1,000 ng/ml in Kupffer cells from both pair- and ethanol-fed rats. However, only LPS-stimulated ERK1/2 phosphorylation was sensitive to 10 ng/ml gAcrp. gAcrp also normalized LPS-stimulated DNA binding activity of early growth response-1 with greater sensitivity in Kupffer cells from rats fed chronic ethanol. In conclusion, these results demonstrate that Kupffer cells from ethanol-fed rats are more sensitive to the anti-inflammatory effects of both gAcrp and flAcrp. Suppression of LPS-stimulated ERK1/2 signaling by low concentrations of gAcrp was associated with normalization of TNF-alpha production by Kupffer cells after chronic ethanol exposure.