胃癌发生中的独特肿瘤标记物—LI-cadherin

LI-cadherin,也被称为cadherin-17,是肠上皮细胞中一种贯穿细胞膜的、钙依赖性的、介导细胞间连接的糖蛋白。与经典钙黏蛋白相比,LI-cadherin是一种具备独特结构和功能的新型钙黏蛋白。LI-cadherin由7个胞外重复序列和一个只包含20个氨基酸残基的较短的胞质尾区组成。在人体中,LI-cadherin特异性的位于肝细胞和肠细胞中的底外侧区。而LI-cadherin介导细胞连接时,既不与链蛋白结合,也不导致β-catenin的上调。一些研究发现,在胃癌中LI-cadherin的过度表达与CDX-2显著相关,而且在肠化生中CDX-2的表达总是与LI-cadherin呈现很强的成对性。最新的研究认为LI-cadherin的表达与胃癌的发生、发展、转移及预后均有关系。在针对胃癌的临床处理方面,LI-cadherin将会是有用的肿瘤标记物。     

The three members of the selectin receptor family recognize a common carbohydrate epitope, the sialyl Lewis(x) oligosaccharide

The selectins (lectin-EGF-complement binding-cell adhesion molecules [LEC-CAMs]) are a family of mammalian receptors implicated in the initial interactions between leukocytes and vascular endothelia, leading to lymphocyte homing, platelet binding, and neutrophil extravasation. The three known selectins, L-selectin (leukocyte adhesion molecule-1 [LECAM-1]), E-selectin (endothelial-leukocyte adhesion molecule-1 [ELAM-1]), and P-selectin (GMP-140) share structural features that include a calcium-dependent lectin domain. The sialyl Lewis(x) carbohydrate epitope has been reported as a ligand for both E- and P-selectins. Although L-selectin has been demonstrated to bind to carbohydrates, structural features of potential mammalian carbohydrate ligand(s) have not been well defined. Using an ELISA developed with a sialyl Lewis(x)-containing glycolipid and an E-selectin-IgG chimera, we have demonstrated the direct binding of the L-selectin-IgG chimera to sialyl Lewis(x). This recognition was calcium dependent, and could be blocked by Mel-14 antibody but not by other antibodies. Recognition was confirmed by the ability of cells expressing the native L-selectin to adhere to immobilized sialyl Lewis(x). These data suggest that the sialyl Lewis(x) oligosaccharide may form the basis of a recognition domain common to all three selectins.     

Interactions required for binding of simian virus 40 T antigen to the viral origin and molecular modeling of initial assembly events

The purified T-antigen origin binding domain binds site specifically to site II, the central region of the simian virus 40 core origin. However, in the context of full-length T antigen, the origin binding domain interacts poorly with DNA molecules containing just site II. Here we investigate the contributions of additional core origin regions, termed the flanking sequences, to origin recognition and the assembly of T-antigen hexamers and double hexamers. Results from these studies indicate that in addition to site-specific binding of the T-antigen origin binding domain to site II, T-antigen assembly requires non-sequence-specific interactions between a basic finger in the helicase domain and particular flanking sequences. Related studies demonstrate that the assembly of individual hexamers is coupled to the distortions in the proximal flanking sequence. In addition, the point in the double-hexamer assembly process that is regulated by phosphorylation of threonine 124, the sole posttranslational modification required for initiation of DNA replication, was further analyzed. Finally, T-antigen structural information is used to model various stages of T-antigen assembly on the core origin and the regulation of this process.     

Intestinal LI-cadherin acts as a Ca2+-dependent adhesion switch

Cadherins are Ca(2+)-dependent transmembrane glycoproteins that mediate cell-cell adhesion and are important for the structural integrity of epithelia. LI-cadherin and the classical E-cadherin are the predominant two cadherins in the intestinal epithelium. LI-cadherin consists of seven extracellular cadherin repeats and a short cytoplasmic part that does not interact with catenins. In contrast, E-cadherin is composed of five cadherin repeats and a large cytoplasmic domain that is linked via catenins to the actin cytoskeleton. Whereas E-cadherin is concentrated in adherens junctions, LI-cadherin is evenly distributed along the lateral contact area of intestinal epithelial cells. To investigate if the particular structural properties of LI-cadherin result in a divergent homotypic adhesion mechanism, we analyzed the binding parameters of LI-cadherin on the single molecule and the cellular level using atomic force microscopy, affinity chromatography and laser tweezer experiments. Homotypic trans-interaction of LI-cadherin exhibits low affinity binding with a short lifetime of only 1.4 s. Interestingly, LI-cadherin binding responds to small changes in extracellular Ca(2+) below the physiological plasma concentration with a high degree of cooperativity. Thus, LI-cadherin might serve as a Ca(2+)-regulated switch for the adhesive system on basolateral membranes of the intestinal epithelium.     

Sialyl Lewis(x) analogs based on a quinic acid scaffold as the fucose mimic

(-)-Quinic acid was used as a starting material for the preparation of sialyl Lewis(x) mimetics in order to target E-selectin. Spatial orientation of the hydroxyl groups of quinic acid could mimic the l-fucose ones. Introduction of a side chain ending with a carboxylic acid was effected to replace the sialic acid interaction at the carbohydrate recognition domain. A first series of derivatives, incorporating amino acids linked to quinic acid, were tested for their affinity and found to interact with E-selectin with IC(50) within the millimolar range.     

Identification of an E-selectin region critical for carbohydrate recognition and cell adhesion [published erratum appears in J Cell Biol 1993 Feb;120(4):1071]

E-selectin elicits cell adhesion by binding to the cell surface carbohydrate, sialyl Lewis X (sLe(x)). We evaluated the effects of mutations in the E-selectin lectin domain on the binding of a panel of anti-E-selectin mAbs and on the recognition of immobilized sLe(x) glycolipid. Functional residues were then superimposed onto a three-dimensional model of the E-selectin lectin domain. This analysis demonstrated that the epitopes recognized by blocking mAbs map to a patch near the antiparallel beta sheet derived from the NH2 and COOH termini of the lectin domain and two adjacent loops. Mutations that affect sLe(x) binding map to this same region. These results thus define a small region of the E-selectin lectin domain that is critical for carbohydrate recognition.     

T-antigen kinase inhibits simian virus 40 DNA replication by phosphorylation of intact T antigen on serines 120 and 123.

Simian virus 40 (SV40) DNA replication begins after two large T-antigen hexamers assemble on the viral minimal origin of replication and locally unwind the template DNA. The activity of T antigen in this reaction is regulated by its phosphorylation state. A form of casein kinase I purified from HeLa nuclear extracts (T-antigen kinase) phosphorylates T antigen on physiologic sites and inhibits its activity in the unwinding reaction (A. Cegielska and D. M. Virshup, Mol. Cell. Biol. 13:1202-1211, 1993). Using a series of mutant T antigens expressed by recombinant baculoviruses in Sf9 cells, we find that the origin unwinding activities of both TS677-->A and TS677,679-->A are inhibited by the T-antigen kinase, as is wild-type T antigen. In contrast, mutants TS120-->A and TS123,679-->A are resistant to inhibition by the kinase. Thus, phosphorylation of serines 120 and 123 is necessary for inhibition of T-antigen activity. Previous studies of casein kinase I substrate specificity have suggested that acidic residues or a phosphorylated amino acid amino terminal to the target residue are required to create a casein kinase I recognition site. However, we find that the T-antigen kinase can add more than 3 mol of Pi per mol to full-length bacterially produced T antigen and that it inhibits the unwinding activity of p34cdc2-activated bacterially produced T antigen. Since no prior phosphorylation is present in this bacterially produced T antigen, and no acidic residues are present immediately amino terminal to serines 120 and 123, other structural elements of T antigen must contribute to the recognition signals for T-antigen kinase. In support of this conclusion, we find that while T-antigen kinase phosphorylates amino-terminal residues in bacterially produced full-length T antigen, it cannot phosphorylate bacterially produced truncated T antigen containing amino acids 1 to 259, a 17-kDa amino-terminal tryptic fragment of T antigen, nor can it phosphorylate denatured T antigen. These findings strongly suggest that the carboxy-terminal domain of T antigen is an important modifier of the recognition signals for phosphorylation of the critical amino-terminal sites by the T-antigen kinase. This conclusion is consistent with previous studies suggesting close apposition of amino- and carboxy-terminal domains of T antigen in the native protein. The three-dimensional conformation of the substrate appears to make a significant contribution to T-antigen kinase substrate specificity.     

Heterotypic trans-interaction of LI- and E-cadherin and their localization in plasmalemmal microdomains

Cadherins are calcium-dependent adhesion molecules important for tissue morphogenesis and integrity. LI-cadherin and E-cadherin are the two prominent cadherins in intestinal epithelial cells. Whereas LI-cadherin belongs to the subfamily of 7D (seven-domain)-cadherins defined by their seven extracellular cadherin repeats and short intracellular domain, E-cadherin is the prototype of classical cadherins with five extracellular domains and a highly conserved cytoplasmic part that interacts with catenins and thereby modulates the organization of the cytoskeleton. Here, we report a specific heterotypic trans-interaction of LI- with E-cadherin, two cadherins of distinct subfamilies. Using atomic force microscopy and laser tweezer experiments, the trans-interaction of LI- and E-cadherin was characterized on the single-molecule level and on the cellular level, respectively. This heterotypic interaction showed similar binding strength (20-52 pN at 200-4000 nm/s) and lifetime (0.8 s) as the respective homotypic interactions of LI- and E-cadherin. VE-cadherin, another classical cadherin, did not bind to LI-cadherin. In enterocytes, LI-cadherin and E-cadherin are located in different membrane regions. LI-cadherin is distributed along the basolateral membrane, whereas the majority of E-cadherin is concentrated in adherens junctions. This difference in membrane distribution was also reflected in Chinese hamster ovary cells stably expressing either LI- or E-cadherin. We found that LI-cadherin is localized almost exclusively in cholesterol-rich fractions, whereas E-cadherin is excluded from these membrane fractions. Given their different membrane localization in enterocytes, the heterotypic trans-interaction of LI- and E-cadherin might play a role during development of the intestinal epithelium when the cells do not yet have elaborate membrane specializations.     

Overexpression of LI-cadherin in gastric cancer is associated with lymph node metastasis

Gastric cancer remains the second leading cause of cancer deaths worldwide. Patients usually present late with local invasion or metastatic diseases. The present study investigated the expression level of liver-intestine cadherin (LI-cadherin) by RT-PCR and its correlation with clinicopathological data in 71 pairs of tumor and non-cancerous gastric mucosa. Protein expression level of LI-cadherin was determined by Western blotting and immunohistochemistry. The mRNA of LI-cadherin was highly expressed in tumor as compared to non-cancerous mucosa. Lymph node metastasis was significantly associated with the expression of LI-cadherin (p=0.038). On multivariate analysis, T staging and LI-cadherin expression were found to be independent factors associated with lymph node metastasis.     

Localization of HIV RNA in mitochondria of infected cells: potential role in cytopathogenicity

A novel member of the cadherin family of cell adhesion molecules has been characterized by cloning from rat liver, sequencing of the corresponding cDNA, and functional analysis after heterologous expression in nonadhesive S2 cells. cDNA clones were isolated using a polyclonal antibody inhibiting Ca(2+)-dependent intercellular adhesion of hepatoma cells. As inferred from the deduced amino acid sequence, the novel molecule has homologies with E-, P-, and N-cadherins, but differs from these classical cadherins in four characteristics. Its extracellular domain is composed of five homologous repeated domains instead of four characteristic for the classical cadherins. Four of the five domains are characterized by the sequence motifs DXNDN and DXD or modifications thereof representing putative Ca(2+)-binding sites of classical cadherins. In its NH2-terminal region, this cadherin lacks both the precursor segment and the endogenous protease cleavage site RXKR found in classical cadherins. In the extracellular EC1 domain, the novel cadherin contains an AAL sequence in place of the HAV sequence motif representing the common cell adhesion recognition sequence of E-, P-, and N-cadherin. In contrast to the conserved cytoplasmic domain of classical cadherins with a length of 150-160 amino acid residues, that of the novel cadherin has only 18 amino acids. Examination of transfected S2 cells showed that despite these structural differences, this cadherin mediates intercellular adhesion in a Ca(2+)-dependent manner. The novel cadherin is solely expressed in liver and intestine and was, hence, assigned the name LI-cadherin. In these tissues, LI- cadherin is localized to the basolateral domain of hepatocytes and enterocytes. These results suggest that LI-cadherin represents a new cadherin subtype and may have a role in the morphological organization of liver and intestine.     

Mechanism of dimerization and structural features of human LI-cadherin

Liver intestine (LI)-cadherin is a member of the cadherin superfamily, which encompasses a group of Ca²⁺-dependent cell-adhesion proteins. The expression of LI-cadherin is observed on various types of cells in the human body, such as normal small intestine and colon cells, and gastric cancer cells. Because its expression is not observed on normal gastric cells, LI-cadherin is a promising target for gastric cancer imaging. However, because the cell adhesion mechanism of LI-cadherin has remained unknown, rational design of therapeutic molecules targeting this cadherin has been hampered. Here, we have studied the homodimerization mechanism of LI-cadherin. We report the crystal structure of the LI-cadherin homodimer containing its first four extracellular cadherin repeats (EC1-4). The EC1-4 homodimer exhibited a unique architecture different from that of other cadherins reported so far, driven by the interactions between EC2 of one protein chain and EC4 of the second protein chain. The crystal structure also revealed that LI-cadherin possesses a noncanonical calcium ion–free linker between the EC2 and EC3 domains. Various biochemical techniques and molecular dynamics simulations were employed to elucidate the mechanism of homodimerization. We also showed that the formation of the homodimer observed in the crystal structure is necessary for LI-cadherin–dependent cell adhesion by performing cell aggregation assays. Taken together, our data provide structural insights necessary to advance the use of LI-cadherin as a target for imaging gastric cancer.     

Strand and face: the topography of interactions between the SV40 origin of replication and T-antigen during the initiation of replication.

The mechanism by which a replicator (origin of replication) becomes denatured during the initiation of replication is not understood for any prokaryotic or eukaryotic system. To address this question, we chemically probed the molecular contacts on the SV40 origin of replication (ori) that are used by the SV40 large T-antigen and a single-stranded DNA-binding protein (SSB) during ori denaturation. Prior to the actual denaturation step, the T-antigen double hexamer bound ori utilizing sugar-phosphate contacts that were located on opposite strands in each flanking domain of ori. Each set of flanking phosphate contacts were also located on approximately opposite faces of the ori duplex. While the phosphate contacts had a 2-fold symmetry with respect to the ori center, T-antigen contacts with nucleotide bases were polar with critical interactions detected in only one of the two flanking domains. During origin denaturation catalyzed by T-antigen and a SSB, numerous new contacts to flanking phosphates were observed on the strand not initially bound by T-antigen, suggesting movement of each T-antigen hexamer outward from ori. These data suggest that T-antigen initially binds ori in a manner that facilitates transfer of each T-antigen hexamer to opposite strands during the initiation of SV40 replication.     

Dissection of H-2Db-restricted cytotoxic T-lymphocyte epitopes on simian virus 40 T antigen by the use of synthetic peptides and H-2Dbm mutants.

Five distinct cytotoxic T-lymphocyte (CTL) recognition sites were identified in the simian virus 40 (SV40) T antigen by using H-2b cells that express the truncated T antigen or antigens carrying internal deletions of various sizes. Four of the CTL recognition determinants, designated sites I, II, III, and V, are H-2Db restricted, while site IV is H-2Kb restricted. The boundaries of CTL recognition sites I, II, and III, clustered in the amino-terminal half of the T antigen, were further defined by use of overlapping synthetic peptides containing amino acid sequences previously determined to be required for recognition by T-antigen site-specific CTL clones by using SV40 deletion mutants. CTL clone Y-1, which recognizes epitope I and whose reactivity is affected by deletion of residues 193 to 211 of the T antigen, responded positively to B6/PY cells preincubated with a synthetic peptide corresponding to T-antigen amino acids 205 to 219. CTL clones Y-2 and Y-3 lysed B6/PY cells preincubated with large-T peptide LT220-233. To distinguish further between epitopes II and III, Y-2 and Y-3 CTL clones were reacted with SV40-transformed cells bearing mutations in the major histocompatibility complex class I antigen. Y-2 CTL clones lysed SV40-transformed H-2Dbm13 cells (bm13SV) which carry several amino acid substitutions in the putative antigen-binding site in the alpha 2 domain of the H-2Db antigen but not bm14SV cells, which contain a single amino acid substitution in the alpha 1 domain. Y-3 CTL clones lysed both mutant transformants. Y-1 and Y-5 CTL clones failed to lyse bm13SV and bm14SV cells; however, these cells could present synthetic peptide LT205-219 to CTL clone Y-1 and peptide SV26(489-503) to CTL clone Y-5, suggesting that the endogenously processed T antigen yields fragments of sizes or sequences different from those of synthetic peptides LT205-219 and SV26(489-503).     

Neurofibromatosis Type 2 Tumor Suppressor Protein,NF2, Induces Proteasome-Mediated Degradation of JC Virus T-Antigen in Human Glioblastoma

Neurofibromatosis type 2 protein (NF2) has been shown to act as tumor suppressor primarily through its functions as a cytoskeletal scaffold. However, NF2 can also be found in the nucleus, where its role is less clear. Previously, our group has identified JC virus (JCV) tumor antigen (T-antigen) as a nuclear binding partner for NF2 in tumors derived from JCV T-antigen transgenic mice. The association of NF2 with T-antigen in neuronal origin tumors suggests a potential role for NF2 in regulating the expression of the JCV T-antigen. Here, we report that NF2 suppresses T-antigen protein expression in U-87 MG human glioblastoma cells, which subsequently reduces T-antigen-mediated regulation of the JCV promoter. When T-antigen mRNA was quantified, it was determined that increasing expression of NF2 correlated with an accumulation of T-antigen mRNA; however, a decrease in T-antigen at the protein level was observed. NF2 was found to promote degradation of ubiquitin bound T-antigen protein via a proteasome dependent pathway concomitant with the accumulation of the JCV early mRNA encoding T-antigen. The interaction between T-antigen and NF2 maps to the FERM domain of NF2, which has been shown previously to be responsible for its tumor suppressor activity. Co-immunoprecipitation assays revealed a ternary complex among NF2, T-antigen, and the tumor suppressor protein, p53 within a glioblastoma cell line. Further, these proteins were detected in various degrees in patient tumor tissue, suggesting that these associations may occur in vivo. Collectively, these results demonstrate that NF2 negatively regulates JCV T-antigen expression by proteasome-mediated degradation, and suggest a novel role for NF2 as a suppressor of JCV T-antigen-induced cell cycle regulation.     

Selectin-carbohydrate interactions in shear flow

Hydrodynamic shear creates mechanical stresses on selectin bonds, modulating affinity and kinetic parameters. Chemical modification of sialyl Lewis(x) increases the strength of L-selectin bonds without altering recognition, suggesting that mechanical and biorecognition characteristics are separable. L-selectin bond formation rates may be strongly influenced by sulfate orientation in sulfo sialyl Lewis(x).     

Selectin–carbohydrate interactions in shear flow

Hydrodynamic shear creates mechanical stresses on selectin bonds, modulating affinity and kinetic parameters. Chemical modification of sialyl Lewis(x) increases the strength of L-selectin bonds without altering recognition, suggesting that mechanical and biorecognition characteristics are separable. L-selectin bond formation rates may be strongly influenced by sulfate orientation in sulfo sialyl Lewis(x).     

Tumor marker disaccharide D-Gal-beta 1, 3-GalNAc complexed to heat-labile enterotoxin from Escherichia coli.

Heat-labile enterotoxin (LT) is part of the cholera toxin (CT) family and consists of a catalytic A subunit and a B pentamer that serves to recognize the oligosaccharide part of the GM1 ganglioside receptor. We report here the crystal structure of heat-labile enterotoxin in complex with the disaccharide portion of the Thomsen-Friedenreich (T-antigen) tumor marker. The toxin:carbohydrate complex is determined to 2.13 A resolution, yielding an R-factor of 18.5%. The T-antigen disaccharide, D-Gal-beta 1,3-GalNAc-Ser/Thr, is present in more than 85% of human carcinomas and monitoring its autoimmune response is used for the early detection of tumors. Insight into the molecular recognition of this tumor antigen by sugar binding proteins can benefit the development of a diagnostic tool for human carcinomas as well as a T-antigen directed anticancer drug delivery system.     

Sialyl Lewis(x)/E-selectin-mediated rolling in a cell-free system.

Selections mediate transient adhesion of neutrophils to stimulated endothelial cells at sites of inflammation by binding counter-receptors that present carbohydrates such as sialyl Lewis(x). We have developed a cell-free adhesion assay using sialyl Lewis(x)-coated microspheres and E-selection-IgG chimera-coated substrates to investigate the premise that rolling primarily results from functional properties of selection-carbohydrate bonds, whereas cellular morphology and signaling act as secondary effects. Sialyl Lewis(x)-coated microspheres attach to and roll over E-selectin-IgG chimera-coated substrates between the physiological wall shear stresses of 0.7 and 2 dynes/cm2. Rolling velocities vary with time and depend on E-selectin-IgG chimera site density and wall shear stress. Our results show that sialyl Lewis(x) is a minimal functional recognition element required for rolling on E-selectin under flow.     

Mutational analysis at Asn-41 in peanut agglutinin. A residue critical for the binding of the tumor-associated Thomsen-Friedenreich antigen.

Peanut agglutinin is a clinically important lectin due to its application in the screening of mature and immature thymocytes as well as in the detection of cancerous malignancies. The basis for these applications is the remarkably strong affinity of the lectin for the tumor-associated Thomsen-Friedenreich antigen (T-antigen) and more so due to its ability to distinguish T-antigen from its cryptic forms. The crystal structure of the complex of peanut agglutinin with T-antigen reveals the basis of this specificity. Among the contacts involved in providing this specificity toward T-antigen is the water-mediated interaction between the side chain of Asn-41 and the carbonyl oxygen of the acetamido group of the second hexopyranose ring of the sugar molecule. Site-directed mutational changes were introduced at this residue with the objective of probing the role of this residue in T-antigen binding and possibly engineering an altered species with increased specificity for T-antigen. Of the three mutants tested, i.e. N41A, N41D, and N41Q, the last one shows improved potency for recognition of T-antigen. The affinities of the mutants can be readily explained on the basis of the crystal structure of the complex and simple modeling. In particular, the change of asparagine to glutamine could lead to a direct interaction of the side chain with the sugar while at the same time retaining the water bridge. This study strengthens the theory that in lectins the nonprimary contacts generally made through water bridges are involved in imparting exquisite specificity.