Mika Kato Kaneko

Molecular analysis of the pathophysiological binding of the platelet aggregation-inducing factor podoplanin to the C-type lectin-like receptor CLEC-2

The mucin‐type sialoglycoprotein podoplanin (aggrus) is involved in tumor cell‐induced platelet aggregation and tumor metastasis. C‐type lectin‐like receptor‐2 (CLEC‐2) was recently identified as an endogenous receptor of podoplanin on platelets. However, the pathophysiological importance and function of CLEC‐2 have not been elucidated. Here we clarified the pathophysiological interaction between podoplanin and CLEC‐2 in vitro and in vivo. Using several deletion mutants of CLEC‐2 expressed as Fc chimeras, we first identified an important podoplanin‐recognition domain in CLEC‐2. Furthermore, the podoplanin–CLEC‐2 interaction was confirmed using several deletion mutants of podoplanin expressed as Fc chimeras. Not only the disialyl‐core1‐attached glycopeptide but also the stereostructure of the podoplanin protein was found to be critical for the CLEC‐2‐binding activity of podoplanin. We next synthesized various glycopeptides of podoplanin that included both the platelet aggregation‐stimulating domain and O‐glycan on Thr52. Interestingly, a disialyl‐core1‐attached glycopeptide was recognized specifically by CLEC‐2. Moreover, the anti‐podoplanin monoclonal antibody NZ‐1 suppressed both the podoplanin–CLEC‐2 interaction and podoplanin‐induced pulmonary metastasis, suggesting that CLEC‐2 is the first pathophysiological receptor of podoplanin to be identified. In summary, we clarified the molecular interaction in vitro and in vivo between a platelet aggregation‐inducing factor, podoplanin, and its specific pathophysiological receptor on platelets, CLEC‐2. Podoplanin and CLEC‐2 might represent promising therapeutic targets in cancer metastasis. (Cancer Sci 2008; 99: 54–61)

Increased expression of podoplanin in malignant astrocytic tumors as a novel molecular marker of malignant progression

Podoplanin (aggrus) is a mucin-like transmembrane sialoglycoprotein that is expressed on lymphatic endothelial cells. Podoplanin is putatively involved in cancer cell migration, invasion, metastasis, and malignant progression and may be involved in platelet aggregation. Previously, we showed upregulated expression of podoplanin in central nervous system (CNS) germinomas, but not in non-germinomatous germ cell tumors, except for parts of immature teratomas in limited numbers. However, little information exists about its role in CNS astrocytic tumors. In this study, 188 astrocytic tumors (30 diffuse astrocytomas, 43 anaplastic astrocytomas, and 115 glioblastomas) were investigated using immunohistochemistry with an anti-podoplanin antibody, YM-1. In 11 of 43 anaplastic astrocytomas (25.6%) and in 54 of 115 glioblastomas (47.0%), podoplanin was expressed on the surface of anaplastic astrocytoma cells and glioblastoma cells, especially around necrotic areas and proliferating endothelial cells. However, the surrounding brain parenchyma was not stained by YM-1. On the other hand, podoplanin expression was not observed in diffuse astrocytoma (0/30: 0%). Furthermore, we investigated the expression of podoplanin using quantitative real-time PCR and Western blot analysis in 54 frozen astrocytic tumors (6 diffuse astrocytomas, 14 anaplastic astrocytomas, and 34 glioblastomas). Podoplanin mRNA and protein expression were markedly higher in glioblastomas than in anaplastic astrocytomas. These data suggest that podoplanin expression might be associated with malignancy of astrocytic tumors.

Focused Differential Glycan Analysis with the Platform Antibody-assisted Lectin Profiling for Glycan-related Biomarker Verification

Protein glycosylation is a critical subject attracting increasing attention in the field of proteomics as it is expected to play a key role in the investigation of histological and diagnostic biomarkers. In this context, an enormous number of glycoproteins have now been nominated as disease-related biomarkers. However, there is no appropriate strategy in the current proteome platform to qualify such marker candidate molecules, which relates their specific expression to particular diseases. Here, we present a new practical system for focused differential glycan analysis in terms of antibody-assisted lectin profiling (ALP). In the developed procedure, (i) a target protein is enriched from clinic samples (e.g. tissue extracts, cell supernatants, or sera) by immunoprecipitation with a specific antibody recognizing a core protein moiety; (ii) the target glycoprotein is quantified by immunoblotting using the same antibody used in (i); and (iii) glycosylation difference is analyzed by means of antibody-overlay lectin microarray, an application technique of an emerging glycan profiling microarray. As model glycoproteins having either N-linked or O-linked glycans, prostate-specific antigen or podoplanin, respectively, were subjected to systematic ALP analysis. As a result, specific signals corresponding to the target glycoprotein glycans were obtained at a sub-picomole level with the aid of specific antibodies, whereby disease-specific or tissue-specific glycosylation changes could be observed in a rapid, reproducible, and high-throughput manner. Thus, the established system should provide a powerful pipeline in support of on-going efforts in glyco-biomarker discovery.

Functional glycosylation of human podoplanin: glycan structure of platelet aggregation-inducing factor.

Podoplanin (Aggrus) is a mucin‐type sialoglycoprotein that plays a key role in tumor cell‐induced platelet aggregation. Podoplanin possesses a platelet aggregation‐stimulating (PLAG) domain, and Thr52 in the PLAG domain of human podoplanin is important for its activity. Endogenous or recombinant human podoplanin were purified, and total glycosylation profiles were surveyed by lectin microarray. Analyses of glycopeptides produced by Edman degradation and mass spectrometry revealed that the disialyl‐corel (NeuAcα2‐3Galβl‐3(NeuAcα2‐6)GalNAcαl‐O‐Thr) structure was primarily attached to a glycosylation site at residue Thr52. Sialic acid‐deficient podoplanin recovered its activity after additional sialylation. These results indicated that the sialylated Corel at Thr52 is critical for podoplanin‐induced platelet aggregation.

Induction of podoplanin by transforming growth factor-β in human fibrosarcoma

Podoplanin/aggrus is increased in tumors and its expression was associated with tumor malignancy. Podoplanin on cancer cells serves as a platelet‐aggregating factor, which is associated with the metastatic potential. However, regulators of podoplanin remain to be determined. Transforming growth factor‐β (TGF‐β) regulates many physiological events, including tumorigenesis. Here, we found that TGF‐β induced podoplanin in human fibrosarcoma HT1080 cells and enhanced the platelet‐aggregating‐ability of HT1080. TGF‐β type I receptor inhibitor (SB431542) and short hairpin RNAs for Smad4 inhibited the podoplanin induction by TGF‐β. These results suggest that TGF‐β is a physiological regulator of podoplanin in tumor cells.

Caenorhabditis elegans galectins LEC-1-LEC-11: structural features and sugar-binding properties.

Galectins form a large family of beta-galactoside-binding proteins in metazoa and fungi. This report presents a comparative study of the functions of potential galectin genes found in the genome database of Caenorhabditis elegans. We isolated full-length cDNAs of eight potential galectin genes (lec-2-5 and 8-11) from a lambdaZAP cDNA library. Among them, lec-2-5 were found to encode 31-35-kDa polypeptides containing two carbohydrate-recognition domains similar to the previously characterized lec-1, whereas lec-8-11 were found to encode 16-27-kDa polypeptides containing a single carbohydrate-recognition domain and a C-terminal tail of unknown function. Recombinant proteins corresponding to lec-1-4, -6, and 8-10 were expressed in Escherichia coli, and their sugar-binding properties were assessed. Analysis using affinity adsorbents with various beta-galactosides, i.e., N-acetyllactosamine (Galbeta1-4GlcNAc), lacto-N-neotetraose (Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc), and asialofetuin, demonstrated that LEC-1-4, -6, and -10 have a significant affinity for beta-galactosides, while the others have a relatively lower affinity. These results indicate that the integrity of key amino acid residues responsible for recognition of lactose (Galbeta1-4Glc) or N-acetyllactosamine in vertebrate galectins is also required in C. elegans galectins. However, analysis of their fine oligosaccharide-binding properties by frontal affinity chromatography suggests their divergence towards more specialized functions.

Podocalyxin expression in malignant astrocytic tumors

Podocalyxin is an anti-adhesive mucin-like transmembrane sialoglycoprotein that has been implicated in the development of aggressive forms of cancer. Podocalyxin is also known as keratan sulfate (KS) proteoglycan. Recently, we revealed that highly sulfated KS or another mucin-like transmembrane sialoglycoprotein podoplanin/aggrus is upregulated in malignant astrocytic tumors. The aim of this study is to examine the relationship between podocalyxin expression and malignant progression of astrocytic tumors. In this study, 51 astrocytic tumors were investigated for podocalyxin expression using immunohistochemistry, Western blot analysis, and quantitative real-time PCR. Immunohistochemistry detected podocalyxin on the surface of tumor cells in six of 14 anaplastic astrocytomas (42.9%) and in 17 of 31 glioblastomas (54.8%), especially around proliferating endothelial cells. In diffuse astrocytoma, podocalyxin expression was observed only in vascular endothelial cells. Podocalyxin might be associated with the malignant progression of astrocytic tumors, and be a useful prognostic marker for astrocytic tumors.

Expression of highly sulfated keratan sulfate synthesized in human glioblastoma cells.

Keratan sulfate (KS) proteoglycan is expressed in the extracellular matrix or cell surface in numerous tissues, predominantly in those of the cornea, cartilage, and brain. However, its structure, function, and regulation remain poorly understood. Our investigation of KS expression in glioblastoma cell lines using Western-blot and flow cytometry with anti-KS antibody (5D4) revealed that LN229 glioblastoma cell highly expresses KS on a cell surface. Real-time PCR analysis showed that LN229 expresses a high level of keratan sulfate Gal-6-sulfotransferase. Results of this study also demonstrate that recombinant 5D4-reactive aggrecan is produced in LN229. Taken together, these results suggest that LN229 produces 5D4-reactive highly sulfated KS and is useful to investigate the KS structure and function in glioblastoma.

Increased expression of highly sulfated keratan sulfate synthesized in malignant astrocytic tumors

Keratan sulfate (KS) proteoglycans are expressed on a subpopulation of microglia in normal adult brain. We previously showed the up-regulated expression of KS in one of glioblastoma cell lines using anti-KS antibody (5D4). However, it has not been clarified whether KS is expressed in brain tumors and is involved in their malignancy. In this study, 54 astrocytic tumors were investigated about KS-expression using Western-blot with 5D4. In six of 14 anaplastic astrocytomas (43%) and 23 of 34 glioblastomas (68%), KS was detected by 5D4. KS was hardly detected by 5D4 in diffuse astrocytoma, suggesting that KS-expression is significantly expressed in malignant astrocytic tumors. In immunohistochemistry, KS is highly expressed in cell surface of malignant astrocytic tumors. Taken together, KS might be associated with the malignancy of astrocytic tumors, and be useful for a prognostic factor of astrocytic tumors.

Neutralization of Blood Group A-Antigen by a Novel Anti-A Antibody: Overcoming ABO-Incompatible Solid-Organ Transplantation

Background. The major barrier to ABO-incompatible solid-organ transplantation is acute humoral rejection. It is known to be triggered by antidonor blood group A/B antibodies, which might bind to A/B-antigen on the endothelium of the graft. Various strategies to reduce antiblood group antibody by overcoming ABO-incompatible transplantation have been tried. However, antigen-suppressing procedures have not been performed. Methods. We produced a novel anti-A antibody (K7508) by immunizing mice with salivary mucin of a blood group A individual, thereby clarifying that blood group A-antigen is expressed in endothelial cells of the liver. We investigated whether K7508 can mask A-antigen on the cells in vitro. Next, we immunized mice with A-antigen-expressing cells coated with K7508 or its Fab fragment, and measured anti-A antibody production in the mice. Results. Blood group A-antigen-expressing cells, such as blood group A-red blood cells (A-RBCs) and A431 cells, coated with K7508 were not recognized by another anti-A antibody in flow cytometry, indicating that A-antigen was masked by K7508 in vitro. The A-antigen on the paraffin-embedded liver tissue was also masked by K7508. Furthermore, the production of anti-A antibody in mice immunized with A-antigen-expressing cells coated with K7508 or its Fab fragment was significantly suppressed compared to that in mice immunized with non-coated cells alone, indicating that A-antigen was neutralized by K7508 in vivo. Conclusions. The neutralization of blood group antigen by antiblood group antibody and especially its Fab fragment might represent one strategy to overcome ABO-incompatible organ transplantation.