-
[show abstract]
[hide abstract]
ABSTRACT: Isocitrate dehydrogenase 1 (IDH1) catalyzes the oxidative carboxylation of isocitrate to α-ketoglutarate in cytosol. IDH1 mutations, which are specific to a single codon in the conserved and functionally important Arginine 132 (R132), result in the ability of the enzyme to catalyze the reduced NADP-dependent reduction of α-ketoglutarate to onco-metabolite R(-)-2-hydroxyglutarate (2-HG). IDH1 mutations, which are early and frequent genetic alterations that occur in gliomas, cartilaginous tumors, and leukemias. We previously established two monoclonal antibodies (MAbs) that are specific for IDH1 mutations: clone HMab-1 against IDH1-R132H and clone SMab-1 against IDH1-R132S. However, specific MAbs against IDH1-R132G or IDH1-R132L have not been reported. To establish IDH1-R132G-specific or IDH1-R132L-specific MAbs, we immunized rats with each mutation-containing IDH1 peptides, and IDH1-R132G-specific or IDH1-R132L-specific MAbs were screened in ELISA. Established MAb GMab-r1 reacted with the IDH1-R132G peptide, but not with IDH1-wild type (WT) in ELISA. In contrast, LMab-1 reacted with the IDH1-R132L peptide, but not with IDH1-WT. Western blot analysis also showed that GMab-r1 and LMab-1 reacted with the IDH1-R132G and IDH1-R132L recombinant proteins, respectively, but not with IDH1-WT or other IDH1 mutants, indicating that GMab-r1 and LMab-1 are IDH1-mutation-specific. Furthermore, GMab-r1 and LMab-1 specifically stained the IDH1-R132G- and IDH1-R132L-expressing cells in immunocytochemistry, respectively. This is the first report to establish anti-IDH1-R132G-specific or IDH1-R132L-specific MAbs, which could be useful in the diagnosis of mutation-bearing tumors.
Monoclonal antibodies in immunodiagnosis and immunotherapy. 06/2013; 32(3):224-8.
-
[show abstract]
[hide abstract]
ABSTRACT: Isocitrate dehydrogenase 2 (IDH2) mutations have been detected in gliomas, cartilaginous tumors, and acute myeloid leukemias. IDH2 mutations are specific to a single codon in the conserved and functionally important Arginine 172 (R172) or Arginine 140 (R140). To date, we have established specific monoclonal antibodies (mAbs) against IDH2-R172K and IDH2-R172M. However, specific mAbs against IDH2-R172W have not been reported. To establish IDH2-R172W-specific mAbs, we immunized rats with IDH2-R172W peptides. Western-blot analysis showed that WMab-1 reacted with the IDH2-R172W recombinant protein, not with IDH2-wild type (WT) or other IDH2 mutants, indicating that WMab-1 is IDH2-R172W-specific. Furthermore, WMab-1 specifically stained the IDH2-R172W-expressing cells in immunocytochemistry, but did not stain IDH2-WT and IDH2-R172M-containing cells. WMab-1 also specifically stained the IDH2-R172W-expressing glioma cells in immunohistochemistry. This is the first report to establish an anti-IDH2-R172W-specific mAb, which could be useful in diagnosis of IDH2-R172W-bearing tumors.
Biochemical and Biophysical Research Communications 03/2013; · 2.48 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Isocitrate dehydrogenase 1 (IDH1) mutations occur in gliomas, acute myeloid leukemias, and cartilaginous tumors. While IDH1 catalyzes the oxidative carboxylation of isocitrate to α-ketoglutarate in cytosol, mutated IDH1 proteins possess the ability to change α -ketoglutarate into onco-metabolite R(-)-2-hydroxyglutarate (2-HG). To data, two monoclonal antibodies (mAbs), which are specific for IDH1 mutations have been established: clone HMab-1 against IDH1-R132H and clone SMab-1 against IDH1-R132S. However, specific mAbs against IDH1-R132G, which are useful for immunohistochemical analysis, have not been reported. To establish IDH1-R132G-specific mAbs, we immunized mice with IDH1-R132G-containing peptides. Established mAb GMab-m1 reacted with the IDH1-R132G peptide, but not with IDH1-wild type (WT) in ELISA. Western-blot analysis also showed that GMab-m1 reacted with the IDH1-R132G recombinant proteins, not with IDH1-WT or other IDH1 mutants, indicating that GMab-m1 is IDH1-R132G-specific. Furthermore, GMab-m1 specifically stained the IDH1-R132G-expressing glioma cells in immunohistochemistry. This is the first report to establish anti-IDH1-R132G-specific mAbs, which is useful in immunohistochemistry of IDH1-R132G-bearing tumors.
Biochemical and Biophysical Research Communications 02/2013; · 2.48 Impact Factor
-
Mika Kato Kaneko,
Shunpei Morita,
Yuta Tsujimoto,
Ryo Yanagiya,
Kana Nasu,
Hiroko Sasaki,
Yasukazu Hozumi,
Kaoru Goto,
Atsushi Natsume,
Mika Watanabe,
Toshihiro Kumabe,
Shingo Takano,
Yukinari Kato
[show abstract]
[hide abstract]
ABSTRACT: Isocitrate dehydrogenase 1/2 (IDH1/2) mutations have been detected in gliomas, cartilaginous tumors, and leukemias. IDH1/2 mutations are early and frequent genetic alterations, are specific to a single codon in the conserved and functionally important Arginine 132 (R132) in IDH1 and Arginine 172 (R172) in IDH2. We previously established several monoclonal antibodies (mAbs), which are specific for IDH1 mutations: clones IMab-1 or HMab-1 against IDH1-R132H or clone SMab-1 against IDH1-R132S. However, specific mAbs against IDH2 mutations have not been reported. To establish IDH2-mutation-specific mAbs, we immunized mice or rats with each mutation-containing IDH2 peptides including IDH2-R172K and IDH2-R172M. After cell fusion, IDH2 mutation-specific mAbs were screened in Enzyme-Linked Immunosorbent Assay (ELISA). Established mAbs KMab-1 and MMab-1 reacted with the IDH2-R172K and IDH2-R172M peptides, respectively, but not with IDH2-wild type (WT) in ELISA. Western-blot analysis also showed that KMab-1 and MMab-1 reacted with the IDH2-R172K and IDH2-R172M recombinant proteins, respectively, not with IDH2-WT or other IDH2 mutants, indicating that KMab-1 and MMab-1 are IDH2-mutation-specific. Furthermore, MMab-1 specifically stained the IDH2-R172M-expressing cells in immunocytochemistry, but did not stain IDH2-WT and other IDH2-mutation-containing cells. In immunohistochemical analysis, MMab-1 specifically stained IDH2-R172M-expressing glioma. This is the first report to establish anti-IDH2-mutation-specific mAbs, which could be useful in diagnosis of mutation-bearing tumors.
Biochemical and Biophysical Research Communications 01/2013; · 2.48 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Current studydemonstrates the glioma tumor antigenpodoplanin to be present at very high levels (>90%) in both glioblastoma (D2159MG, D08-0308MG, and D08-0493MG) and medulloblastoma (D283MED, D425MED, and DAOY) xenografts and cell line. We constructed a novel recombinant single-chain antibody variable region fragment (scFv), NZ-1, specific for podoplanin from the NZ-1 hybridoma. NZ-1-scFv was then fused to Pseudomonas exotoxin A, carrying a C-terminal KDEL peptide (NZ-1-PE38KDEL). The immunotoxin was further stabilized by a disulfide (ds) bond between the heavy-chain and light-chain variable regions as the construct NZ-1-(scdsFv)-PE38KDEL. NZ-1-(scdsFv)-PE38KDEL exhibited significant reactivity to glioblastoma and medulloblastoma cells.The affinity of NZ-1-(scdsFv), NZ-1-(scdsFv)-PE38KDEL and NZ-1 antibody, for podoplanin peptide was 2.1×10(-8) M, 8.0×10(-8) M, and 3.9×10(-10) M, respectively.In a protein stability assay,NZ-1-(scdsFv)-PE38KDELretained33-98% of its activity while that of NZ-1-PE38KDEL declined to 13% of its initial levels after incubation at 37°C for 3 days.In vitro cytotoxicity of the NZ-1-(scdsFv)-PE38KDEL was measured in cells isolated from glioblastoma xenografts, D2159MG, D08-0308MG, D08-0493MG, and in the medulloblastoma D283MED, D425MED, and DOAY xenografts and cell line. The NZ-1-(scdsFv)-PE38KDEL immunotoxin was highly cytotoxic, with an IC(50) in the range of 1.6-29 ng/mL.Significantly, NZ-1-(scdsFv)-PE38KDEL demonstrated tumor-growth delay, averaging 24 days (P<0.001) and 21 days(P<0.001) in D2159MG and D283MEDin vivo tumor models, respectively. Crucially, in the D425MED intracranial tumor model, NZ-1-(scdsFv)-PE38KDEL caused a 41% increase in survival (P≤0.001). In preclinical studies, NZ-1-(scdsFv)-PE38KDEL exhibitedsignificant potential as a targeting agent for malignant brain tumors. © 2012 Wiley Periodicals, Inc.
International Journal of Cancer 11/2012; · 5.44 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: This study aims to develop new monoclonal antibodies (mAbs) against mouse and human podoplanin. Rats were immunized with synthetic peptides, corresponding to amino acids 38-51 of mouse podoplanin or human podoplanin which is 100% homologous to the same site of monkey podoplanin; anti-mouse podoplanin mAb PMab-1 (IgG(2) (a)) and anti-human mAb NZ-1.2 (IgG(2) (a)) were established. In immunocytochemistry, the mouse melanoma B16-F10 and mouse podoplanin (mPDPN)-expressed CHO transfectant were stained by PMab-1; human lymphatic endothelial cells (LEC) and human podoplanin (hPDPN)-expressed squamous cell carcinoma HSC3 transfectant, were stained by NZ-1.2. Western-blot analysis detected an about 40-kDa protein in CHO-mPDPN and B16-F10 by PMab-1, and in HSC3-hPDPN and LEC by NZ-1.2. In frozen sections, PMab-1 reacted with mouse kidney, pulmonary alveoli, pulmonary pleura, and salivary gland myoepithelial cells while NZ-1.2 reacted to the human salivary gland myoepithelial cells. The immunostaining of paraffin-embedded sections also showed the reaction of PMab-1 or NZ-1.2 to the mouse or monkey kidney glomerulus, pulmonary alveoli, and lung lymphatic vessels. These results indicate that the two novel rat mAbs to the mouse and human/monkey podoplanin are useful for Western-blot and immunostaining of somatic tissues on paraffin-embedded sections as well as frozen sections.
ACTA HISTOCHEMICA ET CYTOCHEMICA 08/2012; 45(4):227-37. · 1.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Podoplanin is a platelet aggregation-inducing factor associated with tumor metastasis, malignant progression, and cancer stem cells. We produced a rat-human chimeric anti-podoplanin mAb, NZ-8, from rat anti-podoplanin mAb (NZ-1). Although both NZ-1 and NZ-8 possess high binding affinities and high neutralizing activities of platelet aggregation, the antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity of NZ-8 were much higher than NZ-1. Furthermore, both NZ-1 and NZ-8 inhibited the growth of podoplanin-expressing tumors in vivo. Both NZ-1 and NZ-8 also suppressed hematogenous metastasis of podoplanin-expressing tumors. These results suggest that anti-podoplanin mAbs suppressed hematogenous metastasis by both neutralization and antibody-dependent cellular cytotoxicity/complement-dependent cytotoxicity activities. Targeting therapy to podoplanin-expressing tumors should be useful as a novel immunotherapy.
Cancer Science 07/2012; · 3.33 Impact Factor
-
Shingo Takano,
Yukinari Kato,
Tetsuya Yamamoto, Mika Kato Kaneko,
Eiichi Ishikawa,
Yuta Tsujimoto,
Masahide Matsuda,
Kei Nakai,
Ryo Yanagiya,
Shunpei Morita,
Koji Tsuboi,
Akira Matsumura
[show abstract]
[hide abstract]
ABSTRACT: Isocitrate dehydrogenase 1 (IDH1) mutations, which are early and frequent genetic alterations in astrocytomas, oligodendrogliomas, oligoastrocytomas, and secondary glioblastomas, are specific to arginine 132 (R132). Recently, we established monoclonal antibodies (mAbs) against IDH1 mutations: anti-IDH1-R132H and anti-IDH1-R132S. However, the importance of immunohistochemistry using the combination of those mAbs has not been elucidated. For this study, 164 cases of glioma were evaluated immunohistochemically for IDH1 mutations (R132H and R132S) using anti-IDH1 mAbs (HMab-1 and SMab-1). IDH1 mutation was detected, respectively, in 9.7%, 63.6%, 51.7%, and 77.8% of primary grade IV, secondary grade IV, grade III, and grade II gliomas. For each grade of glioma, prognostic factors for progression-free survival and overall survival were evaluated using clinical and pathological parameters in addition to IDH1 immunohistochemistry. IDH1 mutation, p53 overexpression, and internexin expression, as evaluated using immunohistochemistry with clinical parameters such as degree of surgical removal and preoperative Karnofsky Performance Status (KPS), might be of greater prognostic significance than histological grading alone in grade III as well as IDH1 mutation in grade IV gliomas.
Journal of Neuro-Oncology 03/2012; 108(3):361-73. · 3.21 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Thrombus formation on disrupted atherosclerotic lesion is a key mechanism of cardiovascular events. Podoplanin (Aggrus), expressed on the surface of several tumor cells, is an endogenous ligand for C-type lectin-like receptor 2 (CLEC-2), and is involved in tumor cell-induced platelet aggregation and its malignant potency. Podoplanin, which is also expressed in lymphatic endothelial cells, facilitates blood/lymphatic vessel separation. However, podoplanin expression in atherosclerotic lesion has not been investigated. To clarify podoplanin expression in atherosclerotic lesion and to assess its importance for the onset of cardiovascular events, we examined podoplanin expression in abdominal aortas obtained from 31 autopsy cases. Immunohistochemical analysis indicated that podoplanin was localized to smooth muscle cells and macrophages. Moreover, podoplanin immunoreactivity was increased in advanced atherosclerotic lesions containing necrotic core, many macrophages and smooth muscle cells, compared with early lesions composed of smooth muscle cells and small numbers of macrophages. Furthermore, Western-blot and real time-PCR analyses showed that podoplanin expression was significantly enhanced in advanced atherosclerotic lesions, compared with early lesions. These results suggest that podoplanin contributes to thrombotic property of advanced stages of atherosclerosis and that it might be a novel molecular target for an anti-thrombus drug.
Thrombosis Research 01/2012; 129(4):e70-6. · 2.44 Impact Factor
-
Brain Tumor Pathology 04/2011; · 1.19 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Isocitrate dehydrogenase 1 (IDH1) mutations have recently been identified as early and frequent genetic alterations in astrocytomas, oligodendrogliomas, and oligoastrocytomas, as well as secondary glioblastomas, whereas primary glioblastomas very rarely contain IDH1 mutations. Furthermore, a specific monoclonal antibody, IMab-1, which recognizes IDH1-R132H-the most frequent IDH1 mutation-has been generated. IMab-1 has been reported to react with the IDH1-R132H protein, but not the wild-type IDH1 or the other IDH1 mutant proteins in Western-blot analysis. However, the importance of immunohistochemistry using IMab-1 has not yet been elucidated. In this study, we compared the findings from IMab-1 immunohistochemistry and direct DNA sequencing using 49 glioma samples. IMab-1 detected 12 out of 49 cases; however, only nine cases were found to be IDH1-R132H by direct DNA sequencing because of a small population of IDH1-R132H mutation-possessing tumor cells, indicating that IMab-1 immunohistochemistry is useful for detecting IDH1-R132H. We conducted immunohistochemical detection in 52 cases of grade III astrocytomas. The median time to progression (TTP) was significantly longer in the cases with the IDH1 mutation (86.7 months) compared to the cases without the IDH1 mutation (wild type, 10.4 months) (p < 0.01). In conclusion, the anti-IDH1-R132H-specific monoclonal antibody IMab-1 is very useful for detecting IDH1-R132H in immunohistochemistry, and predicting the time to progression in grade III anaplastic astrocytomas. Therefore, IMab-1 is likely to be useful for the diagnosis of mutation-bearing gliomas and for determining the treatment strategy of grade III gliomas.
Brain Tumor Pathology 02/2011; 28(2):115-23. · 1.19 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Isocitrate dehydrogenase 1 (IDH1) mutations, which are early and frequent genetic alterations in gliomas, are specific to a single codon in the conserved and functionally important Arginine 132 (R132) in IDH1. We earlier established a monoclonal antibody (mAb), IMab-1, which is specific for R132H-containing IDH1 (IDH1-R132H), the most frequent IDH1 mutation in gliomas. To establish IDH1-R132S-specific mAb, we immunized mice with R132S-containing IDH1 (IDH1-R132S) peptide. After cell fusion using Sendai virus envelope, IDH1-R132S-specific mAbs were screened in ELISA. One mAb, SMab-1, reacted with the IDH1-R132S peptide, but not with other IDH1 mutants. Western-blot analysis showed that SMab-1 reacted only with the IDH1-R132S protein, not with IDH1-WT protein or IDH1 mutants, indicating that SMab-1 is IDH1-R132S-specific. Furthermore, SMab-1 specifically stained the IDH1-R132S-expressing glioblastoma cells in immunocytochemistry and immunohistochemistry, but did not react with IDH1-WT or IDH1-R132H-containing glioblastoma cells. We newly established an anti-IDH1-R132S-specific mAb SMab-1 for use in diagnosis of mutation-bearing gliomas.
Biochemical and Biophysical Research Communications 02/2011; 406(4):608-13. · 2.48 Impact Factor
-
Yukinari Kato,
Ganesan Vaidyanathan, Mika Kato Kaneko,
Kazuhiko Mishima,
Nidhi Srivastava,
Vidyalakshmi Chandramohan,
Charles Pegram,
Stephen T Keir,
Chien-Tsun Kuan,
Darell D Bigner,
Michael R Zalutsky
[show abstract]
[hide abstract]
ABSTRACT: Podoplanin/aggrus is a mucin-like sialoglycoprotein that is highly expressed in malignant gliomas. Podoplanin has been reported to be a novel marker to enrich tumor-initiating cells, which are thought to resist conventional therapies and to be responsible for cancer relapse. The purpose of this study was to determine whether an anti-podoplanin antibody is suitable to target radionuclides to malignant gliomas.
The binding affinity of an anti-podoplanin antibody, NZ-1 (rat IgG(2a)), was determined by surface plasmon resonance and Scatchard analysis. NZ-1 was radioiodinated with (125)I using Iodogen [(125)I-NZ-1(Iodogen)] or N-succinimidyl 4-guanidinomethyl 3-[(131)I]iodobenzoate ([(131)I]SGMIB-NZ-1), and paired-label internalization assays of NZ-1 were performed. The tissue distribution of (125)I-NZ-1(Iodogen) and that of [(131)I]SGMIB-NZ-1 were then compared in athymic mice bearing glioblastoma xenografts.
The dissociation constant (K(D)) of NZ-1 was determined to be 1.2 × 10(-10) M by surface plasmon resonance and 9.8 × 10(-10) M for D397MG glioblastoma cells by Scatchard analysis. Paired-label internalization assays in LN319 glioblastoma cells indicated that [(131)I]SGMIB-NZ-1 resulted in higher intracellular retention of radioactivity (26.3 ± 0.8% of initially bound radioactivity at 8 h) compared to that from the (125)I-NZ-1(Iodogen) (10.0 ± 0.1% of initially bound radioactivity at 8 h). Likewise, tumor uptake of [(131)I]SGMIB-NZ-1 (39.9 ± 8.8 %ID/g at 24 h) in athymic mice bearing D2159MG xenografts in vivo was significantly higher than that of (125)I-NZ-1(Iodogen) (29.7 ± 6.1 %ID/g at 24 h).
The overall results suggest that an anti-podoplanin antibody NZ-1 warrants further evaluation for antibody-based therapy against glioblastoma.
Nuclear Medicine and Biology 10/2010; 37(7):785-94. · 3.02 Impact Factor
-
Yukinari Kato,
Chien-Tsun Kuan,
Jinli Chang, Mika Kato Kaneko,
Joanne Ayriss,
Hailan Piao,
Vidyalakshmi Chandramohan,
Charles Pegram,
Roger E McLendon,
Pam Fredman,
Jan-Eric Månsson,
Darell D Bigner
[show abstract]
[hide abstract]
ABSTRACT: The lacto-series gangliosides 3'-isoLM1 and 3',6'-isoLD1 have been identified as tumor-associated antigens whose formation is initiated by the Lc3-synthase. Until now, high-affinity IgG monoclonal antibodies (mAbs) against 3'-isoLM1 and 3',6'-isoLD1, which are highly expressed in gliomas, have not been developed, although mAbs against lacto-series gangliosides are powerful tools for functional studies. We previously produced the Lc3-synthase gene beta3Gn-T5 knockout mice. In this study, we immunized beta3Gn-T5 knockout mice with 3'-isoLM1/3',6'-isoLD1 and produced the anti-3'-isoLM1/3',6'-isoLD1 mAb GMab-1, of the IgG(3) subclass, which should be useful for functional analysis of lacto-series gangliosides and for antibody-based therapy of gliomas.
Biochemical and Biophysical Research Communications 11/2009; 391(1):750-5. · 2.48 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Biochemical and Biophysical Research Communications 09/2008; 374(2):394-8. · 2.48 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Molecular & Cellular Proteomics 09/2008; 8(1):99-108. · 7.40 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Podoplanin (Aggrus) is a mucin-type sialoglycoprotein that is known as a useful marker for lymphatic endothelium and tumor-initiating cells (TICs). Interaction between podoplanin and C-type lectin-like receptor-2 (CLEC-2) is reported to be critical for podoplanin-induced platelet aggregation and cancer metastasis. Recently, several anti-human podoplanin antibodies have been created; however, these anti-podoplanin antibodies have not been well characterized. Five anti-podoplanin antibodies (NZ-1, D2-40, AB3, 18H5, and a rabbit polyclonal antibody) were investigated using ELISA, Western blot, and flow cytometry with synthesized podoplanin peptides and deletion mutants of recombinant podoplanin. The epitope of NZ-1 is platelet aggregation-stimulating (PLAG) domain-2/3; the epitope of D2-40, AB3, and 18H5 is PLAG1/2. The epitopes of D2-40 and AB3 are quite similar, although 18H5 is different from D2-40 and AB3. Using flow cytometric analysis, NZ-1 partially inhibited the interaction between podoplanin and CLEC-2, although other antibodies did not. In conclusion, the two most frequently used anti-podoplanin antibodies, D2-40 and AB3, have similar properties, although several studies have reported differences. NZ-1 neutralizes the interaction between podoplanin and CLEC-2, which may lead to the development of therapeutic antibodies against podoplanin-dependent cancer metastasis.
Hybridoma (2005) 09/2008; 27(4):259-67. · 0.42 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Biochemical and Biophysical Research Communications 06/2008; 369(4):1041-6. · 2.48 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Biochemical and Biophysical Research Communications 05/2008; 368(2):217-22. · 2.48 Impact Factor
-
Koh Amano,
Yasunori Chiba,
Yoshiko Kasahara,
Yukinari Kato, Mika Kato Kaneko,
Atsushi Kuno,
Hiromi Ito,
Kazuo Kobayashi,
Jun Hirabayashi,
Yoshifumi Jigami,
Hisashi Narimatsu
[show abstract]
[hide abstract]
ABSTRACT: Mucin-type O-glycans are the most typical O-glycans found in mammalian cells and assume many different biological roles. Here, we report a genetic engineered yeast strain capable of producing mucin-type sugar chains. Genes encoding Bacillus subtilis UDP-Gal/GalNAc 4-epimerase, human UDP-Gal/GalNAc transporter, human ppGalNAc-T1, and Drosophila melanogaster core1 beta1-3 GalT were introduced into Saccharomyces cerevisiae. The engineered yeast was able to produce a MUC1a peptide containing O-glycan and also a mucin-like glycoprotein, human podoplanin (hPod; also known as aggrus), which is a platelet-aggregating factor that requires a sialyl-core1 structure for activity. After in vitro sialylation, hPod from yeast could induce platelet aggregation. Interestingly, substitution of ppGalNAc-T1 for ppGalNAc-T3 caused a loss of platelet aggregation-inducing activity, despite the fact that the sialyl-core1 was detectable in both hPod proteins on a lectin microarray. Most of O-mannosylation, a common modification in yeast, to MUC1a was suppressed by the addition of a rhodanine-3-acetic acid derivative in the culture medium. The yeast system we describe here is able to produce glycoproteins modified at different glycosylation sites and has the potential for use in basic research and pharmaceutical applications.
Proceedings of the National Academy of Sciences 04/2008; 105(9):3232-7. · 9.68 Impact Factor
