Inhibition of tumor cell-induced platelet aggregation using a novel anti-podoplanin antibody reacting with its platelet-aggregation-stimulating domain

Kato, Yoshinori; Kaneko, Mika K.; Kuno, Atsushi; Uchiyama, Noboru; Amano, Koh; Chiba, Yasunori; Hasegawa, Yasushi; Hirabayashi, Jun; Narimatsu, Hisashi; Mishima, Kazuhiko; Osawa, Motoki

doi:10.1016/j.bbrc.2006.08.171

Cited by 213 publications

(217 citation statements)

References 22 publications

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“…To purify hPod/LN319, the cell lysate was applied on NZ-1 antibody-immobilized resin. After washing the gel, the captured hPod/CHO was eluted with hpp38 -51 peptide (EGGVAMPGAEDDVV) (31).…”

Section: Figmentioning

confidence: 99%

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

Kuno

Kato

Matsuda

et al. 2009

Molecular & Cellular Proteomics

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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 highthroughput manner. Thus, the established system should provide a powerful pipeline in support of ongoing efforts in glyco-biomarker discovery. Molecular & Cellular Proteomics 8:99 -108, 2009.

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“…To purify hPod/LN319, the cell lysate was applied on NZ-1 antibody-immobilized resin. After washing the gel, the captured hPod/CHO was eluted with hpp38 -51 peptide (EGGVAMPGAEDDVV) (31).…”

Section: Figmentioning

confidence: 99%

“…Platelet Aggregation Assay by WBA Carna-Platelet aggregating activity was measured by WBA Carna (IMI, Saitama, Japan) as described previously (31). Heparinized mouse whole blood was drawn from BALB/c mice.…”

Section: Figmentioning

confidence: 99%

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

Kuno

Kato

Matsuda

et al. 2009

Molecular & Cellular Proteomics

Self Cite

106

140

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“…Thus, we used TN-1 and T-1 cells for production of hPod and TN-3 and T-3 cells for controls. The yeast strains were cultivated in YPAD medium for 3 d at 20°C, and production of hPod was confirmed by immunoblotting with an anti-hPod antibody (NZ-1) (14). The hPods produced in each strain had different electrophoretic mobility, presumably due to the addition of different mucin-type glycans (Fig.…”

Section: Production Of Mucin-type Glycosylated Human Podoplanin In Yementioning

confidence: 99%

“…In fact, the anti-human podoplanin antibody NZ-1 has an inhibitory effect on lung colonization of human podoplanintransfected cells (13). Additionally, podoplanin is a potential diagnostic marker for many tumors, including testicular tumors, several squamous cell carcinomas, and brain tumors, and may be associated with malignancy (14,15).Mucins and mucin-like glycoproteins have the potential to be novel cancer markers and thus a new technology for production of a large amount of mucin-like glycoprotein for induction of specific antibodies could prove very useful. Production of a large amount of recombinant mucin-like glycoprotein may also help researchers to analyze the relationship between mucin-type glycans and their functions.…”

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Engineering of mucin-type human glycoproteins in yeast cells

Amano

Chiba

Kasahara

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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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 mucintype sugar chains. Genes encoding Bacillus subtilis UDP-Gal/GalNAc 4-epimerase, human UDP-Gal/GalNAc transporter, human ppGalNAc-T1, and Drosophila melanogaster core1 ␤1-3 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.glycosylation engineering ͉ mucin-type glycan ͉ podoplanin M ucin-type glycosylation is one of the most abundant posttranslational modifications. The modification is initiated by O-linked N-acetylgalactosamine (GalNAc) to Ser or Thr residues on a peptide backbone and is involved in a variety of important biological processes, such as processing of hormones (1), endocytosis (2), and sorting of apical proteins in the Drosophila embryo (3). Mucin-type glycans are sometimes clustered, forming the ''mucin domain'' found on both membrane-bound (4) and secreted mucins (5), which function as a selective molecular barrier at the epithelial surface (6) and are involved in morphogenetic signal transduction (7). It is also known that changes in expression of mucin and in their glycosylation state are closely associated with the development of cancer and cancer-related processes such as cell growth, differentiation, adhesion, invasion, and immune surveillance (8). Immunohistochemical studies have identified several tumor-associated antigens (TAAs) of adenocarcinoma (9), and most TAAs on mucins were originally found as sialylated mucin-type glycans (10). Podoplanin (also called aggrus) (11, 12), one of mucin-type glycoproteins, acts as a platelet-aggregating factor for cancer cells, a finding that has attracted recent attention because it is well known that the platelet-aggregating activity of cancer cells influences tumor metastasis. In fact, the anti-human podoplanin antibody NZ-1 has an inhibitory effect on lung colonization of human podoplanintransfected cells (13). Additionally, podoplanin is a potential diagnostic marker for many tumors, including testicular tumors, several squamous cell carcinomas, and brain tumors, and may be ...

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An in vivo protease activity assay for investigating the functions of the Escherichia coli membrane protease HtpX

2019

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Escherichia coli HtpX is an M48 family zinc metalloproteinase located in the cytoplasmic membrane. Previous studies suggested that it is involved in the quality control of membrane proteins. However, its in vivo proteolytic function has not been characterized in detail, mainly because the physiological substrates have not been identified and no model substrate that allows sensitive detection of the protease activity is available. We constructed a new model substrate of HtpX and established an in vivo semiquantitative and convenient protease activity assay system for HtpX. This system enables detection of differential protease activities of HtpX mutants carrying mutations in conserved regions. This system would also be useful for investigating the functions of HtpX and its homologs in other bacteria.

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Inhibition of tumor cell-induced platelet aggregation using a novel anti-podoplanin antibody reacting with its platelet-aggregation-stimulating domain

Cited by 213 publications

References 22 publications

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

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

Engineering of mucin-type human glycoproteins in yeast cells

An in vivo protease activity assay for investigating the functions of the Escherichia coli membrane protease HtpX

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