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Identification of mesothelioma-specific sialylated epitope recognized with monoclonal antibody SKM9-2 in a mucin-like membrane protein HEG1.

ABSTRACT: The anti-mesothelioma mAb SKM9-2 recognizes the sialylated protein HEG homolog 1 (HEG1). HEG1 is a 400?kDa mucin-like membrane protein found on mesothelioma. SKM9-2 can detect mesothelioma more specifically and sensitively than other antibodies against current mesothelioma markers; therefore, SKM9-2 would be likely useful for the precise detection and diagnosis of malignant mesothelioma. In the present study, we investigated the epitope of SKM9-2. We analyzed the binding of SKM9-2 to truncated HEG1 and candidate epitope-fused glycosylphosphatidylinositol-anchor proteins. The epitope of SKM9-2 was identified as an O-glycosylated region, 893-SKSPSLVSLPT-903, in HEG1. An alanine scanning assay of the epitope showed that SKM9-2 bound to a simple epitope in HEG1, and the SKxPSxVS sequence within the epitope was essential for SKM9-2 recognition. Mass spectrometry analysis and lectin binding analysis of soluble epitope peptides indicated that the SKM9-2 epitope, in which Ser⁸⁹⁷ was not glycosylated, contained two disialylated core 1 O-linked glycan-modified serine residues, Ser⁸⁹³ and Ser⁹⁰⁰. Neuraminidase treatment analysis also confirmed that the epitope in mesothelioma cells contained a similar glycan modification. The specific detection of mesothelioma with SKM9-2 can thus be performed by the recognition of sialylated glycan modification in the specific region of HEG1.

SUBMITTER: Matsuura R

PROVIDER: S-EPMC6155162 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Identification of mesothelioma-specific sialylated epitope recognized with monoclonal antibody SKM9-2 in a mucin-like membrane protein HEG1.

Matsuura Rieko R Kaji Hiroyuki H Tomioka Azusa A Sato Takashi T Narimatsu Hisashi H Moriwaki Yasuhiro Y Misawa Hidemi H Imai Kohzoh K Tsuji Shoutaro S

Scientific reports 20180924 1

The anti-mesothelioma mAb SKM9-2 recognizes the sialylated protein HEG homolog 1 (HEG1). HEG1 is a 400 kDa mucin-like membrane protein found on mesothelioma. SKM9-2 can detect mesothelioma more specifically and sensitively than other antibodies against current mesothelioma markers; therefore, SKM9-2 would be likely useful for the precise detection and diagnosis of malignant mesothelioma. In the present study, we investigated the epitope of SKM9-2. We analyzed the binding of SKM9-2 to truncated H ...[more]

PMID: 30250045

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Project description:The 12E7 murine monoclonal antibody recognizes a protease-sensitive component of human red cells, platelets and lymphocytes which could not be detected on granulocytes. Scatchard analyses indicated that the 125I-labelled antibody binds to 1000, 4000 and 27,000 antigen sites on each red cell, platelet and lymphocyte respectively, with a binding constant ranging from 4 x 10(7) to 9 x 10(7) M-1. The membrane components recognized by the monoclonal antibody were characterized by immunostaining on nitrocellulose sheets. A 28 kDa sialoglycoprotein was visualized following electrophoretic transfer of the red cell and lymphocyte membrane proteins separated by SDS/polyacrylamide-gel electrophoresis. Another component of 25 kDa was also clearly identified in the lymphocyte and platelet lysates, but was barely detectable in the red cell membrane preparations. Enzyme treatment of intact platelets, as well as analysis of the membrane and cytosolic preparations from these cells, have shown that the 25 kDa component was of cytoplasmic origin. The mobility of the 28 kDa membrane component is decreased following neuraminidase treatment of intact blood cells, but these cells still react normally with the monoclonal antibody, indicating that sialic acids are not required for binding. The 28 kDa component is present on red cell membranes prepared from S-s-U-, En(a-) and Gerbich(-) individuals, demonstrating that it is a new sialoglycoprotein not derived from glycophorins A, B, C or D. The 28 kDa component was totally solubilized with 0.1% Triton X-100 from red cell membranes and behaves like the other red cell membrane sialoglycoproteins since it was extracted in the aqueous phase following chloroform/methanol/water or butanol/water partitionings. The 28 kDa component could be partially purified by h.p.l.c. gel permeation chromatography and preparative SDS/polyacrylamide-gel electrophoresis. The material finally obtained strongly inhibits the 12E7 monoclonal as well as human anti-Xga antibodies, suggesting either that the 28 kDa glycoprotein carries both antigens or that the 12E7 and Xga-active molecules copurified.

Dataset Information

Identification of mesothelioma-specific sialylated epitope recognized with monoclonal antibody SKM9-2 in a mucin-like membrane protein HEG1.

Publications

Identification of mesothelioma-specific sialylated epitope recognized with monoclonal antibody SKM9-2 in a mucin-like membrane protein HEG1.

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