2007 Fiscal Year Final Research Report Summary
Gene cloning and functional analysis of α-N-acetyl galactosaminidase derived from salivary gland adenocarcinoma
| Project/Area Number |
17390547
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Surgical dentistry
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| Research Institution | Matsumoto Dental University |
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Principal Investigator |
UEMATSU Takashi Matsumoto Dental University, Graduae School of Oral Medicine Oral Science Course, Associate Professor (40203476)
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| Co-Investigator(Kenkyū-buntansha) |
FURUSAWA Kiyofumi Matsumoto Dental University, Graduate School of Oral Medicine Oral Science Course, Professor (90165481)
LI X Matsumoto Dental University, Institute for Oral Science, Assistant Professor (60350831)
TAKAHASHI Masahiro Matsumoto Dental University, Institute for Oral Science, Assistant Professor (90340059)
DOTO Ryosuke Matsumoto Dental University, Institute for Oral Science, Assistant Professor (40329470)
UCHIHASHI Takayuki Matsumoto Dental Uiversity, Institute for Oral Science, Assistant Professor (70397628)
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| Project Period (FY) |
2005 – 2007
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| Keywords | α-N-acetylgalactosaminidas / macrophage-activating factor / O-glycan / deglycosylation |
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| Research Abstract |
The aim of this study was to clarify whether human salivary gland adenocarcinoma cell-erived α-N-acetylgalactosaminidase (α-NaGalase) affects the bioactivity of an O-linked immune-related biomacromolecule, macrophage-activating factor (GcMAF). High levels of exo-α-NaGalase activity was detected in the cell extract and in the condition medium of the salivary gland cell adenocarcinoma cell line HSG when compared to that of the squamous cell carcinoma cell line, SCCTM. Normal keratinocytes and fibroblasts exhibited low exo-α-NaGalase activities. A protein from HSG was purified by ion exchange and gel filtration chromatography using exo-α-NaGalase activity assay as an indicator of fractionation. The homogeneity of the purified α-NaGalase was demonstrated by SDS-PAGE and its estimated molecular weight was 48 kDa. A characteristic study of the purified enzyme demonstrated that the enzyme hydrolyzed synthetic substrates and had low intrinsic endo-α-NaGalase and α-galactosidase activities as w
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ell as exo-α-NaGalase activity. In the superoxide generation assay, monocytes/macrophages were incubated with the HSG-derived α-NaGalase-treated GcMAF and exhibited a decreased superoxide-generation capacity when compared to those incubated with GcMAF. Phagocytic activity of monocytes/macrophages was increased by incubation with GcMAF, but not with HSG-derived α-NaGalase-treated GcMAF. The in vitro deglycosylation of Gc protein using peanut agglutinin lectin, which recognizes galactose/N-acetylgalactosamine residues, demonstrated that HSG-derived α-NaGalase has both exo-and endo-enzyme activities and facilitates sialidase activity.
These results demonstrate that HSG-derived α-NaGalase possesses unique enzymatic properties when compared to the constitutive enzyme of normal cells and is a candidate cellular immunodeficient factor in the GcMAF related-immune cascade for host defense in cancer patients with salivary gland adenocarcinomas. The Present study is the first report to identify the malignant cell-derived endo-α-N-acetylgalactosaminidase, which affects the bioactivity of an immune-related biomacromolecule, GcMAF. Less
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