Molecular cloning and function analysis of the head and neck cancer cell-derived alpha-N-acetylgalactosaminidase

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【Aims】 The aim of this study was to clone a human salivary gland adenocarcinoma cell-derived α-N-acetylgalactosaminidase (α-NaGalase) gene and to analyze its function.【Methods】 1. A human salivary gland adenocarcinoma cell-derived α-N-acetylgalactosaminidase was purified from HSG cell line. 2. Anti-α-NaGalase antibody was developed in rabbit using a human salivary gland adenocarcinoma cell-derived α-N-acetylgalactosaminidase as an antigen. 3. A head and neck cancer-derived squamous cell carcinoma cell line, HeLa was cultured, and then total RNA was purified with the use of an RNA Isolation System following the manufacturer's instructions. The cDNA synthesis was performed with the use of an oligo dT primer and Reverse transcriptase. The synthesized cDNAs were then used for construction of UniZAP XR vector. 4. Positive clone was screened using Anti-α-NaGalase antibody and ECL method.【Results】 A protein from HSG was purified by ion exchange and gel filtration chromatography using exo-α-Na … More Galase 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 well 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. This clone had DEVD box sequence and nuclear transition signal suggesting that a human salivary gland adenocarcinoma cell-derived α-N-acetylgalactosaminidase retains a distinctive feature of RNA helicase.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