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

[目的]克隆人涎腺腺癌细胞来源的α-N-乙酰半乳糖苷酶(α-NaGalase)基因，并对其功能进行分析。2.以人涎腺腺癌细胞来源的α-N-乙酰半乳糖苷酶为抗原，制备兔抗α-NaGalase抗体。3.培养人头颈部鳞状细胞癌细胞系HeLa，按照生产厂家的说明书使用RNA分离系统纯化总RNA。用寡核苷酸脱氧核糖核酸(DT)引物和逆转录酶进行DNA合成。然后将合成的cDNA用于构建UniZAP XR载体。4.用抗α-NaGalase抗体和ECL法筛选阳性克隆。[结果]经离子交换和凝胶过滤层析，外源α-Na…纯化得到一种蛋白。更多的半乳糖酶活性测定作为分级的指标。纯化的α-NaGalase经十二烷基硫酸钠-PAGE鉴定为均一，其相对分子质量为48 kDa。对纯化酶的性质研究表明，该酶能降解合成底物，内源性α-NaGalase、α-半乳糖苷酶和外源性α-NaGalase活性较低。在超氧化物产生实验中，单核/巨噬细胞与HSG来源的α-NaGalase处理的GcMAF孵育，与GcMAF孵育相比，其产生超氧阴离子的能力降低。GcMAF能提高单核/巨噬细胞的吞噬活性，但不能提高HSG来源的α-NaGalase处理的GcMAF的吞噬活性。花生凝集素可识别半乳糖/N-乙酰半乳糖胺残基，对GC蛋白的体外脱糖作用表明，α-NaGalase同时具有内外酶活性和促进唾液酸酶活性。该克隆具有DEVDbox序列和核转换信号，表明人涎腺腺癌细胞来源的α-N-乙酰氨基半乳糖苷酶保留了α螺旋酶的独特特征。这些结果表明，与正常细胞的构成酶相比，该酶具有独特的酶学性质，是涎腺癌患者GcMAF相关免疫级联反应中的候选细胞免疫缺陷因子。本研究是首次鉴定恶性细胞来源的内切α-N-乙酰半乳糖苷酶，它影响免疫相关生物大分子GcMAF的生物活性。较少

【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