Holmes E H, Greene T G, Tino W T, Boynton A L, Aldape H C, Misrock S L, Murphy G P
Pacific Northwest Cancer Foundation, Department of Cell Surface Biochemistry, Seattle, Washington, USA.
Prostate Suppl. 1996;7:25-9.
Prostate-specific membrane antigen (PSMA) has been detected in human prostatic cancer tissues, serum, and seminal fluid based on Western blot data with the monoclonal antibody 7E11.C5. The reactive protein is very similar in size to that from human prostatic carcinoma LNCaP cells and corresponds to a protein with a molecular size of about 110,000 daltons. Given that PSMA is known to be a 750 amino acid protein of about 84,000 daltons, a substantial portion, perhaps 20-25% of the native molecular weight, is composed of carbohydrates.
In this study, we have begun initial analyses of the glycosylation of the PSMA protein from multiple sources using a variety of exo- and endoglycosidase treatments.
The results indicate that the carbohydrate is primarily N-linked and in each case the deglycosylated protein has an apparent molecular weight of about 86,000 daltons. The glycan present on in vivo-derived PSMA from tumor tissue or serum was found to be primarily N-linked complex type. A small amount of O-linked glycan also appears to be present. In contrast, only high mannose-type N-linked glycans are present on the PSMA from LNCaP cells.
Oligosaccharides present on PSMA derived from both tissue culture LNCaP cells and in vivo specimens are primarily N-linked and comprise about 20-25% of the native molecular weight. N-linked glycans of PSMA derived from in vivo sources were found to be complex type, lacking polylactosamine structures. In contrast, LNCaP cells express only high mannose-type structures. These results will be useful in our ongoing efforts to develop monoclonal antibodies which are specific for protein epitopes present in the extracellular domain of the protein.
基于使用单克隆抗体7E11.C5的蛋白质印迹数据，在人前列腺癌组织、血清和精液中检测到前列腺特异性膜抗原（PSMA）。反应性蛋白的大小与人前列腺癌LNCaP细胞中的蛋白非常相似，对应于一种分子量约为110,000道尔顿的蛋白质。鉴于已知PSMA是一种约84,000道尔顿的750个氨基酸的蛋白质，相当一部分，可能占天然分子量的20 - 25%，由碳水化合物组成。
在本研究中，我们开始使用多种外切糖苷酶和内切糖苷酶处理对来自多个来源的PSMA蛋白的糖基化进行初步分析。
结果表明，碳水化合物主要是N - 连接的，并且在每种情况下，去糖基化蛋白的表观分子量约为86,000道尔顿。在肿瘤组织或血清中体内来源的PSMA上存在的聚糖主要是N - 连接的复合型。也似乎存在少量O - 连接的聚糖。相比之下，LNCaP细胞的PSMA上仅存在高甘露糖型N - 连接聚糖。
来自组织培养LNCaP细胞和体内标本的PSMA上存在的寡糖主要是N - 连接的，约占天然分子量的20 - 25%。发现来自体内来源的PSMA的N - 连接聚糖是复合型的，缺乏多乳糖胺结构。相比之下，LNCaP细胞仅表达高甘露糖型结构。这些结果将有助于我们正在进行的开发针对该蛋白细胞外结构域中存在的蛋白质表位的单克隆抗体的工作。
