Panicot L, Mas E, Pasqualini E, Zerfaoui M, Lombardo D, Sadoulet M O, El Battari A
INSERM U 260, Unité de Recherche de Physiopathologie des Régulations Hormono-Nutritionnelles, Faculté de Médecine-Timone, 27 Boulevard Jean Moulin, 13385 Marseilles-Cedex 5, France.
Glycobiology. 1999 Sep;9(9):935-46. doi: 10.1093/glycob/9.9.935.
The feto-acinar pancreatic protein or FAPP, the oncofetal glycoisoform of bile salt-dependent lipase (BSDL), is characterized by the presence of the J28 glycotope recognized by mAbJ28. This fucosylated epitope is carried out by the O-linked glycans of the C-terminal mucin-like region of BSDL. This glycotope is expressed by human tumoral pancreatic tissues and by human pancreatic tumoral cell lines such as SOJ-6 and BxPC-3 cells. However, it is not expressed by the normal human pancreatic tissues and by MiaPaCa-2 and Panc-1 cells. Due to the presence of many putative sites for O-glycosylation on FAPP and BSDL, the structure of the J28 glycotope cannot be attained by classical physical methods. In the first part of the present study, we have determined which glycosyltransferases were differently expressed in pancreatic tumoral cell lines compared to normal tissues, focusing in part on fucosyltransferases (Fuc-T) and core-2 beta6-N-acetylglucosaminyltransferase (Core2GlcNAc-T). Our data suggested that alpha2-Fuc-T activity was decreased in the four cell lines tested (SOJ-6, BxPC-3, MiaPaCa-2, and Panc-1). The alpha(1-3) and alpha(1-4) fucosylations were decreased in tumor cells that do not express the J28 glycotope whereas alpha4-Fuc-T and Core2GlcNAc-T activities were significantly increased in SOJ-6 cells which best expressed the J28 glycotope. Therefore, we wished to gain information about glycosyltransferases involved in the building of this structure by transfecting the cDNA encoding the mucin-like region of BSDL in CHO-K1 also expressing Core2GlcNAc-T and/or FUT3 and/or FUT7 activities. These CHO-K1 cells have been previously transfected with the cDNA encoding Core2GlcNAc-T and/or FUT3 and/or FUT7. Data indicated that the C-terminal peptide of BSDL (Cter) produced by those cells did not carry out the J28 glycotope unless Core2GlcNAc-T activity is present. Further transfection with FUT3 cDNA, increased the antibody recognition. Nevertheless, transfection with FUT3 or FUT7 alone did not generate the formation of the J28 glycotope on the C-terminal peptide. Furthermore, the Cter peptide produced by CHO-K1 cells expressing Core2GlcNAc-T was more reactive to the mAbJ28 after in vitro fucosylation with the recombinant soluble form of FUT3. These data suggested that the J28 glycotope encompasses structures initiated by Core2GlcNAc-T and further fucosylated by alpha3/4-Fuc-T such as FUT3, likely on GlcNAc residues.
胎儿胰腺腺泡蛋白（FAPP），即胆汁盐依赖性脂肪酶（BSDL）的癌胚糖异构体，其特征在于存在被单克隆抗体J28识别的J28糖表位。这种岩藻糖基化表位由BSDL C末端粘蛋白样区域的O-连接聚糖携带。这种糖表位在人胰腺肿瘤组织以及人胰腺肿瘤细胞系如SOJ-6和BxPC-3细胞中表达。然而，在正常人类胰腺组织以及MiaPaCa-2和Panc-1细胞中不表达。由于FAPP和BSDL上存在许多潜在的O-糖基化位点，经典物理方法无法确定J28糖表位的结构。在本研究的第一部分，我们确定了与正常组织相比，哪些糖基转移酶在胰腺肿瘤细胞系中差异表达，部分聚焦于岩藻糖基转移酶（Fuc-T）和核心2β6-N-乙酰葡糖胺基转移酶（Core2GlcNAc-T）。我们的数据表明，在所测试的四种细胞系（SOJ-6、BxPC-3、MiaPaCa-2和Panc-1）中，α2-Fuc-T活性降低。在不表达J28糖表位的肿瘤细胞中，α(1-3)和α(1-4)岩藻糖基化减少，而在最佳表达J28糖表位的SOJ-6细胞中，α4-Fuc-T和Core2GlcNAc-T活性显著增加。因此，我们希望通过在也表达Core2GlcNAc-T和/或FUT3和/或FUT7活性的CHO-K1细胞中转染编码BSDL粘蛋白样区域的cDNA，来获取有关参与该结构构建的糖基转移酶的信息。这些CHO-K1细胞先前已用编码Core2GlcNAc-T和/或FUT3和/或FUT7的cDNA进行了转染。数据表明，除非存在Core2GlcNAc-T活性，这些细胞产生的BSDL C末端肽（Cter）不会携带J28糖表位。用FUT3 cDNA进一步转染可增加抗体识别。然而，单独用FUT3或FUT7转染不会在C末端肽上产生J28糖表位的形成。此外，用重组可溶性形式的FUT3进行体外岩藻糖基化后，表达Core2GlcNAc-T的CHO-K1细胞产生的Cter肽对单克隆抗体J28的反应性更高。这些数据表明，J28糖表位包含由Core2GlcNAc-T起始并由α3/4-Fuc-T如FUT3进一步岩藻糖基化的结构，可能是在GlcNAc残基上。
