Nakayama J, Yeh J C, Misra A K, Ito S, Katsuyama T, Fukuda M
Department of Laboratory Medicine, Shinshu University School of Medicine, Matsumoto 390-8621, Japan.
Proc Natl Acad Sci U S A. 1999 Aug 3;96(16):8991-6. doi: 10.1073/pnas.96.16.8991.
Among mucus-secreting cells, the gastric gland mucous cells, Brunner's glands, accessory glands of pancreaticobiliary tract, and pancreatic ducts exhibiting gastric metaplasia are unique in that they express class III mucin identified by paradoxical Con A staining composed of periodate oxidation, sodium borohydride reduction, Con A, and horseradish peroxidase reaction. Recently it was shown that these mucous cells secrete glycoproteins having GlcNAcalpha1-->4Galbeta-->R at nonreducing terminals of the carbohydrate moieties. Herein we describe the expression cloning of a cDNA encoding a human alpha1,4-N-acetylglucosaminyltransferase (alpha4GnT), a key enzyme for the formation of GlcNAcalpha1-->4Galbeta1-->R. COS-1 cells were thus cotransfected with a stomach cDNA library and a leukosialin cDNA. Transfected COS-1 cells were screened by using monoclonal antibodies specific for GlcNAcalpha1-->4Galbeta-->R and enriched by fluorescence-activated cell sorting. Sibling selection of recovered plasmids resulted in a cDNA clone that directs the expression of GlcNAcalpha1-->4Galbeta-->R. The deduced amino acid sequence predicts a type II membrane protein with 340 amino acids, showing no significant similarity with any other proteins. The alpha4GnT gene is located at chromosome 3p14.3, and its transcripts are expressed in the stomach and pancreas. An in vitro GlcNAc transferase assay by using a soluble alpha4GnT revealed that alpha1,4-linked GlcNAc residues are transferred most efficiently to core 2 branched O-glycans (Galbeta1-->4GlcNAcbeta1-->6(Galbeta1-->3)GalNAc), forming GlcNAcalpha1-->4Galbeta1-->4GlcNAcbeta1-->6(GlcNAca lpha1-->4Galbeta1- ->3)GalNAc. Transfection of alpha4GnT cDNA into gastric adenocarcinoma AGS cells produced class III mucin, indicating that alpha4GnT is responsible for the formation of class III Con A reactivity. These results indicate that the alpha4GnT is a glycosyltransferase that forms alpha1,4-linked GlcNAc residues, preferentially in O-glycans.
在分泌黏液的细胞中，胃腺黏液细胞、布伦纳腺、胰胆管附属腺以及呈现胃化生的胰管具有独特之处，即它们表达通过高碘酸盐氧化、硼氢化钠还原、伴刀豆球蛋白A（Con A）和辣根过氧化物酶反应进行矛盾性Con A染色鉴定的Ⅲ类黏蛋白。最近研究表明，这些黏液细胞分泌在碳水化合物部分非还原末端具有GlcNAcalpha1→4Galbeta→R的糖蛋白。在此，我们描述了编码人α1,4-N-乙酰氨基葡萄糖转移酶（α4GnT）的cDNA的表达克隆，α4GnT是形成GlcNAcalpha1→4Galbeta1→R的关键酶。因此，用胃cDNA文库和白细胞唾液酸蛋白cDNA共转染COS-1细胞。通过使用对GlcNAcalpha1→4Galbeta→R特异的单克隆抗体筛选转染的COS-1细胞，并通过荧光激活细胞分选进行富集。对回收质粒进行子代选择得到一个指导GlcNAcalpha1→4Galbeta→R表达的cDNA克隆。推导的氨基酸序列预测其为一个含340个氨基酸的Ⅱ型膜蛋白，与其他任何蛋白质均无明显相似性。α4GnT基因位于染色体3p14.3，其转录本在胃和胰腺中表达。通过使用可溶性α4GnT进行的体外GlcNAc转移酶测定表明，α1,4-连接的GlcNAc残基最有效地转移至核心2分支O-聚糖（Galbeta1→4GlcNAcbeta1→6(Galbeta1→3)GalNAc），形成GlcNAcalpha1→4Galbeta1→4GlcNAcbeta1→6(GlcNAca lpha1→4Galbeta1→3)GalNAc。将α4GnT cDNA转染至胃腺癌AGS细胞中产生了Ⅲ类黏蛋白，表明α4GnT负责Ⅲ类Con A反应性的形成。这些结果表明，α4GnT是一种糖基转移酶，优先在O-聚糖中形成α1,4-连接的GlcNAc残基。
