Magnet A D, Fukuda M
Glycobiology Program, La Jolla Cancer Research Center, Burnham Institute, CA 92037, USA.
Glycobiology. 1997 Mar;7(2):285-95. doi: 10.1093/glycob/7.2.285.
Large I antigen is specifically formed by a beta-1,6-N-acetylglucosaminyltransferase (IGnT), which is a Golgi enzyme. IGnT converts a linear carbohydrate structure, the i antigen, to a branched structure, the I antigen in N-acetyllactosamines. This conversion has been shown to be developmentally regulated in human erythrocytes. In mouse embryonic development, it has been shown that poly-N-acetyllactosamine plays a critical role in the compaction process (Rastan,S., Thorpe,S.J., Scudder,P., Brown,S., Gooi,H.C., and Feizi,T. (1985) J. Embryol. Exp. Morphol., 87, 115-128.). In order to understand the regulation of IGnT expression during mouse development, the IGnT transcripts were studied using in situ hybridization. The cDNA encoding IGnT was isolated from a murine PCC4 teratocarcinoma cDNA library by nucleic acid hybridization using probes generated from the human IGnT cDNA. The IGnT cDNA was used to produce a fusion protein, which was then used as an immunogen to produce polyclonal antibodies against the enzyme. Nucleotide sequence data was used to design oligonucleotide primers and cDNA probes. The primers and probes, antibodies specific to the fusion protein, and previously obtained human anti-I or i sera, were used to analyze adult and embryonic mouse tissues for expression of IGnT and I antigen. To detect IGnT mRNA, in situ reverse-transcription and polymerase chain reaction were performed on tissue sections using the oligonucleotide primers. Amplified DNA products were detected by in situ hybridization using the cDNA probes. IGnT protein was detected by immunohistochemistry using the IGnT fusion-protein antibody. Expression of the carbohydrate structures was detected using human anti-I or i sera. The results presented demonstrate that IGnT and the I antigen appear in epithelial cells and dividing cells. The antigen also appears to be expressed on cells exposed to the lumenal surface of tissues. These results support the conclusions obtained by the previous studies that IGnT and the resultant I antigen may play critical roles during development and in adult organisms.
大I抗原由β-1,6-N-乙酰氨基葡萄糖转移酶（IGnT）特异性形成，该酶是一种高尔基体酶。IGnT将线性碳水化合物结构（即i抗原）转化为分支结构（即N-乙酰乳糖胺中的I抗原）。这种转化在人类红细胞中已被证明受发育调控。在小鼠胚胎发育过程中，已表明多聚-N-乙酰乳糖胺在紧密化过程中起关键作用（拉斯坦，S.，索普，S.J.，斯卡德，P.，布朗，S.，古伊，H.C.，和费齐，T.（1985年）《胚胎学与实验形态学杂志》，87，115 - 128）。为了了解小鼠发育过程中IGnT表达的调控，利用原位杂交技术研究了IGnT转录本。通过使用从人IGnT cDNA产生的探针进行核酸杂交，从鼠PCC4畸胎瘤cDNA文库中分离出编码IGnT的cDNA。IGnT cDNA用于产生融合蛋白，然后该融合蛋白用作免疫原以产生针对该酶的多克隆抗体。核苷酸序列数据用于设计寡核苷酸引物和cDNA探针。这些引物和探针、融合蛋白特异性抗体以及先前获得的人抗I或抗i血清，用于分析成年和胚胎小鼠组织中IGnT和I抗原的表达。为了检测IGnT mRNA，使用寡核苷酸引物对组织切片进行原位逆转录和聚合酶链反应。使用cDNA探针通过原位杂交检测扩增的DNA产物。使用IGnT融合蛋白抗体通过免疫组织化学检测IGnT蛋白。使用人抗I或抗i血清检测碳水化合物结构的表达。呈现的结果表明IGnT和I抗原出现在上皮细胞和分裂细胞中。该抗原似乎也在暴露于组织腔表面的细胞上表达。这些结果支持了先前研究得出的结论，即IGnT和产生的I抗原可能在发育过程中和成年生物体中起关键作用。
