ABSTRACT
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.