This technology allows for the study of N-linked glycosylation in the absence of O-linked glycosylation, and may lead to the production of glycoproteins.
UDP-galactose 4-epimerase (GALE) is an enzyme that enables the body to break down the sugar galactose. While the E. Coli form of this enzyme catalyzes only the reversible interconversion of UDPgalactose (UDP-gal) and UDP-glucose (UDP-glc), the human form can also catalyze the interconversion of UDP-N-acetylgalactosamine (UDP-galNAc) and UDP-N-acetylglucosamine (UDP-glcNAc). GALE is therefore an important regulator of these glucose and galactose-derivative substrate pools, all of which can be added to growing sugar chains during N-linked and O-linked glycosylation. Methods currently exist to study mammalian cells that lack GALE activity or in which N-linked glycosylation is specifically blocked; however, no clean mechanism exists for specifically inhibiting O-linked glycosylation. This invention provides genetically modified cell lines that express a GALE capable of interconverting UPD-gal and UDP-glc, but essentially incapable of interconverting UDP-galNAc and UDP-glcNAc. Because UDPgalNAc is the obligate first sugar donor for all mucin-type O-linked glycosylation reactions in mammals, this modification inhibits O-linked glycosylation while allowing galactose metabolism and glycosylation of N-linked sites to proceed normally. These cell lines can be used to produce glycoproteins containing N-linked modifications but essentially no mucin-type O-linked modifications.