Remodeling of Mouse Milk Glycoconjugates by Transgenic Expression of a Human Glycosyltransferase

Abstract

The mammary gland is a unique biosynthetic tissue that produces a variety of species-specific glycoconjugates, but the factors regulating the production of specific glycoconjugates are not well understood. To explore the underlying regulation, a fusion gene containing a cDNA encoding the human ␣1,2-fucosyltransferase (␣1,2FT), which generates the H-blood group antigen, flanked by the murine whey acidic protein promoter and a polyadenylation signal, was introduced into mice. Milk samples from transgenic animals contained soluble forms of the ␣1,2FT, as revealed by Western blots of milk samples using an anti-␣1,2FT antiserum and by the demonstration of ␣1,2FT enzyme activity. Milk from transgenic animals also contained large quantities of 2 -fucosyllactose (Fuc␣1-2Gal␤1-4Glc) and modified glycoproteins containing the H-antigen, whereas milk from control animals lacked these glycoconjugates. Expression levels of 2 -fucosyllactose were high in most animals and represented 1 ⁄3 to nearly 1 ⁄2 of the total milk oligosaccharides. These results demonstrate that heterologous transgenic expression of a glycosyltransferase can result in the expression of both the transgene and its secondary gene products and that the structures of milk oligosaccharides can be remodeled depending on expression of the appropriate enzyme. Furthermore, these results suggest that the lactating mammary gland may be a unique biosynthetic reactor for the production of biologically active oligosaccharides and glycoconjugates.Despite the fact that animal oligosaccharides are important in many biological and pathological processes (Karlsson, 1989;Kornfeld, 1992; Drickamer and Taylor, 1993;McEver et al., 1995), little is known about the regulation of animal cell glycoconjugate biosynthesis. Biosynthesis is influenced by expression of appropriate glycosyltransferases, correct targeting of the enzymes to the Golgi apparatus, sugar nucleotide levels, competition between enzymes, residence time of glycoconjugates in intracellular compartments, acceptor specificity of the enzymes, and many other factors (Kornfeld and Kornfeld, 1985;Kobata and Takasaki, 1992;Cummings, 1992;Baenziger, 1994). Experimental modulation of these factors has been difficult. One approach that has proven somewhat successful in vitro is to attempt to alter oligosaccharide biosynthesis by genetic manipulation of glycosyltransferases (Lee et al., 1989;Smith et al., 1990;Lowe et al., 1990). This approach has so far been limited to cultured and immortalized cell lines and has been useful in producing neoglycoconjugates for biological studies .One major site for glycoconjugate biosynthesis in animals, and one that has been of enormous historical importance in the field of glycobiology, is the lactating mammary gland. Milk contains, in addition to lactose, numerous glycoproteins (Patton et al., 1990;Fiat and Jollès, 1989) and a variety of free oligosaccharides (Kobata et al., 1972). The structure and composition of the free oligosaccharides in milk differ between mammals, but the human lac...

Secretion of Human Furin into Mouse Milk

“…4). Similarly, Prieto et al (1995) described the presence of active human ␣1,2-fucosyltransferase in the milk of mice (38). Thus, it is possible to produce significant amounts of secreted forms of intracellular enzymes in milk.…”

Glycoproteins and Their Relationship to Human Disease

“…Transgenic mice which overexpress α2-Fuc-transferase produce quite different structures of their glycoproteins with higher amounts of the H antigen, less α3-sialylation and less α3-Gal determinants while sialyl-Tn expression is increased (table 4) [Prieto et al, 1995;Shinkel et al, 1997]. When blood group H α2-Fuc-transferase was transfected into rat colon carcinoma REGb cells, the cells became more aggressive and tumorigenic and, unexpectedly, wound healing improved.…”

Local over-expression of prolactin in differentiating mouse mammary gland induces functional defects and benign lesions, but no carcinoma

“…At day 8 of lactation, normalized litters (eight pups) from lactating females (WT or Tg) were removed 4 h before milk collection was performed as described (Prieto et al 1995). Briefly, females were anaesthetized using isoflurane gas (Abbot Laboratories), and 15 min prior to milking, they were injected intraperitoneally with 5 units of oxytocin (Sigma) to facilitate milk extrusion through the nipple.…”