Expression and Characterization of Human ß-1, 4-Galactosyltransferase 1 (ß4GalT1) Using Silkworm-Baculovirus Expression System.

Baculovirus expression vector system (BEVS) is widely known as a mass-production tool to produce functional recombinant glycoproteins except that it may not be always suitable for medical practice due to the differences in the structure of N-linked glycans between insects and mammalian. Currently, various approaches have been reported to alter N-linked glycan structures of glycoproteins derived from insects into terminally sialylated complex-type N-glycans. In the light of those studies, we also proposed in vitro maturation of N-glycan with mass-produced and purified glycosyltransferases by silkworm-BEVS. ß-1,4-Galactosyltransferase 1 (ß4GalT1) is known as one of type II transmembrane enzymes that transfer galactose in a ß-1, 4 linkage to accepter sugars, and a key enzyme for further sialylation of N-glycans. In this study, we developed a large-scale production of recombinant human ß4GalT1 (rhß4GalT1) with N- or C-terminal tags in silkworm-BEVS. We demonstrated that rhß4GalT1 is N-glycosylated and without mucin-type glycosylation. Interestingly, we found that purified rhß4GalT1 from silkworm serum presented higher galactosyltransferase activity than that expressed from cultured mammalian cells. We also validated the UDP-galactose transferase activity of produced rhß4GalT1 proteins by using protein subtracts from silkworm silk gland. Taken together, rhß4GalT1 from silkworms can become a valuable tool for producing high-quality recombinant glycoproteins with mammalian-like N-glycans.

Immunocytochemical localization of the Golgi apparatus using protein-specific antibodies to galactosyltransferase.

Polyclonal antisera to human milk galactosyltransferase have been widely used as immunocytochemical reagent to visualize the Golgi apparatus. Immunochemical analysis revealed partial specificity of these antisera to glycan epitopes expressed on galactosyltransferase and other gene products (R. A. Childs et al., Biochem. J. 238, 605-611 (1986)). Since glycan-specific reagents such as lectins are known to label the Golgi apparatus by virtue of their exclusive glycan specificity, Golgi localization of galactosyltransferase by use of these polyclonal antisera remained elusive. In order to demonstrate authentic Golgi localization of the galactosyltransferase peptide, we expressed the enzyme in Escherichia coli as non-glycosylated beta-galactosidase-fusion proteins and used them as affinity matrix to protein-epitope purified polyclonal antibodies from antisera to the milk enzyme, and to induce protein-specific antisera by injecting them into rabbits, respectively. A short fusion protein comprising most of the substrate binding sites and a protein which included the stem region of galactosyltransferase were expressed. The short fusion protein was poorly immunogenic whereas the long fusion protein induced a high-titer antiserum. Protein-epitope purified antibodies derived from polyclonal antisera to the milk enzyme as well as antibodies to the long fusion protein cross-reacted with milk galactosyltransferase and with fusion protein as demonstrated by immunoblotting and enzyme-linked immunosorbent assay (ELISA) and produced typical Golgi morphologies in both HeLa and CaCo2 cells when used for immunocytochemical localization of galactosyltransferase. We conclude that previous immunolocalization studies have correctly been interpreted as Golgi localization of the galactosyltransferase protein and that antigenicity to the protein moiety is confirmed to the N-terminal third of the enzyme.