N-glycosylation of recombinant human fucosyltransferase III is required for its in vivo folding in mammalian and insect cells.
Human alpha3/4fucosyltransferase (FT3) catalyses the synthesis of fucosylated glycoconjugates involved in cell-cell interactions. FT3 has two potential N-glycosylation sites at Asn(154) and Asn(185). Soluble secretory forms of the enzyme (SFT3) and mutant forms with the first, second and both glycosylation sites (SFT3DN1, SFT3DN2, SFT3DN) mutated have been expressed in baby hamster kidney (BHK) and Spodoptera frugiperda (Sf9) cells. Deletion of the first or both sites caused total enzyme inactivation. Deletion of the second site caused 99% and 75% decrease of secretory enzyme expression in BHK and Sf9 cells, respectively. Sf9 cells produced 1 mg/l SFT3 and 0.3 mg/l SFT3DN2; these values were 175- and 3750-fold higher, respectively, than those observed for BHK cells. A significant amount of protein was accumulated intracellularly in Sf9 cells which for SFT3 was active and for SFT3DN2 was inactive, indicating the importance of the glycans from the second glycosylation site for protein folding. The corresponding full-length forms FT3, FT3DN1 and FT3DN2 associated with calnexin as observed by immunoprecipitation studies, which indicated the possible role of this chaperon in the folding of glycosylated glycosyltransferases.
Morais
Vanessa A
VA
Instituto de Tecnologia Química e Biológica, Avenida da República, Apartado 127, 2781-901 Oeiras, Portugal.
Costa
Maria T
MT
Costa
Júlia
J
D000818:Animals; D016367:Baculoviridae; D001665:Binding Sites / Q000235:genetics; D037281:Calnexin / Q000737:chemistry / Q000378:metabolism; D002460:Cell Line; D006224:Cricetinae; D005647:Fucosyltransferases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D017353:Gene Deletion; D015971:Gene Expression Regulation, Enzymologic; D006031:Glycosylation; D006801:Humans; D009154:Mutation; D011485:Protein Binding; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis; D018411:Spodoptera; D014162:Transfection
D011994:Recombinant Proteins; D037281:Calnexin; D005647:Fucosyltransferases; C019818:3-galactosyl-N-acetylglucosaminide 4-alpha-L-fucosyltransferase