The two N-glycans present on bovine Pofut1 are differently involved in its solubility and activity.
O-Fucosylation is a post-translational glycosylation in which an O-fucose is covalently attached to the hydroxyl group of a specific serine or threonine residue. This modification occurs within the consensus sequence C2X(4-5)(S/T)C3 present on epidermal growth factor-like repeats of several proteins, including the Notch receptors and their ligands. The enzyme responsible for the addition of O-fucose to epidermal growth factor-like repeats is protein O-fucosyltransferase 1. Protein O-fucosyltransferase 1-mediated O-fucosylation is essential in Notch signaling, folding and targeting to the cell surface. Here, we studied the expression pattern of protein O-fucosyltransferase 1 in cattle and showed that the active enzyme is present in all tissues examined from embryo and adult as a glycoprotein with two N-glycans. By comparing protein O-fucosyltransferase 1 sequences available in databases, we observed that mammalian protein O-fucosyltransferase 1 enzymes possess two putative N-glycosylation sites, and that only the first is conserved among bilaterians. To gain more insight regarding the significance of N-glycans on protein O-fucosyltransferase 1, we substituted, by site-directed mutagenesis, bovine protein O-fucosyltransferase 1 N65, N163 or both, with L or Q. We demonstrated that the loss of N-glycan on N163 caused a slight decrease in protein O-fucosyltransferase 1 activity. In contrast, glycosylation of N65 was crucial for protein O-fucosyltransferase 1 functionality. Loss of glycosylation at N65 resulted in aggregation of protein O-fucosyltransferase 1, suggesting that N-glycosylation at this site is essential for proper folding of the enzyme.
Loriol
Céline
C
INRA, UMR1061 Unité de Génétique Moléculaire Animale, GDR-CNRS 2590, Université de Limoges, IFR 45, Faculté des Sciences et Techniques, 87060 Limoges, France.
Audfray
Aymeric
A
Dupuy
Fabrice
F
Germot
Agnès
A
Maftah
Abderrahman
A
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002417:Cattle; D002522:Chlorocebus aethiops; D017124:Conserved Sequence; D005647:Fucosyltransferases / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism; D006031:Glycosylation; D007930:Leucine / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011134:Polysaccharides / Q000737:chemistry*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012995:Solubility; D014162:Transfection
D011134:Polysaccharides; D011994:Recombinant Proteins; D018698:Glutamic Acid; D005647:Fucosyltransferases; C105148:polypeptide fucosyltransferase; D007930:Leucine