title: Engineering β1,4-galactosyltransferase I to reduce secretion and enhance N-glycan elongation in insect cells.
abstract: β1,4-galactosyltransferase I (B4GALT1) is a Golgi-resident enzyme that elongates glycoprotein glycans, but a subpopulation of this enzyme is secreted following proteolytic cleavage in its stem domain. We hypothesized that engineering B4GALT1 to block cleavage and secretion would enhance its retention and, therefore, its function. To test this hypothesis, we replaced the cytoplasmic/transmembrane/stem (CTS) domains of B4GALT1 with those from human α1,3-fucosyltransferase 7 (FUT7), which is not cleaved and secreted. Expression of FUT7-CTS-B4GALT1 in insect cells produced lower levels of secreted and higher levels of intracellular B4GALT1 activity than the native enzyme. We also noted that the B4GALT1 used in our study had a leucine at position 282, whereas all other animal B4GALT1 sequences have an aromatic amino acid at this position. Thus, we examined the combined impact of changing the CTS domains and the amino acid at position 282 on intracellular B4GALT1 activity levels and N-glycan processing in insect cells. The results demonstrated a correlation between the levels of intracellular B4GALT1 activity and terminally galactosylated N-glycans, N-glycan branching, the appearance of hybrid structures, and reduced core fucosylation. Thus, engineering B4GALT1 to reduce its cleavage and secretion is an approach that can be used to enhance N-glycan elongation in insect cells.
authors.affiliation: Department of Molecular Biology, University of Wyoming, Laramie, WY 82071, USA; GlycoBac, LLC, Laramie, WY 82072, USA.
authors.forename: Christoph
authors.initials: C
authors.lastname: Geisler
authors.affiliation: Department of Molecular Biology, University of Wyoming, Laramie, WY 82071, USA.
authors.forename: Hideaki
authors.initials: H
authors.lastname: Mabashi-Asazuma
authors.affiliation: Institute of Biological Chemistry, Academia Sinica 128, Nankang, Taipei 115, Taiwan.
authors.forename: Chu-Wei
authors.initials: CW
authors.lastname: Kuo
authors.affiliation: Institute of Biological Chemistry, Academia Sinica 128, Nankang, Taipei 115, Taiwan.
authors.forename: Kay-Hooi
authors.initials: KH
authors.lastname: Khoo
authors.affiliation: Department of Molecular Biology, University of Wyoming, Laramie, WY 82071, USA; GlycoBac, LLC, Laramie, WY 82072, USA. Electronic address: dljarvis@uwyo.edu.
authors.forename: Donald L
authors.initials: DL
authors.lastname: Jarvis
mesh_terms: D000818:Animals; D001483:Base Sequence; D002417:Cattle; D005647:Fucosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005700:Galactosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000378:metabolism; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D061987:Sf9 Cells
chemical_list: D011134:Polysaccharides; D011993:Recombinant Fusion Proteins; C502597:FUT7 protein, human; D005647:Fucosyltransferases; D005700:Galactosyltransferases; C119701:beta-1,4-galactosyltransferase I
keywords: Galactosyltransferase; Glycoengineering; Glycosylation; Glycosyltransferase; Insect cells