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Engineering β1,4-galactosyltransferase I to reduce secretion and enhance N-glycan elongation in insect cells

Paper ID Volume ID Publish Year Pages File Format Full-Text
22972 43408 2015 14 PDF Available
Title
Engineering β1,4-galactosyltransferase I to reduce secretion and enhance N-glycan elongation in insect cells
Abstract

•B4GALT1 is a resident Golgi enzyme with a key role in N-glycan elongation.•The natural process of B4GALT1 cleavage and secretion attenuates its Golgi function.•B4GALT1 was engineered to block its secretion and enhance its specific activity.•Engineered B4GALT1 had higher intracellular activity levels and Golgi localization.•Engineering B4GALT1 for increased intracellular activity enhanced N-glycan elongation.

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.

Keywords
AcMNPV, Autographa californica multiple nucleopolyhedrosis virus; CTS, cytoplasmic tail/transmembrane domain/stem region; FDL, Fused Lobes, the insect cell N-glycan processing β-N-acetylglucosaminidase; FUT7, GDP-fucose: β-galactoside: α1,3-fucosyltransfe
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Engineering β1,4-galactosyltransferase I to reduce secretion and enhance N-glycan elongation in insect cells
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 193, 10 January 2015, Pages 52–65
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us