Inhibition of hybrid- and complex-type glycosylation reveals the presence of the GlcNAc transferase I-independent fucosylation pathway.
A mammalian N-acetylglucosamine (GlcNAc) transferase I (GnT I)-independent fucosylation pathway is revealed by the use of matrix-assisted laser desorption/ionization (MALDI) and negative-ion nano-electrospray ionization (ESI) mass spectrometry of N-linked glycans from natively folded recombinant gly...
| Main Authors: | , , , , , , , , |
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| Format: | Journal article |
| Language: | English |
| Published: |
2006
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| _version_ | 1826291829065121792 |
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| author | Crispin, M Harvey, D Chang, V Yu, C Aricescu, A Jones, E Davis, S Dwek, R Rudd, P |
| author_facet | Crispin, M Harvey, D Chang, V Yu, C Aricescu, A Jones, E Davis, S Dwek, R Rudd, P |
| author_sort | Crispin, M |
| collection | OXFORD |
| description | A mammalian N-acetylglucosamine (GlcNAc) transferase I (GnT I)-independent fucosylation pathway is revealed by the use of matrix-assisted laser desorption/ionization (MALDI) and negative-ion nano-electrospray ionization (ESI) mass spectrometry of N-linked glycans from natively folded recombinant glycoproteins, expressed in both human embryonic kidney (HEK) 293S and Chinese hamster ovary (CHO) Lec3.2.8.1 cells deficient in GnT I activity. The biosynthesis of core fucosylated Man5GlcNAc2 glycans was enhanced in CHO Lec3.2.8.1 cells by the alpha-glucosidase inhibitor, N-butyldeoxynojirimycin (NB-DNJ), leading to the increase in core fucosylated Man5GlcNAc2 glycans and the biosynthesis of a novel core fucosylated monoglucosylated oligomannose glycan, Glc1Man7GlcNAc2Fuc. Furthermore, no fucosylated Man9GlcNAc2 glycans were detected following inhibition of alpha-mannosidase I with kifunensine. Thus, core fucosylation is prevented by the presence of terminal alpha1-2 mannoses on the 6-antennae but not the 3-antennae of the trimannosyl core. Fucosylated Man5GlcNAc2 glycans were also detected on recombinant glycoprotein from HEK 293T cells following inhibition of Golgi alpha-mannosidase II with swainsonine. The paucity of fucosylated oligomannose glycans in wild-type mammalian cells is suggested to be due to kinetic properties of the pathway rather than the absence of the appropriate catalytic activity. The presence of the GnT I-independent fucosylation pathway is an important consideration when engineering mammalian glycosylation. |
| first_indexed | 2024-03-07T03:05:20Z |
| format | Journal article |
| id | oxford-uuid:b24ff7bb-651e-4601-9a55-da26fbae2315 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T03:05:20Z |
| publishDate | 2006 |
| record_format | dspace |
| spelling | oxford-uuid:b24ff7bb-651e-4601-9a55-da26fbae23152022-03-27T04:10:50ZInhibition of hybrid- and complex-type glycosylation reveals the presence of the GlcNAc transferase I-independent fucosylation pathway.Journal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:b24ff7bb-651e-4601-9a55-da26fbae2315EnglishSymplectic Elements at Oxford2006Crispin, MHarvey, DChang, VYu, CAricescu, AJones, EDavis, SDwek, RRudd, PA mammalian N-acetylglucosamine (GlcNAc) transferase I (GnT I)-independent fucosylation pathway is revealed by the use of matrix-assisted laser desorption/ionization (MALDI) and negative-ion nano-electrospray ionization (ESI) mass spectrometry of N-linked glycans from natively folded recombinant glycoproteins, expressed in both human embryonic kidney (HEK) 293S and Chinese hamster ovary (CHO) Lec3.2.8.1 cells deficient in GnT I activity. The biosynthesis of core fucosylated Man5GlcNAc2 glycans was enhanced in CHO Lec3.2.8.1 cells by the alpha-glucosidase inhibitor, N-butyldeoxynojirimycin (NB-DNJ), leading to the increase in core fucosylated Man5GlcNAc2 glycans and the biosynthesis of a novel core fucosylated monoglucosylated oligomannose glycan, Glc1Man7GlcNAc2Fuc. Furthermore, no fucosylated Man9GlcNAc2 glycans were detected following inhibition of alpha-mannosidase I with kifunensine. Thus, core fucosylation is prevented by the presence of terminal alpha1-2 mannoses on the 6-antennae but not the 3-antennae of the trimannosyl core. Fucosylated Man5GlcNAc2 glycans were also detected on recombinant glycoprotein from HEK 293T cells following inhibition of Golgi alpha-mannosidase II with swainsonine. The paucity of fucosylated oligomannose glycans in wild-type mammalian cells is suggested to be due to kinetic properties of the pathway rather than the absence of the appropriate catalytic activity. The presence of the GnT I-independent fucosylation pathway is an important consideration when engineering mammalian glycosylation. |
| spellingShingle | Crispin, M Harvey, D Chang, V Yu, C Aricescu, A Jones, E Davis, S Dwek, R Rudd, P Inhibition of hybrid- and complex-type glycosylation reveals the presence of the GlcNAc transferase I-independent fucosylation pathway. |
| title | Inhibition of hybrid- and complex-type glycosylation reveals the presence of the GlcNAc transferase I-independent fucosylation pathway. |
| title_full | Inhibition of hybrid- and complex-type glycosylation reveals the presence of the GlcNAc transferase I-independent fucosylation pathway. |
| title_fullStr | Inhibition of hybrid- and complex-type glycosylation reveals the presence of the GlcNAc transferase I-independent fucosylation pathway. |
| title_full_unstemmed | Inhibition of hybrid- and complex-type glycosylation reveals the presence of the GlcNAc transferase I-independent fucosylation pathway. |
| title_short | Inhibition of hybrid- and complex-type glycosylation reveals the presence of the GlcNAc transferase I-independent fucosylation pathway. |
| title_sort | inhibition of hybrid and complex type glycosylation reveals the presence of the glcnac transferase i independent fucosylation pathway |
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