Expression system for structural and functional studies of human glycosylation enzymes

Kelley W. Moremen, Annapoorani Ramiah, Melissa Stuart, Jason Steel, Lu Meng, Farhad Forouhar, Heather A. Moniz, Gagandeep Gahlay, Zhongwei Gao, Digantkumar Chapla, Shuo Wang, Jeong Yeh Yang, Pradeep Kumar Prabhakar, Roy Johnson, Mitche Dela Rosa, Christoph Geisler, Alison V. Nairn, Jayaraman Seetharaman, Sheng Cheng Wu, Liang TongHarry J. Gilbert, Joshua LaBaer, Donald L. Jarvis

Research output: Contribution to journalArticle

33 Citations (Scopus)

Abstract

Vertebrate glycoproteins and glycolipids are synthesized in complex biosynthetic pathways localized predominantly within membrane compartments of the secretory pathway. The enzymes that catalyze these reactions are exquisitely specific, yet few have been extensively characterized because of challenges associated with their recombinant expression as functional products. We used a modular approach to create an expression vector library encoding all known human glycosyltransferases, glycoside hydrolases, and sulfotransferases, as well as other glycan-modifying enzymes. We then expressed the enzymes as secreted catalytic domain fusion proteins in mammalian and insect cell hosts, purified and characterized a subset of the enzymes, and determined the structure of one enzyme, the sialyltransferase ST6GalNAcII. Many enzymes were produced at high yields and at similar levels in both hosts, but individual protein expression levels varied widely. This expression vector library will be a transformative resource for recombinant enzyme production, broadly enabling structure-function studies and expanding applications of these enzymes in glycochemistry and glycobiology.

Original languageEnglish (US)
Pages (from-to)156-162
Number of pages7
JournalNature Chemical Biology
Volume14
Issue number2
DOIs
StatePublished - Feb 1 2018

Fingerprint

Glycosylation
Enzymes
Libraries
Glycomics
Sialyltransferases
Sulfotransferases
Glycosyltransferases
Glycoside Hydrolases
Secretory Pathway
Glycolipids
Biosynthetic Pathways
Polysaccharides
Insects
Vertebrates
Catalytic Domain
Glycoproteins
Proteins
Membranes

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

Cite this

Moremen, K. W., Ramiah, A., Stuart, M., Steel, J., Meng, L., Forouhar, F., ... Jarvis, D. L. (2018). Expression system for structural and functional studies of human glycosylation enzymes. Nature Chemical Biology, 14(2), 156-162. https://doi.org/10.1038/nchembio.2539

Expression system for structural and functional studies of human glycosylation enzymes. / Moremen, Kelley W.; Ramiah, Annapoorani; Stuart, Melissa; Steel, Jason; Meng, Lu; Forouhar, Farhad; Moniz, Heather A.; Gahlay, Gagandeep; Gao, Zhongwei; Chapla, Digantkumar; Wang, Shuo; Yang, Jeong Yeh; Prabhakar, Pradeep Kumar; Johnson, Roy; Rosa, Mitche Dela; Geisler, Christoph; Nairn, Alison V.; Seetharaman, Jayaraman; Wu, Sheng Cheng; Tong, Liang; Gilbert, Harry J.; LaBaer, Joshua; Jarvis, Donald L.

In: Nature Chemical Biology, Vol. 14, No. 2, 01.02.2018, p. 156-162.

Research output: Contribution to journalArticle

Moremen, KW, Ramiah, A, Stuart, M, Steel, J, Meng, L, Forouhar, F, Moniz, HA, Gahlay, G, Gao, Z, Chapla, D, Wang, S, Yang, JY, Prabhakar, PK, Johnson, R, Rosa, MD, Geisler, C, Nairn, AV, Seetharaman, J, Wu, SC, Tong, L, Gilbert, HJ, LaBaer, J & Jarvis, DL 2018, 'Expression system for structural and functional studies of human glycosylation enzymes', Nature Chemical Biology, vol. 14, no. 2, pp. 156-162. https://doi.org/10.1038/nchembio.2539
Moremen, Kelley W. ; Ramiah, Annapoorani ; Stuart, Melissa ; Steel, Jason ; Meng, Lu ; Forouhar, Farhad ; Moniz, Heather A. ; Gahlay, Gagandeep ; Gao, Zhongwei ; Chapla, Digantkumar ; Wang, Shuo ; Yang, Jeong Yeh ; Prabhakar, Pradeep Kumar ; Johnson, Roy ; Rosa, Mitche Dela ; Geisler, Christoph ; Nairn, Alison V. ; Seetharaman, Jayaraman ; Wu, Sheng Cheng ; Tong, Liang ; Gilbert, Harry J. ; LaBaer, Joshua ; Jarvis, Donald L. / Expression system for structural and functional studies of human glycosylation enzymes. In: Nature Chemical Biology. 2018 ; Vol. 14, No. 2. pp. 156-162.
@article{b6bcf5294c8f41bd867745e1d9c00493,
title = "Expression system for structural and functional studies of human glycosylation enzymes",
abstract = "Vertebrate glycoproteins and glycolipids are synthesized in complex biosynthetic pathways localized predominantly within membrane compartments of the secretory pathway. The enzymes that catalyze these reactions are exquisitely specific, yet few have been extensively characterized because of challenges associated with their recombinant expression as functional products. We used a modular approach to create an expression vector library encoding all known human glycosyltransferases, glycoside hydrolases, and sulfotransferases, as well as other glycan-modifying enzymes. We then expressed the enzymes as secreted catalytic domain fusion proteins in mammalian and insect cell hosts, purified and characterized a subset of the enzymes, and determined the structure of one enzyme, the sialyltransferase ST6GalNAcII. Many enzymes were produced at high yields and at similar levels in both hosts, but individual protein expression levels varied widely. This expression vector library will be a transformative resource for recombinant enzyme production, broadly enabling structure-function studies and expanding applications of these enzymes in glycochemistry and glycobiology.",
author = "Moremen, {Kelley W.} and Annapoorani Ramiah and Melissa Stuart and Jason Steel and Lu Meng and Farhad Forouhar and Moniz, {Heather A.} and Gagandeep Gahlay and Zhongwei Gao and Digantkumar Chapla and Shuo Wang and Yang, {Jeong Yeh} and Prabhakar, {Pradeep Kumar} and Roy Johnson and Rosa, {Mitche Dela} and Christoph Geisler and Nairn, {Alison V.} and Jayaraman Seetharaman and Wu, {Sheng Cheng} and Liang Tong and Gilbert, {Harry J.} and Joshua LaBaer and Jarvis, {Donald L.}",
year = "2018",
month = "2",
day = "1",
doi = "10.1038/nchembio.2539",
language = "English (US)",
volume = "14",
pages = "156--162",
journal = "Nature Chemical Biology",
issn = "1552-4450",
publisher = "Nature Publishing Group",
number = "2",

}

TY - JOUR

T1 - Expression system for structural and functional studies of human glycosylation enzymes

AU - Moremen, Kelley W.

AU - Ramiah, Annapoorani

AU - Stuart, Melissa

AU - Steel, Jason

AU - Meng, Lu

AU - Forouhar, Farhad

AU - Moniz, Heather A.

AU - Gahlay, Gagandeep

AU - Gao, Zhongwei

AU - Chapla, Digantkumar

AU - Wang, Shuo

AU - Yang, Jeong Yeh

AU - Prabhakar, Pradeep Kumar

AU - Johnson, Roy

AU - Rosa, Mitche Dela

AU - Geisler, Christoph

AU - Nairn, Alison V.

AU - Seetharaman, Jayaraman

AU - Wu, Sheng Cheng

AU - Tong, Liang

AU - Gilbert, Harry J.

AU - LaBaer, Joshua

AU - Jarvis, Donald L.

PY - 2018/2/1

Y1 - 2018/2/1

N2 - Vertebrate glycoproteins and glycolipids are synthesized in complex biosynthetic pathways localized predominantly within membrane compartments of the secretory pathway. The enzymes that catalyze these reactions are exquisitely specific, yet few have been extensively characterized because of challenges associated with their recombinant expression as functional products. We used a modular approach to create an expression vector library encoding all known human glycosyltransferases, glycoside hydrolases, and sulfotransferases, as well as other glycan-modifying enzymes. We then expressed the enzymes as secreted catalytic domain fusion proteins in mammalian and insect cell hosts, purified and characterized a subset of the enzymes, and determined the structure of one enzyme, the sialyltransferase ST6GalNAcII. Many enzymes were produced at high yields and at similar levels in both hosts, but individual protein expression levels varied widely. This expression vector library will be a transformative resource for recombinant enzyme production, broadly enabling structure-function studies and expanding applications of these enzymes in glycochemistry and glycobiology.

AB - Vertebrate glycoproteins and glycolipids are synthesized in complex biosynthetic pathways localized predominantly within membrane compartments of the secretory pathway. The enzymes that catalyze these reactions are exquisitely specific, yet few have been extensively characterized because of challenges associated with their recombinant expression as functional products. We used a modular approach to create an expression vector library encoding all known human glycosyltransferases, glycoside hydrolases, and sulfotransferases, as well as other glycan-modifying enzymes. We then expressed the enzymes as secreted catalytic domain fusion proteins in mammalian and insect cell hosts, purified and characterized a subset of the enzymes, and determined the structure of one enzyme, the sialyltransferase ST6GalNAcII. Many enzymes were produced at high yields and at similar levels in both hosts, but individual protein expression levels varied widely. This expression vector library will be a transformative resource for recombinant enzyme production, broadly enabling structure-function studies and expanding applications of these enzymes in glycochemistry and glycobiology.

UR - http://www.scopus.com/inward/record.url?scp=85040782404&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85040782404&partnerID=8YFLogxK

U2 - 10.1038/nchembio.2539

DO - 10.1038/nchembio.2539

M3 - Article

C2 - 29251719

AN - SCOPUS:85040782404

VL - 14

SP - 156

EP - 162

JO - Nature Chemical Biology

JF - Nature Chemical Biology

SN - 1552-4450

IS - 2

ER -