A Pipeline for Studying and Engineering Single-Subunit Oligosaccharyltransferases

Thapakorn Jaroentomeechai, Xiaolu Zheng, Jasmine Hershewe, Jessica C. Stark, Michael Christopher Jewett, Matthew P. DeLisa*

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapter

1 Citation (Scopus)

Abstract

Asparagine-linked (N-linked) protein glycosylation is one of the most abundant types of posttranslational modification, occurring in all domains of life. The central enzyme in N-linked glycosylation is the oligosaccharyltransferase (OST), which catalyzes the covalent attachment of preassembled glycans to specific asparagine residues in target proteins. Whereas in higher eukaryotes the OST is comprised of eight different membrane proteins, of which the catalytic subunit is STT3, in kinetoplastids and prokaryotes the OST is a monomeric enzyme bearing homology to STT3. Given their relative simplicity, these single-subunit OSTs (ssOSTs) have emerged as important targets for mechanistic dissection of poorly understood aspects of N-glycosylation and at the same time hold great potential for the biosynthesis of custom glycoproteins. To take advantage of this utility, this chapter describes a multipronged approach for studying and engineering ssOSTs that integrates in vivo screening technology with in vitro characterization methods, thereby creating a versatile and readily adaptable pipeline for virtually any ssOST of interest.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
PublisherAcademic Press Inc.
Pages55-81
Number of pages27
DOIs
StatePublished - Jan 1 2017

Publication series

NameMethods in Enzymology
Volume597
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Fingerprint

Glycosylation
Pipelines
Asparagine
Bearings (structural)
Dissection
Biosynthesis
Enzymes
Post Translational Protein Processing
Eukaryota
Polysaccharides
Catalytic Domain
Glycoproteins
Screening
Membrane Proteins
Proteins
Technology
dolichyl-diphosphooligosaccharide - protein glycotransferase

Keywords

  • Asparagine-linked (N-linked) protein glycosylation
  • Bacterial glycoengineering
  • Cell-free glycosylation
  • Cell-free protein synthesis
  • Directed evolution
  • Enzyme engineering
  • Nanodisc technology
  • Posttranslational modification

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

Jaroentomeechai, T., Zheng, X., Hershewe, J., Stark, J. C., Jewett, M. C., & DeLisa, M. P. (2017). A Pipeline for Studying and Engineering Single-Subunit Oligosaccharyltransferases. In Methods in Enzymology (pp. 55-81). (Methods in Enzymology; Vol. 597). Academic Press Inc.. https://doi.org/10.1016/bs.mie.2017.07.011
Jaroentomeechai, Thapakorn ; Zheng, Xiaolu ; Hershewe, Jasmine ; Stark, Jessica C. ; Jewett, Michael Christopher ; DeLisa, Matthew P. / A Pipeline for Studying and Engineering Single-Subunit Oligosaccharyltransferases. Methods in Enzymology. Academic Press Inc., 2017. pp. 55-81 (Methods in Enzymology).
@inbook{c46f735be8cf4a43b08aa8d499f22d67,
title = "A Pipeline for Studying and Engineering Single-Subunit Oligosaccharyltransferases",
abstract = "Asparagine-linked (N-linked) protein glycosylation is one of the most abundant types of posttranslational modification, occurring in all domains of life. The central enzyme in N-linked glycosylation is the oligosaccharyltransferase (OST), which catalyzes the covalent attachment of preassembled glycans to specific asparagine residues in target proteins. Whereas in higher eukaryotes the OST is comprised of eight different membrane proteins, of which the catalytic subunit is STT3, in kinetoplastids and prokaryotes the OST is a monomeric enzyme bearing homology to STT3. Given their relative simplicity, these single-subunit OSTs (ssOSTs) have emerged as important targets for mechanistic dissection of poorly understood aspects of N-glycosylation and at the same time hold great potential for the biosynthesis of custom glycoproteins. To take advantage of this utility, this chapter describes a multipronged approach for studying and engineering ssOSTs that integrates in vivo screening technology with in vitro characterization methods, thereby creating a versatile and readily adaptable pipeline for virtually any ssOST of interest.",
keywords = "Asparagine-linked (N-linked) protein glycosylation, Bacterial glycoengineering, Cell-free glycosylation, Cell-free protein synthesis, Directed evolution, Enzyme engineering, Nanodisc technology, Posttranslational modification",
author = "Thapakorn Jaroentomeechai and Xiaolu Zheng and Jasmine Hershewe and Stark, {Jessica C.} and Jewett, {Michael Christopher} and DeLisa, {Matthew P.}",
year = "2017",
month = "1",
day = "1",
doi = "10.1016/bs.mie.2017.07.011",
language = "English (US)",
series = "Methods in Enzymology",
publisher = "Academic Press Inc.",
pages = "55--81",
booktitle = "Methods in Enzymology",
address = "United States",

}

Jaroentomeechai, T, Zheng, X, Hershewe, J, Stark, JC, Jewett, MC & DeLisa, MP 2017, A Pipeline for Studying and Engineering Single-Subunit Oligosaccharyltransferases. in Methods in Enzymology. Methods in Enzymology, vol. 597, Academic Press Inc., pp. 55-81. https://doi.org/10.1016/bs.mie.2017.07.011

A Pipeline for Studying and Engineering Single-Subunit Oligosaccharyltransferases. / Jaroentomeechai, Thapakorn; Zheng, Xiaolu; Hershewe, Jasmine; Stark, Jessica C.; Jewett, Michael Christopher; DeLisa, Matthew P.

Methods in Enzymology. Academic Press Inc., 2017. p. 55-81 (Methods in Enzymology; Vol. 597).

Research output: Chapter in Book/Report/Conference proceedingChapter

TY - CHAP

T1 - A Pipeline for Studying and Engineering Single-Subunit Oligosaccharyltransferases

AU - Jaroentomeechai, Thapakorn

AU - Zheng, Xiaolu

AU - Hershewe, Jasmine

AU - Stark, Jessica C.

AU - Jewett, Michael Christopher

AU - DeLisa, Matthew P.

PY - 2017/1/1

Y1 - 2017/1/1

N2 - Asparagine-linked (N-linked) protein glycosylation is one of the most abundant types of posttranslational modification, occurring in all domains of life. The central enzyme in N-linked glycosylation is the oligosaccharyltransferase (OST), which catalyzes the covalent attachment of preassembled glycans to specific asparagine residues in target proteins. Whereas in higher eukaryotes the OST is comprised of eight different membrane proteins, of which the catalytic subunit is STT3, in kinetoplastids and prokaryotes the OST is a monomeric enzyme bearing homology to STT3. Given their relative simplicity, these single-subunit OSTs (ssOSTs) have emerged as important targets for mechanistic dissection of poorly understood aspects of N-glycosylation and at the same time hold great potential for the biosynthesis of custom glycoproteins. To take advantage of this utility, this chapter describes a multipronged approach for studying and engineering ssOSTs that integrates in vivo screening technology with in vitro characterization methods, thereby creating a versatile and readily adaptable pipeline for virtually any ssOST of interest.

AB - Asparagine-linked (N-linked) protein glycosylation is one of the most abundant types of posttranslational modification, occurring in all domains of life. The central enzyme in N-linked glycosylation is the oligosaccharyltransferase (OST), which catalyzes the covalent attachment of preassembled glycans to specific asparagine residues in target proteins. Whereas in higher eukaryotes the OST is comprised of eight different membrane proteins, of which the catalytic subunit is STT3, in kinetoplastids and prokaryotes the OST is a monomeric enzyme bearing homology to STT3. Given their relative simplicity, these single-subunit OSTs (ssOSTs) have emerged as important targets for mechanistic dissection of poorly understood aspects of N-glycosylation and at the same time hold great potential for the biosynthesis of custom glycoproteins. To take advantage of this utility, this chapter describes a multipronged approach for studying and engineering ssOSTs that integrates in vivo screening technology with in vitro characterization methods, thereby creating a versatile and readily adaptable pipeline for virtually any ssOST of interest.

KW - Asparagine-linked (N-linked) protein glycosylation

KW - Bacterial glycoengineering

KW - Cell-free glycosylation

KW - Cell-free protein synthesis

KW - Directed evolution

KW - Enzyme engineering

KW - Nanodisc technology

KW - Posttranslational modification

UR - http://www.scopus.com/inward/record.url?scp=85029232452&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85029232452&partnerID=8YFLogxK

U2 - 10.1016/bs.mie.2017.07.011

DO - 10.1016/bs.mie.2017.07.011

M3 - Chapter

C2 - 28935112

AN - SCOPUS:85029232452

T3 - Methods in Enzymology

SP - 55

EP - 81

BT - Methods in Enzymology

PB - Academic Press Inc.

ER -

Jaroentomeechai T, Zheng X, Hershewe J, Stark JC, Jewett MC, DeLisa MP. A Pipeline for Studying and Engineering Single-Subunit Oligosaccharyltransferases. In Methods in Enzymology. Academic Press Inc. 2017. p. 55-81. (Methods in Enzymology). https://doi.org/10.1016/bs.mie.2017.07.011