Refolded recombinant Siglec5 for NMR investigation of complex carbohydrate binding

Adam W. Barb, Xu Wang, James H. Prestegard

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

Sialic-acid-binding immunoglobulin-like lectin (Siglec5) is a carbohydrate-binding surface receptor expressed on neutrophils, monocytes and B cells in human lymphoid and myeloid cell lineages. Existing structural and functional data fail to define the clear ligand specificity of Siglec5, though like other Siglec family members, it binds a variety of complex carbohydrates containing a sialic acid at the non-reducing terminus. Prokaryotic expression of this protein has proven challenging due to disulfide bonds and Asn-linked glycosylation. We developed an expression and purification protocol that uses an on-column strategy to refold Escherichia coli expressed protein that produced a high yield (2 mg/L) of the single N-terminal Siglec5 carbohydrate recognition domain (CRD). A 2D heteronuclear single-quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectrum showed this material was folded, and a secondary structure prediction based on the assigned chemical shifts of backbone atoms was consistent with a previously determined X-ray model. NMR chemical shift mapping of Siglec5 binding to three carbohydrate ligands revealed similarities in binding interfaces and affinities. In addition, the role of alternate protein conformations identified by NMR in ligand binding is discussed. These studies demonstrate the Siglec5 CRD alone is sufficient for binding sialylated carbohydrates and provide a foundation for further investigation of Siglec5 structure and function.

Original languageEnglish (US)
Pages (from-to)183-189
Number of pages7
JournalProtein Expression and Purification
Volume88
Issue number2
DOIs
StatePublished - 2013

Fingerprint

Magnetic Resonance Spectroscopy
Carbohydrates
Sialic Acid Binding Immunoglobulin-like Lectins
Ligands
Protein Conformation
Escherichia coli Proteins
N-Acetylneuraminic Acid
Cell Lineage
Myeloid Cells
Glycosylation
Disulfides
Monocytes
Neutrophils
B-Lymphocytes
X-Rays
Lymphocytes
Proteins

Keywords

  • Carbohydrate recognition
  • Glycoprotein
  • NMR chemical shift perturbation
  • On-column refolding
  • Sialoside binding

ASJC Scopus subject areas

  • Biotechnology

Cite this

Refolded recombinant Siglec5 for NMR investigation of complex carbohydrate binding. / Barb, Adam W.; Wang, Xu; Prestegard, James H.

In: Protein Expression and Purification, Vol. 88, No. 2, 2013, p. 183-189.

Research output: Contribution to journalArticle

@article{f0e56811c45642b6bb6da21addf4245a,
title = "Refolded recombinant Siglec5 for NMR investigation of complex carbohydrate binding",
abstract = "Sialic-acid-binding immunoglobulin-like lectin (Siglec5) is a carbohydrate-binding surface receptor expressed on neutrophils, monocytes and B cells in human lymphoid and myeloid cell lineages. Existing structural and functional data fail to define the clear ligand specificity of Siglec5, though like other Siglec family members, it binds a variety of complex carbohydrates containing a sialic acid at the non-reducing terminus. Prokaryotic expression of this protein has proven challenging due to disulfide bonds and Asn-linked glycosylation. We developed an expression and purification protocol that uses an on-column strategy to refold Escherichia coli expressed protein that produced a high yield (2 mg/L) of the single N-terminal Siglec5 carbohydrate recognition domain (CRD). A 2D heteronuclear single-quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectrum showed this material was folded, and a secondary structure prediction based on the assigned chemical shifts of backbone atoms was consistent with a previously determined X-ray model. NMR chemical shift mapping of Siglec5 binding to three carbohydrate ligands revealed similarities in binding interfaces and affinities. In addition, the role of alternate protein conformations identified by NMR in ligand binding is discussed. These studies demonstrate the Siglec5 CRD alone is sufficient for binding sialylated carbohydrates and provide a foundation for further investigation of Siglec5 structure and function.",
keywords = "Carbohydrate recognition, Glycoprotein, NMR chemical shift perturbation, On-column refolding, Sialoside binding",
author = "Barb, {Adam W.} and Xu Wang and Prestegard, {James H.}",
year = "2013",
doi = "10.1016/j.pep.2013.01.005",
language = "English (US)",
volume = "88",
pages = "183--189",
journal = "Protein Expression and Purification",
issn = "1046-5928",
publisher = "Academic Press Inc.",
number = "2",

}

TY - JOUR

T1 - Refolded recombinant Siglec5 for NMR investigation of complex carbohydrate binding

AU - Barb, Adam W.

AU - Wang, Xu

AU - Prestegard, James H.

PY - 2013

Y1 - 2013

N2 - Sialic-acid-binding immunoglobulin-like lectin (Siglec5) is a carbohydrate-binding surface receptor expressed on neutrophils, monocytes and B cells in human lymphoid and myeloid cell lineages. Existing structural and functional data fail to define the clear ligand specificity of Siglec5, though like other Siglec family members, it binds a variety of complex carbohydrates containing a sialic acid at the non-reducing terminus. Prokaryotic expression of this protein has proven challenging due to disulfide bonds and Asn-linked glycosylation. We developed an expression and purification protocol that uses an on-column strategy to refold Escherichia coli expressed protein that produced a high yield (2 mg/L) of the single N-terminal Siglec5 carbohydrate recognition domain (CRD). A 2D heteronuclear single-quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectrum showed this material was folded, and a secondary structure prediction based on the assigned chemical shifts of backbone atoms was consistent with a previously determined X-ray model. NMR chemical shift mapping of Siglec5 binding to three carbohydrate ligands revealed similarities in binding interfaces and affinities. In addition, the role of alternate protein conformations identified by NMR in ligand binding is discussed. These studies demonstrate the Siglec5 CRD alone is sufficient for binding sialylated carbohydrates and provide a foundation for further investigation of Siglec5 structure and function.

AB - Sialic-acid-binding immunoglobulin-like lectin (Siglec5) is a carbohydrate-binding surface receptor expressed on neutrophils, monocytes and B cells in human lymphoid and myeloid cell lineages. Existing structural and functional data fail to define the clear ligand specificity of Siglec5, though like other Siglec family members, it binds a variety of complex carbohydrates containing a sialic acid at the non-reducing terminus. Prokaryotic expression of this protein has proven challenging due to disulfide bonds and Asn-linked glycosylation. We developed an expression and purification protocol that uses an on-column strategy to refold Escherichia coli expressed protein that produced a high yield (2 mg/L) of the single N-terminal Siglec5 carbohydrate recognition domain (CRD). A 2D heteronuclear single-quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectrum showed this material was folded, and a secondary structure prediction based on the assigned chemical shifts of backbone atoms was consistent with a previously determined X-ray model. NMR chemical shift mapping of Siglec5 binding to three carbohydrate ligands revealed similarities in binding interfaces and affinities. In addition, the role of alternate protein conformations identified by NMR in ligand binding is discussed. These studies demonstrate the Siglec5 CRD alone is sufficient for binding sialylated carbohydrates and provide a foundation for further investigation of Siglec5 structure and function.

KW - Carbohydrate recognition

KW - Glycoprotein

KW - NMR chemical shift perturbation

KW - On-column refolding

KW - Sialoside binding

UR - http://www.scopus.com/inward/record.url?scp=84873358322&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84873358322&partnerID=8YFLogxK

U2 - 10.1016/j.pep.2013.01.005

DO - 10.1016/j.pep.2013.01.005

M3 - Article

VL - 88

SP - 183

EP - 189

JO - Protein Expression and Purification

JF - Protein Expression and Purification

SN - 1046-5928

IS - 2

ER -