Refolded recombinant Siglec5 for NMR investigation of complex carbohydrate binding

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

Research output: Contribution to journalArticle

9 Scopus citations

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 - Feb 11 2013

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Keywords

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

ASJC Scopus subject areas

  • Biotechnology

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