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.
authors: Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602, USA.
authors: Adam W
authors: AW
authors: Barb
authors: Xu
authors: X
authors: Wang
authors: James H
authors: JH
authors: Prestegard
mesh_terms: D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015214:Antigens, Differentiation, Myelomonocytic / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D037102:Lectins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D058849:Protein Refolding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012794:Sialic Acids / Q000378:metabolism*
chemical_list: D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D037102:Lectins; D011994:Recombinant Proteins; C114531:SIGLEC5 protein, human; D012794:Sialic Acids; D004247:DNA