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Human Siglec-5 inhibitory receptor and immunoglobulin A (IgA) have separate binding sites in streptococcal beta protein.

ABSTRACT: Sialic acid-binding immunoglobulin-like lectins (Siglecs) are receptors believed to be important for regulation of cellular activation and inflammation. Several pathogenic microbes bind specific Siglecs via sialic acid-containing structures at the microbial surface, interactions that may result in modulation of host responses. Recently, it was shown that the group B Streptococcus (GBS) binds to human Siglec-5 (hSiglec-5), an inhibitory receptor expressed on macrophages and neutrophils, via the IgA-binding surface ? protein, providing the first example of a protein/protein interaction between a pathogenic microbe and a Siglec. Here we show that the hSiglec-5-binding part of ? resides in the N-terminal half of the protein, which also harbors the previously determined IgA-binding region. We constructed bacterial mutants expressing variants of the ? protein with non-overlapping deletions in the N-terminal half of the protein. Using these mutants and recombinant ? fragments, we showed that the hSiglec-5-binding site is located in the most N-terminal part of ? (B6N region; amino acids 1-152) and that the hSiglec-5- and IgA-binding domains in ? are completely separate. We showed with BIAcore(TM) analysis that tandem variants of the hSiglec-5- and IgA-binding domains bind to their respective ligands with high affinity. Finally, we showed that the B6N region, but not the IgA-binding region of ?, triggers recruitment of the tyrosine phosphatase SHP-2 to hSiglec-5 in U937 monocytes. Taken together, we have identified and isolated the first microbial non-sialic acid Siglec-binding region that can be used as a tool in studies of the ?/hSiglec-5 interaction.

SUBMITTER: Nordstrom T

PROVIDER: S-EPMC3190825 | biostudies-other | 2011 Sep

REPOSITORIES: biostudies-other

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Human Siglec-5 inhibitory receptor and immunoglobulin A (IgA) have separate binding sites in streptococcal beta protein.

Nordström Therése T Movert Elin E Olin Anders I AI Ali Syed R SR Nizet Victor V Varki Ajit A Areschoug Thomas T

The Journal of biological chemistry 20110727 39

Sialic acid-binding immunoglobulin-like lectins (Siglecs) are receptors believed to be important for regulation of cellular activation and inflammation. Several pathogenic microbes bind specific Siglecs via sialic acid-containing structures at the microbial surface, interactions that may result in modulation of host responses. Recently, it was shown that the group B Streptococcus (GBS) binds to human Siglec-5 (hSiglec-5), an inhibitory receptor expressed on macrophages and neutrophils, via the I ...[more]

PMID: 21795693

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Project description:BACKGROUND:Siglec-8 is a CD33 subfamily cell-surface receptor selectively expressed on human eosinophils. After cytokine priming, Siglec-8 mAb or glycan ligand binding causes eosinophil apoptosis associated with reactive oxygen species (ROS) production. Most CD33-related Siglecs function as inhibitory receptors, but the ability of Siglec-8 to stimulate eosinophil ROS production and apoptosis suggests that Siglec-8 might instead function as an activating receptor. OBJECTIVE:We sought to determine the role of IL-5 priming and identify the signaling molecules involved in Siglec-8 function for human eosinophils. METHODS:We used an mAb and/or a multimeric synthetic sulfated sialoglycan ligand recognizing Siglec-8 in combination with integrin blocking antibodies, pharmacologic inhibitors, phosphoproteomics, and Western blot analysis to define the necessity of various proteins involved in Siglec-8 function for human eosinophils. RESULTS:Cytokine priming was required to elicit the unanticipated finding that Siglec-8 engagement promotes rapid ?2-integrin-dependent eosinophil adhesion. Also novel was the finding that this adhesion was necessary for subsequent ROS production and apoptosis. Siglec-8-mediated ROS was generated through reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation because pretreatment of eosinophils with catalase (an extracellular superoxide scavenger) or NSC 23766 (a Rac GTPase inhibitor) completely inhibited Siglec-8-mediated eosinophil apoptosis. Finally, engagement of Siglec-8 on IL-5-primed eosinophils resulted in increased phosphorylation of Akt, p38, and c-Jun N-terminal kinase 1 that was also ?2-integrin dependent; pharmacologic inhibition of these kinases completely prevented Siglec-8-mediated eosinophil apoptosis. CONCLUSIONS:These data demonstrate that Siglec-8 functions uniquely as an activating receptor on IL-5-primed eosinophils through a novel pathway involving regulation of ?2-integrin-dependent adhesion, NADPH oxidase, and a subset of protein kinases.

Dataset Information

Human Siglec-5 inhibitory receptor and immunoglobulin A (IgA) have separate binding sites in streptococcal beta protein.

Publications

Human Siglec-5 inhibitory receptor and immunoglobulin A (IgA) have separate binding sites in streptococcal beta protein.

Similar Datasets

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