The M/GP(5) glycoprotein complex of porcine reproductive and respiratory syndrome virus binds the sialoadhesin receptor in a sialic acid-dependent manner.

The porcine reproductive and respiratory syndrome virus (PRRSV) is a major threat to swine health worldwide and is considered the most significant viral disease in the swine industry today. In past years, studies on the entry of the virus into its host cell have led to the identification of a number of essential virus receptors and entry mediators. However, viral counterparts for these molecules have remained elusive and this has made rational development of new generation vaccines impossible. The main objective of this study was to identify the viral counterparts for sialoadhesin, a crucial PRRSV receptor on macrophages. For this purpose, a soluble form of sialoadhesin was constructed and validated. The soluble sialoadhesin could bind PRRSV in a sialic acid-dependent manner and could neutralize PRRSV infection of macrophages, thereby confirming the role of sialoadhesin as an essential PRRSV receptor on macrophages. Although sialic acids are present on the GP(3), GP(4) and GP(5) envelope glycoproteins, only the M/GP(5) glycoprotein complex of PRRSV was identified as a ligand for sialoadhesin. The interaction was found to be dependent on the sialic acid binding capacity of sialoadhesin and on the presence of sialic acids on GP(5). These findings not only contribute to a better understanding of PRRSV biology, but the knowledge and tools generated in this study also hold the key to the development of a new generation of PRRSV vaccines.

Surface plasmon resonance imaging for real-time, label-free analysis of protein interactions with carbohydrate microarrays.

Plant lectin recognition of glycans was evaluated by SPR imaging using a model array of N-biotinylated aminoethyl glycosides of beta-D-glucose (negative control), alpha-D: -mannose (conA-responsive), beta-D-galactose (RCA(120)-responsive) and N-acetyl-beta-D-: glucosamine (WGA-responsive) printed onto neutravidin-coated gold chips. Selective recognition of the cognate ligand was observed when RCA(120) was passed over the array surface. Limited or no binding was observed for the non-cognate ligands. SPR imaging of an array of 40 sialylated and unsialylated glycans established the binding preference of hSiglec7 for alpha2-8-linked disialic acid structures over alpha2-6-sialyl-LacNAcs, which in turn were recognized and bound with greater affinity than alpha2-3-sialyl-LacNAcs. Affinity binding data could be obtained with as little as 10-20 microg of lectin per experiment. The SPR imaging technique was also able to establish selective binding to the preferred glycan ligand when analyzing crude culture supernatant containing 10-20 microg of recombinant hSiglec7-Fc. Our results show that SPR imaging provides results that are in agreement with those obtained from fluorescence based carbohydrate arrays but with the added advantage of label-free analysis.

The murine inhibitory receptor mSiglec-E is expressed broadly on cells of the innate immune system whereas mSiglec-F is restricted to eosinophils.

Murine (m) Siglec-E and mSiglec-F are recently discovered murine sialic acid-binding Ig-like lectins with tyrosine-based inhibitory signaling motifs. They are postulated to be the orthologs of human (h) siglec-7, -8 or -9 and siglec-5, respectively. We report here the first detailed characterization of mSiglec-E, and compare its expression pattern with mSiglec-F. Similar to hSiglec-7, mSiglec-E preferred alpha 2-8-linked disialic acid over alpha 2-3- and alpha 2-6-linked sialic acids. Using a specific Ab, FACS analysis demonstrated that mSiglec-E was expressed mainly on neutrophils in blood and their immature precursors in bone marrow. mSiglec-E was present on peritoneal cavity macrophages and on subsets of mature NK cells and splenic dendritic cells. mSiglec-E was also found ona novel population of peritoneal cavity B-1a-like cells and a subset of splenic B cells enriched in transitional T2 and marginal zone B cells. In striking contrast to mSiglec-E, mSiglec-F was expressed predominantly on eosinophils in blood and their precursors in the bone marrow. The distinct and largely non-overlapping expression profiles of mSiglec-E and mSiglec-F suggest that they play non-redundant roles in the innate immune system. mSiglec-E is likely to modulate the functions of several types of effector cells, whereas mSiglec-F is likely to be more restricted to eosinophil biology.