James C. Paulson

Siglecs and their roles in the immune system.

Cell surfaces in the immune system are richly equipped with a complex mixture of glycans, which can be recognized by diverse glycan-binding proteins. The Siglecs are a family of sialic-acid-binding immunoglobulin-like lectins that are thought to promote cell–cell interactions and regulate the functions of cells in the innate and adaptive immune systems through glycan recognition. In this Review, we describe recent studies on signalling mechanisms and discuss the potential role of Siglecs in triggering endocytosis and in pathogen recognition. Finally, we discuss the postulated functions of the recently discovered CD33-related Siglecs and consider the factors that seem to be driving their rapid evolution.

Structure and Receptor Specificity of the Hemagglutinin from an H5N1 Influenza Virus.

The hemagglutinin (HA) structure at 2.9 angstrom resolution, from a highly pathogenic Vietnamese H5N1 influenza virus, is more related to the 1918 and other human H1 HAs than to a 1997 duck H5 HA. Glycan microarray analysis of this Viet04 HA reveals an avian α2-3 sialic acid receptor binding preference. Introduction of mutations that can convert H1 serotype HAs to human α2-6 receptor specificity only enhanced or reduced affinity for avian-type receptors. However, mutations that can convert avian H2 and H3 HAs to human receptor specificity, when inserted onto the Viet04 H5 HA framework, permitted binding to a natural human α2-6 glycan, which suggests a path for this H5N1 virus to gain a foothold in the human population.

Receptor determinants of human and animal influenza virus isolates: differences in receptor specificity of the H3 hemagglutinin based on species of origin.

The binding of influenza virus to erythrocytes and host cells is mediated by the interaction of the viral hemagglutinin (H) with cell surface receptors containing sialic acid (SA). The specificity of this interaction for 19 human and animal influenza isolates was examined using human erythrocytes enzymatically modified to contain cell surface sialyloligosaccharides with the sequence SA alpha 2,6Gal beta 1,4GlcNAc; SA alpha 2,3Gal beta 1,4(3)GlcNAc; SA alpha 2,3Gal beta 1,3GalNAc; or SA alpha 2,6GalNAc. Although none of the viruses agglutinated cells containing the SA alpha 2,6GalNAc linkage, differential agglutination of cells containing the other three sequences revealed at least three distinct receptor binding types. Several virus isolates exhibited marked receptor specificity, binding only to cells containing the SA alpha 2,6Gal or the SA alpha 2,3Gal linkage, while others bound equally well to cells containing either linkage. Moreover, some viruses could distinguish between two oligosaccharide receptor determinants containing the terminal SA alpha 2,3Gal linkage when present in the SA alpha 2,3Gal beta 1,4(3)GlcNAc sequence or the SA alpha 2,3Gal beta 1,3GalNAc sequence binding cells containing only the former. The observed receptor specificities were not significantly influenced by the viral neuraminidases as shown by the use of the potent neuraminidase inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid. Receptor specificity appeared, to some extent, to be dependent on the species from which the virus was isolated. In particular, human isolates of the H3 serotype all agglutinated cells containing the SA alpha 2,6Gal linkage, but not cells bearing the SA alpha 2,3Gal beta 1,3GalNAc sequence. In contrast, antigenically similar (H3) isolates from avian and equine species preferentially bound erythrocytes containing the SA alpha 2,3Gal linkage. This is of particular interest in view of the identification of the avian virus H3 hemagglutinin as the progenitor of the H3 hemagglutinin present on the current human Hong Kong viruses.

Recapitulation of IVIG Anti-Inflammatory Activity with a Recombinant IgG Fc

It is well established that high doses of monomeric immunoglobulin G (IgG) purified from pooled human plasma [intravenous immunoglobulin (IVIG)] confer anti-inflammatory activity in a variety of autoimmune settings. However, exactly how those effects are mediated is not clear because of the heterogeneity of IVIG. Recent studies have demonstrated that the anti-inflammatory activity of IgG is completely dependent on sialylation of the N-linked glycan of the IgG Fc fragment. Here we determine the precise glycan requirements for this anti-inflammatory activity, allowing us to engineer an appropriate IgG1 Fc fragment, and thus generate a fully recombinant, sialylated IgG1 Fc with greatly enhanced potency. This therapeutic molecule precisely defines the biologically active component of IVIG and helps guide development of an IVIG replacement with improved activity and availability.

New World Bats Harbor Diverse Influenza A Viruses

Aquatic birds harbor diverse influenza A viruses and are a major viral reservoir in nature. The recent discovery of influenza viruses of a new H17N10 subtype in Central American fruit bats suggests that other New World species may similarly carry divergent influenza viruses. Using consensus degenerate RT-PCR, we identified a novel influenza A virus, designated as H18N11, in a flat-faced fruit bat (Artibeus planirostris) from Peru. Serologic studies with the recombinant H18 protein indicated that several Peruvian bat species were infected by this virus. Phylogenetic analyses demonstrate that, in some gene segments, New World bats harbor more influenza virus genetic diversity than all other mammalian and avian species combined, indicative of a long-standing host-virus association. Structural and functional analyses of the hemagglutinin and neuraminidase indicate that sialic acid is not a ligand for virus attachment nor a substrate for release, suggesting a unique mode of influenza A virus attachment and activation of membrane fusion for entry into host cells. Taken together, these findings indicate that bats constitute a potentially important and likely ancient reservoir for a diverse pool of influenza viruses.

Single amino acid substitutions in influenza haemagglutinin change receptor binding specificity

The haemagglutinin (HA) glycoproteins of influenza virus membranes are responsible for binding viruses to cells by interacting with membrane receptor molecules which contain sialic acid (for review see ref. 1). This interaction is known to vary in detailed specificity for different influenza viruses (see, for example, refs 2–4) and we have attempted to identify the sialic acid binding site of the haemagglutinin by comparing the amino acid sequences of haemagglutinins with different binding specificities. We present here evidence that haemagglutinins which differ in recognizing either NeuAcα2→3Gal- or NeuAcα2→6Gal-linkages in glycoproteins also differ at amino acid 226 of HA1. This residue is located in a pocket on the distal tip of the molecule, an area previously proposed from considerations of the three-dimensional structure of the haemagglutinin to be involved in receptor binding5.

A potent and broad neutralizing antibody recognizes and penetrates the HIV glycan shield.

An HIV antibody achieves potency and breadth by binding simultaneously to two conserved glycans on the viral envelope protein. The HIV envelope (Env) protein gp120 is protected from antibody recognition by a dense glycan shield. However, several of the recently identified PGT broadly neutralizing antibodies appear to interact directly with the HIV glycan coat. Crystal structures of antigen-binding fragments (Fabs) PGT 127 and 128 with Man9 at 1.65 and 1.29 angstrom resolution, respectively, and glycan binding data delineate a specific high mannose-binding site. Fab PGT 128 complexed with a fully glycosylated gp120 outer domain at 3.25 angstroms reveals that the antibody penetrates the glycan shield and recognizes two conserved glycans as well as a short β-strand segment of the gp120 V3 loop, accounting for its high binding affinity and broad specificify. Furthermore, our data suggest that the high neutralization potency of PGT 127 and 128 immunoglobulin Gs may be mediated by cross-linking Env trimers on the viral surface.

Recurrent Severe Infections Caused by a Novel Leukocyte Adhesion Deficiency

THE recruitment of neutrophils to sites of inflammation is initiated by the local production of bacteria-derived attractants, inflammatory cytokines, and other host-derived factors. These factors i...

Glycoproteins: what are the sugar chains for?

For many glycoproteins, the carbohydrate groups confer important physical properties such as conformational stability, protease resistance, charge and water-binding capacity. Equally important, however, are the roles of carbohydrate groups in biological recognition, where sequence diversity provides signals for protein targeting and cell-cell interactions.

Influenza virus strains selectively recognize sialyloligosaccharides on human respiratory epithelium; the role of the host cell in selection of hemagglutinin receptor specificity

The complement of sialyloligosaccharides present on the surface of human tracheal epithelium has been implicated as an important factor in the selection of hemagglutinin receptor specificity of human influenza A virus. Human strains of influenza A virus preferentially recognize host cell receptors bearing SA alpha 2,6Gal sequences, a sequence which is found on the surface of ciliated tracheal epithelium. A fluorescently-labelled H3 human virus strain bound avidly to the apical surface of human tracheal epithelium, while a fluorescently-labelled receptor variant strain, which preferentially binds SA alpha 2,3Gal sequences, showed little binding to the epithelial surface and localized primarily to intracellular mucin droplets. Extracts of human bronchial mucin, which is known to contain sialic acid primarily in the SA alpha 2,3Gal linkage, was a potent inhibitor of the binding of the receptor variant strain to trachea sections, while the binding of the parent strain was unaffected by the presence of mucin. Human bronchial mucin also inhibited the binding of the receptor variant strains, but not the parent virus strains, to human erythrocytes derivatized to contain SA alpha 2,6Gal sequences. These results suggest that a combination of selection pressures present in the respiratory tract environment have resulted in the evolution of a hemagglutinin receptor specificity in human influenza A virus strains which optimizes recognition of, binding to and infection of host cells.