Antiresorptive activity of osteoprotegerin requires an intact heparan sulfate-binding site

Osteoprotegerin (OPG), a secreted decoy receptor for receptor activator of nuclear factor B ligand (RANKL), plays an essential role in regulating bone resorption. While much is known about the function of the N-terminal domains of OPG, which is responsible for binding to RANKL, the exact biological functions of the three C-terminal domains of OPG remain uncertain. We have previously shown that one likely function of the C-terminal domains of OPG is to bind cell surface heparan sulfate (HS), but the in vivo evidence was lacking. To investigate the biological significance of OPG–HS interaction in bone remodeling, we created OPG knock-in mice (opgAAA). The mutated OPG is incapable of binding to HS but binds RANKL normally. Surprisingly,opgAAA/AAAmice displayed a severe osteoporotic phenotype that is very similar toopg-null mice, suggesting that the antiresorption activity of OPG requires HS. Mechanistically, we propose that the HS immobilizes secreted OPG at the surface of osteoblasts lineage cells, which facilitates binding of OPG to membrane-anchored RANKL. To further support this model, we altered the structure of osteoblast HS genetically to make it incapable of binding to OPG. Interestingly,osteocalcin-Cre;Hs2stf/fmice also displayed osteoporotic phenotype with similar severity toopgAAA/AAAmice. Combined, our data provide strong genetic evidence that OPG–HS interaction is indispensable for normal bone homeostasis.

Intestinal bile acids directly modulate the structure and function ofC. difficileTcdB toxin

Intestinal bile acids are known to modulate the germination and growth ofClostridioides difficile. Here we describe a role for intestinal bile acids in directly binding and neutralizing TcdB toxin, the primary determinant ofC. difficiledisease. We show that individual primary and secondary bile acids reversibly bind and inhibit TcdB to varying degrees through a mechanism that requires the combined oligopeptide repeats region to which no function has previously been ascribed. We find that bile acids induce TcdB into a compact “balled up” conformation that is no longer able to bind cell surface receptors. Lastly, through a high-throughput screen designed to identify bile acid mimetics we uncovered nonsteroidal small molecule scaffolds that bind and inhibit TcdB through a bile acid-like mechanism. In addition to suggesting a role for bile acids inC. difficilepathogenesis, these findings provide a framework for development of a mechanistic class ofC. difficileantitoxins.

Properties of Subviral Particles of Hepatitis B Virus

ABSTRACT In the sera of patients infected with hepatitis B virus (HBV), in addition to infectious particles, there is an excess (typically 1,000- to 100,000-fold) of empty subviral particles (SVP) composed solely of HBV envelope proteins in the form of relatively smaller spheres and filaments of variable length. Hepatitis delta virus (HDV) assembly also uses the envelope proteins of HBV to produce an infectious particle. Rate-zonal sedimentation was used to study the particles released from liver cell lines that produced SVP only, HDV plus SVP, and HBV plus SVP. The SVP made in the absence of HBV or HDV were further examined by electron microscopy. They bound efficiently to heparin columns, consistent with an ability to bind cell surface glycosaminoglycans. However, unlike soluble forms of HBV envelope protein that were potent inhibitors, the SVP did not inhibit the ability of HBV and HDV to infect primary human hepatocytes.

Membrane binding of zebrafish actinoporin-like protein: AF domains, a novel superfamily of cell membrane binding domains

Actinoporins are potent eukaryotic pore-forming toxins specific for sphingomyelin-containing membranes. They are structurally similar to members of the fungal fruit-body lectin family that bind cell-surface exposed Thomsen–Friedenreich antigen. In the present study we found a number of sequences in public databases with similarity to actinoporins. They originate from three animal and two plant phyla and can be classified in three families according to phylogenetic analysis. The sequence similarity is confined to a region from the C-terminal half of the actinoporin molecule and comprises the membrane binding site with a highly conserved P-[WYF]-D pattern. A member of this novel actinoporin-like protein family from zebrafish was cloned and expressed in Escherichia coli. It displays membrane-binding behaviour but does not have permeabilizing activity or sphingomyelin specificity, two properties typical of actinoporins. We propose that the three families of actinoporin-like proteins and the fungal fruit-body lectin family comprise a novel superfamily of membrane binding proteins, tentatively called AF domains (abbreviated from actinoporin-like proteins and fungal fruit-body lectins).

High-Resolution Molecular and Antigen Structure of the VP8* Core of a Sialic Acid-Independent Human Rotavirus Strain

ABSTRACT The most intensively studied rotavirus strains initially attach to cells when the “heads” of their protruding spikes bind cell surface sialic acid. Rotavirus strains that cause disease in humans do not bind this ligand. The structure of the sialic acid binding head (the VP8* core) from the simian rotavirus strain RRV has been reported, and neutralization epitopes have been mapped onto its surface. We report here a 1.6-Å resolution crystal structure of the equivalent domain from the sialic acid-independent rotavirus strain DS-1, which causes gastroenteritis in humans. Although the RRV and DS-1 VP8* cores differ functionally, they share the same galectin-like fold. Differences between the RRV and DS-1 VP8* cores in the region that corresponds to the RRV sialic acid binding site make it unlikely that DS-1 VP8* binds an alternative carbohydrate ligand in this location. In the crystals, a surface cleft on each DS-1 VP8* core binds N-terminal residues from a neighboring molecule. This cleft may function as a ligand binding site during rotavirus replication. We also report an escape mutant analysis, which allows the mapping of heterotypic neutralizing epitopes recognized by human monoclonal antibodies onto the surface of the VP8* core. The distribution of escape mutations on the DS-1 VP8* core indicates that neutralizing antibodies that recognize VP8* of human rotavirus strains may bind a conformation of the spike that differs from those observed to date.

Residues in the C-terminal region of activin A determine specificity for follistatin and type II receptor binding

Activin is a secreted growth factor that signals by binding two related classes of single transmembrane receptors at the cell surface. The interaction of activin with its receptors is highly regulated by other cell surface receptors, antagonistic ligands, and high affinity extracellular binding proteins such as follistatin. Two activin A mutants, the deletion mutant des[85-109]-activin A and the point mutant K102E-activin A (K102E), were investigated with respect to their ability to bind cell surface receptors and the binding protein follistatin. The deletion mutant exhibits low affinity for both receptors and follistatin whereas the point mutant fails to bind cell surface receptors but binds follistatin-288 with high affinity. K102E is able to compete with wild type activin to bind to follistatin and can thus increase the concentration of activin available for receptor binding and signaling. These findings underline the importance of the C-terminal region of activin for binding interactions and show that different residues in this region are involved in cell surface receptor and follistatin interactions.