Search

The glycosylphosphatidylinositol (GPI)-anchored prion protein (PrP(C)), usually associated with neurodegenerative diseases, modulates various cellular responses and may scaffold multiprotein cell surface signaling complexes. Engagement of PrP(C) with the secretable cochaperone hop/STI1 induces neurotrophic transmembrane signals through unknown molecular mechanisms. We addressed whether interaction of PrP(C) and hop/STI1 entails structural rearrangements relevant for signaling. Using recombinant wild-type and mutant mouse proteins and binding peptides, we measured circular dichroism (CD), fluorescence spectroscopy, and small angle X-ray scattering (SAXS). PrP(C):hop/STI1 interaction triggers loss of PrP helical structures, involving at least a perturbation of the PrP(143-153) alpha-helix, but no secondary structural modification of hop/STI1 was detected. Novel SAXS models revealed a significant C-terminal compaction of hop/STI1 when bound to PrP. Differing from a recent dimeric model of human hop/STI1, both size-exclusion chromatography and SAXS data support a monomeric form of free murine hop/STI1. Changes in the PrP(143-153) alpha-helix may engage the transmembrane signaling proteins laminin receptor precursor and neural cell adhesion molecule, both of which bind that domain of PrP(C), and further ligands may be engaged by the tertiary structural changes of hop/STI1. These reciprocal structural modifications indicate a versatile mechanism for signaling mediated by PrP(C):hop/STI1 interaction, consistent with the hypothesis of PrP(C)-dependent multiprotein signaling complexes.

We investigated the mechanisms of anticoagulant activity mediated by sulfated galactans. The anticoagulant activity of sulfated polysaccharides is achieved mainly through potentiation of plasma cofactors, which are the natural inhibitors of coagulation proteases. Our results indicated the following. 1) Structural requirements for the interaction of sulfated galactans with coagulation inhibitors and their target proteases are not merely a consequence of their charge density. 2) The structural basis of this interaction is complex because it involves naturally heterogeneous polysaccharides but depends on the distribution of sulfate groups and on monosaccharide composition. 3) Sulfated galactans require significantly longer chains than heparin to achieve anticoagulant activity. 4) Possibly, it is the bulk structure of the sulfated galactan, and not a specific minor component as in heparin, that determines its interaction with antithrombin. 5) Sulfated galactans of approximately 15 to approximately 45 kDa bind to antithrombin but are unable to link the plasma inhibitor and thrombin. This last effect requires a molecular size above 45 kDa. 6) Sulfated galactan and heparin bind to different sites on antithrombin. 7) Sulfated galactans are less effective than heparin at promoting antithrombin conformational activation. Overall, these observations indicate that a different mechanism predominates over the conformational activation of antithrombin in ensuring the antithrombin-mediated anticoagulant activity of the sulfated galactans. Possibly, sulfated galactan connects antithrombin and thrombin, holding the protease in an inactive form. The conformational activation of antithrombin and the consequent formation of a covalent complex with thrombin appear to be less important for the anticoagulant activity of sulfated galactan than for heparin. Our results demonstrate that the paradigm of heparin-antithrombin interaction cannot be extended to other sulfated polysaccharides. Each type of polysaccharide may form a particular complex with the plasma inhibitor and the target protease.