Kathy Wehrly

Conversion of raft associated prion protein to the protease-resistant state requires insertion of PrP-res (PrPSc) into contiguous membranes

Prion protein (PrP) is usually attached to membranes by a glycosylphosphatidylinositol‐anchor that associates with detergent‐resistant membranes (DRMs), or rafts. To model the molecular processes that might occur during the initial infection of cells with exogenous transmissible spongiform encephalopathy (TSE) agents, we examined the effect of membrane association on the conversion of the normal protease‐sensitive PrP isoform (PrP‐sen) to the protease‐resistant isoform (PrP‐res). A cell‐free conversion reaction approximating physiological conditions was used, which contained purified DRMs as a source of PrP‐sen and brain microsomes from scrapie‐infected mice as a source of PrP‐res. Interestingly, DRM‐associated PrP‐sen was not converted to PrP‐res until the PrP‐sen was either released from DRMs by treatment with phosphatidylinositol‐specific phospholipase C (PI‐PLC), or the combined membrane fractions were treated with the membrane‐fusing agent polyethylene glycol (PEG). PEG‐assisted conversion was optimal at pH 6–7, and acid pre‐treating the DRMs was not sufficient to permit conversion without PI‐PLC or PEG, arguing against late endosomes/lysosomes as primary compartments for PrP conversion. These observations raise the possibility that generation of new PrP‐res during TSE infection requires (i) removal of PrP‐sen from target cells; (ii) an exchange of membranes between cells; or (iii) insertion of incoming PrP‐res into the raft domains of recipient cells.

Sulfated glycans and elevated temperature stimulate PrPSc-dependent cell-free formation of protease-resistant prion protein

A conformational conversion of the normal, protease‐ sensitive prion protein (PrP‐sen or PrPC) to a protease‐resistant form (PrP‐res or PrPSc) is commonly thought to be required in transmissible spongiform encephalopathies (TSEs). Endogenous sulfated glycosaminoglycans are associated with PrP‐res deposits in vivo, suggesting that they may facilitate PrP‐res formation. On the other hand, certain exogenous sulfated glycans can profoundly inhibit PrP‐res accumulation and serve as prophylactic anti‐TSE compounds in vivo. To investigate the seemingly paradoxical effects of sulfated glycans on PrP‐res formation, we have assayed their direct effects on PrP conversion under physiologically compatible cell‐free conditions. Heparan sulfate and pentosan polysulfate stimulated PrP‐res formation. Conversion was stimulated further by increased temperature. Both elevated temperature and pentosan polysulfate promoted interspecies PrP conversion. Circular dichroism spectropolarimetry measurements showed that pentosan polysulfate induced a conformational change in PrP‐sen that may potentiate its PrP‐res‐induced conversion. These results show that certain sulfated glycosaminoglycans can directly affect the PrP conversion reaction. Therefore, depending upon the circumstances, sulfated glycans may be either cofactors or inhibitors of this apparently pathogenic process.

ANALYSIS OF TWO MONOCLONAL ANTIBODIES REACTIVE WITH ENVELOPE PROTEINS OF MURINE RETROVIRUSES: ONE PAN SPECIFIC ANTIBODY AND ONE SPECIFIC FOR MOLONEY LEUKEMIA VIRUS

Many monoclonal antibodies (MAbs) reactive with various proteins of murine leukemia viruses (MuLVs) have been developed. In this report two additional MAbs with differing and unusual specificities are described. MAb 573 is reactive with the envelope protein of all MuLVs tested including viruses in the ecotropic, xenotropic, polytropic and amphotropic classes. Notably, MAb 573 is one of only two reported MAbs that react with the envelope protein of amphotropic MuLVs. This MAb appears to recognize a conformational epitope within the envelope protein, as it reacts strongly with live virus and live infected cells, but does not react with formalin-fixed or alcohol-fixed infected cells or denatured viral envelope protein in immunoblots. In contrast, Mab 538 reacts only with an epitope unique to the envelope protein of the Moloney (Mo-) strain of MuLV, a prototypic ecotropic MuLV that is the basis for many retroviral tools used in molecular biology. MAb 538 can react with live cells and viruses, or detergent denatured or fixed envelope protein. The derivation of these antibodies as well as their characterization with regard to their isotype, range of reactivity with different MuLVs and utility in different immunological procedures are described in this study.