The binding of prion proteins to serum components is affected by detergent extraction conditions

Abstract

As many GPI anchored proteins, PrP C and its abnormal conformer PrP Sc , are inserted into membrane microdomains known as rafts. Upon raft disruption, PrP C becomes soluble, while PrP Sc aggregates into insoluble structures. It was recently published that, as opposed to PrP C , PrP Sc , as well as its protease resistant core PrP27-30, can bind specifically to plasminogen and other serum components. These findings were suggested to have important physiological implications in transmissible spongiform encephalopathies (TSE) diagnosis and pathogenesis. In this work, we show that the binding of PrP Sc or PrP 27-30 to serum proteins occurs only at specific detergent combinations, in which disease associated PrPs are present in aggregated structures. At detergent conditions in which rafts are intact, it is actually PrP C. that binds to blood proteins, albeit not directly, but through neighboring rafts components. Our results therefore indicate that the binding of PrP Sc to blood components has no physiological relevance.

Non‐infectious aggregates of the prion protein react with several PrPSc‐directed antibodies

“…aggregates through non-specific, epitope-independent interactions (Morel et al 2004). Such interactions may be promoted by particular detergent solubilization conditions (Shaked et al 2002). These results raise the question of whether the antibody recognition of infectious and noninfectious PrP aggregates observed in our experiments is to some extent attributable to epitope-independent binding.…”

Plasminogen

“…[30][31][32][33] However, the ability of plasminogen to bind to PrP Sc was dependent on conditions of the lipid rafts and plasminogen was actually associated with PrP C in the intact lipid rafts. 34 To determine the functional relevance of this interaction for PrP Sc replication, we explored whether plasminogen enhances PrP Sc propagation using cell culture models and the in vitro PrP conversion assay, termed protein misfolding cyclic amplification (PMCA). 35 The addition of plasminogen in PMCA resulted in the generation of significantly more PrP Sc in a concentration-dependent manner (Fig.…”

Toward Molecular Dissection of PrPC-PrPSc Interactions

“…Throughout these binding experiments, we have attempted to reduce the unwanted possibility of identifying nonspecific interactions between the PrP graftedantibodies and abnormal PrP conformers. This is an important consideration for this study; Shaked et al (22) have clearly demonstrated that an unsuitable detergent environment can promote artifactual binding interactions with disease-associated PrP conformers. These authors posited that the observed nonspecific binding derives largely from the propensity of PrP Sc to aggregate under detergent extraction conditions that disrupt lipid membrane rafts within which the disease-associated PrP conformers are thought to exist (22).…”

“…This is an important consideration for this study; Shaked et al (22) have clearly demonstrated that an unsuitable detergent environment can promote artifactual binding interactions with disease-associated PrP conformers. These authors posited that the observed nonspecific binding derives largely from the propensity of PrP Sc to aggregate under detergent extraction conditions that disrupt lipid membrane rafts within which the disease-associated PrP conformers are thought to exist (22). For example, the use of detergents such as Nonidet P-40 in combination with deoxycholate or Tween 20 was observed to promote aggregation of PrP Sc , thereby abetting nonspecific binding interactions, possibly through avidity effects resulting from multiple low affinity interactions with individual constituents of the PrP aggregate.…”