Synthesis and structural characterization of a mimetic membrane‐anchored prion protein

Abstract

During pathogenesis of transmissible spongiform encephalopathies (TSEs) an abnormal form (PrPSc) of the host encoded prion protein (PrPC) accumulates in insoluble fibrils and plaques. The two forms of PrP appear to have identical covalent structures, but differ in secondary and tertiary structure. Both PrPC and PrPSc have glycosylphospatidylinositol (GPI) anchors through which the protein is tethered to cell membranes. Membrane attachment has been suggested to play a role in the conversion of PrPC to PrPSc, but the majority of in vitro studies of the function, structure, folding and stability of PrP use recombinant protein lacking the GPI anchor. In order to study the effects of membranes on the structure of PrP, we synthesized a GPI anchor mimetic (GPIm), which we have covalently coupled to a genetically engineered cysteine residue at the C‐terminus of recombinant PrP. The lipid anchor places the protein at the same distance from the membrane as does the naturally occurring GPI anchor. We demonstrate that PrP coupled to GPIm (PrP–GPIm) inserts into model lipid membranes and that structural information can be obtained from this membrane‐anchored PrP. We show that the structure of PrP–GPIm reconstituted in phosphatidylcholine and raft membranes resembles that of PrP, without a GPI anchor, in solution. The results provide experimental evidence in support of previous suggestions that NMR structures of soluble, anchor‐free forms of PrP represent the structure of cellular, membrane‐anchored PrP. The availability of a lipid‐anchored construct of PrP provides a unique model to investigate the effects of different lipid environments on the structure and conversion mechanisms of PrP.

Structural changes of membrane-anchored native PrP C

“…Thus, the effect of membrane binding on the structure of PrP is naturally of interest. Recent studies used recPrP with synthetic membrane anchors and followed the secondary structure upon lipid contact by using either UV-CD spectroscopy (19) or FTIR measurements (20). Both studies concluded that the structure of recPrP, with a synthetic membrane anchor, is identical upon lipid contact to the structure of anchorless PrP in lipid-free solutions.…”

A C-terminal Membrane Anchor Affects the Interactions of Prion Proteins with Lipid Membranes

“…Thus, generating membrane-anchored PrP using recombinant expression in combination with selective chemical modifications is a viable alternative, which has been used by several groups so far (9,20,21). Here, we utilize a strategy developed previously in our group that relies on expressed protein ligation to prepare PrP con-taining a C-terminal membrane anchor that mimics a GPI anchor and conveys strong membrane attachment (22).…”

Development of fluorescent cholesterol derivatives for the exogenous introduction of proteins to the plasma membrane

“…100,101,105,106 Beyond, the fluorescent protein GFP was also used to demonstrate the membrane anchoring properties of such synthetic lipids. 93,94,100,101,103,[105][106][107][108][109] It was found that the removal of the carbohydrate moiety or its truncation can retain the anchoring function of the GPIs.…”

“…97 Even though the membrane attachment seems to be crucial for the pathogenesis, previous studies have only been performed with recombinant, soluble PrP lacking any membrane anchor 98,99 or with PrP having anchor mimics structurally not related to the native GPI anchor. 93,94,100,101 The structural consequences of the membrane association of these PrP derivatives have not been investigated on cellular or model membranes.…”

“…Semisynthetic GPI anchor mimetics attached to PrP or GFP. 93,94,100,101,[105][106][107] recycling endosomes similarly to GFP bearing a native GPI anchor. 110 However, the deletion of the glycan core significantly altered the lateral diffusion kinetics of these lipoproteins in the plasma membrane.…”