Semisynthesis of a Glycosylphosphatidylinositol‐Anchored Prion Protein

Abstract

Pinning down the role of the anchor: The chemical synthesis of a cysteine‐modified glycosylphosphatidylinositol (GPI) anchor provides access to homogeneous GPI‐anchored prion protein through expressed protein ligation (see scheme). By this method, it should be possible to investigate the influence of the complex posttranslational GPI modification on protein structure and function.

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

“…We have successfully extended our previously developed expressed protein ligation strategy to generate multimilligram amounts of three different PrP variants. Our previous results demonstrate that homogeneous, folded PrPs equipped with a membrane anchor behave similarly to GPI-modified PrP in vitro (23), and they have allowed us to address several questions about the membrane interaction and conformational changes of PrPs.…”

“…7). These results suggest that attaching PrP constructs via their C-terminal membrane anchor helps the N-terminal polybasic region of PrP (residues [23][24][25][26][27], which is lacking in the T_PrP construct, to enhance the electrostatic interaction of PrP with anionic phospholipid membranes. Indeed, Wang et al (33) already determined that this region initiates (electrostatic) interaction between PrP and anionic phospholipids.…”

“…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). We demonstrated previously that PrP with the GPI anchor mimic performs similarly to recombinant PrP equipped with a synthetic GPI anchor in comparative aggregation assays (23). This finding implies that the effects of both membrane anchors on PrP conversion are very similar and that the lipidated peptide constitutes a useful and easily accessible GPI anchor mimic.…”

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

“…26,35,37,102 A possible solution is the chemical synthesis of representative diacylglycerophosphoinositolglycan species, but currently these methods are complicated and not well applicable for protein anchoring. 103,104 An example of this problem is to set up appropriate experiments for answering how important the GPI-anchoring is in the basic PrP Sc replication process. The demand for membrane associated PrP analogues inspired huge efforts to prepare semisynthetic lipo-PrP derivatives (Figure 6).…”

“…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.…”