A chemical approach to unraveling the biological function of the glycosylphosphatidylinositol anchor

Abstract

The glycosylphosphatidylinositol (GPI) anchor is a C-terminal posttranslational modification found on many eukaryotic proteins that reside in the outer leaflet of the cell membrane. The complex and diverse structures of GPI anchors suggest a rich spectrum of biological functions, but few have been confirmed experimentally because of the lack of appropriate techniques that allow for structural perturbation in a cellular context. We previously synthesized a series of GPI anchor analogs with systematic deletions within the glycan core and coupled them to the GFP by a combination of expressed protein ligation and native chemical ligation Here we investigate the behavior of these GPI-protein analogs in living cells. These modified proteins integrated into the plasma membranes of a variety of mammalian cells and were internalized and directed to recycling endosomes similarly to GFP bearing a native GPI anchor. The GPI-protein analogs also diffused freely in cellular membranes. However, changes in the glycan structure significantly affected membrane mobility, with the loss of monosaccharide units correlating to decreased diffusion. Thus, this cellular system provides a platform for dissecting the contributions of various GPI anchor components to their biological function. cellular diffusion ͉ glycosylphosphatidylinositol-protein analogs ͉ intracellular trafficking ͉ GFP-GPI ͉ GPI glycan core

Mechanical Compressibility of the Glycosylphosphatidylinositol (GPI) Anchor Backbone Governed by Independent Glycosidic Linkages

“…High lateral mobility and diffusivity of the GPI anchored proteins has been attributed to the rigidity of the GPI anchor and its ability to prevent intermittent interactions with the lipid bilayer. 6 Flexibility of the anchor, on the other hand, is thought to secure access to multiple spatial orientations of the anchored protein and facilitate its interactions with other membrane-associated molecules. 7 There is a general consensus that assigning the biological function of an oligosaccharide requires not only a concise structural characterization, but also the overall dynamic picture of the carbohydrate topology.…”

The Protein Lipidation and Its Analysis

“…The relationship between structural diversity and function is difficult to establish due to the lack of appropriate methods. Bertozzi´s group has reported important contributions in this area, studying the role of different component of the glycan core on GPI function [23,24]. Additionally, the structural differences detected in GPI-units from parasites in contrast to mammals have been also exploited for the generation of vaccines [25].…”

Encapsulation and Surface Engineering of Pancreatic Islets: Advances and Challenges

“…Islet surface modification strategy involves covalent conjugation of molecules to islet cell surfaces. However, this technology is limited to the introduction of specified functional small molecules to cells and might interfere with cell physiology (Rabuka et al, 2008;Paulick et al, 2007). Layer-by-layer (LbL) technique has been recently applied as a new approach to modify islet surfaces (Krol et al, 2006;.…”