Structure and Orientation of a Glycosylphosphatidyl Inositol Anchored Protein at the Air/Water Interface

Abstract

The monolayer organization of intestinal alkaline phosphatase (AP), a glycosylphosphatidyl inositol (GPI) anchored dimeric protein (AP-GPI), was monitored using polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) and Brewster angle microscopy (BAM). The behavior of the monolayer was reversible during the compression−expansion cycles as shown by BAM. At high surface pressure, we observed formation of condensed protein domains. Upon decompression, the protein clusters remained stable over a period of several tenths of a minute, and then the condensed domain disappeared. These results suggest strong interactions between proteins forming clusters, which could be involved in the formation of membrane microdomains. The protein did not unfold as evidenced by PM-IRRAS, whatever the initial spreading surface pressure used. The PM-IRRAS spectra obtained during compression of a native AP-GPI monolayer induced slight reversible modifications on PM-IRRAS spectra. From the amide I/amide II ratio and the α-helix band position, it can be deduced that the protein was anchored at the interface with the GPI standing up and the great axis of the ellipsoid model of AP-GPI remained parallel to the interface. The PM-IRRAS spectra obtained during compression and their simulated spectra were consistent with the existence of small movements of intramolecular protein domains. Such movements may be related to the allosteric properties of mammalian alkaline phosphatases.

Characterizing the interactions between GPI-anchored alkaline phosphatases and membrane domains by AFM

“…The minimal size of bright dots was compatible with structural data [56] and the time course of their visualization at the membrane surface agreed with previous work done on direct incorporation of AP-GPI into liposomes [15]. The presence of larger spots at the membrane surface also gave direct support for BIAP having a marked tendency to form clusters, a hypothesis formulated from indirect biochemical [13] and physicochemical [78] evidences. It is worth noting that pretreatment of BIAP with phosphatidylinositol-specific phospholipase C to remove the GPI anchor prevented the imaging of the protein at the SLBs surface.…”

“…The bovine intestinal alkaline phosphatase (BIAP) presents about 90% sequence similarity with PLAP and is made of two identical monomers containing 486 amino acids and one GPI anchor. The apparent molecular weight from electrophoresis determination is about 130 kDa [78]. This size, associated with the possibility to purify reasonable amount of eukaryotic protein from a single source, makes AP-GPI attractive candidates for studying GPI-anchored proteins/model membrane domains interactions by AFM.…”

“…Most of the available biomimetic systems like LangmuirBlodgett films (LB), supported lipid bilayers (SLBs), large (LUVs), and giant unilamellar vesicles (GUVs) have been used to better characterize the formation of in-plane lipiddependent microdomains and lipid/AP-GPI interactions in membranes [15,17,21,52,55,67,78].…”

Adsorption kinetics and dilatational rheological studies for the soluble and anchored forms of alkaline phosphatase at the air/water interface

“…In this sense, to rebuild the surface structure with a high mechanical stability, the conformational and orientation changes must not be highly restricted. In our case, the presence of a phospholipid (hydrophobic anchor) covalently bound to the polypeptidic moiety of the protein gives the GPI-anchored alkaline phosphatase extremely peculiar surface properties, with the GPI-anchor driving the processes of adsorption, orientation, 10,25,[46][47][48] and packing 9 and, as stated by the present work, also influencing the surface elasticity properties. To the best of our knowledge, this is the first report concerning the elasticity properties of a GPI-anchored protein.…”

Subgel Phase Structure in Monolayers of Glycosylphosphatidylinositol Glycolipids

“…[11] Insights into the behavior of GPIs and GPI-APs in cell membranes could contribute to the understanding of the roles GPIs play in these processes. [12] Monolayers formed with GPI-APs have been investigated, [13] but the limited scope did not provide fundamental insights into the behavior of GPIs in cellular membranes. We aim to elucidate the structural characteristics and conformational behavior of GPIs in well-defined membrane models.…”