The glycolipid transfer protein (GLTP) domain of phosphoinositol 4-phosphate adaptor protein-2 (FAPP2): Structure drives preference for simple neutral glycosphingolipids

Abstract

Phosphoinositol 4-phosphate adaptor protein-2 (FAPP2) plays a key role in glycosphingolipid (GSL) production using its C-terminal domain to transport newly synthesized glucosylceramide away from the cytosol-facing glucosylceramide synthase in the cis-Golgi for further anabolic processing. Structural homology modeling against human glycolipid transfer protein (GLTP) predicts a GLTP-fold for FAPP2 C-terminal domain, but no experimental support exists to warrant inclusion in the GLTP superfamily. Here, the biophysical properties and glycolipid transfer specificity of FAPP2-C-terminal domain have been characterized and compared with other established GLTP-folds. Experimental evidence for a GLTP-fold includes: i) far-UV circular dichroism (CD) showing secondary structure with high alpha-helix content and a low thermally-induced unfolding transition (~41 °C); ii) near-UV-CD indicating only subtle tertiary conformational change before/after interaction with membranes containing/lacking glycolipid; iii) Red-shifted tryptophan (Trp) emission wavelength maximum (λmax~352 nm) for apo-FAPP2-C-terminal domain consistent with surface exposed intrinsic Trp residues; iv) ‘signature’ GLTP-fold Trp fluorescence response, i.e., intensity decrease (~30%) accompanied by strongly blue-shifted λmax (~14 nm) upon interaction with membranes containing glycolipid, supporting direct involvement of Trp in glycolipid binding and enabling estimation of partitioning affinities. A structurally-based preference for other simple uncharged GSLs, in addition to glucosylceramide, makes human FAPP2-GLTP more similar to fungal HET-C2 than to plant AtGLTP1 (glucosylceramide-specific) or to broadly GSL-selective human GLTP. These findings along with the distinct mRNA exon/intron organizations originating from single-copy genes on separate human chromosomes suggest adaptive evolutionary divergence by these two GLTP-folds.

Modulation of Hepatitis C Virus Genome Replication by Glycosphingolipids and Four-Phosphate Adaptor Protein 2

“…7 and 8), further implying a direct role for LacCer in HCV replication. Hence, we propose that the supplemented LacCer contributes to HCV replication via nonvesicular and vesicular trafficking, as a recent report indicates that FAPP2 also binds to LacCer (80,81). However, we were surprised that GlcCer also would rescue HCV replication, as FAPP2 KD slightly increased the GlcCer level (Fig.…”

Phosphatidylserine Stimulates Ceramide 1-Phosphate (C1P) Intermembrane Transfer by C1P Transfer Proteins

“…A saturation response is eventually achieved, as max blue-shifts to ϳ342 nm and the total Trp emission intensity declines by ϳ20 -30%. A similar but stronger response occurs during glycolipid uptake by GLTP and related orthologs that differ from ACD11 by having a Trp residue within the glycolipid binding site (33)(34)(35). Titration of ACD11 with sphingosine 1-phosphate or phosphatidic acid, which contain phosphate headgroups but are not transferred (13), induced no significant change in Trp emission max (Fig.…”

“…Our experimental set-up takes these physiological factors into account. (38,44,45), the fungal HET-C2 GLTP (46), and the GLTPH domain of FAPP2 (35). With GLTP, including anionic phosphoglycerides, such as PA, PG, PS, and PI, in sphingolipid source vesicles impedes GalCer intermembrane transfer at low ionic strength due to enhanced membrane association by GLTP (i.e.…”

Arabidopsis Accelerated Cell Death 11, ACD11, Is a Ceramide-1-Phosphate Transfer Protein and Intermediary Regulator of Phytoceramide Levels

“…The clustered Lys/Arg residues of ACD11 form a positively-charged triad that is ideally arranged for binding phosphate, explaining the inability of ACD11 to bind sugar headgroups and transfer glycolipids (Petersen et al, 2008). It is noteworthy that Arg103 occupies the same position where Trp acts as a stacking plate for the Cer-linked headgroup sugar in human GLTP, fungal HET-C2, plant GLTP1, and human FAPP2 (Kamlekar et al, 2013; West et al, 2008). Conversely, residues analogous to Asp60 and His143 of ACD11 are absolutely conserved in the lipid headgroup recognition centers of all known GLTP-folds.…”