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.
|Original language||English (US)|
|Number of pages||11|
|Journal||Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids|
|State||Published - Feb 2013|
Bibliographical noteFunding Information:
We are grateful for the support by NIH/NIGMS GM45928 & GM34847 , NIH/NCI CA121493 , Spanish Ministerio de Ciencia e Innovacion (MICINN BFU2010-17711 ), Russian Foundation for Basic Research 012-04-00168 ; Abby Rockefeller Mauzé Trust , and the Dewitt Wallace, Maloris, Mayo, and Hormel Foundations . The molecular graphic images shown in Figs. 5, 7 , and S1 were produced using UCSF Chimera ( NIH P41 RR-01081 ) provided by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco .
- Divergent evolution
- GLTP superfamily
- Glycosphingolipid binding and transfer
- Membrane interaction
- Near-UV and far-UV circular dichroism
- Tryptophan fluorescence