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.
Original language | English (US) |
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Pages (from-to) | 417-427 |
Number of pages | 11 |
Journal | Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids |
Volume | 1831 |
Issue number | 2 |
DOIs | |
State | Published - Feb 2013 |
Bibliographical note
Funding 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 .
Keywords
- Divergent evolution
- GLTP superfamily
- Glycosphingolipid binding and transfer
- Keywords
- Membrane interaction
- Near-UV and far-UV circular dichroism
- Tryptophan fluorescence