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

• Published in: Biochimica et biophysica acta 2013-02, Vol.1831 (2), p.417-427
• Main Authors: Kamlekar, Ravi Kanth, Simanshu, Dhirendra K., Gao, Yong-guang, Kenoth, Roopa, Pike, Helen M., Prendergast, Franklyn G., Malinina, Lucy, Molotkovsky, Julian G., Venyaminov, Sergei Yu, Patel, Dinshaw J., Brown, Rhoderick E.
• Format: Article
• Language: English
• Subjects: Adaptor Proteins, Signal Transducing - chemistry; Adaptor Proteins, Signal Transducing - metabolism; Amino Acid Sequence; Carrier Proteins - chemistry; Carrier Proteins - metabolism; chromosomes; Circular Dichroism; Divergent evolution; exons; fluorescence; fungi; genes; GLTP superfamily; Glycosphingolipid binding and transfer; glycosphingolipids; Glycosphingolipids - metabolism; humans; introns; Membrane interaction; messenger RNA; Molecular Sequence Data; Near-UV and far-UV circular dichroism; Sequence Homology, Amino Acid; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet; tryptophan; Tryptophan fluorescence; wavelengths
• ISSN: 1388-1981; 0006-3002; 1879-2618
• DOI: 10.1016/j.bbalip.2012.10.010

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~352nm) 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 (~14nm) 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. ► FAPP2 enables glycosphingolipid synthesis in the Golgi by transfer of glucosylceramide. ► FAPP2 contains a modified GLTP-fold that can also transfer other neutral glycosphingolipids. ► Glycolipid selectivity of FAPP2-GLTP is more focused than human GLTP. ► Glycolipid compartmentation appears to mute evolutionary selection pressure and divergence.