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

Ravi Kanth Kamlekar, Dhirendra K. Simanshu, Yong-Guang Gao, Roopa Kenoth, Helen M. Pike, Franklyn G. Prendergast, Lucy Malinina, Julian G. Molotkovsky, Sergei Yu Venyaminov, Dinshaw J. Patel, Rhoderick E Brown

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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 languageEnglish (US)
Pages (from-to)417-427
Number of pages11
JournalBiochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
Volume1831
Issue number2
DOIs
StatePublished - Feb 1 2013

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Keywords

  • Divergent evolution
  • GLTP superfamily
  • Glycosphingolipid binding and transfer
  • Keywords
  • Membrane interaction
  • Near-UV and far-UV circular dichroism
  • Tryptophan fluorescence

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