title: 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 (λ(max)~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 λ(max) (~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.
authors.affiliation: The Hormel Institute, University of Minnesota, Austin, MN, USA.
authors.forename: Ravi Kanth
authors.initials: RK
authors.lastname: Kamlekar
authors.forename: Dhirendra K
authors.initials: DK
authors.lastname: Simanshu
authors.forename: Yong-guang
authors.initials: YG
authors.lastname: Gao
authors.forename: Roopa
authors.initials: R
authors.lastname: Kenoth
authors.forename: Helen M
authors.initials: HM
authors.lastname: Pike
authors.forename: Franklyn G
authors.initials: FG
authors.lastname: Prendergast
authors.forename: Lucy
authors.initials: L
authors.lastname: Malinina
authors.forename: Julian G
authors.initials: JG
authors.lastname: Molotkovsky
authors.forename: Sergei Yu
authors.initials: SY
authors.lastname: Venyaminov
authors.forename: Dinshaw J
authors.initials: DJ
authors.lastname: Patel
authors.forename: Rhoderick E
authors.initials: RE
authors.lastname: Brown
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002942:Circular Dichroism; D006028:Glycosphingolipids / Q000378:metabolism*; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; C511922:GLTP protein, human; D006028:Glycosphingolipids; C487038:PLEKHA8 protein, human