The lipid transfer protein Saposin B does not directly bind CD1d for lipid antigen loading

Abstract

Lipid antigens are presented on the surface of cells by the CD1 family of glycoproteins, which have structural and functional similarity to MHC class I molecules. The hydrophobic lipid antigens are embedded in membranes and inaccessible to the lumenal lipid-binding domain of CD1 molecules. Therefore, CD1 molecules require lipid transfer proteins for lipid loading and editing. CD1d is loaded with lipids in late endocytic compartments, and lipid transfer proteins of the saposin family have been shown to play a crucial role in this process. However, the mechanism by which saposins facilitate lipid binding to CD1 molecules is not known and is thought to involve transient interactions between protein components to ensure CD1-lipid complexes can be efficiently trafficked to the plasma membrane for antigen presentation. Of the four saposin proteins, the importance of Saposin B (SapB) for loading of CD1d is the most well-characterised. However, a direct interaction between CD1d andSapB has yet to be described. In order to determine how SapB might load lipids onto CD1d, we used purified, recombinant CD1d and SapB and carried out a series of highly sensitive binding assays to monitor direct interactions. Using equilibrium binding analysis, chemical cross-linking and cocrystallisation experiments, under a range of different conditions, we could not demonstrate a direct interaction. This work establishes comprehensively that the role of SapB in lipid loading does not involve direct binding to CD1d. We discuss the implication of this for our understanding of lipid loading of CD1d and propose several factors that may influence this process.

A tetrameric assembly of saposin A: increasing structural diversity in lipid transfer proteins

“…PCR product encoding human AC (residues 22-395) was produced from this cDNA using primers (5'-GAAACTAGTCAGCACGCGCCGCCGTGG-3' and 5'-GTGCTTAAGCCAACCTATACAAGGGTCAGGGC-3'). For production of an inducible, stable cell line, this PCR product was cloned, using SpeI and AflII, into our modified version of the piggyBac target protein plasmid, PB-T-H6, encoding an N-terminal secretion signal and C-terminal hexahistidine tag (Shamin et al, 2019). HEK293F cells were triple-transfected with PB-T-H6 containing AC, PB-RN and PBase using a DNA mass ratio of 5:1:1 and transfected cells were selected with geneticin using the established protocol for the piggyBac expression system (Li et al, 2013).…”