Abstract
Although the bioactive sphingolipid ceramide is an important cell signaling molecule, relatively few direct ceramide-interacting proteins are known. We used an approach combining yeast surface cDNA display and deep sequencing technology to identify novel proteins binding directly to ceramide. We identified 234 candidate ceramide-binding protein fragments and validated binding for 20. Most (17) bound selectively to ceramide, although a few (3) bound to other lipids as well. Several novel ceramide-binding domains were discovered, including the EF-hand calcium-binding motif, the heat shock chaperonin-binding motif STI1, the SCP2 sterol-binding domain, and the tetratricopeptide repeat region motif. Interestingly, four of the verified ceramide-binding proteins (HPCA, HPCAL1, NCS1, and VSNL1) and an additional three candidate ceramide-binding proteins (NCALD, HPCAL4, and KCNIP3) belong to the neuronal calcium sensor family of EF hand-containing proteins. We used mutagenesis to map the ceramide-binding site in HPCA and to create a mutant HPCA that does not bind to ceramide. We demonstrated selective binding to ceramide by mammalian cell-produced wild type but not mutant HPCA. Intriguingly, we also identified a fragment from prostaglandin D2synthase that binds preferentially to ceramide 1-phosphate. The wide variety of proteins and domains capable of binding to ceramide suggests that many of the signaling functions of ceramide may be regulated by direct binding to these proteins. Based on the deep sequencing data, we estimate that our yeast surface cDNA display library covers ∼60% of the human proteome and our selection/deep sequencing protocol can identify target-interacting protein fragments that are present at extremely low frequency in the starting library. Thus, the yeast surface cDNA display/deep sequencing approach is a rapid, comprehensive, and flexible method for the analysis of protein-ligand interactions, particularly for the study of non-protein ligands.
Highlights
From the Department of Anesthesia, UCSF Helen Diller Family Comprehensive Cancer Center, University of California at San Francisco, San Francisco, California 94110
Based on the deep sequencing data, we estimate that our yeast surface cDNA display library covers ϳ60% of the human proteome and our selection/deep sequencing protocol can identify targetinteracting protein fragments that are present at extremely low frequency in the starting library
Generation of Polyclonal Yeast Display Selection Outputs Enriched for Ceramide Binding—We have previously described the construction and application of large (Ͼ30 million clones) yeast surface display libraries expressing human protein fragments derived from cDNA from multiple human tissue sources (13–15, 20 –24)
Summary
Generation of Polyclonal Populations of Yeast Enriched with Clones Displaying Ceramide-binding Protein Fragments—The construction of yeast displayed human cDNA libraries and protocols, and media recipes for their use have been described previously (13, 20 –25). Yeast cells from 3 ml of the overnight galactose-induced cDNA display library culture were collected by centrifugation, washed twice with PBS, resuspended in 1 ml of PBS, added to the ceramide-coated Dynabeads, and incubated for 2 h with rotation at 25 °C. Induced yeast clones were washed twice with PBS and incubated with rotation for 1 h at room temperature with 200 nM biotinylated ceramide, C1P, sphingomyelin (SM), phosphatidylcholine (PC), or phosphatidylethanolamine (PE) (Echelon Biosciences) and 1:1000 mouse anti-Xpress antibody (Thermo Fisher/ Life Technologies, Inc./Invitrogen) in PBS to monitor surface display levels. The HPCA 1 ϩ 2 and HPCA 3 ϩ 4 mutagenized yeast display libraries were induced as described above, and 2 ml of the induced cultures were washed twice with PBS and resuspended in 1 ml of PBS with 1 M ceramide-biotin and 1:500 mouse anti-V5 antibody (Pierce/Thermo Scientific, Waltham, MA) and incubated for 1 h with rotation at 25 °C. Images were captured using a C-DiGit blot scanner (LI-COR Biosciences, Lincoln, NE)
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