Sortase-A-based strategy for the analysis of CD1d-associated lipids — ASN Events
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Sortase-A-based strategy for the analysis of CD1d-associated lipids (#96)

Maren Thomaier 1 , Yuting Wang 1 , Emilie Huc-Claustre 2 , Laura Trautenberg 1 , Sandrine Baltzer 1 , Lingyun Dai 3 , Gijs Grotenberg 3 , Andrej Shevchenko 4 , Sebastian Zeissig 1 5
  1. DFG-Center for Regenerative Therapies Dresden Technische Universität Dresden, Dresden, SAXONY, Germany
  2. CRTC Cancer Research Centre Toulouse, Toulouse, France
  3. Singapore-MIT Alliance Department, National University of Singapore, Singapore
  4. Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
  5. Universitäts Klinkum Karl Gustav Carus, Dresden, Germany

The analysis of lipids associated with CD1 is technically challenging as detergents required for the extraction of CD1 from membranes lead to the loss of bound lipids. The majority of studies that previously analyzed CD1-associated lipids therefore used genetically modified CD1 proteins lacking the transmembrane domain and that are directly secreted into the cell culture supernatant after passage through the secretory pathway1-3. These studies gave important information on the endogenous lipids bound to CD11-3. However, lack of the cytoplasmic tail of soluble CD1 proteins is associated with defect endolysosomal trafficking and thus does not allow the study of lipids that associate with CD1 in these compartments. We therefore aimed to build on recent studies that developed cleavable CD1 proteins with cytoplasmic tails3, 4 to study CD1-associated lipids.

To this end, we developed an engineered CD1d protein carrying the recognition motif of Sortase-A (SortA) from S. aureus in its extracellular juxtamembrane domain. The transpeptidase SortA cleaves its recognition sequence, which is not present in eukaryotes, forming an acyl-enzyme intermediate and subsequently links the cleaved protein covalently to an N-terminal triglycine motif6, 7. This strategy leads to a surface cleavable CD1d protein with a C-terminal tag allowing for detergent-free extraction and tag-based purification of CD1d that is suitable for the analysis of associated lipids using shotgun and HPLC-MS based lipidomics.

Mouse CD1d carrying the SortA recognition motif could be expressed in various cell lines, showed expression levels comparable to wildtype CD1d and was able to load and present exogenous lipid antigens to NKT cells. Moreover, nearly complete shaving of cell-surface CD1d was achieved without alterations in cell viability. CD1d could be coupled to various tags and purified using affinity chromatography. As such, SortA-based strategies allow for detergent-free, enzymatic harvest of CD1d proteins from the cell surface for subsequent analysis of associated lipids.

  1. 1) Cox, D., Fox, L., Tian, R., Bardet, W., Skaley, M., Mojsilovic, D., Gumperz, J. & Hildebrand, W. Determination of cellular lipids bound to human CD1d molecules. PLoS One 4, e5325 (2009).
  2. 2) Huang, S., Cheng, T.Y., Young, D.C., Layre, E., Madigan, C.A., Shires, J., Cerundolo, V., Altman, J.D. & Moody, D.B. Discovery of deoxyceramides and diacylglycerols as CD1b scaffold lipids among diverse groove-blocking lipids of the human CD1 system. Proc Natl Acad Sci U S A 108, 19335-19340 (2011).
  3. 3) Yuan, W., Kang, S.J., Evans, J.E. & Cresswell, P. Natural lipid ligands associated with human CD1d targeted to different subcellular compartments. J Immunol 182, 4784-4791 (2009).
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  5. 5) Olszak T, Neves JF, Dowds CM, Baker K, Glickman J, Davidson NO, Lin CS, Jobin C, Brand S, Sotlar K, Wada K, Katayama K, Nakajima A, Mizuguchi H, Kawasaki K, Nagata K, Müller W, Snapper SB, Schreiber S, Kaser A, Zeissig S, Blumberg RS. Protective mucosal immunity mediated by epithelial CD1d and IL-10. Nature. 2014 May 22;509(7501):497-502.
  6. 6) Popp, M.W., Antos, J.M., Grotenbreg, G.M., Spooner, E. & Ploegh, H.L. Sortagging: a versatile method for protein labeling. Nat Chem Biol 3, 707-708 (2007).
  7. Witte, M.D., Wu, T., Guimaraes, C.P., Theile, C.S., Blom, A.E.M., Ingram, J.R., Li, Z., Kundrat, L., Goldberg, S.D. & Ploegh, H.L. Site-specific protein modification using immobilized sortase in batch and continuous-flow systems. Nat. Protocols 10, 508-516 (2015).