Eleven residues determine the acyl chain specificity of ceramide synthases.

Rotem Tidhar, Iris D Zelnik, Giora Volpert, Shifra Ben-Dor, Samuel Kelly, Alfred H Merrill, Anthony H Futerman

The Journal of biological chemistry 2018 Jun 22

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Lipids display large structural complexity, with ∼40,000 different lipids identified to date, ∼4000 of which are sphingolipids. A critical factor determining the biological activities of the sphingolipid, ceramide, and of more complex sphingolipids is their N-acyl chain length, which in mammals is determined by a family of six ceramide synthases (CerS). Little information is available about the CerS regions that determine specificity toward different acyl-CoA substrates. We previously demonstrated that substrate specificity resides in a region of ∼150 residues in the Tram-Lag-CLN8 domain. Using site-directed mutagenesis and biochemical analyses, we now narrow specificity down to an 11-residue sequence in a loop located between the last two putative transmembrane domains (TMDs) of the CerS. The specificity of a chimeric protein, CerS5(299-309→CerS2), based on the backbone of CerS5 (which generates C16-ceramide), but containing 11 residues from CerS2 (which generates C22-C24-ceramides), was altered such that it generated C22-C24 and other ceramides. Moreover, a chimeric protein, CerS4(291-301→CerS2), based on CerS4 (which normally generates C18-C22 ceramides) displayed significant activity toward C24:1-CoA. Additional data supported the notion that substitutions of these 11 residues alter the specificities of the CerS toward their cognate acyl-CoAs. Our findings may suggest that this short loop may restrict adjacent TMDs, leading to a more open conformation in the membrane, and that the CerS acting on shorter acyl-CoAs may have a longer, more flexible loop, permitting TMD flexibility. In summary, we have identified an 11-residue region that determines the acyl-CoA specificity of CerS. © 2018 Tidhar et al.

Citation

Rotem Tidhar, Iris D Zelnik, Giora Volpert, Shifra Ben-Dor, Samuel Kelly, Alfred H Merrill, Anthony H Futerman. Eleven residues determine the acyl chain specificity of ceramide synthases. The Journal of biological chemistry. 2018 Jun 22;293(25):9912-9921