De novo phytosterol synthesis in animals

Abstract

Sterols are vital for nearly all eukaryotes. Their distribution differs in plants and animals, with phytosterols commonly found in plants whereas most animals are dominated by cholesterol. We show that sitosterol, a common sterol of plants, is the most abundant sterol in gutless marine annelids. Using multiomics, metabolite imaging, heterologous gene expression, and enzyme assays, we show that these animals synthesize sitosterol de novo using a noncanonical C-24 sterol methyltransferase (C₂₄-SMT). This enzyme is essential for sitosterol synthesis in plants, but not known from most bilaterian animals. Our phylogenetic analyses revealed that C₂₄-SMTs are present in representatives of at least five animal phyla, indicating that the synthesis of sterols common to plants is more widespread in animals than currently known.

Figure 1 ∣. Olavius algarvensis has an unusual sterol profile dominated by sitosterol, a common plant sterol.

A, Extracted-ion chromatograms (XIC) of cholesterol ([M-H₂O+H]⁺ C₂₇H₄₅ at m/z 369.352 (red)) and sitosterol ([M-H₂O+H]⁺ C₂₉H₄₉ at m/z 397.383 (blue)). The XICs were generated from lipid extracts of (from top to bottom): the gutless marine annelid O. algarvensis, the freshwater annelid Tubifex tubifex, and the seagrass Posidonia oceanica. B, Chemosynthetic symbiotic bacteria are located just below the cuticle of O. algarvensis.16S rRNA fluorescence in situ hybridization (FISH) image of a cross section through a worm showing the symbionts in yellow (general eubacterial probe) and host nuclei in blue (DAPI). C and D, Distribution of sitosterol and cholesterol in O. algarvensis measured by TOF-SIMS C, Summed intensity of cholesterol ions (m/z 369.38, 385.34, 401.35) measured with TOF-SIMS. D, Summed intensity of sitosterol ions (m/z 397.47, 383.37, 413.45) as measured by TOF-SIMS. E, The ¹³C isotopic composition of sterols in gutless annelids differed from that of the neighboring seagrass (P. oceanica) and sediment porewater. Scale bar in B-D 100 μm.

Figure 2 ∣. Olavius algarvensis encodes and expresses a C₂₄-SMT which catalyzes two consecutive methylations, using desmosterol as the first and 24-methylene-cholesterol as the second substrate.

A, The O. algarvensis C₂₄-SMT gene consists of four exons, separated by three introns. The exons form a 1071 bp open reading frame encoding a 356 amino-acid polypeptide. The four conserved regions of the enzyme are highlighted by red arrows. B, Chromatograms of enzymatic assays with desmosterol (top) and 24-methylene-cholesterol (bottom) as substrates. O. algarvensis C₂₄-SMT, after overexpression in E. coli, added a methyl group to the side chains of desmosterol and 24-methylene-cholesterol. In the first methylation desmosterol, an intermediate of cholesterol synthesis, was methylated to produce 24-methylene-cholesterol (C₂₈ sterol). In the second methylation, 24-methylene-cholesterol was methylated to produce a C-29 sterol, most likely (epi)clerosterol (C₂₉ sterol). C, Mass spectra of the different substrates and alkylated products from the enzymatic assays. Sterol intermediates differ by the number of methyl groups (CH₂ at m/z 14) attached to their side chain. The side chain of desmosterol is not alkylated, 24-methylene-cholesterol has a methyl group at C-24, and (epi)clerosterol has two methyl groups at C-24 and C-28. The substrates and alkylated products were identified by MS, retention time and comparison with standardsThe fragmentation pattern suggests that the methyl groups were added to the side chain of the sterols. The stereochemistry of the methyl-groups was not determined. D, Structural representation of the two methylation steps in O. algarvensis. E, Comparison of the enzyme used in the proposed sterol synthesis pathways in Olavius to the canonical cholesterol and sitosterol synthesis pathways (similar enzymatic reactions are colored similarly). The first six steps are common to both cholesterol and sitosterol synthesis pathways. This trunk pathway branches off after the synthesis of desmosterol. For sitosterol synthesis, desmosterol is first methylated by C₂₄-SMT to 24-methylene-cholesterol, which is then methylated in a second, consecutive step by C₂₄-SMT to (epi)clerosterol. (Epi)clerosterol is reduced to sitosterol by a sterol C24-reductase (DHCR24, DIM). Squalene monooxygenase (SQE), oxydosqualene cyclase (LAS, CAS), sterol 14 demethylase (CYP51), sterol 14-reductase (LBR, FK), C-4 demethylation (C-4 dem.), Sterol Δ7-Δ8 isomerase (EBP, HYD1), sterol 5-desaturase (SC5DL, DWF7), sterol Δ7 reductase (DHCR7, DWF5), and C-24 sterol methyltransferase (C₂₄-SMT, SMT1, SMT2).

Figure 3 ∣. C₂₄-SMT homologs are widely distributed across the tree of life including at least five animal phyla.

A, Maximum likelihood amino acid tree of eukaryotic and bacterial C₂₄-SMTs. Sequences were clustered at 90% identity. Nodes with ultrafastbootstrap (UFB) values > 95 % are shown with grey circles. Nematode C₄-SMTs were used as an outgroup. The homologs found in animal datasets are highlighted in green. Clades with experimentally verified C₂₄-SMTs are outlined in black . B, Collapsed version of the tree shown in A. C₂₄-SMTs fall into nine well supported clades (UFB >95%). C, Most eukaryotic clades had several phylogenetically distinct homologs. D, Detailed view of the Annelida (clade 3.I from A). The phylogeny of annelid C₂₄-SMTs largely corresponds to their evolutionary history, exceptions are shown in red.