Cell surface–associated glycans mediate many cellular processes, including adhesion, migration, signaling, and extracellular matrix organization. The galactosylation of core fucose (GalFuc epitope) in paucimannose and complex-type N-glycans is characteristic of protostome organisms, including flatworms (planarians). Although uninvestigated, the structures of these glycans may play a role in planarian regeneration. Whole-organism MALDI-MS analysis of N-linked oligosaccharides from the planarian Schmidtea mediterranea revealed the presence of multiple isomeric high-mannose and paucimannose structures with unusual mono-, di-, and polygalactosylated (n = 3–5) core fucose structures; the latter structures have not been reported in other systems. Di- and trigalactosylated core fucoses were the most dominant glycomers. N-Glycans showed extensive, yet selective, methylation patterns, ranging from non-methylated to polymethylated glycoforms. Although the majority of glycoforms were polymethylated, a small fraction also consisted of non-methylated glycans. Remarkably, monogalactosylated core fucose remained unmethylated, whereas its polygalactosylated forms were methylated, indicating structurally selective methylation. Using database searches, we identified two potential homologs of the Galβ1–4Fuc–synthesizing enzyme from nematodes (GALT-1) that were expressed in the prepharyngeal, pharyngeal, and mesenchymal regions in S. mediterranea. The presence of two GALT-1 homologs suggests different requirements for mono- and polygalactosylation of core fucose for the formation of multiple isomers. Furthermore, we observed variations in core fucose glycosylation patterns in different planarian strains, suggesting evolutionary adaptation in fucose glycosylation. The various core chitobiose modifications and methylations create >60 different glycoforms in S. mediterranea. These results contribute greatly to our understanding of N-glycan biosynthesis and suggest the presence of a GlcNAc-independent biosynthetic pathway in S. mediterranea.
Methionine and cysteine metabolisms are important for the survival and pathogenesis of
Mycobacterium tuberculosis
(
Mtb
). The transsulfuration pathway converts methionine to cysteine and represents an important link between antioxidant and methylation metabolism in diverse organisms. Using a combination of biochemistry and cryo–electron microscopy, we characterized the first enzyme of the transsulfuration pathway, cystathionine β-synthase (
Mtb
Cbs) in
Mtb
. We demonstrated that
Mtb
Cbs is a heme-less, pyridoxal-5′-phosphate–containing enzyme, allosterically activated by
S
-adenosylmethionine (SAM). The atomic model of
Mtb
Cbs in its native and SAM-bound conformations revealed a unique mode of SAM-dependent allosteric activation. Further, SAM stabilized
Mtb
Cbs by sterically occluding proteasomal degradation, which was crucial for supporting methionine and redox metabolism in
Mtb
. Genetic deficiency of
Mtb
Cbs reduced
Mtb
survival upon homocysteine overload in vitro, inside macrophages, and in mice coinfected with HIV. Thus, the
Mtb
Cbs-SAM axis constitutes an important mechanism of coordinating sulfur metabolism in
Mtb
.
Planaria is an ideal system to study factors involved in regeneration and tissue homeostasis. Little is known about the role of metabolites and small molecules in stem cell maintenance and lineage specification in planarians. Using liquid chromatography and mass spectrometry (LC‐MS)‐based quantitative metabolomics, we determined the relative levels of metabolites in stem cells, progenitors, and differentiated cells of the planarian Schmidtea mediterranea. Tryptophan and its metabolic product serotonin are significantly enriched in stem cells and progenitor population. Serotonin biosynthesis in these cells is brought about by a noncanonical enzyme, phenylalanine hydroxylase. Knockdown of Smed‐pah leads to complete disappearance of eyes in regenerating planaria, while exogenous supply of serotonin and its precursor rescues the eyeless phenotype. Our results demonstrate a key role for serotonin in eye regeneration.
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