Fig. 8: Impact of the gut microbiome on the mouse brain glycoproteome.

a, Three groups of six adult germ-free mice (three males and three females) were colonized by B. uniformis, by a defined eight-member community of human gut commensal or remained germ-free for 2 weeks. b, Spearman correlation of glycopeptide intensities between biological replicates after protein abundance normalization. c, PCA of normalized reporter intensities of the 18 brain samples. d, N-glycopeptide regulation in the mouse brain depending on gut microbiome composition. e, Overlap of significantly regulated proteins at the glycoproteome and proteome level. f, Mitochondrial proteins involved in cellular respiration and G protein γ-subunits regulated in protein abundance. g, Cytoskeleton proteins change in thermal stability upon gut microbiome colonization (n, number of proteins in each family; actins: Actb, Acta2 and Actg1; dyneins: Dnm1, Dnm2 and Dnm3; myosins: Myh10, Myh11, Myh9 and Myl12b; α-tubulins: Tuba1b, Tuba4a, Tuba8 and Tubal3; β-tubulins: Tubb1, Tubb2, Tubb2b, Tubb3, Tubb4a, Tubb4b, Tubb5 and Tubb6; γ-tubulins: Tubg1 and Tubg2). h, ΔAUC of thermal stability of proteins in brains of germ-free and eight-member colonized mice. Glycoproteins with at least one glycopeptide changing significantly in abundance upon colonization and proteins with GO term ‘structural molecule activity’ had higher thermal stability compared to other glycoproteins (two-sided t-test) The horizontal lines represent the median. i, N-glycosylation modulation on proteins involved in neurotransmission. j, Cntnap1 changes in thermal stability upon gut microbiome colonization. k, Site-specific regulation of N-glycosylation on the Grin2a glutamate receptor. Each vertical line represents one glycopeptide and each dot represents one replicate (3 conditions × 6 biological replicates). l, Eaat2 encodes a membrane-bound transporter, glycosylated at the N205 and N215 sites on an extracellular loop (blue). Human structure combined with AlphaFold predictions for the extracellular region is shown. The light-gray structure represents the homotrimer (Protein Data Bank 7VR8). Yellow planes depict the membrane orientation; dark-blue and dark-gray regions depict the predicted structures aligned to the template structure. Spheres indicate the N205 and N215 glycosites. m, The glycoforms on the two glycosylation sites of the Eaat2 protein are differentially modulated upon gut microbiota colonization. n, Solubility of the Eaat2 glycoforms from the solubility experiment. Gut-microbiome-modulated glycoforms are highlighted. The horizontal lines represent the median.