Cohen et al Based on data from the Human Protein Atlas(HPA) GPCRs targeted by human microbial acyl amides are localized to the gastrointestinal tract and its associated immune cells. In mouse models, this collection of gastrointestinal tract localized GPCRs have been reported to affect diverse mucosal functions including metabolism(GPRI19), immune cell differentiation (SIPR4, PTGIR, PTGER4), immune cell trafficking(SIPR4, G2A)and tissue repair(Ptgir) It is not possible at this time to look for co-localization of GPCR and hm-NAS gene expression in specific gastrointestinal niches, as neither the HMP nor the HPa are sufficiently comprehensive in their survey of human body sites. Nonetheless, 16S and metagenomic deep sequencing studies link bacteria containing hm-NAS genes or hm- NAS genes themselves to specific locations in the gastrointestinal tract where GPCRs of nterest are expressed (Extended Data Fig 4) Bacterial and human ligands share structure and function Human microbiota-encoded N-acyl amides bear structural similarity to endogenous GPCr- active ligands(Fig. 4). The clearest overlap in structure and function between bacterial and human GPCR-active ligands is for the endocannabinoid receptor GPR119(Fig. 4 and 5) Endogenous GPRI19 ligands include oleoylethanolamide(OEA)and the dietary lipid derivative 2-oleoyl glycerol(2-0G) 15, 16 In our heterologous expression experiment we isolated both the palmitoyl and oleoyl analogs of N-acyl serinol. The latter only differs from 2-OG by the presence of an amide instead of an ester and from Oea by the presence of an dditional ethanol substituent. N-oleoyl serinol is a similarly potent GPR119 agonist compared to the endogenous ligand OEa(EC50 12 AM vS. 7 uM) but elicits almost a 2-fold greater maximum GPRI19 activation(Fig 5a). N-palmitoyl derivatives of all 20 natural amino acids were synthesized and none activated GPri19 by more than 37% relative to OEA (Fig. 5b). The generation of a potent and specific long-chain N-acyl-based GPRI ligand therefore necessitates a more complex biosynthesis than the simple N-acylation of an amino acid as is commonly seen for characterized NAs enzymes. In this case, the biosynthesis of N-acyl serinols is achieved through the coupling of an NAs domain with an aminotransferase that is predicted to generate serinol from glycerol(Extended Data Fig. 2 The endogenous agonist for SIPR4, sphingosine-l-phosphate(SIP)and the M-3- hydroxypalmitoyl ornithine/lysine family of bacterial agonists share similar head group charges. SIP is a significantly more potent agonist(EC500.09 HM vS EC50 32 uM) however, the bacterial agonists are more specific for SIPR4. The bacterial N-3- hydroxypalmitoyl ornithine did not activate SIPRl, 2, or 3 in our GPCR screen, whereas SIP activates all four members of the SIP receptor family tested No direct comparison could be made between the microbiota-derived and endogenous ligands for PTGiR or PtgeR4 as there are no known endogenous antagonists for these receptors. Many human GPCRs remain orphan receptors lacking known endogenous ligands. Ligands for at least some of these receptors will undoubtedly be found among the small molecules produced by the human microbiota. G2A is an orphan receptor and therefore does not have a well-defined endogenous agonist, although it has been reported to respond to lysophosphatidylcholine. 7, 18 We found that the bacterial metabolites N-3- hydroxypalmitoyl glycine(commendamide) and N-palmitoyl alanine, both activate g2A Nature. Author manuscript; available in PMC 2018 February 28Based on data from the Human Protein Atlas (HPA) GPCRs targeted by human microbial N￾acyl amides are localized to the gastrointestinal tract and its associated immune cells. In mouse models, this collection of gastrointestinal tract localized GPCRs have been reported to affect diverse mucosal functions including metabolism (GPR119), immune cell differentiation (S1PR4, PTGIR, PTGER4), immune cell trafficking (S1PR4, G2A) and tissue repair (PTGIR).9–14 It is not possible at this time to look for co-localization of GPCR and hm-NAS gene expression in specific gastrointestinal niches, as neither the HMP nor the HPA are sufficiently comprehensive in their survey of human body sites. Nonetheless, 16S and metagenomic deep sequencing studies link bacteria containing hm-NAS genes or hm￾NAS genes themselves to specific locations in the gastrointestinal tract where GPCRs of interest are expressed (Extended Data Fig. 4). Bacterial and human ligands share structure and function Human microbiota-encoded N-acyl amides bear structural similarity to endogenous GPCR￾active ligands (Fig. 4). The clearest overlap in structure and function between bacterial and human GPCR-active ligands is for the endocannabinoid receptor GPR119 (Fig. 4 and 5). Endogenous GPR119 ligands include oleoylethanolamide (OEA) and the dietary lipid derivative 2-oleoyl glycerol (2-OG).15,16 In our heterologous expression experiment we isolated both the palmitoyl and oleoyl analogs of N-acyl serinol. The latter only differs from 2-OG by the presence of an amide instead of an ester and from OEA by the presence of an additional ethanol substituent. N-oleoyl serinol is a similarly potent GPR119 agonist compared to the endogenous ligand OEA (EC50 12 µM vs. 7 µM) but elicits almost a 2-fold greater maximum GPR119 activation (Fig. 5a). N-palmitoyl derivatives of all 20 natural amino acids were synthesized and none activated GPR119 by more than 37% relative to OEA (Fig. 5b). The generation of a potent and specific long-chain N-acyl-based GPR119 ligand therefore necessitates a more complex biosynthesis than the simple N-acylation of an amino acid as is commonly seen for characterized NAS enzymes. In this case, the biosynthesis of N-acyl serinols is achieved through the coupling of an NAS domain with an aminotransferase that is predicted to generate serinol from glycerol (Extended Data Fig. 2). The endogenous agonist for S1PR4, sphingosine-1-phosphate (S1P) and the N-3- hydroxypalmitoyl ornithine/lysine family of bacterial agonists share similar head group charges. S1P is a significantly more potent agonist (EC50 0.09 µM vs. EC50 32 µM); however, the bacterial agonists are more specific for S1PR4. The bacterial N-3- hydroxypalmitoyl ornithine did not activate S1PR1, 2, or 3 in our GPCR screen, whereas S1P activates all four members of the S1P receptor family tested. No direct comparison could be made between the microbiota-derived and endogenous ligands for PTGIR or PTGER4, as there are no known endogenous antagonists for these receptors. Many human GPCRs remain orphan receptors lacking known endogenous ligands. Ligands for at least some of these receptors will undoubtedly be found among the small molecules produced by the human microbiota. G2A is an orphan receptor and therefore does not have a well-defined endogenous agonist, although it has been reported to respond to lysophosphatidylcholine.17,18 We found that the bacterial metabolites N-3- hydroxypalmitoyl glycine (commendamide) and N-palmitoyl alanine, both activate G2A. Cohen et al. Page 5 Nature. Author manuscript; available in PMC 2018 February 28. Author Manuscript Author Manuscript Author Manuscript Author Manuscript