Genetic and functional diversity of ß-N-acetylgalactosamine-targeting glycosidases expanded by deep-sea metagenome analysis.

ß-N-Acetylgalactosamine-containing glycans play essential roles in several biological processes, including cell adhesion, signal transduction, and immune responses. ß-N-Acetylgalactosaminidases hydrolyze ß-N-acetylgalactosamine linkages of various glycoconjugates. However, their biological significance remains ambiguous, primarily because only one type of enzyme, exo-ß-N-acetylgalactosaminidases that specifically act on ß-N-acetylgalactosamine residues, has been documented to date. In this study, we identify four groups distributed among all three domains of life and characterize eight ß-N-acetylgalactosaminidases and ß-N-acetylhexosaminidase through sequence-based screening of deep-sea metagenomes and subsequent searching of public protein databases. Despite low sequence similarity, the crystal structures of these enzymes demonstrate that all enzymes share a prototype structure and have diversified their substrate specificities (oligosaccharide-releasing, oligosaccharide/monosaccharide-releasing, and monosaccharide-releasing) through the accumulation of mutations and insertional amino acid sequences. The diverse ß-N-acetylgalactosaminidases reported in this study could facilitate the comprehension of their structures and functions and present evolutionary pathways for expanding their substrate specificity.

Identification and characterization of a PL35 GAGs lyase with 4-O-sulfated N-acetylgalactosamine (A-type)-rich structures producing property.

Glycosaminoglycan (GAG) lyases are important tools for investigating the structure of GAGs and preparing low-molecular-weight GAGs. The PL35 family, a recently established polysaccharide lyase family, should be further investigated. In this study, we discovered a new GAG lyase, CHa1, which belongs to the PL35 family. When expressed heterologously in Escherichia coli (BL21), CHa1 exhibited high expression levels and solubility. The optimal activity was observed in Tris-HCl buffer (pH 7.0) or sodium phosphate buffer (pH 8.0) at 30 °C. The specific activities towards HA, CSA, CSC, CSD, CSE, and HS were 3.81, 13.03, 36.47, 18.46, 6.46, and 0.50 U/mg protein, respectively. CHa1 digests substrate chains randomly that acting as an endolytic lyase and shows a significant preference for GlcA-containing structures, prefers larger oligosaccharides (≥UDP8) and can generate a series of oligosaccharides composed mainly of the A unit when digesting CSA. These oligosaccharides include ΔC-A, ΔC-A-A, ΔC-A-A-A, ΔC-A-A-A-A, and ΔC-A-A-A-A-A. The residues Tyr257 and His421 play crucial roles in the catalytic process, and Ser211, Asn212, Asn213, Trp214, Gln216, Lys360, Arg460 and Gln462 may participate in the binding process of CHa1. This study on CHa1 contributes to our understanding of the PL35 family and provides valuable tools for investigating the structure of GAGs.

Research progress on asialoglycoprotein receptor-targeted radiotracers designed for hepatic nuclear medicine imaging.

Asialoglycoprotein receptor (ASGPR) specifically recognizes glycans terminated with ß-d-galactose or N-acetylgalactosamine. Its exclusive expression in mammalian hepatocytes renders it an ideal hepatic-targeted biomarker. To date, ASGPR-targeted ligands have been actively developed for drug delivery and hepatic imaging. This review provides a comprehensive summary of the progress achieved to-date in the field of developing ASGPR-targeted nuclear medicine imaging (NMI) radiotracers, highlighting the recent advancements over the last decade in terms of structure, radionuclides and labeling strategies. The biodistribution patterns, imaging characteristics, challenges and future prospective are discussed.

Host genetic regulation of human gut microbial structural variation.

Although the impact of host genetics on gut microbial diversity and the abundance of specific taxa is well established1-6, little is known about how host genetics regulates the genetic diversity of gut microorganisms. Here we conducted a meta-analysis of associations between human genetic variation and gut microbial structural variation in 9,015 individuals from four Dutch cohorts. Strikingly, the presence rate of a structural variation segment in Faecalibacterium prausnitzii that harbours an N-acetylgalactosamine (GalNAc) utilization gene cluster is higher in individuals who secrete the type A oligosaccharide antigen terminating in GalNAc, a feature that is jointly determined by human ABO and FUT2 genotypes, and we could replicate this association in a Tanzanian cohort. In vitro experiments demonstrated that GalNAc can be used as the sole carbohydrate source for F. prausnitzii strains that carry the GalNAc-metabolizing pathway. Further in silico and in vitro studies demonstrated that other ABO-associated species can also utilize GalNAc, particularly Collinsella aerofaciens. The GalNAc utilization genes are also associated with the host's cardiometabolic health, particularly in individuals with mucosal A-antigen. Together, the findings of our study demonstrate that genetic associations across the human genome and bacterial metagenome can provide functional insights into the reciprocal host-microbiome relationship.

Plasma Pharmacokinetics of N-Acetylgalactosamine-Conjugated Small-Interfering Ribonucleic Acids (GalNAc-Conjugated siRNAs).

Small-interfering ribonucleic acids (siRNAs) with N-acetylgalactosamine (GalNAc) conjugation for improved liver uptake represent an emerging class of drugs that modulate liver-expressed therapeutic targets. The pharmacokinetics of GalNAc-siRNAs are characterized by a rapid distribution from plasma to tissue (hours) and a long terminal plasma half-life, analyzed in the form of the antisense strand, driven by redistribution from tissue (weeks). Understanding how clinical pharmacokinetics relate to the dose and type of siRNA chemical stabilizing method used is critical, e.g., to design studies, to investigate safety windows, and to predict the pharmacokinetics of new preclinical assets. To this end, we collected and analyzed pharmacokinetic data from the literature regarding nine GalNAc-siRNAs. Based on this analysis, we showed that the clinical plasma pharmacokinetics of GalNAc-siRNAs are approximately dose proportional and similar between chemical stabilizing methods. This holds for both the area under the concentration-time curve (AUC) and the maximum plasma concentration (Cmax). Corresponding rat and monkey pharmacokinetic data for a subset of the nine GalNAc-siRNAs show dose-proportional Cmax, supra-dose-proportional AUC, and similar pharmacokinetics between chemical stabilizing methods​. Together, the animal and human pharmacokinetic data indicate that plasma clearance divided by bioavailability follows allometric principles and scales between species with an exponent of 0.75. Finally, the clinical plasma concentration-time profiles can be empirically described by standard one-compartment kinetics with first-order absorption up to 24 h after subcutaneous dosing, and by three-compartment kinetics with first-order absorption in general. To describe the system more mechanistically, we report a corrected and unambiguously defined version of a previously published physiologically based pharmacokinetic model.

O-GalNAc glycosylation determines intracellular trafficking of APP and Aß production.

A primary pathology of Alzheimer's disease (AD) is amyloid ß (Aß) deposition in brain parenchyma and blood vessels, the latter being called cerebral amyloid angiopathy (CAA). Parenchymal amyloid plaques presumably originate from neuronal Aß precursor protein (APP). Although vascular amyloid deposits' origins remain unclear, endothelial APP expression in APP knock-in mice was recently shown to expand CAA pathology, highlighting endothelial APP's importance. Furthermore, two types of endothelial APP-highly O-glycosylated APP and hypo-O-glycosylated APP-have been biochemically identified, but only the former is cleaved for Aß production, indicating the critical relationship between APP O-glycosylation and processing. Here, we analyzed APP glycosylation and its intracellular trafficking in neurons and endothelial cells. Although protein glycosylation is generally believed to precede cell surface trafficking, which was true for neuronal APP, we unexpectedly observed that hypo-O-glycosylated APP is externalized to the endothelial cell surface and transported back to the Golgi apparatus, where it then acquires additional O-glycans. Knockdown of genes encoding enzymes initiating APP O-glycosylation significantly reduced Aß production, suggesting this non-classical glycosylation pathway contributes to CAA pathology and is a novel therapeutic target.

N-glycan antennal modifications are altered in Caenorhabditis elegans lacking the HEX-4 N-acetylgalactosamine-specific hexosaminidase.

Simple organisms are often considered to have simple glycomes, but plentiful paucimannosidic and oligomannosidic glycans overshadow the less abundant N-glycans with highly variable core and antennal modifications; Caenorhabditis elegans is no exception. By use of optimized fractionation and assessing wildtype in comparison to mutant strains lacking either the HEX-4 or HEX-5 ß-N-acetylgalactosaminidases, we conclude that the model nematode has a total N-glycomic potential of 300 verified isomers. Three pools of glycans were analyzed for each strain: either PNGase F released and eluted from a reversed-phase C18 resin with either water or 15% methanol or PNGase Ar released. While the water-eluted fractions were dominated by typical paucimannosidic and oligomannosidic glycans and the PNGase Ar-released pools by glycans with various core modifications, the methanol-eluted fractions contained a huge range of phosphorylcholine-modified structures with up to three antennae, sometimes with four N-acetylhexosamine residues in series. There were no major differences between the C. elegans wildtype and hex-5 mutant strains, but the hex-4 mutant strains displayed altered sets of methanol-eluted and PNGase Ar-released pools. In keeping with the specificity of HEX-4, there were more glycans capped with N-acetylgalactosamine in the hex-4 mutants, as compared with isomeric chito-oligomer motifs in the wildtype. Considering that fluorescence microscopy showed that a HEX-4::enhanced GFP fusion protein colocalizes with a Golgi tracker, we conclude that HEX-4 plays a significant role in late-stage Golgi processing of N-glycans in C. elegans. Furthermore, finding more "parasite-like" structures in the model worm may facilitate discovery of glycan-processing enzymes occurring in other nematodes.

Multivalent Lactobionic Acid and N-Acetylgalactosamine-Conjugated Peptide Nucleic Acids for Efficient In Vivo Targeting of Hepatocytes.

Peptide nucleic acids (PNAs) are used/applied in various studies to target genomic DNA and RNA to modulate gene expression. Non-specific targeting and rapid elimination always remain a challenge for PNA-based applications. Here, the synthesis, characterization, in vitro and in vivo study of di lactobionic acid (diLBA) and tris N-acetyl galactosamine (tGalNAc) conjugated PNAs for liver-targeted delivery are reported. For proof of concept, diLBA, and tGalNAc conjugated PNAs (anti-miR-122 PNAs) were synthesized to target microRNA-122 (miR-122) which is over-expressed in the hepatic tissue. Different lengths of anti-miR-122 PNAs conjugated with diLBA and tGalNAc are tested. Cell culture and in vivo analyses to determine biodistribution, efficacy, and toxicity profile are performed. This work indicates that diLBA conjugates show significant retention in hepatocytes in addition to tGalNAc conjugates after in vivo delivery. Full-length PNA conjugates show significant downregulation of miR-122 levels and subsequent de-repression of its downstream targets with no evidence of toxicity. The results provide a robust framework for ligand-conjugated delivery systems for PNAs that can be explored for broader biomedical applications.

Synthesis of O-Linked Glycoconjugates in the Nervous System.

Glycoproteins carrying O-linked N-acetylgalactosamine, N-acetylglucosamine, mannose, fucose, glucose, and xylose are found in the nervous system. Lipids are glycosylated by distinct glycosylation enzymes as well. Membrane lipid, ceramide, is modified by the addition of either glucose or galactose to form glycosphingolipid, galactosylceramide, or glucosylceramide. Recent careful analyses by MS have identified glucosylated lipids of cholesterol and phosphatidic acid. These O-linked carbohydrate residues are found primarily on the outer surface of the plasma membrane or in the extracellular space. Their expression is cell or tissue specific and developmentally regulated. Due to their structural diversity, they play important roles in a variety of biological processes such as membrane transport, metabolic stress responses, cell-cell interactions and so on. Discoveries of human diseases associated with glycosylation enzyme deficits have proved modification of lipids and proteins with carbohydrates play critical roles in human health and disease in the nervous systems.

Mutation Hotspot for Changing the Substrate Specificity of ß-N-Acetylhexosaminidase: A Library of GlcNAcases.

ß-N-Acetylhexosaminidase from Talaromyces flavus (TfHex; EC 3.2.1.52) is an exo-glycosidase with dual activity for cleaving N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) units from carbohydrates. By targeting a mutation hotspot of the active site residue Glu332, we prepared a library of ten mutant variants with their substrate specificity significantly shifted towards GlcNAcase activity. Suitable mutations were identified by in silico methods. We optimized a microtiter plate screening method in the yeast Pichia pastoris expression system, which is required for the correct folding of tetrameric fungal ß-N-acetylhexosaminidases. While the wild-type TfHex is promiscuous with its GalNAcase/GlcNAcase activity ratio of 1.2, the best single mutant variant Glu332His featured an 8-fold increase in selectivity toward GlcNAc compared with the wild-type. Several prepared variants, in particular Glu332Thr TfHex, had significantly stronger transglycosylation capabilities than the wild-type, affording longer chitooligomers - they behaved like transglycosidases. This study demonstrates the potential of mutagenesis to alter the substrate specificity of glycosidases.

Identification of Multiple Domains of Entamoeba histolytica Intermediate Subunit Lectin-1 with Hemolytic and Cytotoxic Activities.

Galactose and N-acetyl-D-galactosamine-inhibitable lectin of Entamoeba histolytica have roles in the pathogenicity of intestinal amoebiasis. Igl1, the intermediate subunit lectin-1 of E. histolytica, has been shown to have both hemolytic and cytotoxic activities that reside in the C-terminus of the protein. To identify the amino acid regions responsible for these activities, recombinant proteins were prepared and used in hemolytic and cytotoxic assays. The results revealed that Igl1 has multiple domains with hemolytic and cytotoxic activities and that amino acids 787-846, 968-1028 and 1029-1088 are involved in these activities. The hemolytic activities of the fragments were partly inhibited by mannose, galactose and N-acetylgalactosamine, and glucose showed lower or negligible inhibitory effects for the activities. This is the first report of a protozoan protein with hemolytic and cytotoxic activities in multiple domains.

Mn-doped bimetallic synergistic catalysis boosts for enzymatic phosphorylation of N-Acetylglucosamine/ N-Acetylgalactosamine and their derivatives.

Metal-organic frameworks (MOFs), as advanced enzyme immobilization platforms for improving biocatalysis and protein biophysics, are rarely investigated as solid supports in the enzymatic synthesis of carbohydrate and derivatives, which can be attributed to the complex biochemical reaction mechanisms and the adverse interactions between the high polarity of substrate sugars, glycoenzymes and traditional MOFs. Here, we introduced divalent metal ion Mn2+ into MOF to prepare bimetallic MOF microreactor that encapsulated N-acetylhexosamine 1-Kinase (NahK), a critical anomeric kinase involved in the enzymatic synthesis of sugar nucleotide. The introduced Mn ions not only adjusted the microstructure of MOFs, but also participated in the enzymatic catalysis as cofactor, thus facilitated the N-acetylglucosamine/ N-acetylgalactosamine (GlcNAc/GalNAc) phosphorylation. The Mn-doped NahK@Zn-metal organic material (MOM), integrated with high catalytic activity, high stability, and high recoverability, solved the issues of immobilization related to glucokinase activity. These features significantly improved the operability and reduced the processing cost, assuring industrial application prospects for sugar nucleotides synthesis.

Preclinical Toxicological Assessment of A Novel siRNA, SLN360, Targeting Elevated Lipoprotein (a) in Cardiovascular Disease.

SLN360 is a liver-targeted N-acetyl galactosamine (GalNAc)-conjugated small interfering RNA (siRNA) with a promising profile for addressing lipoprotein (a)-related cardiovascular risk. Here, we describe the findings from key preclinical safety studies. In vitro, SLN360 specifically reduced LPA expression in primary human hepatocytes with no relevant off-target effects. In rats, 10 mg/kg subcutaneous SLN360 was distributed specifically to the liver and kidney (peak 126 or 246 mg/g tissue at 6 h, respectively), with <1% of peak liver levels observed in all other tested organs. In vitro, no genotoxicity and no effect on human Ether-a-go-go Related Gene currents or proinflammatory cytokine production was observed, whereas in vivo, no SLN360-specific antibodies were detected in rabbit serum. In rat and nonhuman primate 29-day toxicology studies, SLN360 was well tolerated at all doses. In both species, known GalNAc-conjugated siRNA-induced microscopic changes were observed in the kidney and liver, with small increases in alanine aminotransferase and alkaline phosphatase observed in the high dose rats. Findings were in line with previously described siRNA-GalNAc platform-related effects and all observations were reversible and considered nonadverse. In cynomolgus monkeys, liver LPA messenger RNA and serum lipoprotein (a) were significantly reduced at day 30 and after an 8-week recovery period. No dose-related changes in safety assessment endpoints were noted. No SLN360-induced cytokine production, complement activation, or micronucleus formation was observed in vivo. The toxicological profile of SLN360 presented here is restricted to known GalNAc siRNA effects and no other toxicity associated with SLN360 has been noted. The preclinical profile of SLN360 confirmed suitability for entry into clinical studies.

ABO genotype alters the gut microbiota by regulating GalNAc levels in pigs.

The composition of the intestinal microbiome varies considerably between individuals and is correlated with health1. Understanding the extent to which, and how, host genetics contributes to this variation is essential yet has proved to be difficult, as few associations have been replicated, particularly in humans2. Here we study the effect of host genotype on the composition of the intestinal microbiota in a large mosaic pig population. We show that, under conditions of exacerbated genetic diversity and environmental uniformity, microbiota composition and the abundance of specific taxa are heritable. We map a quantitative trait locus affecting the abundance of Erysipelotrichaceae species and show that it is caused by a 2.3 kb deletion in the gene encoding N-acetyl-galactosaminyl-transferase that underpins the ABO blood group in humans. We show that this deletion is a ≥3.5-million-year-old trans-species polymorphism under balancing selection. We demonstrate that it decreases the concentrations of N-acetyl-galactosamine in the gut, and thereby reduces the abundance of Erysipelotrichaceae that can import and catabolize N-acetyl-galactosamine. Our results provide very strong evidence for an effect of the host genotype on the abundance of specific bacteria in the intestine combined with insights into the molecular mechanisms that underpin this association. Our data pave the way towards identifying the same effect in rural human populations.

Protein Engineering of PhUGT, a Donor Promiscuous Glycosyltransferase, for the Improved Enzymatic Synthesis of Antioxidant Quercetin 3-O-N-Acetylgalactosamine.

Quercetin 3-O-N-acetylgalactosamine (Q3GalNAc), a derivative of dietary hyperoside, had never been enzymatically synthesized due to the lack of well-identified N-acetylgalactosamine-transferase (GalNAc-T). Herein, PhUGT, an identified flavonoid 3-O-galactosyltransferase from Petunia hybrida, was demonstrated to display quercetin GalNAc-T activity, transferring a N-acetylgalactosamine (GalNAc) from UDP-N-acetylgalactosamine (UDP-GalNAc) to the 3-OH of quercetin to form Q3GalNAc with a low conversion of 11.7% at 40 °C for 2 h. Protein engineering was thus performed, and the resultant PhUGT variant F368T got an enhanced conversion of 75.5% toward UDP-GalNAc. The enzymatically synthesized Q3GalNAc exhibited a comparable antioxidant activity with other quercetin 3-O-glycosides. Further studies revealed that PhUGT was a donor promiscuous glycosyltransferase (GT), recognizing seven sugar donors. This finding overturned a previous notion that PhUGT exclusively recognized UDP-galactose (UDP-Gal). The reason why PhUGT was mistaken for a UDP-Gal-specific GT was demonstrated to be a shorter reaction time, in which many quercetin 3-O-glycosides, except hyperoside, could not be effectively synthesized. The fact that the microbial cell factory expressing PhUGT could yield an array of Q3Gs further confirmed the donor promiscuity of PhUGT. This study laid a foundation for the scale production of Q3GalNAc and provided a potent biocatalyst capable of glycodiversifying quercetin as well.

Cooperativity of catalytic and lectin-like domain of Trypanosoma congolense trans-sialidase modulates its catalytic activity.

Trans-sialidases (TS) represent a multi-gene family of unusual enzymes, which catalyse the transfer of terminal sialic acids (Sia) from sialoglycoconjugates to terminal galactose or N-acetylgalactosamine residues of oligosaccharides without the requirement of CMP-Neu5Ac, the activated Sia used by typical sialyltransferases. Enzymes comprise a N-terminal catalytic domain (CD) followed by a lectin-like domain (LD). Most work on trypanosomal TS has been done on enzymatic activities focusing on the CD of TS from Trypanosoma cruzi (causing Chagas disease in Latin America), subspecies of Trypanosoma brucei, (causing human sleeping sickness in Africa) and Trypanosoma congolense (causing African Animal Trypanosomosis in livestock). Previously, we demonstrated that T. congolense TS (TconTS)-LD binds to several carbohydrates, such as 1,4-ß-mannotriose. In this study we investigated the influence of TconTS3-LD on Sia transfer efficiency of TconTS1a-CD by swapping domains. in silico analysis on structure models of TconTS enzymes revealed the potential of domain swaps between TconTS1a and TconTS3 without structural disruptions of the enzymes overall topologies. Recombinant domain swapped TconTS1a/TS3 showed clear Sia transfer activity, when using fetuin and lactose as Sia donor and acceptor substrates, respectively. While Sia transfer activity remained unchanged from the level of TconTS1a, hydrolytic release of free Neu5Ac as a side product was suppressed resulting in increased transfer efficiency. Presence of 1,4-ß-mannotriose during TS reactions modulates enzyme activities enhancing transfer efficiency possibly due to occupation of the binding site in TconTS1a-LD. Interestingly this effect was in the same range as that observed when swapping TconTS1a-CD and TconTS3-LD. In summary, this study demonstrate the proof-of-principle for swapping CDs and LDs of TconTS and that TconTS3-LD influences enzymatic activity of TconTS1a-CD providing evidence that LDs play pivotal roles in modulating activities and biological functions of TconTS and possibly other TS.

Loss of the N-acetylgalactosamine side chain of the GPI-anchor impairs bone formation and brain functions and accelerates the prion disease pathology.

Glycosylphosphatidylinositol (GPI) is a posttranslational glycolipid modification of proteins that anchors proteins in lipid rafts on the cell surface. Although some GPI-anchored proteins (GPI-APs), including the prion protein PrPC, have a glycan side chain composed of N-acetylgalactosamine (GalNAc)-galactose-sialic acid on the core structure of GPI glycolipid, in vivo functions of this GPI-GalNAc side chain are largely unresolved. Here, we investigated the physiological and pathological roles of the GPI-GalNAc side chain in vivo by knocking out its initiation enzyme, PGAP4, in mice. We show that Pgap4 mRNA is highly expressed in the brain, particularly in neurons, and mass spectrometry analysis confirmed the loss of the GalNAc side chain in PrPC GPI in PGAP4-KO mouse brains. Furthermore, PGAP4-KO mice exhibited various phenotypes, including an elevated blood alkaline phosphatase level, impaired bone formation, decreased locomotor activity, and impaired memory, despite normal expression levels and lipid raft association of various GPI-APs. Thus, we conclude that the GPI-GalNAc side chain is required for in vivo functions of GPI-APs in mammals, especially in bone and the brain. Moreover, PGAP4-KO mice were more vulnerable to prion diseases and died earlier after intracerebral inoculation of the pathogenic prion strains than wildtype mice, highlighting the protective roles of the GalNAc side chain against prion diseases.

Towards Mapping of the Human Brain N-Glycome with Standardized Graphitic Carbon Chromatography.

The brain N-glycome is known to be crucial for many biological functions, including its involvement in neuronal diseases. Although large structural studies of brain N-glycans were recently carried out, a comprehensive isomer-specific structural analysis has still not been achieved, as indicated by the recent discovery of novel structures with galactosylated bisecting GlcNAc. Here, we present a detailed, isomer-specific analysis of the human brain N-glycome based on standardized porous graphitic carbon (PGC)-LC-MS/MS. To achieve this goal, we biosynthesized glycans with substitutions typically occurring in the brain N-glycome and acquired their normalized retention times. Comparison of these values with the standardized retention times of neutral and desialylated N-glycan fractions of the human brain led to unambiguous isomer specific assignment of most major peaks. Profound differences in the glycan structures between naturally neutral and desialylated glycans were found. The neutral and sialylated N-glycans derive from diverging biosynthetic pathways and are biosynthetically finished end products, rather than just partially processed intermediates. The focus on structural glycomics defined the structure of human brain N-glycans, amongst these are HNK-1 containing glycans, a bisecting sialyl-lactose and structures with fucose and N-acetylgalactosamine on the same arm, the so-called LDNF epitope often associated with parasitic worms.

A single sulfatase is required to access colonic mucin by a gut bacterium.

Humans have co-evolved with a dense community of microbial symbionts that inhabit the lower intestine. In the colon, secreted mucus creates a barrier that separates these microorganisms from the intestinal epithelium1. Some gut bacteria are able to utilize mucin glycoproteins, the main mucus component, as a nutrient source. However, it remains unclear which bacterial enzymes initiate degradation of the complex O-glycans found in mucins. In the distal colon, these glycans are heavily sulfated, but specific sulfatases that are active on colonic mucins have not been identified. Here we show that sulfatases are essential to the utilization of distal colonic mucin O-glycans by the human gut symbiont Bacteroides thetaiotaomicron. We characterized the activity of 12 different sulfatases produced by this species, showing that they are collectively active on all known sulfate linkages in O-glycans. Crystal structures of three enzymes provide mechanistic insight into the molecular basis of substrate specificity. Unexpectedly, we found that a single sulfatase is essential for utilization of sulfated O-glycans in vitro and also has a major role in vivo. Our results provide insight into the mechanisms of mucin degradation by a prominent group of gut bacteria, an important process for both normal microbial gut colonization2 and diseases such as inflammatory bowel disease3.

A Drug-Drug Interaction Study Evaluating the Effect of Givosiran, a Small Interfering Ribonucleic Acid, on Cytochrome P450 Activity in the Liver.

Givosiran (trade name GIVLAARI) is a small interfering ribonucleic acid that targets hepatic delta-aminolevulinic acid synthase 1 (ALAS1) messenger RNA for degradation through RNA interference (RNAi) that has been approved for the treatment of acute hepatic porphyria (AHP). RNAi therapeutics, such as givosiran, have a low liability for drug-drug interactions (DDIs) because they are not metabolized by cytochrome 450 (CYP) enzymes, and do not directly inhibit or induce CYP enzymes in the liver. The pharmacodynamic effect of givosiran (lowering of hepatic ALAS1, the first and rate limiting enzyme in the heme biosynthesis pathway) presents a unique scenario where givosiran could potentially impact heme-dependent activities in the liver, such as CYP enzyme activity. This study assessed the impact of givosiran on the pharmacokinetics of substrates of 5 major CYP450 enzymes in subjects with acute intermittent porphyria (AIP), the most common type of AHP, by using the validated "Inje cocktail," comprised of caffeine (CYP1A2), losartan (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6), and midazolam (CYP3A4). We show that givosiran treatment had a differential inhibitory effect on CYP450 enzymes in the liver, resulting in a moderate reduction in activity of CYP1A2 and CYP2D6, a minor effect on CYP3A4 and CYP2C19, and a similar weak effect on CYP2C9. To date, this is the first study evaluating the DDI for an oligonucleotide therapeutic and highlights an atypical drug interaction due to the pharmacological effect of givosiran. The results of this study suggest that givosiran does not have a large effect on heme-dependent CYP enzyme activity in the liver.