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• W2894048250 abstract "Glycosaminoglycans (GAGs) and GAG-degrading enzymes have wide-ranging applications in the medical and biotechnological industries. The former are also an important nutrient source for select species of the human gut microbiota (HGM), a key player in host–microbial interactions. How GAGs are metabolized by the HGM is therefore of interest and has been extensively investigated in the model human gut microbe Bacteroides thetaiotaomicron. The presence of as-yet uncharacterized GAG-inducible genes in its genome and of related species, however, is testament to our incomplete understanding of this process. Nevertheless, it presents a potential opportunity for the discovery of additional GAG-degrading enzymes. Here, we investigated a gene of unknown function (BT_3328) from the chondroitin sulfate (CS) utilization locus of B. thetaiotaomicron. NMR and UV spectroscopic assays revealed that it encodes a novel polysaccharide lyase (PL), hereafter referred to as BtCDH, reflecting its source (B. thetaiotaomicron (Bt)) and its ability to degrade the GAGs CS, dermatan sulfate (DS), and hyaluronic acid (HA). When incubated with HA, BtCDH generated a series of unsaturated HA sugars, including Δ4,5UA-GlcNAc, Δ4,5UA-GlcNAc-GlcA-GlcNac, Δ4,5UA-[GlcNAc-GlcA]2-GlcNac, and Δ4,5UA-[GlcNAc-GlcA]3-GlcNac, as end products and hence was classed as endo-acting. A combination of genetic and biochemical assays revealed that BtCDH localizes to the cell surface of B. thetaiotaomicron where it enables extracellular GAG degradation. BtCDH homologs were also detected in several other HGM species, and we therefore propose that it represents the founding member of a new polysaccharide lyase family (PL29). The current discovery also contributes new insights into CS metabolism by the HGM. Glycosaminoglycans (GAGs) and GAG-degrading enzymes have wide-ranging applications in the medical and biotechnological industries. The former are also an important nutrient source for select species of the human gut microbiota (HGM), a key player in host–microbial interactions. How GAGs are metabolized by the HGM is therefore of interest and has been extensively investigated in the model human gut microbe Bacteroides thetaiotaomicron. The presence of as-yet uncharacterized GAG-inducible genes in its genome and of related species, however, is testament to our incomplete understanding of this process. Nevertheless, it presents a potential opportunity for the discovery of additional GAG-degrading enzymes. Here, we investigated a gene of unknown function (BT_3328) from the chondroitin sulfate (CS) utilization locus of B. thetaiotaomicron. NMR and UV spectroscopic assays revealed that it encodes a novel polysaccharide lyase (PL), hereafter referred to as BtCDH, reflecting its source (B. thetaiotaomicron (Bt)) and its ability to degrade the GAGs CS, dermatan sulfate (DS), and hyaluronic acid (HA). When incubated with HA, BtCDH generated a series of unsaturated HA sugars, including Δ4,5UA-GlcNAc, Δ4,5UA-GlcNAc-GlcA-GlcNac, Δ4,5UA-[GlcNAc-GlcA]2-GlcNac, and Δ4,5UA-[GlcNAc-GlcA]3-GlcNac, as end products and hence was classed as endo-acting. A combination of genetic and biochemical assays revealed that BtCDH localizes to the cell surface of B. thetaiotaomicron where it enables extracellular GAG degradation. BtCDH homologs were also detected in several other HGM species, and we therefore propose that it represents the founding member of a new polysaccharide lyase family (PL29). The current discovery also contributes new insights into CS metabolism by the HGM. Mammalian glycosaminoglycans (GAGs) 2The abbreviations used are: GAGglycosaminoglycanCSchondroitin sulfatePLpolysaccharide lyaseCSchondroitin sulfateDSdermatan sulfateHAhyaluronic acidHGMhuman gut microbiotaPULpolysaccharide utilization locusHPAEChigh-performance anion-exchange chromatographySECsize-exclusion chromatographyUAuronic acidDPdegree of polymerizationPKproteinase K. are complex carbohydrates consisting of repeating disaccharide units of uronic acids or galactose linked to a hexosamine sugar (1Pomin V.H. Mulloy B. Glycosaminoglycans and proteoglycans.Pharmaceuticals (Basel). 2018; 11 (29495527): E2710.3390/ph11010027Crossref PubMed Scopus (103) Google Scholar, 2Hong S.W. Kim B.T. Shin H.Y. Kim W.S. Lee K.S. Kim Y.S. Kim D.H. Purification and characterization of novel chondroitin ABC and AC lyases from Bacteroides stercoris HJ-15, a human intestinal anaerobic bacterium.Eur. J. Biochem. 2002; 269 (12071957): 2934-294010.1046/j.1432-1033.2002.02967.xCrossref PubMed Scopus (38) Google Scholar). These include chondroitin sulfate (CS) made up of GlcA and GalNAc, dermatan sulfate (DS), or chondroitin sulfate B made up of IdoA and GalNAc, hyaluronic acid (HA) made up of GlcA and GlcNAc, heparin/heparan sulfate (HS) made up of a mixture of GlcA and IdoA linked to GlcNAc and keratan sulfate made up of galactose linked to GlcNAc. GAGs such as CS, DS, and HS are also characterized by highly diverse sulfation patterns (introduced during biosynthesis by specific sulfotransferase enzymes) that define various molecular subtypes of each glycan (3Kusche-Gullberg M. Kjellén L. Sulfotransferases in glycosaminoglycan biosynthesis.Curr. Opin. Struct. Biol. 2003; 13 (14568616): 605-61110.1016/j.sbi.2003.08.002Crossref PubMed Scopus (243) Google Scholar, 4Gama C.I. Tully S.E. Sotogaku N. Clark P.M. Rawat M. Vaidehi N. Goddard 3rd, W.A. Nishi A. Hsieh-Wilson L.C. Sulfation patterns of glycosaminoglycans encode molecular recognition and activity.Nat. Chem. Biol. 2006; 2 (16878128): 467-47310.1038/nchembio810Crossref PubMed Scopus (445) Google Scholar). CS, for example, can be sulfated on GalNAc at different positions including C4, C6, or both to yield major CS subtypes namely CSA, CSC, and CSE, respectively (5Vázquez J.A. Rodríguez-Amado I. Montemayor M.I. Fraguas J. González Mdel P. Murado M.A. Chondroitin sulfate, hyaluronic acid and chitin/chitosan production using marine waste sources: characteristics, applications and eco-friendly processes: a review.Mar. Drugs. 2013; 11 (23478485): 747-77410.3390/md11030747Crossref PubMed Scopus (174) Google Scholar, 6Sugahara K. Mikami T. Uyama T. Mizuguchi S. Nomura K. Kitagawa H. Recent advances in the structural biology of chondroitin sulfate and dermatan sulfate.Curr. Opin. Struct. Biol. 2003; 13 (14568617): 612-62010.1016/j.sbi.2003.09.011Crossref PubMed Scopus (588) Google Scholar) or at both C2 of GlcA and C6 of GalNAc, yielding CSD (7Davidson S. Gilead L. Amira M. Ginsburg H. Razin E. Synthesis of chondroitin sulfate D and heparin proteoglycans in murine lymph node-derived mast cells. The dependence on fibroblasts.J. Biol. Chem. 1990; 265 (2115517): 12324-12330Abstract Full Text PDF PubMed Google Scholar). In contrast, HA is not sulfated and hence less structurally complex compared with the other GAG types. glycosaminoglycan chondroitin sulfate polysaccharide lyase chondroitin sulfate dermatan sulfate hyaluronic acid human gut microbiota polysaccharide utilization locus high-performance anion-exchange chromatography size-exclusion chromatography uronic acid degree of polymerization proteinase K. GAGs are ubiquitously distributed in the human body and implicated in several important physiological processes such as cell signaling, inflammation, neuronal development, adhesion, and tumor progression (8Kim S.H. Turnbull J. Guimond S. Extracellular matrix and cell signalling: the dynamic cooperation of integrin, proteoglycan and growth factor receptor.J. Endocrinol. 2011; 209 (21307119): 139-15110.1530/JOE-10-0377Crossref PubMed Scopus (795) Google Scholar9Wight T.N. Kinsella M.G. Qwarnström E.E. The role of proteoglycans in cell adhesion, migration and proliferation.Curr. Opin. Cell Biol. 1992; 4 (1419056): 793-80110.1016/0955-0674(92)90102-ICrossref PubMed Scopus (335) Google Scholar, 10Gill S. Wight T.N. Frevert C.W. Proteoglycans: key regulators of pulmonary inflammation and the innate immune response to lung infection.Anat. Rec. (Hoboken). 2010; 293 (20503391): 968-98110.1002/ar.21094Crossref PubMed Scopus (83) Google Scholar11Iozzo R.V. Sanderson R.D. Proteoglycans in cancer biology, tumour microenvironment and angiogenesis.J. Cell Mol. Med. 2011; 15 (21155971): 1013-103110.1111/j.1582-4934.2010.01236.xCrossref PubMed Scopus (407) Google Scholar). Dietary GAGs are also a nutrient source for the human gut microbiota (HGM), which is known to impact greatly on human health and disease status (12Ulmer J.E. Vilén E.M. Namburi R.B. Benjdia A. Beneteau J. Malleron A. Bonnaffé D. Driguez P.A. Descroix K. Lassalle G. Le Narvor C. Sandström C. Spillmann D. Berteau O. Characterization of glycosaminoglycan (GAG) sulfatases from the human gut symbiont Bacteroides thetaiotaomicron reveals the first GAG-specific bacterial endosulfatase.J. Biol. Chem. 2014; 289 (25002587): 24289-2430310.1074/jbc.M114.573303Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar13Pudlo N.A. Urs K. Kumar S.S. German J.B. Mills D.A. Martens E.C. Symbiotic human gut bacteria with variable metabolic priorities for host mucosal glycans.MBio. 2015; 6 (26556271): e01282-15Crossref PubMed Scopus (102) Google Scholar, 14Raghavan V. Groisman E.A. Species-specific dynamic responses of gut bacteria to a mammalian glycan.J. Bacteriol. 2015; 197 (25691527): 1538-154810.1128/JB.00010-15Crossref PubMed Scopus (25) Google Scholar, 15Tuncil Y.E. Xiao Y. Porter N.T. Reuhs B.L. Martens E.C. Hamaker B.R. Reciprocal prioritization to dietary glycans by gut bacteria in a competitive environment promotes stable coexistence.MBio. 2017; 8 (29018117): e01068-17Crossref PubMed Scopus (91) Google Scholar, 16Cartmell A. Lowe E.C. Baslé A. Firbank S.J. Ndeh D.A. Murray H. Terrapon N. Lombard V. Henrissat B. Turnbull J.E. Czjzek M. Gilbert H.J. Bolam D.N. How members of the human gut microbiota overcome the sulfation problem posed by glycosaminoglycans.Proc. Natl. Acad. Sci. U.S.A. 2017; 114 (28630303): 7037-704210.1073/pnas.1704367114Crossref PubMed Scopus (71) Google Scholar17Desai M.S. Seekatz A.M. Koropatkin N.M. Kamada N. Hickey C.A. Wolter M. Pudlo N.A. Kitamoto S. Terrapon N. Muller A. Young V.B. Henrissat B. Wilmes P. Stappenbeck T.S. Núñez G. et al.A Dietary fiber-deprived gut microbiota degrades the colonic mucus barrier and enhances pathogen susceptibility.Cell. 2016; 167 (27863247): 1339-135310.1016/j.cell.2016.10.043Abstract Full Text Full Text PDF PubMed Scopus (1299) Google Scholar). Indeed, recent evidence suggests that CS is a priority nutrient for prominent gut microbes such as Bacteroides thetaiotaomicron and Bacteroides ovatus and that only a subset of HGM species are capable of metabolizing the GAG (13Pudlo N.A. Urs K. Kumar S.S. German J.B. Mills D.A. Martens E.C. Symbiotic human gut bacteria with variable metabolic priorities for host mucosal glycans.MBio. 2015; 6 (26556271): e01282-15Crossref PubMed Scopus (102) Google Scholar, 15Tuncil Y.E. Xiao Y. Porter N.T. Reuhs B.L. Martens E.C. Hamaker B.R. Reciprocal prioritization to dietary glycans by gut bacteria in a competitive environment promotes stable coexistence.MBio. 2017; 8 (29018117): e01068-17Crossref PubMed Scopus (91) Google Scholar, 17Desai M.S. Seekatz A.M. Koropatkin N.M. Kamada N. Hickey C.A. Wolter M. Pudlo N.A. Kitamoto S. Terrapon N. Muller A. Young V.B. Henrissat B. Wilmes P. Stappenbeck T.S. Núñez G. et al.A Dietary fiber-deprived gut microbiota degrades the colonic mucus barrier and enhances pathogen susceptibility.Cell. 2016; 167 (27863247): 1339-135310.1016/j.cell.2016.10.043Abstract Full Text Full Text PDF PubMed Scopus (1299) Google Scholar). Understanding how GAGs are metabolized could therefore inform strategies including nutrient-based approaches to manipulate the HGM and hence improve human health. In line with this, several studies (reviewed by Ndeh and Gilbert (18Ndeh D. Gilbert H.J. Biochemistry of complex glycan depolymerisation by the human gut microbiota.FEMS Microbiol. Rev. 2018; 42 (29325042): 146-16410.1093/femsre/fuy002Crossref PubMed Scopus (127) Google Scholar)) have examined the metabolism of dietary GAGs by the HGM. The results demonstrate that GAG degradation is largely orchestrated by a combination of three major enzyme types including polysaccharide lyases (PLs, EC 4.2.2.-), sulfatases (EC 3.1.6.-), and glycoside hydrolases (EC 3.2.1.-). In the model human gut microbe B. thetaiotaomicron, as well as other gut Bacteroidetes, the genes encoding these enzymes are typically located within polysaccharide utilization loci (PULs) (14Raghavan V. Groisman E.A. Species-specific dynamic responses of gut bacteria to a mammalian glycan.J. Bacteriol. 2015; 197 (25691527): 1538-154810.1128/JB.00010-15Crossref PubMed Scopus (25) Google Scholar, 19Martens E.C. Chiang H.C. Gordon J.I. Mucosal glycan foraging enhances fitness and transmission of a saccharolytic human gut bacterial symbiont.Cell Host Microbe. 2008; 4 (18996345): 447-45710.1016/j.chom.2008.09.007Abstract Full Text Full Text PDF PubMed Scopus (553) Google Scholar, 20Raghavan V. Lowe E.C. Townsend 2nd, G.E. Bolam D.N. Groisman E.A. Tuning transcription of nutrient utilization genes to catabolic rate promotes growth in a gut bacterium.Mol. Microbiol. 2014; 93 (25041429): 1010-102510.1111/mmi.12714Crossref PubMed Scopus (35) Google Scholar21Terrapon N. Lombard V. Gilbert H.J. Henrissat B. Automatic prediction of polysaccharide utilization loci in Bacteroidetes species.Bioinformatics. 2015; 31 (25355788): 647-65510.1093/bioinformatics/btu716Crossref PubMed Scopus (142) Google Scholar). These genetic loci are diverse and encode a variety of cell envelope–associated multiprotein systems involved in complex glycan metabolism. Although the mechanisms of various GAG PULs in these organisms have been extensively investigated, knowledge of their functioning is incomplete, evident by the presence of as-yet uncharacterized GAG-inducible genes within these PULs (21Terrapon N. Lombard V. Gilbert H.J. Henrissat B. Automatic prediction of polysaccharide utilization loci in Bacteroidetes species.Bioinformatics. 2015; 31 (25355788): 647-65510.1093/bioinformatics/btu716Crossref PubMed Scopus (142) Google Scholar). In the CS PUL of B. thetaiotaomicron, three of such genes, BT_3328, BT_3329, and BT_3330 (all of unknown function), appear in a distinct operon (BT_3328–30) conserved in several HGM Bacteroidetes (14Raghavan V. Groisman E.A. Species-specific dynamic responses of gut bacteria to a mammalian glycan.J. Bacteriol. 2015; 197 (25691527): 1538-154810.1128/JB.00010-15Crossref PubMed Scopus (25) Google Scholar, 20Raghavan V. Lowe E.C. Townsend 2nd, G.E. Bolam D.N. Groisman E.A. Tuning transcription of nutrient utilization genes to catabolic rate promotes growth in a gut bacterium.Mol. Microbiol. 2014; 93 (25041429): 1010-102510.1111/mmi.12714Crossref PubMed Scopus (35) Google Scholar, 21Terrapon N. Lombard V. Gilbert H.J. Henrissat B. Automatic prediction of polysaccharide utilization loci in Bacteroidetes species.Bioinformatics. 2015; 31 (25355788): 647-65510.1093/bioinformatics/btu716Crossref PubMed Scopus (142) Google Scholar). The latter suggests that they might be an important adaptation for CS metabolism in these microbes. Here, we characterized the largest member of this operon (BT_3328), revealing that it encodes a novel polysaccharide lyase hereafter referred to as BtCDH, reflecting its source (B. thetaiotaomicron (Bt)) and ability to degrade the GAGs CS, DS, and HA. We also provide evidence suggesting that BtCDH is not only the founding member of a novel enzyme family but also a cell surface GAG-degrading enzyme, the first to be reported in B. thetaiotaomicron and related species. Taken together, our findings contribute new insights into our knowledge of CS metabolism by the HGM, a key player in host–microbial interactions. The gene encoding native BtCDH lyase (BT_3328 from B. thetaiotaomicron VPI-5482) is 2607 bp in length and has a GC content of 51%. BtCDH is a large protein of 868 amino acid residues, a molecular mass of 96 kDa, and an isoelectric pI of 5.33. A majority of the top hits (>50% identity) from a BLASTp search with native BtCDH are hypothetical/putative uncharacterized proteins mainly annotated as DUF4955 domain–containing. The DUF4955 domain whose function is unknown is present at the C terminus of the protein, whereas another domain DUF4988 is located directly after the putative lipoprotein signal peptide sequence at the N terminus of the protein (Fig. 1A). Top BtCDH lyase homologs were mostly detected in species from the Bacteroides, Alistipes, and Prevotella genera, all highly represented in the HGM. They were also prominent in species from marine environments including Saccharophagus degradans, Zobellia galactinovorans, Vibrio harveyi, and Coraliomargarita akajimensis. A phylogenetic tree of selected homologs of BtCDH is presented in Fig. 1B. Purified recombinant BtCDH was initially assayed spectrophotometrically using CS from bovine trachea (which is mainly CS-4 sulfate or CSA (22Shaya D. Hahn B.-S. Park N.Y. Sim J.-S. Kim Y.S. Cygler M. Characterization of chondroitin sulfate lyase ABC from Bacteroides thetaiotaomicron WAL2926.Biochemistry. 2008; 47 (18512954): 6650-666110.1021/bi800353gCrossref PubMed Scopus (25) Google Scholar)) (Fig. 2A) as substrate. BtCDH degraded CSA generating unsaturated products that were detected by TLC and high-performance anion-exchange chromatography (HPAEC) using a UV detector at 235 nm (A235) (Fig. 2, B and C, respectively). Limit products of the reaction were also resolved by size-exclusion chromatography (SEC) and analyzed by 1H NMR (Fig. 2D and Fig. S1). Distinct resonance signals were detected at 5.9 and 5.2 ppm in the spectra of the digested products as opposed to undigested CSA. The former is evidence of a H4 proton chemical shift consistent with glyosidic bond cleavage and the generation of C4,5 double bonds (Δ4,5) in the terminal nonreducing end GlcA or uronic acid (UA) residue (Δ4,5UA) of various products, whereas the latter peak at 5.2 ppm corresponds to resonance signals of the H1 anomeric proton of the same sugar (Fig. 2D) (23Rani A. Goyal A. A new member of family 8 polysaccharide lyase chondroitin AC lyase (Ps PL8A) from Pedobacter saltans displays endo- and exo-lytic catalysis.J. Mol. Catalysis B Enzymatic. 2016; 134: 215-22410.1016/j.molcatb.2016.11.001Crossref Scopus (12) Google Scholar24Lemmnitzer K. Riemer T. Groessl M. Süss R. Knochenmuss R. Schiller J. Comparison of ion mobility-mass spectrometry and pulsed-field gradient nuclear magnetic resonance spectroscopy for the differentiation of chondroitin sulfate isomers.Anal. Methods. 2016; 8: 8483-849110.1039/C6AY02531ECrossref Google Scholar, 25Huckerby T.N. Lauder R.M. Brown G.M. Nieduszynski I.A. Anderson K. Boocock J. Sandall P.L. Weeks S.D. Characterization of oligosaccharides from the chondroitin sulfates. 1H-NMR and 13C-NMR studies of reduced disaccharides and tetrasaccharides.Eur. J. Biochem. 2001; 268 (11231269): 1181-118910.1046/j.1432-1327.2001.01948.xCrossref PubMed Scopus (34) Google Scholar26Silva C. Novoa-Carballal R. Reis R.L. Pashkuleva I. Following the enzymatic digestion of chondroitin sulfate by a simple GPC analysis.Anal. Chim. Acta. 2015; 885 (26231907): 207-21310.1016/j.aca.2015.05.034Crossref PubMed Scopus (20) Google Scholar). To explore the substrate specificity in BtCDH in more detail, the enzyme was also tested against a series of commercially available uronic acid-based substrates including CS from shark cartilage (mainly CS-6 sulfate or CSC), DS from porcine intestinal mucosa (mainly DS-4 sulfate), high and low molecular weight HA sodium salt from Streptococcus equi (10,000–30,000 (HAL) and 90,000–110,000 Da (HAH), respectively), heparin, alginate, ulvan, and polygalacturonic acid. BtCDH was active against CS DS and HA (in the order CSC > CSA/HAL > DS > HAH) but not heparin, alginate, ulvan, and polygalacturonic acid (Table 1). Previous studies analyzing CSA and CSC have reported major differences in the sulfated disaccharide composition of both substrates with the most prominent being the levels of the ΔUA-GalNAc4S, which is higher in CSA, and ΔUA-GalNAc6S, which is higher in CSC (12Ulmer J.E. Vilén E.M. Namburi R.B. Benjdia A. Beneteau J. Malleron A. Bonnaffé D. Driguez P.A. Descroix K. Lassalle G. Le Narvor C. Sandström C. Spillmann D. Berteau O. Characterization of glycosaminoglycan (GAG) sulfatases from the human gut symbiont Bacteroides thetaiotaomicron reveals the first GAG-specific bacterial endosulfatase.J. Biol. Chem. 2014; 289 (25002587): 24289-2430310.1074/jbc.M114.573303Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 22Shaya D. Hahn B.-S. Park N.Y. Sim J.-S. Kim Y.S. Cygler M. Characterization of chondroitin sulfate lyase ABC from Bacteroides thetaiotaomicron WAL2926.Biochemistry. 2008; 47 (18512954): 6650-666110.1021/bi800353gCrossref PubMed Scopus (25) Google Scholar, 27Muthusamy A. Achur R.N. Valiyaveettil M. Madhunapantula S.V. Kakizaki I. Bhavanandan V.P. Gowda C.D. Structural characterization of the bovine tracheal chondroitin sulfate chains and binding of Plasmodium falciparum–infected erythrocytes.Glycobiology. 2004; 14 (15044390): 635-64510.1093/glycob/cwh077Crossref PubMed Scopus (20) Google Scholar, 28Malavaki C.J. Asimakopoulou A.P. Lamari F.N. Theocharis A.D. Tzanakakis G.N. Karamanos N.K. Capillary electrophoresis for the quality control of chondroitin sulfates in raw materials and formulations.Anal. Biochem. 2008; 374 (18054774): 213-22010.1016/j.ab.2007.11.006Crossref PubMed Scopus (54) Google Scholar). BtCDH activity thus appears to be affected by the type of sulfation in the GAG substrates.Table 1Activity of BtCDH lyase against various uronate substrates To determine whether BtCDH was endo- or exo-acting, we performed time-course experiments with HAL, a less chemically complex GAG compared with its sulfated counterparts. HAL was incubated with BtCDH over different times (0, 1, 5, and 60 min and ∼12 h (overnight), after which reactions were stopped by boiling and analyzed by TLC. BtCDH degraded HAL within 10 min, generating a fast migrating band (band A) (Fig. 3A). After 1 h of the reaction, the original HAL spot at the origin disappeared accompanied by the appearance another major band (band B), as well as low staining intermittent bands (Fig. 3A). The pattern of products remained unchanged even after overnight digestion, and hence the generated sugars represented the final limit products of HAL digestion. Bands A and B limit products were purified by SEC and analyzed by MS (HILIC LC-MS). Purified Band A yielded incremental masses of 380.117, 381.120, and 382.123 Da (differing by a mass of 1 Da) equivalent to the mass of singly charged ions of the unsaturated Δ4,5UA-GlcNAc disaccharide, whereas band B yielded incremental masses of 759.227, 759.729, and 760.230 Da (differing by a mass of 0.5 Da) etc. (Fig. 3B), equivalent to the mass of doubly charged fragments derived from the unsaturated Δ4,5UA-[GlcNAc-GlcA]3-GlcNAc octasaccharide parent ion. To detect underrepresented sugars, all SEC fractions were pooled in one sample and analyzed. The spectra revealed the presence of new products with incremental masses of 759.226, 760.230, and 761.232 (differing by a mass of 1 Da) and 1138.337, 1139.340, and 1140.344 (differing by a mass of 1 Da), corresponding to singly charged ions of the unsaturated Δ4,5UA-GlcNAc-GlcA-GlcNAc tetrasaccharide and Δ4,5UA-[GlcNAc-GlcA]2-GlcNAc hexasaccharide sugars, respectively (Fig. 3B). The generation of diverse molecular weight limit products thus suggests that BtCDH is an endo-acting enzyme. In addition, the accumulation of the high molecular weight HA octasaccharide suggests that BtCDH preferentially cleaves sugars with a degree of polymerization (DP) typically ≥10. The requirement for substrates with a DP ≥ 10 was also demonstrated using sulfated CS oligosaccharides as substrates with HPAEC data showing a major shift in the DP10 oligosaccharide profile (as opposed to a minor shift for DP8) after treatment with BtCDH (Fig. 4). The proportion of each HA product generated was also determined by UV with the results showing that over 70% of the generated products were Δ4,5UA-GlcNAc disaccharides (Fig. S2). Time-course experiments with sulfated GAGs CSA and CSC as substrates also yielded fairly similar patterns observed for HAL (Fig. S3). As with HAL, high amounts of the basic disaccharide building block (Δ4,5UA-GalNAc) were detected, suggesting high processivity in the enzyme.Figure 4Activity of BtCDH against various chondroitin sulfate oligosaccharides. Substrates (1 mg/ml each) were treated with 0.5 μm of BtCDH (shown as DPx +, with x being the number of constituent monosaccharides) overnight or diluted in equivalent volume of buffer as control (DPx). HPAEC chromatograms show major shifts in the signals of sulfated DP10 and DP12 oligosaccharides following treatment, accompanied by the appearance of a strong signals for the Δ4,5UA-GalNAc disaccharide in each case.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The effects of temperature, pH and metal ions on BtCDH activity were tested with CSA as substrate. BtCDH preferred higher reaction temperatures with the activity increasing linearly from 20 °C to a maximum rate of ∼15 μm/min at 60 °C (Fig. 5A). After this optimum temperature, the enzyme activity dropped sharply. To determine the optimum pH of BtCDH, enzyme activity was examined against CSA in different buffers over a pH range of 4–9. BtCDH activity rose gradually with increase pH between 4 and 6 and then sharply to an optimum pH of 7.0 in 10 mm MES buffer (Fig. 5B). BtCDH activity against CS was also tested in the presence of 5 mm EDTA and metal ions including Mg2+, Ca2+, Mn2+, Co2+, Zn2+, and Ni2+. Its activity was only slightly affected by metals with the most inhibition (27.42%) observed in the presence of Co2+ ions (Fig. 5C). A genetic mutant ΔBtCDH lacking the entire ORF for BtCDH (the BT_3328 gene) was created by counter selectable allelic exchange. Growth experiments with both B. thetaiotaomicron WT and ΔBtCDH strains were carried out with four different GAG substrates CSA, DS, CSC, HAL, and glucose as sole carbon source for at least 24 h. B. thetaiotaomicron WT and BtCDH cells grew at a very similar rates on various substrates, except for CSC where a minor defect in late phase growth was observed (Fig. S4). Rabbit anti-BtCDH polyclonal antibodies where generated using the recombinant BtCDH protein as immunogen. On Western blots containing cell-free extracts of B. thetaiotaomicron grown on CSA, two immunosensitive bands (BtCDHH and BtCDHL of high and low molecular weights, respectively) were detected (Fig. 6A). Despite its poor enrichment relative to BtCDHL, BtCDHH’s molecular weight was close to 96 kDa, corresponding to the mass of native BtCDH, whereas the mass of BtCDHL was ∼30 kDa. Interestingly, when cell extracts of the ΔBtCDH strain lacking BtCDH were tested with the same antibodies, both bands were not detected, suggesting that BtCDHL is a subcomponent possibly a truncated form of BtCDH (Fig. 6A, top panel). To confirm this, BtCDH was FLAG-tagged (BtCDH FLAG) by genetically introducing a FLAG peptide sequence (DYKDDDK) at the C-terminal of the protein, thus allowing for its detection using rabbit anti-FLAG specific antibodies. Under these circumstances, only BtCDHH was detected by immunoblotting (Fig. 6A), suggesting that BtCDHL was an N-terminal truncated version of BtCDH. As indicated earlier, BtCDH contains an N-terminal lipidation sequence (Fig. 1A) and hence is potentially localized extracellularly at the cell surface of B. thetaiotaomicron. To confirm this experimentally, we performed proteinase K (PK) and immunofluorescence assays. B. thetaiotaomicron WT cells were grown to exponential phase in 1% CSA, harvested, and treated with 0.5 mg/ml PK. Treated and untreated cells (controls) were lysed under reducing conditions, and cell extracts were analyzed by Western blotting using anti-BtCDH antibodies. The results showed degradation of BtCDHL and BtCDHH in PK-treated but not control cells (Fig. 6B). The experiment was also controlled by immunoblotting the same extracts with polyclonal antibodies targeting the constitutively expressed periplasmic heparin lyase BT4657 (16Cartmell A. Lowe E.C. Baslé A. Firbank S.J. Ndeh D.A. Murray H. Terrapon N. Lombard V. Henrissat B. Turnbull J.E. Czjzek M. Gilbert H.J. Bolam D.N. How members of the human gut microbiota overcome the sulfation problem posed by glycosaminoglycans.Proc. Natl. Acad. Sci. U.S.A. 2017; 114 (28630303): 7037-704210.1073/pnas.1704367114Crossref PubMed Scopus (71) Google Scholar) whose bands remained intact in test and control extracts (Fig. 6B). The data thus suggest that BtCDH is localized to cell surface of B. thetaiotaomicron. The cell-surface localization of BtCDH was finally confirmed through immunofluorescence assays using anti-BtCDH antibodies (Fig. 6C). We also performed whole-cell aerobic assays on B. thetaiotaomicron WT and ΔBtCDH following growth on CSA. Under these conditions, transport across the outer membrane is limited, and only surface enzyme activity is detected. The cells were harvested at exponential phase and reincubated with CSA and CSC for 2 h, and the reaction was examined by TLC. The results showed extensive degradation of both CSA and CSC by the WT strain, visible as a smear of products (Fig. 6D). This was significantly reduced in the knockout strain especially for the CSA substrate in which just a single band of generated product was observed (Fig. 6D). Supernatants at exponential phase were also analyzed for secreted BtCDH by Western blotting with polyclonal rabbit anti-BtCDH ant" @default.
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• W2894048250 date "2018-11-01" @default.
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• W2894048250 title "The human gut microbe Bacteroides thetaiotaomicron encodes the founding member of a novel glycosaminoglycan-degrading polysaccharide lyase family PL29" @default.
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