Key points are not available for this paper at this time.
Akkermansia muciniphila is key member of the human gut microbiota that impacts many features of host health. A major characteristic of this bacterium is its interaction with host mucin, which is abundant in the gut environment, and its ability to metabolize mucin as a nutrient source. The machinery deployed by A. muciniphila to enable this interaction appears to be extensive and sophisticated, yet it is incompletely defined. The uncharacterized protein AMUC₁438 is encoded by a gene that was previously shown to be upregulated when the bacterium is grown on mucin. This uncharacterized protein has features suggestive of carbohydrate-recognition and peptidase activity, which led us to hypothesize that it has a role in mucin depolymerization. Here, we provide structural and functional support for the assignment of AMUC₁438 as a unique O-glycopeptidase with mucin-degrading capacity. O-glycopeptidase enzymes recognize glycans but hydrolyze the peptide backbone and are common in host-adapted microbes that colonize or invade mucus layers. Structural, kinetic, and mutagenic analyses point to a metzincin metalloprotease catalytic motif but with an active site that specifically recognizes a GalNAc residue α-linked to serine or threonine (i. e. , the Tn-antigen). The enzyme catalyzes hydrolysis of the bond immediately N-terminal to the glycosylated residue. Additional modeling analyses suggest the presence of a carbohydrate-binding module that may assist in substrate recognition. We anticipate that these results will be fundamental to a wider understanding of the O-glycopeptidase class of enzymes and how they may contribute to host adaptation. Akkermansia muciniphila is key member of the human gut microbiota that impacts many features of host health. A major characteristic of this bacterium is its interaction with host mucin, which is abundant in the gut environment, and its ability to metabolize mucin as a nutrient source. The machinery deployed by A. muciniphila to enable this interaction appears to be extensive and sophisticated, yet it is incompletely defined. The uncharacterized protein AMUC₁438 is encoded by a gene that was previously shown to be upregulated when the bacterium is grown on mucin. This uncharacterized protein has features suggestive of carbohydrate-recognition and peptidase activity, which led us to hypothesize that it has a role in mucin depolymerization. Here, we provide structural and functional support for the assignment of AMUC₁438 as a unique O-glycopeptidase with mucin-degrading capacity. O-glycopeptidase enzymes recognize glycans but hydrolyze the peptide backbone and are common in host-adapted microbes that colonize or invade mucus layers. Structural, kinetic, and mutagenic analyses point to a metzincin metalloprotease catalytic motif but with an active site that specifically recognizes a GalNAc residue α-linked to serine or threonine (i. e. , the Tn-antigen). The enzyme catalyzes hydrolysis of the bond immediately N-terminal to the glycosylated residue. Additional modeling analyses suggest the presence of a carbohydrate-binding module that may assist in substrate recognition. We anticipate that these results will be fundamental to a wider understanding of the O-glycopeptidase class of enzymes and how they may contribute to host adaptation. The mammalian gastrointestinal tract is protected by a mucosal barrier that has inner and outer layers. The dense inner layer is tightly adhered to epithelial cells and effectively bacteria free, whereas the outer layer is a loose matrix that is colonized by bacteria (see Ref. (1Martens E. C. Neumann M. Desai M. S. Interactions of commensal and pathogenic microorganisms with the intestinal mucosal barrier. Nat. Rev. Microbiol. 2018; 16: 457-470Crossref PubMed Scopus (391) Google Scholar) for a review). Both layers largely comprise mucins, densely O-glycosylated proteins that have 60 to 70% complex carbohydrate chains by weight, with the inner layer having membrane-associated mucins and the outer layer having unattached gel-forming mucins. The diverse carbohydrate chains of mucin promote colonization of bacterial species that possess the proper metabolic capabilities to forage for glycans as a nutrient source. Akkermansia muciniphila is a Gram-negative bacterium of the phylum Verrucomicrobia (2Derrien M. Vaughan E. E. Plugge C. M. de Vos W. M. Akkermansia muciniphila gen. nov. , sp. nov. , a human intestinal mucin-degrading bacterium. Int. J. Syst. Evol. Microbiol. 2004; 54: 1469-1476Crossref PubMed Scopus (1353) Google Scholar). It is a common gastrointestinal commensal in animals and found comprising 3 to 5% of a healthy human gut microbiota (3Belzer C. de Vos W. M. Microbes inside—from diversity to function: the case of Akkermansia. ISME J. 2012; 6: 1449-1458Crossref PubMed Scopus (473) Google Scholar). Since the initial description of this bacterium (2Derrien M. Vaughan E. E. Plugge C. M. de Vos W. M. Akkermansia muciniphila gen. nov. , sp. nov. , a human intestinal mucin-degrading bacterium. Int. J. Syst. Evol. Microbiol. 2004; 54: 1469-1476Crossref PubMed Scopus (1353) Google Scholar), its abundance in the human microbiome has been correlated with an enormous array of healthy or disease states, highlighting the importance of its role in gut homeostasis and overall host health (see Ref. (4Cirstea M. Radisavljevic N. Finlay B. B. Good bug, bad bug: breaking through microbial stereotypes. Cell Host Microbe. 2018; 23: 10-13Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) for an overview). A. muciniphila is known for its capacity to deconstruct and utilize mucin as a nutrient source. Genomic analysis of this bacterium combined with transcriptomic studies when grown on mucin indicate that approximately 3% of the genes in the A. muciniphila genome contribute to mucin degradation (5van Passel M. W. J. Kant R. Zoetendal E. G. Plugge C. M. Derrien M. Malfatti S. A. et al. The Genome of Akkermansia muciniphila, a dedicated intestinal mucin degrader, and its use in exploring intestinal metagenomes. PLoS One. 2011; 6e16876Crossref Scopus (295) Google Scholar, 6Ottman N. Davids M. Suarez-Diez M. Boeren S. Schaap P. J. Martins dos Santos V. A. P. et al. Genome-scale model and omics analysis of metabolic capacities of Akkermansia muciniphila reveal a preferential mucin-degrading lifestyle. Appl. Environ. Microbiol. 2017; 83e01014-17Crossref PubMed Scopus (140) Google Scholar). A large number of these genes are carbohydrate-active enzymes devoted to carbohydrate processing, which reflects the abundance of glycans in mucins. However, notable amongst the mucin-processing enzymes are three proteins classified in the MEROPS satabase (7Rawlings N. D. Barrett A. J. Finn R. Twenty years of the MEROPS database of proteolytic enzymes, their substrates and inhibitors. Nucl. Acids Res. 2016; 44: D343-D350Crossref PubMed Scopus (527) Google Scholar) as family M60 peptidases (AMUC₀627, AMUC₀908, and AMUC₂001), one that is classified as M98 (AMUC₁514), and the unclassified metzincin-like peptidase OgpA (AMUC₁119). The amuc₀627, amuc₀908, and amuc₂001 genes are upregulated when A. muciniphila is grown on mucin, supporting their role in mucin metabolism, whereas AMUC₀627, AMUC₀908, AMUC₁514, and OgpA have demonstrated in vitro mucinase activity (8Shon D. J. Malaker S. A. Pedram K. Yang E. Krishnan V. Dorigo O. et al. An enzymatic toolkit for selective proteolysis, detection, and visualization of mucin-domain glycoproteins. Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 21299-21307Crossref PubMed Scopus (52) Google Scholar, 9Trastoy B. Naegeli A. Anso I. Sjögren J. Guerin M. E. Structural basis of mammalian mucin processing by the human gut O-glycopeptidase OgpA Akkermansia 2020; PubMed Scopus Google Scholar). proteins to the of and the of the of an on the substrate and of the peptide bond the site of immediately N-terminal to the glycosylated residue I. E. B. C. et of glycans as a of peptidase Natl. Acad. Sci. U. S. A. 2017; PubMed Scopus Google Scholar). are of these or of which host-adapted microbes S. A. A by and pathogenic One. 2012; PubMed Scopus Google Scholar, D. J. A. Malaker S. A. structural and of mucin J. PubMed Scopus Google Scholar). the A. muciniphila genes upregulated when the bacterium is grown on mucin is one that The encoded protein is as family carbohydrate-binding by of an module of that is as to family The N-terminal is as a of the presence of an motif N. and of catalytic Sci. 23: PubMed Scopus Google Scholar). However, this is with family as the motif are classified the basis of these for on mucin, carbohydrate-binding and peptidase that this protein is an O-glycopeptidase that an as yet uncharacterized of We the of AMUC₁438 through structural and activity on a of and O-glycosylated The results reveal that the enzyme is an O-glycopeptidase that immediately the N-terminal of serine or threonine a α-linked that the The catalytic of AMUC₁438 to the class of peptidases and is to OgpA A. muciniphila, they are the The of OgpA is to that of the known peptidases A. muciniphila (AMUC₀627, AMUC₀908, and which a of (8Shon D. J. Malaker S. A. Pedram K. Yang E. Krishnan V. Dorigo O. et al. An enzymatic toolkit for selective proteolysis, detection, and visualization of mucin-domain glycoproteins. Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 21299-21307Crossref PubMed Scopus (52) Google Scholar). The AMUC₁438 is unique amongst known mucin-degrading enzymes of A. muciniphila, the interaction of A. muciniphila with its in the J. et and a of protein database Acids Res. PubMed Scopus Google Scholar) with AMUC₁438 reveal with of the with the presence of a peptide and an to an motif N. and of catalytic Sci. 23: PubMed Scopus Google Scholar, M. C. et protein PubMed Scopus Google Scholar). The this motif are to to an protein and the a the MEROPS database with classified peptidase (7Rawlings N. D. Barrett A. J. Finn R. Twenty years of the MEROPS database of proteolytic enzymes, their substrates and inhibitors. Nucl. Acids Res. 2016; 44: D343-D350Crossref PubMed Scopus (527) Google Scholar). of the of AMUC₁438 the the with carbohydrate-binding module family Yang J. a for carbohydrate-active enzyme Acids Res. 2012; PubMed Scopus Google Scholar), which is with the of the in family to which the module However, this module is in the as to an unclassified family E. S. V. B. N. The carbohydrate-active enzyme and Acids Res. PubMed Scopus Google Scholar). enable a functional analysis of we analysis for in and with their in We to and to be we it in initial The and be to as by the presence of the motif in the and we these for peptidase activity a This to reveal on the of when A. muciniphila is grown on mucin, and the presence of a we that the protein may have activity on mucins. and but or to a in the of mucin in with the proteins This activity on which we by a mucinase mucin degradation is as the of the of the proteins to activity on or The of was by with a and a by a in (see and The to protein in the of which that be and to to to of of for are shown in to in a for are shown in The of the protein a with a on one and of on the of the this is a comprising and to the was a that was as a A of this active O-glycopeptidase is an that the of the motif the is the catalytic residue The residue the the motif and is found on a to the supporting the of the catalytic this is the which a the these features the catalytic as having a metzincin motif U. et al. The of PubMed Scopus Google Scholar). this is the as that for the protein to of of and was in previously B. Naegeli A. Anso I. Sjögren J. Guerin M. E. Structural basis of mammalian mucin processing by the human gut O-glycopeptidase OgpA Akkermansia 2020; PubMed Scopus Google Scholar) We with the of OgpA in complex with the The catalytic by the site and catalytic residue this in the of the in of the peptide of by OgpA in the However, in and with a and a in of which are in in the of OgpA B. Naegeli A. Anso I. Sjögren J. Guerin M. E. Structural basis of mammalian mucin processing by the human gut O-glycopeptidase OgpA Akkermansia 2020; PubMed Scopus Google Scholar). This that the in AMUC₁438 may comprise an site in this enzyme to the enzyme an O-glycopeptidase to A of with the peptide the OgpA the comprising the site However, with the of in the the and the and of the indicate that the AMUC₁438 substrate in a that is that of the basis of the of we an with for of an active catalytic We this to that a of AMUC₁438 O-glycopeptidase We by the activity of on a of on a peptide activity on the peptide a and activity on the peptide an that the enzyme is an activity on with glycans a for the The is a family O-glycopeptidase of the It has for peptide and that is known to immediately N-terminal to site of I. E. B. C. et of glycans as a of peptidase Natl. Acad. Sci. U. S. A. 2017; PubMed Scopus Google Scholar, S. E. C. C. et that analysis of and complex PubMed Scopus Google Scholar). The of activity on the peptide to that of the that N-terminal to the site of support a previously (8Shon D. J. Malaker S. A. Pedram K. Yang E. Krishnan V. Dorigo O. et al. An enzymatic toolkit for selective proteolysis, detection, and visualization of mucin-domain glycoproteins. Proc. Natl. Acad. Sci. U. S. A. 2020; 117: 21299-21307Crossref PubMed Scopus (52) Google Scholar, S. A. Pedram K. Krishnan V. C. et al. The and functional analysis of human Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), we the of in a of of the immediately N-terminal to the of the results with the in to the known abundance of on the the of the in the the for the the site results reveal for the the site of However, we activity on and this that was active on a peptide an as yet of activity on of the of the substrate of the of with of the results and of the in the AMUC₁438 O-glycopeptidase activity, we a (see the for The substrate was on an A peptide with the the and serine the α-linked residue. of the substrate of in a and and substrate of hydrolysis by and proteins and a of and a of The for and The of the with OgpA a role of and in recognition. a a for a We to of of AMUC₁438 to this we of the three and their activity on the We an of the catalytic residue as an the their as by they activity on the peptide This the role of three in substrate the with the GalNAc residue. for the AMUC₁438 we to of the We to of but we to the of to this the in the in The that be and which to the in the a three the as in the O-glycopeptidase B. 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PubMed Scopus Google Scholar, Finn R. of by a family of carbohydrate-binding Full Text Full Text PDF PubMed Scopus Google Scholar). The model of the AMUC₁438 structural the to the the C. family Finn R. of by a family of carbohydrate-binding Full Text Full Text PDF PubMed Scopus Google Scholar). The in is as a in the AMUC₁438 one of the is the of a in the AMUC₁438 is to the the major features of to be in the AMUC₁438 this may have a role in recognition. The active of by the has features that it as a This was by the structural analysis of the which a metzincin catalytic and mucinase activity that was by the However, the of the this catalytic with of A the for database the of AMUC₁438 as a with A. muciniphila and the of a and species of bacteria are the and A of the microbes that the proteins to AMUC₁438 to be found in the bacterial MEROPS and are found largely in host-adapted bacteria S. A. A by and pathogenic One. 2012; PubMed Scopus Google Scholar). 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