Loss of the disease-associated glycosyltransferase Galnt3 alters Muc10 glycosylation and the composition of the oral microbiome

Abstract

The importance of the microbiome in health and its disruption in disease is continuing to be elucidated. However, the multitude of host and environmental factors that influence the microbiome are still largely unknown. Here, we examined UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 3 (Galnt3)-deficient mice, which serve as a model for the disease hyperphosphatemic familial tumoral calcinosis (HFTC). In HFTC, loss of GALNT3 activity in the bone is thought to lead to altered glycosylation of the phosphate-regulating hormone fibroblast growth factor 23 (FGF23), resulting in hyperphosphatemia and subdermal calcified tumors. However, GALNT3 is expressed in other tissues in addition to bone, suggesting that systemic loss could result in other pathologies. Using semiquantitative real-time PCR, we found that Galnt3 is the major O-glycosyltransferase expressed in the secretory cells of salivary glands. Additionally, 16S rRNA gene sequencing revealed that the loss of Galnt3 resulted in changes in the structure, composition, and stability of the oral microbiome. Moreover, we identified the major secreted salivary mucin, Muc10, as an in vivo substrate of Galnt3. Given that mucins and their O-glycans are known to interact with various microbes, our results suggest that loss of Galnt3 decreases glycosylation of Muc10, which alters the composition and stability of the oral microbiome. Considering that oral findings have been documented in HFTC patients, our study suggests that investigating GALNT3-mediated changes in the oral microbiome may be warranted. The importance of the microbiome in health and its disruption in disease is continuing to be elucidated. However, the multitude of host and environmental factors that influence the microbiome are still largely unknown. Here, we examined UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 3 (Galnt3)-deficient mice, which serve as a model for the disease hyperphosphatemic familial tumoral calcinosis (HFTC). In HFTC, loss of GALNT3 activity in the bone is thought to lead to altered glycosylation of the phosphate-regulating hormone fibroblast growth factor 23 (FGF23), resulting in hyperphosphatemia and subdermal calcified tumors. However, GALNT3 is expressed in other tissues in addition to bone, suggesting that systemic loss could result in other pathologies. Using semiquantitative real-time PCR, we found that Galnt3 is the major O-glycosyltransferase expressed in the secretory cells of salivary glands. Additionally, 16S rRNA gene sequencing revealed that the loss of Galnt3 resulted in changes in the structure, composition, and stability of the oral microbiome. Moreover, we identified the major secreted salivary mucin, Muc10, as an in vivo substrate of Galnt3. Given that mucins and their O-glycans are known to interact with various microbes, our results suggest that loss of Galnt3 decreases glycosylation of Muc10, which alters the composition and stability of the oral microbiome. Considering that oral findings have been documented in HFTC patients, our study suggests that investigating GALNT3-mediated changes in the oral microbiome may be warranted. The importance of the human microbiome throughout the body is only now being fully appreciated. In health, the microbiome interacts synergistically with the host to contribute to normal tissue functions as well as to protect from pathogen colonization (1Kamada N. Chen G.Y. Inohara N. Núñez G. Control of pathogens and pathobionts by the gut microbiota.Nat. Immunol. 2013; 14 (23778796): 685-69010.1038/ni.2608Crossref PubMed Scopus (915) Google Scholar, 2Lamont R.J. Koo H. Hajishengallis G. The oral microbiota: dynamic communities and host interactions.Nat. Rev. Microbiol. 2018; 16 (30301974): 745-75910.1038/s41579-018-0089-xCrossref PubMed Scopus (710) Google Scholar). Alterations in the composition of the microbiome have been shown to be involved in various disease states, including inflammatory diseases of the digestive tract (3Huttenhower C. Kostic A.D. Xavier R.J. Inflammatory bowel disease as a model for translating the microbiome.Immunity. 2014; 40 (24950204): 843-85410.1016/j.immuni.2014.05.013Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 4Lamont R.J. Hajishengallis G. Polymicrobial synergy and dysbiosis in inflammatory disease.Trends Mol. Med. 2015; 21 (25498392): 172-18310.1016/j.molmed.2014.11.004Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar) and diseases of the oral cavity. At this site, dysbiotic changes in the microbiome are associated with dysregulated immune responses that lead to the common oral disease periodontitis (2Lamont R.J. Koo H. Hajishengallis G. The oral microbiota: dynamic communities and host interactions.Nat. Rev. Microbiol. 2018; 16 (30301974): 745-75910.1038/s41579-018-0089-xCrossref PubMed Scopus (710) Google Scholar, 5Abusleme L. Dupuy A.K. Dutzan N. Silva N. Burleson J.A. Strausbaugh L.D. Gamonal J. Diaz P.I. The subgingival microbiome in health and periodontitis and its relationship with community biomass and inflammation.ISME J. 2013; 7 (23303375): 1016-102510.1038/ismej.2012.174Crossref PubMed Scopus (594) Google Scholar, 6Dutzan N. Kajikawa T. Abusleme L. Greenwell-Wild T. Zuazo C.E. Ikeuchi T. Brenchley L. Abe T. Hurabielle C. Martin D. Morell R.J. Freeman A.F. Lazarevic V. Trinchieri G. Diaz P.I. et al.A dysbiotic microbiome triggers TH17 cells to mediate oral mucosal immunopathology in mice and humans.Sci. Transl. Med. 2018; 10 (30333238): eaat079710.1126/scitranslmed.aat0797Crossref PubMed Scopus (190) Google Scholar, 7Griffen A.L. Beall C.J. Campbell J.H. Firestone N.D. Kumar P.S. Yang Z.K. Podar M. Leys E.J. Distinct and complex bacterial profiles in human periodontitis and health revealed by 16S pyrosequencing.ISME J. 2012; 6 (22170420): 1176-118510.1038/ismej.2011.191Crossref PubMed Scopus (620) Google Scholar). The secreted mucosal membrane is a key point of interaction with the microbiome. This protective layer, which is present along most internal epithelial surfaces of the body, forms a barrier that mediates interaction with the microbiota and confers physical protection (8Johansson M.E. Hansson G.C. Immunological aspects of intestinal mucus and mucins.Nat. Rev. Immunol. 2016; 16 (27498766): 639-64910.1038/nri.2016.88Crossref PubMed Scopus (434) Google Scholar, 9Johansson M.E. Sjövall H. Hansson G.C. The gastrointestinal mucus system in health and disease.Nat. Rev. Gastroenterol. Hepatol. 2013; 10 (23478383): 352-36110.1038/nrgastro.2013.35Crossref PubMed Scopus (801) Google Scholar). The main components of this barrier include mucins, proteins with unique rheological properties conferred by the abundant O-glycans present throughout their repetitive domains. O-glycans on mucins bind water to form hydrated gels that line and protect internal epithelial surfaces (8Johansson M.E. Hansson G.C. Immunological aspects of intestinal mucus and mucins.Nat. Rev. Immunol. 2016; 16 (27498766): 639-64910.1038/nri.2016.88Crossref PubMed Scopus (434) Google Scholar, 9Johansson M.E. Sjövall H. Hansson G.C. The gastrointestinal mucus system in health and disease.Nat. Rev. Gastroenterol. Hepatol. 2013; 10 (23478383): 352-36110.1038/nrgastro.2013.35Crossref PubMed Scopus (801) Google Scholar). Additionally, mucins and their associated O-glycans bind microorganisms to modulate clearance, attachment, and the ability to form biofilms (10Culp D.J. Robinson B. Cash M.N. Bhattacharyya I. Stewart C. Cuadra-Saenz G. Salivary mucin 19 glycoproteins: innate immune functions in Streptococcus mutans-induced caries in mice and evidence for expression in human saliva.J. Biol. Chem. 2015; 290 (25512380): 2993-300810.1074/jbc.M114.597906Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 11Frenkel E.S. Ribbeck K. Salivary mucins protect surfaces from colonization by cariogenic bacteria.Appl. Environ. Microbiol. 2015; 81 (25344244): 332-33810.1128/AEM.02573-14Crossref PubMed Scopus (75) Google Scholar, 12Frenkel E.S. Ribbeck K. Salivary mucins promote the coexistence of competing oral bacterial species.ISME J. 2017; 11 (28117832): 1286-129010.1038/ismej.2016.200Crossref PubMed Scopus (24) Google Scholar). The importance of mucins and O-glycans in digestive system health is illustrated by studies in model organisms demonstrating that loss of the major intestinal mucin Muc2 or disruption of O-glycan biosynthesis results in spontaneous colitis, colon cancer, and susceptibility to inflammatory conditions of the digestive tract (13An G. Wei B. Xia B. McDaniel J.M. Ju T. Cummings R.D. Braun J. Xia L. Increased susceptibility to colitis and colorectal tumors in mice lacking core 3-derived O-glycans.J. Exp. Med. 2007; 204 (17517967): 1417-142910.1084/jem.20061929Crossref PubMed Scopus (255) Google Scholar, 14Bergstrom K. Liu X. Zhao Y. Gao N. Wu Q. Song K. Cui Y. Li Y. McDaniel J.M. McGee S. Chen W. Huycke M.M. Houchen C.W. Zenewicz L.A. West C.M. et al.Defective intestinal mucin-type O-glycosylation causes spontaneous colitis-associated cancer in mice.Gastroenterology. 2016; 151 (27059389): 152-164.e1110.1053/j.gastro.2016.03.039Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 15Fu J. Wei B. Wen T. Johansson M.E. Liu X. Bradford E. Thomsson K.A. McGee S. Mansour L. Tong M. McDaniel J.M. Sferra T.J. Turner J.R. Chen H. Hansson G.C. et al.Loss of intestinal core 1-derived O-glycans causes spontaneous colitis in mice.J. Clin. Invest. 2011; 121 (21383503): 1657-166610.1172/JCI45538Crossref PubMed Scopus (252) Google Scholar, 16Gao N. Bergstrom K. Fu J. Xie B. Chen W. Xia L. Loss of intestinal O-glycans promotes spontaneous duodenal tumors.Am. J. Physiol. Gastrointest. Liver Physiol. 2016; 311 (27229122): G74-G8310.1152/ajpgi.00060.2016Crossref PubMed Scopus (23) Google Scholar, 17Velcich A. Yang W. Heyer J. Fragale A. Nicholas C. Viani S. Kucherlapati R. Lipkin M. Yang K. Augenlicht L. Colorectal cancer in mice genetically deficient in the mucin Muc2.Science. 2002; 295 (11872843): 1726-172910.1126/science.1069094Crossref PubMed Scopus (715) Google Scholar, 18Wenzel U.A. Magnusson M.K. Rydström A. Jonstrand C. Hengst J. Johansson M.E. Velcich A. Öhman L. Strid H. Sjövall H. Hansson G.C. Wick M.J. Spontaneous colitis in Muc2-deficient mice reflects clinical and cellular features of active ulcerative colitis.PLoS One. 2014; 9 (24945909): e10021710.1371/journal.pone.0100217Crossref PubMed Scopus (72) Google Scholar). However, little is known regarding how individual mucins influence the establishment and composition of the microbiome. Mucins and O-glycans have been conserved throughout evolution, suggesting important roles in many diverse biological processes (19Aoki K. Porterfield M. Lee S.S. Dong B. Nguyen K. McGlamry K.H. Tiemeyer M. The diversity of O-linked glycans expressed during Drosophila melanogaster development reflects stage- and tissue-specific requirements for cell signaling.J. Biol. Chem. 2008; 283 (18725413): 30385-3040010.1074/jbc.M804925200Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 20Lang T. Hansson G.C. Samuelsson T. Gel-forming mucins appeared early in metazoan evolution.Proc. Natl. Acad. Sci. U.S.A. 2007; 104 (17911254): 16209-1621410.1073/pnas.0705984104Crossref PubMed Scopus (204) Google Scholar, 21Schwientek T. Bennett E.P. Flores C. Thacker J. Hollmann M. Reis C.A. Behrens J. Mandel U. Keck B. Schäfer M.A. Haselmann K. Zubarev R. Roepstorff P. Burchell J.M. Taylor-Papadimitriou J. Hollingsworth M.A. Clausen H. Functional conservation of subfamilies of putative UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases in DrosophilaCaenorhabditis elegans, and mammals: one subfamily composed of l(2)35Aa is essential in Drosophila.J. Biol. Chem. 2002; 277 (11925450): 22623-2263810.1074/jbc.M202684200Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 22Ten Hagen K.G. Tran D.T. Gerken T.A. Stein D.S. Zhang Z. Functional characterization and expression analysis of members of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase family from Drosophila melanogaster.J. Biol. Chem. 2003; 278 (12829714): 35039-3504810.1074/jbc.M303836200Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). O-Glycosylation is controlled by a family of enzymes known as the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (Galnts) 2The abbreviations used are: GalntUDP-GalNAc:polypeptide N-acetylgalactosaminyltransferasesPNApeanut agglutininSMGsubmandibular glandMAAMaackia amurensis agglutininOTUoperational taxonomic unitHFTChyperphosphatemic familial tumoral calcinosisNMneuraminidaseqPCRsemiquantitative real-time PCRLEfSelinear discriminant analysis effect sizeEembryonic dayPpostnatal dayERendoplasmic reticulumPCoAprincipal coordinates analysisAMOVAanalysis of molecular variancePFAparaformaldehydeDIGdigoxigeninTRITCtetramethylrhodamine isothiocyanatecontiggroup of overlapping clonesθ YCθ Yue and ClaytonTSAtyramide signal amplificationRIPAradioimmune precipitation assay. that catalyze the addition of GalNAc to the hydroxyl group of serine or threonine, the first committed step in O-glycan biosynthesis (23Bennett E.P. Mandel U. Clausen H. Gerken T.A. Fritz T.A. Tabak L.A. Control of mucin-type O-glycosylation: a classification of the polypeptide GalNAc-transferase gene family.Glycobiology. 2012; 22 (22183981): 736-75610.1093/glycob/cwr182Crossref PubMed Scopus (531) Google Scholar, 24Tran D.T. Ten Hagen K.G. Mucin-type O-glycosylation during development.J. Biol. Chem. 2013; 288 (23329828): 6921-692910.1074/jbc.R112.418558Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). Members of this large family display differential tissue- and cell-specific expression patterns and unique substrate preferences, suggesting that each family member may be responsible for the proper glycosylation of specific proteins in vivo (23Bennett E.P. Mandel U. Clausen H. Gerken T.A. Fritz T.A. Tabak L.A. Control of mucin-type O-glycosylation: a classification of the polypeptide GalNAc-transferase gene family.Glycobiology. 2012; 22 (22183981): 736-75610.1093/glycob/cwr182Crossref PubMed Scopus (531) Google Scholar, 25Revoredo L. Wang S. Bennett E.P. Clausen H. Moremen K.W. Jarvis D.L. Ten Hagen K.G. Tabak L.A. Gerken T.A. Mucin-type O-glycosylation is controlled by short- and long-range glycopeptide substrate recognition that varies among members of the polypeptide GalNAc transferase family.Glycobiology. 2016; 26 (26610890): 360-37610.1093/glycob/cwv108Crossref PubMed Scopus (54) Google Scholar, 26Tian E. Ten Hagen K.G. Expression of the UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase family is spatially and temporally regulated during Drosophila development.Glycobiology. 2006; 16 (16251381): 83-9510.1093/glycob/cwj051Crossref PubMed Scopus (51) Google Scholar). Studies in model organisms have shown that certain Galnts are required for viability and involved in conserved biological processes, such as cell adhesion, secretion, and cell-signaling events (21Schwientek T. Bennett E.P. Flores C. Thacker J. Hollmann M. Reis C.A. Behrens J. Mandel U. Keck B. Schäfer M.A. Haselmann K. Zubarev R. Roepstorff P. Burchell J.M. Taylor-Papadimitriou J. Hollingsworth M.A. Clausen H. Functional conservation of subfamilies of putative UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases in DrosophilaCaenorhabditis elegans, and mammals: one subfamily composed of l(2)35Aa is essential in Drosophila.J. Biol. Chem. 2002; 277 (11925450): 22623-2263810.1074/jbc.M202684200Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 27Ten Hagen K.G. Tran D.T. A UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase is essential for viability in Drosophila melanogaster.J. Biol. Chem. 2002; 277 (11925446): 22616-2262210.1074/jbc.M201807200Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 28Tian E. Hoffman M.P. Ten Hagen K.G. O-Glycosylation modulates integrin and FGF signalling by influencing the secretion of basement membrane components.Nat. Commun. 2012; 3 (22643896): 86910.1038/ncomms1874Crossref PubMed Scopus (46) Google Scholar, 29Tian E. Stevens S.R. Guan Y. Springer D.A. Anderson S.A. Starost M.F. Patel V. Ten Hagen K.G. Tabak L.A. Galnt1 is required for normal heart valve development and cardiac function.PLoS One. 2015; 10 (25615642): e011586110.1371/journal.pone.0115861PubMed Google Scholar, 30Tian E. Ten Hagen K.G. A UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase is required for epithelial tube formation.J. Biol. Chem. 2007; 282 (17098739): 606-61410.1074/jbc.M606268200Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 31Tran D.T. Zhang L. Zhang Y. Tian E. Earl L.A. Ten Hagen K.G. Multiple members of the UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase family are essential for viability in Drosophila.J. Biol. Chem. 2012; 287 (22157008): 5243-525210.1074/jbc.M111.306159Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 32Zhang L. Syed Z.A. van Dijk Härd I. Lim J.M. Wells L. Ten Hagen K.G. O-Glycosylation regulates polarized secretion by modulating Tango1 stability.Proc. Natl. Acad. Sci. U.S.A. 2014; 111 (24799692): 7296-730110.1073/pnas.1322264111Crossref PubMed Scopus (49) Google Scholar, 33Zhang L. Tran D.T. Ten Hagen K.G. An O-glycosyltransferase promotes cell adhesion during development by influencing secretion of an extracellular matrix integrin ligand.J. Biol. Chem. 2010; 285 (20371600): 19491-1950110.1074/jbc.M109.098145Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 34Zhang L. Turner B. Ribbeck K. Ten Hagen K.G. Loss of the mucosal barrier alters the progenitor cell niche via Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling.J. Biol. Chem. 2017; 292 (29127201): 21231-2124210.1074/jbc.M117.809848Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). In humans, mutations in one family member, GALNT3, are responsible for the disease hyperphosphatemic familial tumoral calcinosis (HFTC), characterized by hyperphosphatemia, altered bone density, and the development of subdermal calcified tumors (35Topaz O. Shurman D.L. Bergman R. Indelman M. Ratajczak P. Mizrachi M. Khamaysi Z. Behar D. Petronius D. Friedman V. Zelikovic I. Raimer S. Metzker A. Richard G. Sprecher E. Mutations in GALNT3, encoding a protein involved in O-linked glycosylation, cause familial tumoral calcinosis.Nat. Genet. 2004; 36 (15133511): 579-58110.1038/ng1358Crossref PubMed Scopus (463) Google Scholar). GALNT3-mediated glycosylation of the phosphate-regulating hormone FGF23 within osteocytes of the bone is presumed to protect FGF23 from protease cleavage under normal conditions; loss of the GALNT3 glycosyltransferase results in inactivating cleavage of FGF23 and dysregulation of phosphate homeostasis. A mouse model of this disease that is deficient for Galnt3 recapitulates many disease phenotypes, including Fgf23 inactivating cleavage and hyperphosphatemia (36Ichikawa S. Sorenson A.H. Austin A.M. Mackenzie D.S. Fritz T.A. Moh A. Hui S.L. Econs M.J. Ablation of the Galnt3 gene leads to low-circulating intact fibroblast growth factor 23 (Fgf23) concentrations and hyperphosphatemia despite increased Fgf23 expression.Endocrinology. 2009; 150 (19213845): 2543-255010.1210/en.2008-0877Crossref PubMed Scopus (125) Google Scholar). Whereas both males and females displayed these HFTC hallmark phenotypes, only males displayed growth retardation, infertility, and increased bone density (36Ichikawa S. Sorenson A.H. Austin A.M. Mackenzie D.S. Fritz T.A. Moh A. Hui S.L. Econs M.J. Ablation of the Galnt3 gene leads to low-circulating intact fibroblast growth factor 23 (Fgf23) concentrations and hyperphosphatemia despite increased Fgf23 expression.Endocrinology. 2009; 150 (19213845): 2543-255010.1210/en.2008-0877Crossref PubMed Scopus (125) Google Scholar, 37Miyazaki T. Mori M. Yoshida C.A. Ito C. Yamatoya K. Moriishi T. Kawai Y. Komori H. Kawane T. Izumi S. Toshimori K. Komori T. Galnt3 deficiency disrupts acrosome formation and leads to oligoasthenoteratozoospermia.Histochem. Cell Biol. 2013; 139 (23052838): 339-35410.1007/s00418-012-1031-3Crossref PubMed Scopus (26) Google Scholar). The reasons for these gender-specific differences are currently unknown. UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases peanut agglutinin submandibular gland Maackia amurensis agglutinin operational taxonomic unit hyperphosphatemic familial tumoral calcinosis neuraminidase semiquantitative real-time PCR linear discriminant analysis effect size embryonic day postnatal day endoplasmic reticulum principal coordinates analysis analysis of molecular variance paraformaldehyde digoxigenin tetramethylrhodamine isothiocyanate group of overlapping clones θ Yue and Clayton tyramide signal amplification radioimmune precipitation assay. Interestingly, GALNT3 (Galnt3 in mice) is abundantly expressed in other tissues and thus may have a role in other parts of the body that could contribute to disease pathology (21Schwientek T. Bennett E.P. Flores C. Thacker J. Hollmann M. Reis C.A. Behrens J. Mandel U. Keck B. Schäfer M.A. Haselmann K. Zubarev R. Roepstorff P. Burchell J.M. Taylor-Papadimitriou J. Hollingsworth M.A. Clausen H. Functional conservation of subfamilies of putative UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases in DrosophilaCaenorhabditis elegans, and mammals: one subfamily composed of l(2)35Aa is essential in Drosophila.J. Biol. Chem. 2002; 277 (11925450): 22623-2263810.1074/jbc.M202684200Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 23Bennett E.P. Mandel U. Clausen H. Gerken T.A. Fritz T.A. Tabak L.A. Control of mucin-type O-glycosylation: a classification of the polypeptide GalNAc-transferase gene family.Glycobiology. 2012; 22 (22183981): 736-75610.1093/glycob/cwr182Crossref PubMed Scopus (531) Google Scholar). Here, we identify Galnt3 as the major family member expressed in adult mouse salivary glands, which are responsible for producing the components of saliva, including mucins. Interestingly, we show that loss of Galnt3 results in dramatic changes in the composition and structure of the oral microbiome. We further identify the major secreted mucin Muc10 as an in vivo and in vitro substrate of Galnt3. Our results demonstrate for the first time that the loss of a single glycosyltransferase can alter the oral microbiome, possibly through altered glycosylation of a major salivary mucin. Given the importance of the microbiome in health and disease, our study suggests that investigation of the oral microbiome in patients with mutations in GALNT3 may be warranted to have a more comprehensive understanding of disease pathology. Previous studies have shown that Galnt3 is abundantly expressed in other tissues throughout the body, including the salivary glands (23Bennett E.P. Mandel U. Clausen H. Gerken T.A. Fritz T.A. Tabak L.A. Control of mucin-type O-glycosylation: a classification of the polypeptide GalNAc-transferase gene family.Glycobiology. 2012; 22 (22183981): 736-75610.1093/glycob/cwr182Crossref PubMed Scopus (531) Google Scholar, 38Zara J. Hagen F.K. Ten Hagen K.G. Van Wuyckhuyse B.C. Tabak L.A. Cloning and expression of mouse UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase-T3.Biochem. Biophys. Res. Commun. 1996; 228 (8912633): 38-4410.1006/bbrc.1996.1613Crossref PubMed Scopus (52) Google Scholar). To begin to interrogate the relative abundance and temporal expression of Galnt3 in the submandibular glands (SMGs), we investigated expression levels of each Galnt family member at three embryonic stages (embryonic day 13 (E13), E15, and E17), two early postnatal stages (postnatal day 2 (P2) and 1 week), and three adult stages (4, 8, and 12 weeks) by performing qPCR (Fig. 1 and Fig. S1). At E13, Galnt1 is the most abundantly expressed isoform, followed by Galnt2 and Galnt11 (Fig. 1A). At E15 and E17, Galnt2 becomes the most abundantly expressed isoform, followed by Galnt1 at E15 and Galnt4 and Galnt1 at E17 (Fig. 1A). Interestingly, a dramatic change in Galnt expression occurs after birth, as Galnt3 and Galnt12 are the most abundantly expressed isoforms at P2 and 1 week of age (Fig. 1B and Fig. S1A). Moreover, Galnt3 expression continues to increase into adulthood, where it is by far the most abundant isoform at 4, 8, and 12 weeks of age (Fig. 1B and Fig. S1A), in both males and females (Fig. S1B). In situ hybridization revealed that Galnt3 expression is confined to the acinar cells (detected with Aqp5) of the SMGs, the cells responsible for producing the secreted components of the saliva (Fig. 1C). Previous work has shown that one of the most abundantly expressed Galnts during embryonic stages (Galnt1) plays key roles during SMG growth and development (28Tian E. Hoffman M.P. Ten Hagen K.G. O-Glycosylation modulates integrin and FGF signalling by influencing the secretion of basement membrane components.Nat. Commun. 2012; 3 (22643896): 86910.1038/ncomms1874Crossref PubMed Scopus (46) Google Scholar). This led us to examine the possibility that Galnt3, the most abundant isoform in adult SMGs, may play a role in adult SMG function. To investigate this, we crossed heterozygous Galnt3-deficient (Galnt3+/−) animals to generate homozygous Galnt3-deficient (Galnt3−/−) animals and WT littermate controls. Galnt3−/− mice have been previously generated as a mouse model for HFTC and were found to have hyperphosphatemia, inappropriately normal levels of 1,25-dihydroxyvitamin D, and decreased levels of circulating intact Fgf23, similar to what is seen in HFTC patients (36Ichikawa S. Sorenson A.H. Austin A.M. Mackenzie D.S. Fritz T.A. Moh A. Hui S.L. Econs M.J. Ablation of the Galnt3 gene leads to low-circulating intact fibroblast growth factor 23 (Fgf23) concentrations and hyperphosphatemia despite increased Fgf23 expression.Endocrinology. 2009; 150 (19213845): 2543-255010.1210/en.2008-0877Crossref PubMed Scopus (125) Google Scholar). As it is well-documented that rodent SMGs become morphologically distinct between males and females beginning at ∼6 weeks of age (with males developing many more granular convoluted tubules than females) (39Gresik E.W. The postnatal development of the sexually dimorphic duct system and of amylase activity in the submandibular glands of mice.Cell Tissue Res. 1975; 157 (1122548): 411-42210.1007/bf00225529Crossref PubMed Scopus (86) Google Scholar), all future experiments analyzed male and female animals separately. Upon examination of SMGs at 8 weeks of age, we found no significant differences in gland weight (Fig. S2, A and B) or morphology (Fig. S2C) between WT and Galnt3−/− mice for either sex. Previous studies from our laboratory investigating the role of another member of this family in the SMG found an induction of ER stress upon loss of Galnt1 (28Tian E. Hoffman M.P. Ten Hagen K.G. O-Glycosylation modulates integrin and FGF signalling by influencing the secretion of basement membrane components.Nat. Commun. 2012; 3 (22643896): 86910.1038/ncomms1874Crossref PubMed Scopus (46) Google Scholar). To determine whether the loss of Galnt3 also induced ER stress and the unfolded protein response (similar to that seen in Galnt1−/− SMGs (28Tian E. Hoffman M.P. Ten Hagen K.G. O-Glycosylation modulates integrin and FGF signalling by influencing the secretion of basement membrane components.Nat. Commun. 2012; 3 (22643896): 86910.1038/ncomms1874Crossref PubMed Scopus (46) Google Scholar)), we next examined Xbp1 mRNA splicing. Quantification of the ratio of spliced Xbp1 (Xbp1s) to unspliced Xbp1 (Xbp1u) revealed no statistically significant differences between WT and Galnt3−/− SMGs for either sex (Fig. S2, D–G), indicating that the loss of Galnt3 does not result in ER stress. Additionally, we performed a number of analyses of adult SMG function. Ex vivo analysis of SMG salivary flow rate, volume of saliva secreted, and ion content of saliva revealed no significant differences between WT and Galnt3−/− animals for either sex (Fig. S3, A–F). These results indicate that the loss of Galnt3 does not result in overt differences in SMG size, morphology, or function via these assays. In addition to its role in hydration and lubrication of the oral cavity, saliva is believed to play a role in regulating the oral microbiota (40Tabak L.A. In defense of the oral cavity: structure, biosynthesis, and function of salivary mucins.Annu. Rev. Physiol. 1995; 57 (7778877): 547-56410.1146/annurev.ph.57.030195.002555Crossref PubMed Scopus (297) Google Scholar). This led us to investigate the effect of loss of Galnt3 on the microbial communities within the oral cavity through the characterization of the local microbiome. Microbiome studies analyze the numbers and types of distinct microbial taxa as well as their relative abundances to provide assessments of microbial community structures. We collected oral mucosal samples as described previously (41Abusleme L. Hong B.Y. Hoare A. Konkel J.E. Diaz P.I. Moutsopoulos N.M. Oral microbiome characterization in murine models.Bio. Protoc. 2017; 7 (29333479): e265510.21769/BioProtoc.2655Crossref PubMed Google Scholar) from individual WT and Galnt3−/− mice at both 8 and 12 weeks of age and performed 16S rRNA gene sequencing. β-diversity comparisons of the microbial community structure within the oral cavity across genotypes and age groups were assessed using the θ Yue and Clayton (θ YC) distance (which takes into account the specific bacterial species present as well as their relative abundances within the communities being compared) and visualized on two-dimensional principal coordinates analysis (PCoA) plots (Fig. 2). We also analyzed α-diversity within each genotype and age group (which measures the number of bacterial taxa and their relative proportions within a particular group),