Ameloblastin Amphipathic Helix Motif Mediates Ameloblast Polarization and Prismatic Enamel Formation via A RoA Signaling Pathway

Ameloblastin Amphipathic Helix Motif Mediates Ameloblast Polarization and Prismatic Enamel Formation via A RoA Signaling Pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Ameloblastin Amphipathic Helix Motif Mediates Ameloblast Polarization and Prismatic Enamel Formation via A RoA Signaling Pathway Janet Moradian-Oldak, Gayathri Visakan, Rucha Arun Bapat , Jing Cai, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8492927/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Ameloblastin (Ambn) is a tooth-specific multifunctional protein crucial for enamel biomineralization and its prismatic structure. To examine the function of the evolutionarily conserved cell-binding Ambn amphipathic helix (AH) motif, we deleted the hydrophobic residues within Ambn AH motif in genetically engineered mice. Enamel in the homozygous (AmbnΛL76-P86) mutants had normal thickness but was hypo-mineralized and lacked prismatic structure. Micro-CT analysis using conventional neural network revealed loss in mineral density and a delay in the initiation of secretory stage of amelogenesis. Ameloblasts in the mutants were stunted and exhibited loss of cell polarity, as demonstrated by the mis-localization of Pard3, Claudin-1 and GM130 immunosignals. In the AmbnΔL76-P86 mutants, a loss of Ambn-ameloblast distal membrane interaction was observed, with nuclear localization of Β-catenin and p-Smad2/3, and a decrease in RhoA immunolabeling intensity. Our study demonstrates a novel signaling role of Ambn AH motif in mediating cell polarization and forming enamel prismatic structure. Biological sciences/Cell biology/Cell signalling/Extracellular signalling molecules Health sciences/Diseases/Dental diseases Full Text Additional Declarations There is no conflict of interest Supplementary Files SupplementaryInformation.pdf Suplementary Material Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: revise 05 Mar, 2026 Review # 3 received at journal 22 Feb, 2026 Review # 2 received at journal 03 Feb, 2026 Review # 1 received at journal 01 Feb, 2026 Reviewer # 3 agreed at journal 26 Jan, 2026 Reviewer # 2 agreed at journal 23 Jan, 2026 Reviewer # 1 agreed at journal 23 Jan, 2026 Reviewers invited by journal 23 Jan, 2026 Submission checks completed at journal 19 Jan, 2026 Editor assigned by journal 31 Dec, 2025 First submitted to journal 31 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8492927","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":579238415,"identity":"e64eb101-ae04-4bd4-b99f-96aea7fc7a28","order_by":0,"name":"Janet Moradian-Oldak","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsUlEQVRIiWNgGAWjYBACxgbmBgmGAzYQHg9xWhhBWtJI0ALSBNRymAQtzO2NjTd+nDmfOL/9AOODt23E2NFzsNmy58btxA1nEpgN5xKlZUZimwTPB6AWCQY2aV5itUj++XAucf4MBvbfRGuR5rlxILHhBgMbM3FagH6xljmTbLzhTGKz5JxzRGgxbG8+ePPNMTvZ+e2HD354U0aMlgaEhQ04VaEAeeKUjYJRMApGwYgGACVsPQQWMjzxAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-5777-6297","institution":"USC","correspondingAuthor":true,"prefix":"","firstName":"Janet","middleName":"","lastName":"Moradian-Oldak","suffix":""},{"id":579238416,"identity":"84f0ab5e-57ba-4df9-8469-dffd1e36e16f","order_by":1,"name":"Gayathri Visakan","email":"","orcid":"","institution":"USC","correspondingAuthor":false,"prefix":"","firstName":"Gayathri","middleName":"","lastName":"Visakan","suffix":""},{"id":579238417,"identity":"b60500be-dd33-43bc-835b-5e5effee75ec","order_by":2,"name":"Rucha Arun Bapat ","email":"","orcid":"","institution":"USC","correspondingAuthor":false,"prefix":"","firstName":"Rucha ","middleName":"","lastName":"Arun Bapat ","suffix":""},{"id":579238418,"identity":"8ddb5acf-79ec-4384-acd7-7fc81178dcda","order_by":3,"name":"Jing Cai","email":"","orcid":"https://orcid.org/0000-0002-9207-7907","institution":"USC","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Cai","suffix":""},{"id":579238419,"identity":"b4b14b33-a6bd-4784-bedb-5badb667a560","order_by":4,"name":"Ethan Trevor Suwandi ","email":"","orcid":"","institution":"Northwestern University","correspondingAuthor":false,"prefix":"","firstName":"Ethan ","middleName":"","lastName":"Trevor Suwandi ","suffix":""},{"id":579238420,"identity":"b59ca89a-1bc1-4b8e-8e70-b722d5f77ab5","order_by":5,"name":"Derk Joester","email":"","orcid":"https://orcid.org/0000-0002-9663-3309","institution":"Northwestern University","correspondingAuthor":false,"prefix":"","firstName":"Derk","middleName":"","lastName":"Joester","suffix":""},{"id":579238421,"identity":"ac479858-d5ce-4d83-acc6-42efbff6d909","order_by":6,"name":"Natalie Kegulian ","email":"","orcid":"","institution":"USC","correspondingAuthor":false,"prefix":"","firstName":"Natalie ","middleName":"","lastName":"Kegulian ","suffix":""},{"id":579238422,"identity":"654d7c43-f515-4c4f-8bfc-d7c880cc5351","order_by":7,"name":"Edwin Sarkisians ","email":"","orcid":"","institution":"USC","correspondingAuthor":false,"prefix":"","firstName":"Edwin ","middleName":"","lastName":"Sarkisians ","suffix":""},{"id":579238423,"identity":"320bbe40-6118-4a34-a560-12b5d99cd221","order_by":8,"name":"Marziyeh Aghazadeh","email":"","orcid":"","institution":"USC","correspondingAuthor":false,"prefix":"","firstName":"Marziyeh ","middleName":"","lastName":"Aghazadeh","suffix":""},{"id":579238424,"identity":"63cece8e-c5fd-4bfb-bfae-e5ead4d6f19d","order_by":9,"name":"Simon Webster Webster","email":"","orcid":"","institution":"USC","correspondingAuthor":false,"prefix":"","firstName":"Simon Webster ","middleName":"","lastName":"Webster","suffix":""}],"badges":[],"createdAt":"2026-01-01 03:30:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8492927/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8492927/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101278774,"identity":"600deb7a-bac2-4835-bf00-5a6743751339","added_by":"auto","created_at":"2026-01-28 04:22:07","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1666783,"visible":true,"origin":"","legend":"Article File","description":"","filename":"Dec31ManuscriptVisakanOldaketal.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8492927/v1_covered_0bd37dd4-ad14-4106-9e08-3190b9bb9933.pdf"},{"id":101278773,"identity":"2e622744-9efa-4233-9581-c59e6ebe9d18","added_by":"auto","created_at":"2026-01-28 04:22:01","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":7680017,"visible":true,"origin":"","legend":"Suplementary Material","description":"","filename":"SupplementaryInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8492927/v1/699eba5f70d34f8acb58e42a.pdf"}],"financialInterests":"There is no conflict of interest","formattedTitle":"Ameloblastin Amphipathic Helix Motif Mediates Ameloblast Polarization and Prismatic Enamel Formation via A RoA Signaling Pathway","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"international-journal-of-oral-science","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"ijos2","sideBox":"Learn more about [International Journal of Oral Science](http://www.nature.com/ijos/)","snPcode":"41368","submissionUrl":"https://mts-ijos.nature.com/cgi-bin/main.plex","title":"International Journal of Oral Science","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8492927/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8492927/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Ameloblastin (Ambn) is a tooth-specific multifunctional protein crucial for enamel biomineralization and its prismatic structure. To examine the function of the evolutionarily conserved cell-binding Ambn amphipathic helix (AH) motif, we deleted the hydrophobic residues within Ambn AH motif in genetically engineered mice. Enamel in the homozygous (AmbnΛL76-P86) mutants had normal thickness but was hypo-mineralized and lacked prismatic structure. Micro-CT analysis using conventional neural network revealed loss in mineral density and a delay in the initiation of secretory stage of amelogenesis. Ameloblasts in the mutants were stunted and exhibited loss of cell polarity, as demonstrated by the mis-localization of Pard3, Claudin-1 and GM130 immunosignals. In the AmbnΔL76-P86 mutants, a loss of Ambn-ameloblast distal membrane interaction was observed, with nuclear localization of Β-catenin and p-Smad2/3, and a decrease in RhoA immunolabeling intensity. Our study demonstrates a novel signaling role of Ambn AH motif in mediating cell polarization and forming enamel prismatic structure.","manuscriptTitle":"Ameloblastin Amphipathic Helix Motif Mediates Ameloblast Polarization and Prismatic Enamel Formation via A RoA Signaling Pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-28 04:21:56","doi":"10.21203/rs.3.rs-8492927/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2026-03-05T08:21:50+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-02-23T01:54:38+00:00","index":3,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-02-04T02:51:52+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-02-01T13:26:17+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-01-27T02:34:17+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-01-23T10:37:47+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-01-23T10:26:43+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2026-01-23T09:19:29+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-20T02:16:35+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-01T03:26:06+00:00","index":"","fulltext":""},{"type":"submitted","content":"International Journal of Oral Science","date":"2026-01-01T03:26:05+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"international-journal-of-oral-science","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"ijos2","sideBox":"Learn more about [International Journal of Oral Science](http://www.nature.com/ijos/)","snPcode":"41368","submissionUrl":"https://mts-ijos.nature.com/cgi-bin/main.plex","title":"International Journal of Oral Science","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"daa7edae-484f-44b5-a771-d8783e9e0c49","owner":[],"postedDate":"January 28th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":61632321,"name":"Biological sciences/Cell biology/Cell signalling/Extracellular signalling molecules"},{"id":61632322,"name":"Health sciences/Diseases/Dental diseases"}],"tags":[],"updatedAt":"2026-05-05T20:55:18+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-28 04:21:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8492927","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8492927","identity":"rs-8492927","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}