Supporting Information

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

Gabriella Peluso1, E Tian1, Loreto Abusleme2, Takashi Munemasa4,5, Taro Mukaibo4,5, and Kelly G. Ten Hagen1*

From the 1Developmental Glycobiology Section, NIDCR, National Institutes of Health, Bethesda, Maryland 20892-4370 2Laboratory of Oral Microbiology, and 3Laboratory of Craniofacial Translational Research, Faculty of Dentistry,

University of Chile, Santiago, Chile 4Secretory Mechanisms and Dysfunctions Section, National Institute of Dental and Craniofacial Research, National

Institutes of Health, Bethesda, MD 20892, USA 5Division of Oral Reconstruction and Rehabilitation, Kyushu Dental University, Kitakyushu, Fukuoka 803-8580, Japan

*To whom correspondence should be addressed: Kelly G. Ten Hagen, Ph.D., Building 30, Room 407, 30 Convent Drive, MSC 4370, Bethesda, MD 20892-4370; Kelly.Tenhagen@nih.gov ; Tel: 301-451-6318; Fax: 301-402-0897. File contents: Supporting information, Figure S1 Supporting information, Figure S2 Supporting information, Figure S3 Supporting information, Figure S4 Supporting information, Figure S5 Supporting information, Figure S6 Supporting information, Figure S7 Supporting information, Figure S8 Supporting information, Figure S9 Supporting information, Figure S10 Supporting information, Table S1 Supporting information, Table S2 Supporting information, Table S3 Supporting information, References

Figure S1. Galnt gene expression in SMGs. (A) qPCR analysis of entire murine Galnt family in early postnatal and adult

SMGs. (B) Gene expression of Galnts in 12 wk SMGs separated by sex. No difference in expression of Galnt3 was observed

between male and female SMGs. Values represent mean from three or more animals. Expression was normalized to 29S

rRNA and is represented as percentage of total Galnt expression at each individual stage of development in (A) or for each

gender in (B). Error bars are SD.

Figure S2. Loss of Galnt3 does not affect SMG weight, SMG morphology, or the ER stress response. SMG weight is

unchanged in 8 wk Galnt3-/- animals as compared to WT for both males (A) and females (B). Values represent mean ± SD.

Each dot represents one mouse. (C) Hematoxylin and eosin (H&E) staining of 8 wk SMGs. No obvious differences were

seen between WT and Galnt3-/- SMGs for either sex. Scale bar, 100 μm. (D-G) ER stress-dependent Xbp1 mRNA splicing.

Spliced Xbp1 mRNA was variably detected in 8 wk WT and Galnt3-/- SMGs from male (D) and female (E) animals. 10

ng/μl tunicamycin-treated E12.5 SMGs (TM) were used as a positive control and DMSO-treated SMGs (DMSO) were used

as a negative control. H2O was used as a no cDNA control. Quantification of spliced Xbp1 (Xbp1s) to unspliced Xbp1

(Xbp1u) (WT ratio set to 1) revealed no significant differences between WT and Galnt3-/- SMGs for males (F) or females

(G). Values represent mean ± SD. Each dot represents one mouse.

Figure S3. Loss of Galnt3 does not affect SMG saliva secretion or ion concentrations. Ex vivo salivary flow assay

using carbachol/isoproterenol-stimulated 12 wk SMGs. Salivary flow rate (μl/min) (A), volume of saliva secreted (μl) (B),

and ion concentrations (mM) (C) for male SMGs is shown. Salivary flow rate (μl/min) (D), volume of saliva secreted (μl)

(E), and ion concentrations (mM) (F) for female SMGs is shown. No differences were observed between WT and Galnt3-/-

SMGs for either sex. Values represent mean ± SD from three or more animals.

Figure S4. Alpha diversity analyses for all groups. Non-parametric Shannon diversity Index was used to assess alpha

diversity of all groups analyzed in the main text. Each dot represents one mouse. Boxes extend from the 25th to 75th

percentiles, and the whiskers were plotted from the minimum to maximum value. All outlying values are shown. (A) 8

wk WT and Galnt3-/- males, (B) 12 wk WT and Galnt3-/- males, (C) 8 wk WT and Galnt3-/- females, (D) 12 wk WT and

Galnt3-/- females, (E) WT males at 8 wks and 12 wks, (F) WT females at 8 wks and 12 wks, (G) Galnt3-/- males at 8 wks

and 12 wks, (H) Galnt3-/- females at 8 wks and 12 wks, (I) 8 wk WT males and females, (J) 12 wk WT males and

females. ns, not significant; *, p

Figure S5. Relative abundance of taxa in oral swab samples. Relative abundance of bacterial taxa at genus level in oral

swab samples from 8 wk WT and Galnt3-/- females (A); 12 wk WT and Galnt3-/- females (B); 8 wk and 12 wk WT males

(C); and 8 wk and 12 wk WT females (D). Each bar represents one mouse. Unclassified genera are shown at family level.

Species level taxonomy is reported in parenthesis when > 97% similarity achieved with BLAST. No significant differences

were found for any of the comparisons shown.

Figure S6. Comparison of WT male and female oral microbiomes. Principle coordinates analysis (PCoA) plots

comparing 8 wk WT male and female (A), and 12 wk WT male and female oral microbiomes based on θ YC distances (to

analyze microbial community structure) with 95% confidence ellipses. The oral microbiome was sampled at both 8 wks and

12 wks for each individual animal. Statistically significant differences in community structure were seen at 8wks (as

determined by AMOVA) but not at 12 wks. Relative abundance of bacterial taxa at genus level from 8 wk WT male and

female (C), and from 12 wk WT male and female (D) oral swab samples. Each bar represents one mouse. Unclassified

genera are shown at the family level. Species level taxonomy is reported in parenthesis when > 97% similarity achieved

with BLAST. * indicates species over-represented in female samples and # represents species over-represented in

male samples according to LEfSe analyses.

Figure S7. PNA staining specificity in male and female SMGs. Immunofluorescent staining of WT male (A) and female

(B) SMGs with the lectin PNA (red) or with PNA competed with the sugar galactose (PNA+0.2M Gal) to demonstrate

specificity. Male SMGs were not treated with NM but female SMGs were treated with NM to remove sialic acid and allow

PNA detection. Nuclei are shown in blue. Scale bars, 10 μm.

Figure S8. Immunoprecipitation of Muc10 from WT SMGs. Immunoprecipitation of Muc10 from 8 wk WT male SMGs

using a Muc10 antibody, followed by western blotting with PNA lectin (to detect O-glycans, red) and Muc10 antibody

(green). Immunoprecipitated Muc10 is O-glycosylated (detected with PNA). IP = immunoprecipitation; FT = flow through;

Ab = Muc10 antibody. Molecular weight markers (kDa) are shown to the left.

Figure S9. Peptide and full-length Muc10 amino acid sequences. Two degenerate full-length amino acid sequences

(273 aa and 264 aa) of Muc10 showing location of Muc10 peptide sequences used for Galnt3 enzyme assays. Peptide

names in the table are color-coded to corresponding sequences within full-length Muc10. The repeat domain of Muc10 is

underlined.

Figure S10. Muc10 is a substrate for Galnt3. Two additional repeats of the in vitro enzymatic activity experiments of

Galnt3 against Muc10 acceptor peptides are shown in A and B. EA2 was used as a positive control. Galnt3 was able to

A

B

glycosylate the Muc10-s, Muc10-273, Muc10-264, and Muc10-l peptides. Galnt3 showed no activity toward the Muc10-

10 peptide. Each data point represents an individual assay. Error bars are SD.

Mutation Phenotype Sex Ethnicity HFTC Dental Phenotype Other Dental/Oral Cavity Findings Reference(s)

1

p.K465_y508del; p.R162X

HFTC M

African American

Yes

In 6 of 7 affected siblings: • Normal mucosal coloring and architecture• Normal salivary flow from all major glands• Caries and mild-to-moderate gingivitis

Baldursson et al. 1969Clarke et al. 1984Viegas et al. 1985Witcher et al. 1989

Slavin et al. 1993Topaz et al. 2004Carmichael et al. 2009

2 HFTC + HHS F Yes

3 HFTC + HHS M Yes

4 HFTC M Yes

5 HFTC F Yes

6 HFTC + HHS F N/A

7 HFTC F Yes

8

p.C173VfsX4

HFTC M

African American

Edentulous • Edentulous

McPhaul et al. 1961Lyles et al. 1985Martinez et al. 1990Ichikawa et al. 2005

9 HFTC + HHS M Yes• Significant periodontal and periapical disease• Clinically hypoplastic teeth with fully developed enamel of normal

color

10 p.C173VfsX4; p.R162X

HFTC + HHS M Yes• Clinically hypoplastic teeth with fully developed enamel of normal

color

11 p.K463X HFTC M Caucasian N/A • Dense mandibular inclusions Campagnoli et al. 2006

12 p.Q592X HFTC M Caucasian Yes • Thin dental enamel Specktor et al. 2006

13

p.C173VfsX4

HFTC M

African

Yes Both:• Generalized gingivitis• Multiple tooth decay• Hypoplastic teeth

• Attack of enamel à amelogenesis imperfecta• Visible tooth resorption

Laleye et al. 200814 HFTC M Yes

15 p.R438C; p.Q592X HFTC + HHS F Caucasian Yes • Firm nodule on right lower gum line• Solid submental calcification of the salivary glandDumitrescu et al. 2009Ramnitz et al. 2016

16 p.R438C HFTC F Pakistani Yes • Abnormal gum root structure Yancovitch et al. 2011

17p.G256V

HFTC M

Caucasian

Yes• Dental abscesses and spontaneous tooth loss started at age 25• At age 42, all permanent teeth substituted by implants but dental

health otherwise good Rafaelsen et al. 2014

18 HFTC + HHS F Yes • Dental problems similar to her brother in her 20s• At age 55, some dental implants but good dental health

19 N/A HFTC M N/A Yes

• Enamel hypoplasia• Maxillary and mandibular hypoplasia• Skeletal class II malocclusion• Dental deep-bite

• Multiple impacted teeth• Calcified deposits in minute nerve bundles in the buccal mucosa

Favia et al. 2014

20 p.R162X; p.A440E HFTC + HHS M African American

N/A • Painful and recurrent calcified lesions of right mandible Finer et al. 2014

21 p.R162X HFTC + HHS M African N/A • Chronic recurrent multifocal osteomyelitis of maxilla and mandible Demellawy et al. 2015

Oral cavity involvement in HFTC patients with GALNT3 mutations

Supplementary Table S1

Gene Primers

Galnt1 Sense: 5'-TCATCAAGAGCAGCGGCAAAGC-3'Anti-sense: 5'- ACAAGGCACATTCAGCAGAAACGG-3'

Galnt2 Sense: 5'-CGCCCTCTGCCTCCCTCTTTC-3'Anti-sense: 5'-TGATTGCTGCTTGCCCACTTGTTC-3'

Galnt3 Sense: 5'-TGCTACTCAGGGTGTCGTCCAG-3'Anti-sense: 5'-GCGTCACATGGCACTAAGTTTGG-3'

Galnt4 Sense: 5'-CCGCAATCGTATGTCCTGTCATCG-3'Anti-sense: 5'-AACGCCAGTCAAACCCACCAATC-3'

Galnt5 Sense: 5'-GCCGAGCAGAGATGGAAAGAAGG-3'Anti-sense: 5'-CTGGTGGTTGGGAGGTCATTGTG-3'

Galnt6 Sense: 5'-GTGTTGACCAGAAGTTCCG-3'Anti-sense: 5'-GATTTCATTCAGCAAGATGGC-3'

Galnt7 Sense: 5'-GGCTCGTGGTCCTCTGGTCTTC-3'Anti-sense: 5'-TCTCTGTCTTCCCTCATCCTGCTC-3'

Galnt9 Sense: 5’-CATCTGGAGGAGGTGGTCTACAAC-3’ Antisense: 5’-CTCGGTCTCTTGGCTGTCATCTTG-3’

Galnt10 Sense: 5'-CCGAGGCGAGGCTGCTTGG-3'Anti-sense: 5'-GGGTGACTGGGCTGGTGTGG-3'

Galnt11 Sense: 5'-CAGCAGTGGACCTTTGGGAAGAAC-3'Anti-sense: 5'-TGTTGAGAGGAGGAGCCATCGC-3'

Galnt12 Sense: 5'-CCGAGAGACCGTCCCAGAGAAC-3'Anti-sense: 5'-ACATTTCCTGCTGTGCTTGTGAAC-3'

Galnt13 Sense: 5'-CACCCGTCTTCAGTCTCCGTATTG-3'Anti-sense: 5'-GACATCAACAAGCACCCACATCAG-3'

Galnt14 Sense: 5'-GATGAGCGGCGGTATCTGAATGC-3'Anti-sense: 5'-GGTGATGATGATGCTGGTGTGAGG-3'

Galnt15 Sense: 5'-GGACTGGAGGACCGAAGAGGATG-3'Anti-sense: 5'-AGAGGATGACGCTGGCTGTAGG-3'

Galnt16 Sense: 5'-CGCCAATGCCATCGCCATCC-3'Anti-sense: 5'-GCTCGGTTGTCCTGCCATAAGTAG-3'

Galnt18 Sense: 5'-CCTGCCCTGCTCTCGGATTGC-3'Anti-sense: 5'-GCCTTGCGTGCGGTGATGTC-3'

Galnt19 Sense: 5'-TGGGTGTATGTTTGCGTGCTTGAG-3'Anti-sense: 5'-GCGTCCTTGTCCCTATCCACTGAG-3'

Galnt20 Sense: 5’-TCGGGCTCTGGGCATCTATGTTAC-3’Antisense: 5’-CTGAGTGACGGGTTTGCTGGTG-3’

Supplementary Table S2

Gene Primers

Mucin 1 Sense: 5’-GACCACTTCTGCCAACTTGT-3’Anti-sense: 5’-GGCTTCACCAGGCTTACG-3’

Mucin 2 Sense: 5’-GCTGACGAGTGGTTGGTGAATGAC-3’Anti-sense: 5’-GATGAGGTGGCAGACAGGAGACA-3’

Mucin 4 Sense: 5’-GAGGAGCAGGAGGCACAGAA-3’Anti-sense: 5’-CCGTTGAAGGTGAAGTTGGCATTA-3’

Mucin 5ac Sense: 5’-AGCCTCCTCTTGTTCTGAGATGTC-3’Anti-sense: 5’-GCTCACAGAGTGGCGATGGT-3’

Mucin 6 Sense: 5’-ACGGACCGCAGCACTTCTC-3’Anti-sense: 5’-CCTGGCAACGAGTTAGAGTCACAT-3’

Mucin 10 Sense: 5’-AACCACACCAGCAACAACCACAA-3’Anti-sense: 5’-GGCTGTAGAGGTGCTAGGCTTAGG-3’

Mucin 13 Sense: 5’-GGTAGCAGGTGGCGTCTT-3’Anti-sense: 5’-AGTGAAGCATCATTGAGTGGACA-3’

Mucin 20 Sense: 5’-CTAAGGACCTCACTGAGCACAAC-3’Anti-sense: 5’-TGGCATCACCGTTCTTCTGG-3’

Supplementary Table S3

References

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