-
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; [email protected] ; 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
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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.
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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
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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.
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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.
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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.
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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.
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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.
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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
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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
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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
-
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