Boston University
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Theses & Dissertations Boston University Theses & Dissertations
2017
PERIOSTIN is a direct binding
partner of FAM20C
https://hdl.handle.net/2144/21970 Boston University
BOSTON UNIVERSITY
HENRY M. GOLDMAN SCHOOL OF DENTAL MEDICINE THESIS
PERIOSTIN IS A DIRECT BINDING PARTNER OF FAM20C JU-HSIEN LIN
Doctoral of Dental Surgery (D.D.S), Dentistry of Chung Shan Medical University, 2010
Submitted in partial fulfillment of the requirements for the degree of Master of Science in Dentistry
In the Department of Periodontology and Oral Biology 2017
Approved by:
First
Reader _______________________________________________Date_______________ Dr. Yoshiyuki Mochida, DDS, Ph.D.
Clinical Assistant Professor
Department of Molecular and Cell Biology
Second Reader _______________________________________________Date_______________ Dr. Wayne A Gonnerman, Ph.D. Assistant Professor Department of Periodontology Department Chariman_______________________________________________Date_____________ Dr. Serge Dibart, DMD Professor Department of Periodontology
Dedication
This thesis is dedicated to my father and mother, who have shown me unconditional love and sacrifice and helped me emotionally and financially in all aspects of my life. They are the reason for all my success and my mother is truly a remarkable woman. To Dr. Wei-chen Lu the best husband, best friend, and the inspiration of my life since the day we met. I’m very thankful for all the wonderful people in my life, I’m truly blessed.
Acknowledgments
I thank Dr. Serge Dibart D.M.D for giving me the opportunity to continue my dental education in the field of Periodontology and Oral Biology.
I was privileged to be under the instruction of Dr. Mochida D.D.S, Ph.D. He has taught me to be exceptional in my work and to always strive for
perfection. I attribute the success of this research to Dr. Mochida. I was truly blessed to be under his supervision.
PERIOSTIN IS A DIRECT BINDING PARTNER OF FAM20C
JU-HSIEN LIN
Henry M. Goldman School of Dental Medicine, Boston University, 2017 Major Professor: Dr. Yoshiyuki Mochida, DDS, Ph.D.,
Clinical Assistant Professor of Molecular and Cell Biology
ABSTRACT
Periodontium consists of gingiva, alveolar mucosa, alveolar bone, cementum and periodontal ligament (PDL). PERIOSTIN, which is expressed in fibroblastic cells in PDL and in osteoblastic cells on the alveolar bone surface, is a key
extracellular matrix protein that maintains homeostasis of periodontal tissue. During the process for FAM20C protein purification, we discovered that
PERIOSTIN is co-purified with FAM20C. Immunolocalization of both FAM20C and PERIOSTIN were observed in the PDL extracellular matrix of murine periodontal tissues. PERIOSTIN bound with FAM20C in vitro. We further identified the binding domain of FAM20C in PERIOSTIN, which mapped within Fasciclin (FAS) I domain 1-4 (RD 1-4) of PERIOSTIN. PERIOSTIN has four conserved S-X-E motifs in both human and mouse FAS domain, which allowed us to use the murine model determine whether PERIOSTIN is phosphorylated by FAM20C and whether this phosphorylation might contribute to the integrity of periodontium tissues.
Table of Contents
Title
Page……….i
Reader’s approval page ………....……….ii
Abstract………..v
Table of contents……… vii
List of figures …………...………..viii
List of tables….……….……...ix
List of abbreviation………...x
List of illustration………..xi
1 Introduction………...……….1
1.1 Periodontium………..……….1
1.2 Periostin……….……….…….5
1.3 FAM20C………...………...8
2 Objective………..9
3 Materials and Methods………10
3.1 Reagents and antibodies………10
3.2 Plasmid construction………12
3.3 Cell culture………12
3.4 Transfection, Immunoprecipitation, and Western Blot Analysis…...…13
3.6 Purification of FAM20C proteins………14
3.7 In vitro Binding assay………..15
3.8 Mapping of Fam20c-V5 binding domain to Periostin………...15
3.9 In vitro kinase assay………16
3.10 Phosphoprotome analyses using Biotinylated phos-tag™ …………..16
4 Results………...17
4.1 Binding of Fam20c to Periostin………..18
4.2 Mapping of FAM20C-binding domain in PERIOSTIN………19
4.3 Phosphorylation of Periostin by Fam20C……….19
4.4 Secretion of Periostin………...20
5 Discussion……….21
6 Figures…...………26
7 Reference………..37
List of Figures
Figure 1: Localization of FAM20C and PERIOSTIN in mouse periodontium……26
Figure 2: Binding of FAM20C to PEIOSTIN………...…29
Figure 3: Fasciclin domain in PERIOSTIN is the FAM20C-binding region……...31
Figure 4: Phosphorylation of PERIOSTIN by FAM20C………33
Figure 5: Secretion of PERIOSTIN………..34
List of Table
List of abbreviations
List of Illustration
Illustration 1. Structure of dentogingival junction……….1
1. Introduction
1.1 Periodontium
The periodontium consists of root cementum, periodontal ligament, bone lining the tooth socket (alveolar bone), and a part of the gingiva facing the tooth
(dentogingival junction) defined as those tissues supporting and investing the tooth (CHO & GARANT, 2000; NANCI & BOSSHARDT, 2006).
Dentogingival junction
The dentogingival junction, gingiva facing the tooth, is an adaptation of the oral mucosa that contains epithelial and connective tissue components. There are three functional compartments- gingival, sulcular, and junctional epithelium (CHO & GARANT, 2000; NANCI & BOSSHARDT, 2006).
Illustration 1. Structures of dentogingival junction. There are three main components of dentogingival junction: (1) free gingiva and attached gingiva, (2) gingival sulcus, (3) juctional epithelium (JE).
The function of junctional epithelium is to seal off periodontal tissues from the oral environment. Its integrity is thus essential for maintaining a healthy
periodontium. Periodontal disease sets in when the sealing fails. The junctional epithelium originates from the reduced enamel epithelium when the tooth erupts into the oral cavity. Structurally, the junctional epithelium is a nondifferentiated, stratified squamous epithelium with a very high turnover. It is derived from a layer of cuboidal basal cells situated away from the tooth surface that rest on a
basement membrane. The gingiva of the epithelial attachment, consists of a basal lamina-like structure that is adherent to the tooth surface and to which the superficial cell layer is attached by hemidesmosomes. Compared to gingival cells junctional epithelial cells contain more cytoplasm, rough endoplasmic reticulum, and Golgi bodies. They contain fewer tonofilaments and desmosomes, and have wider intercellular spaces. The latter fluid-filled spaces normally contain
polymorphonuclear leukocytes, PMN and monocytes that penetrate the subepithelial connective tissue through the junctional epithelium and into the gingival sulcus. The PMNs and mononuclear cells and tissue fluid are the first line of defense to control the perpetual microbial challenge (CHO & GARANT, 2000; NANCI & BOSSHARDT, 2006; Takei & Carranza, 2012).
Cementum
Cementum is the hard, avascular connective tissues that coats the roots of teeth and to which the principal periodontal ligament fibers attach. Based on the presence or absence of cementocytes it is classified as cellular or acellualr cementum.
Acellular fiber cementum Cellular fiber cementum
Location Cervical Apical and furcation
Component Cells absent Cementocytes present
Function Anchoring root to PDL Reshape the root
Table 1. Comparison of two type of cementum (NANCI & BOSSHARDT,
2006).
The composition of cementum is about 50% inorganic substances and 50% organic matrix. Type I collagen is the predominant organic component,
constituting up to 90% of the organic matrix. Other collagens include type III, a less cross-linked collagen found in high concentrations during development and repair ⁄ regeneration of mineralized tissues, and type XII which binds to type I collagen and also to non-collagenous matrix proteins. In mature cementum, type V, VI, and XIV collagens are found in trace amounts.(Bosshardt, 2005; CHO & GARANT, 2000; NANCI & BOSSHARDT, 2006).
Alveolar bone
There are three parts to jaw alveolar processes. The outer cortical plates (buccal, lingual, and palatal) of compact bone are the first. Second is the central spongiosa. Third is bone lining the alveolus. The cortical plate and bone lining the alveolus meet at the alveolar crest. Bone lining the socket provides
attachment for the periodontal ligament fiber bundles and is referred to as bundle bone (NANCI & BOSSHARDT, 2006). The non-collagenous composition of alveolar bone include type I and III collagens and polypeptides including bone sialoprotein and osteopontin (BARTOLD & Narayanan, 2006).
Periodontal ligament
The periodontal ligament is a soft, specialized connective tissue between the cementum covering the root of the tooth and the bone forming the socket wall. The width ranges from 0.15 to 0.38 mm, with its thinnest portion around the middle third of the root. It shows a progressive decrease in thickness with increasing age. It supports the teeth in their sockets and permits them to resist the forces generated by mastication. The periodontal ligament also has the capacity to act as a sensory receptor and modurates tissue homeostasis and repair ⁄ regeneration (CHO & GARANT, 2000; NANCI & BOSSHARDT, 2006). The periodontal ligament has a very high turnover rate. It is a fibrous tissue containing collagen type I, III, V, VI, and XII. The ligament also contains
2006). Furthermore, it has been shown that PDL cells have the capacity to differentiate into osteoblasts and cementoblasts (Chien, Lin, & Cho, 1999). The cells in periodontal ligament regulate the extent of demineralization and prevent ankylosis. Bone sialoprotein and osteopontin may play important roles in balancing this status. Matrix ‘Gla’ protein acts as an inhibitor of mineralization and is also present in periodontal tissues which may also act to preserve the periodontal ligament width. At the molecular level, it has been reported that Msx2 prevents the osteogenic differentiation of periodontal ligament fibroblasts by repressing Runx2 transcriptional activity (Yoshizawa et al., 2004). It has also been reported that glycosaminoglycans or RGD–cementum attachment protein may also play a role in maintaining the unmineralized state (J, SJ, RC, WA, & C, 1995; Ohno et al., 2002). Currently, further study is needed to determine how the periodontal ligament remains unmineralized.
1.2 PERIOSTIN
PERIOSTIN was first known as osteoblast-specific factor 2, but was renamed based on the expression patterns specific to periosteum and PDL. A potential function of PERIOSTIN is to act as a cell adhesion molecule for preosteoblasts and to induce osteoblast attachment and spreading (Horiuchi et al., 1999). In humans, the PERIOSTIN gene is located on chromosome 13 at map position 13q13.3. The gene product has 835 amino acids (Hamilton, 2008). PERIOSTIN
(POSTIN) is a 90-kDa secreted protein that has several domains composed of an amino-terminal EMI domain, tandem repeats of 4 Fas I domains, and a carboxyl-terminal region (CTR) including a heparin-binding site at its C-carboxyl-terminal end. Based on these typical Fas I domains, POSTIN is classified as a member of the fasciclin I family. Fasciclin I is a GPI-anchored Drosophila protein containing 4 tandem Fas I domains composed of about 150 amino acid residues each. They are not related to any other protein domain of known structures. It functions in guidance of axon growth [8]. It is reported that PERIOSTIN directly interacts with Type I collagen, FIBRONECTIN and NOTCH1 through its EMI domain, while TENASCIN-C and BMP-1 through its Fas I domains (Kii et al., 2006; 2010; Maruhashi, Kii, Saito, & Kudo, 2010; Norris et al., 2007; G. Takayama et al., 2006; Tanabe et al., 2010).
Illustration 2. Structure of PERIOSTIN. PERIOSTIN is a 90-KDa secreted protein, induced by TGFβ. The protein structure contains an amino-terminal EMI domain, a tandem 4 repeat of Fas1 domains, and a carboxyl-terminal domain CTR (I. Takayama & Kudo, 2012).
POSTN is highly expressed in collagen-rich connective tissues. It has been shown to be associated with various tissues and pathologies including bone,
PDLs, skin, cardiac valves, muscle injury, vascular injury, myocardial infarction, epithelial ovarian cancer, colorectal cancer, pulmonary vascular remodeling, bronchial asthma, and oral cancer.
PERIOSTIN functions in cardiac repair, tumorigenesis, and obesity. In cardiac repair, PERIOSTIN plays an important role in the development of the embryonic heart by increasing collagen fibrillogenesis and compaction. With regard to tumorigenesis, PERIOSTIN plays a role in tumor development and metastasis. The Fas 1–2 domain involved in cell adhesion facilitates tumor growth and angiogenesis. In obesity, PERIOSTIN is highly expressed in adipose tissue suggesting a role in repair or expansion of adipose tissue (Romanos, Asnani, Hingorani, & Deshmukh, 2013).
In this report, I will focus on possible functions of PERIOSTIN in the periodontium.
PERIOSTIN helps to maintain tissue integrity by enhancing the proteolytic activation of lysyl oxidase (LOX) which is responsible for collagen cross-link formation. It thus determines the diameter of collagen fibrils to give different tissue strength. (Kudo, 2011; Romanos et al., 2013) Mechanical loading has a positive relationship to bone growth and development. Under occlusional stress, PERIOSTIN stimulates osteoblast adhesion, differentiation, and survival through the FAK signaling pathway and suppresses the action of sclerostin by osteocytes (Horiuchi et al., 1999; Romanos et al., 2013; I. Takayama & Kudo, 2012). PDL
plays a role in transmission of masticatory force. PERIOSTIN expression level in PDL is proportionally up-regulated with mechanical stress (Kii et al., 2006;
Romanos et al., 2013). These findings suggest that PERIOSTIN has effects on the formation of the meshwork structure of the PDL fibers and consequent changes in periodontal structures.
1.3 FAM20C
FAM20C, also known as ‘‘dentin matrix protein 4’’ (DMP4) in mice, contains 579 amino acids in the human form. There is a 26-amino acid signal peptide at the N-terminus and approximately 350 amino acids at C- terminal region (D. Nalbant, Youn, & Nalbant, 2005). FAM20C is highly expressed in the mineralized tissues; i.e. osteocytes, odontoblasts, ameloblasts, and cementoblasts, as well as in the dentin and enamel. The high expression level of FAM20C in these tissues suggests that it has an important role in the formation and mineralization of these tissues (Wang et al., 2010).
Golgi casein kinase (G-CK) was first described in lactating mammary glands. It is capable of enzymatically phosphorylating endogenous Casein (Bingham & Farrell, 1974). Recently, FAM20C was identified as the Golgi casein kinase. It phosphorylates secretory pathway proteins within Ser-X-Glu/phospho-Ser (SxE/pS) motifs, observed in the small integrin-binding ligand, N-linked glycoproteins (SIBLINGs) (Tagliabracci, Engel, Wen, & Wiley, 2012).
In mammals, FAM20C is a member of the ‘‘family with sequence similarity 20’’; consisting of three members: FAM20A, FAM20B and FAM20C (D. Nalbant et al., 2005). FAM20C is also known as a causative gene for Raine syndrome (Hao, Narayanan, Muni, Ramachandran, & George, 2007). Raine syndrome is an autosomal recessive disorder characterized by generalized osteosclerosis with periosteal bone formation and a distinctive facial phenotype (Ishikawa, Xu, Ogura, Manning, & Irvine, 2012; Kinoshita, Hori, Taguchi, & Fukumoto, 2014). FAM20C kinase activity is essential for limiting bone formation in humans. Since SIBLING proteins as FAM20C substrates can act as nucleaters or inhibitors of
biomineralization, Raine syndrome’s feature could be explained by decreased phosphorylation of SIBLING proteins. To support this notion, it was been reported that ASARM peptide products inhibit mineralization by binding to
hydroxyapatite in a phosphorylation-dependent manner. (Addison, Masica, Gray, & McKee, 2009; Ishikawa et al., 2012).
2. Objectives
Studies in our lab previously found that PERIOSTIN co-purified with FAM20C. FAM20C as Golgi casein kinase (GCK) has the ability to phosphorylate secretory proteins. Therefore, the purposes of the current study were to investigate 1) where PERIOSTIN and FAM20C are located within craniofacial tissues and 2) whether FAM20C can bind to PERIOSTIN either directly or indirectly, and 3) to further map the binding domain of FAM20C in PERIOSTIN.
3. Materials and Methods 3.1 Reagents and antibodies
• Dulbecco's Modified Eagle Medium (DMEM) (1X), liquid (4.5 g/L
D-Glucose,L-Glutamine,110mg/L Sodium Pyruvate) was purchased from Life Technologies (Carlsbad, CA, USA).
• Penicillin and streptomycin mixture was added to DMEM using
Penicillin-Streptomycin Solution, 100X, 10,000 I.U. Penicillin 10,000 µg/mL and Streptomycin purchased from Mediatech, Inc. (Manassas, VA., USA).
• Fetal Bovine Serum, was purchased from Invitrogen (Carlsbad, CA, USA)
and added to DMEM.
• X-tremeGENE 9 DNA transfection reagent was purchased from Roche
• Cells were detached from culture plates using Trypsin, 0.25% 1X, with
2.5g porcine trypsin (1:250/L gamma irradiated) in HBSS with 1g/L EDTA, without calcium or magnesium purchased from Thermo Scientific
(Waltham, MA, USA).
• SDS NuPAGE® MOPS SDS Running Buffer (for Bis-Tris Gels only) (20X)
was purchased from Invitrogen (Carlsbad, CA, USA).
• NuPAGE® Novex 4-12% Bis-Tris Gel 1.0 mm, 12 well was purchased
from Invitrogen (Carlsbad, CA, USA).
• Immobilon-P Membrane purchased from Millipore (Billerica, MA, USA)
was used to transfer Western blot gel.
• Nonfat dry milk was purchased from Lab Scientific (Livingston, NJ, USA). • ECL™ Western Blotting Detection Reagents from GE Healthcare.
• HyBlot CL autoradiography film was purchased from Denville Scientific
Incorporation.
• Western Blot Stripping Buffer was purchased from Thermo Scientific. • Lysis buffer was prepared containing 150mM NaCl, 20mM Tris-HCl
(pH7.5), 10mM EDTA ,1% Triton-X 100 and 1% deoxycholate.
• Transfer buffer was prepared containing 0.025M Tris-HCl, 0.192M glycine,
and 20% methanol.
• Tris-buffered saline was prepared (TBS) (1X) containing 20mM Tris (pH
• TBS-T was prepared contains 20 mM Tris (pH 7.5), 137 mM NaCl, and
0.1% tween.
• SDS-Sample buffer was prepared containing 100mM Tris-HCL (pH8.8),
0.01% bromphenol blue, 36% glycerol and 4% SDS.
• Recombinant mouse PERIOSTIN protein (2955-F2) was obtained from
R&D Systems.
• The antibodies used in this study were as follows; anti-V5 (Life
Technologies), anti-HA (clone 12CA5, Roche Life Science), anti-HA high affinity (clone 3F10, Roche Life Science), and rabbit polyclonal anti-PERIOSTIN (ab14041, Abcam) antibody.
3.2 Plasmid construction
Human FAM20C expression vector constructs including wild-type (WT) and a mutant form lacking its kinase activity (D478A, {Tagliabracci, 2012 #213})
expression vector were generated by PCR methods. The plasmids containing the full length coding sequence for human FAM20C was purchased from Open Biosystems and used as a PCR template. The sequences of the primers were; (for FAM20C-WT) forward primer: 5’GCGGTACCGCCATGAAGATGATGCTGG-3’ and reverse primer: 5’-GCCTCGAGCCTCGCCGAGGCGGCTCTG-5’GCGGTACCGCCATGAAGATGATGCTGG-3’, (for FAM20C-D478A) mutagenesis forward primer:
CACTTAGCCAATGGAAGAGGG-3’ and mutagenesis reverse primer: 5’-CCCTCTTCCATTGGCTAAGTG-3’. The PCR products of each FAM20C form
were ligated into pcDNA3.1-V5/His mammalian expression vector, sequenced and the plasmids harboring FAM20C-WT and FAM20C-D478A cDNAs followed by V5/His- (pcDNA3.1-FAM20C-WT-V5/His and pcDNA3.1-FAM20C-D478A-V5/His) were successfully generated.
3.3 Cell culture
Human embryonic kidney HEK 293 cells purchased from Clontech (Mountain View, CA, USA) were maintained in DMEM containing a high concentration of glucose (4.5 mg/ml) supplemented with 10% FBS, and 100 units/ml of penicillin and 100 µg/ml of streptomycin in a 5% CO2 atmosphere at 37 oC.
3.4 Transfection, Immunoprecipitation, and Western Blot Analysis
HEK 293 cells were plated onto 6-well culture plates at a concentration of 3 x105 cells/well. On the following day, cells were transiently transfected in duplicate using X-treme GENE 9 DNA transfection reagent (Roche Applied Sciences, Indianapolis, IN, USA ) . After 24 hours of transfection, cells were lysed using the lysis buffer(150mM NaCl, 20mM Tris-HCl (pH7.5), 10mM EDTA ,1% Triton-X 100 and 1% deoxycholate). Cell lysates were centrifuged and the supernatant collected. Samples were incubated with SDS sample buffer for 5 min. at room temperature and separated by SDS-polyacrylamide gel
Samples were transferred to Immobilon-P Membrane in transfer buffer using Mini-PROTEAN® Tetra Cell system at 30 volts and 0.04 ampere overnight. Western blot analysis was then performed as follows: the membrane was
blocked with TBS (20 mM Tris-HCl, pH 7.5, 137 mM NaCl) containing 10% skim milk overnight at 4oC. The membrane was then washed 3 times with TBS and incubated with the respective antibody overnight at 4 oC. After washing the membrane 3 times with TBS the membrane was incubated with anti-HA, anti-V5 or anti-PERIOSTIN antibody for one hour at room temperature. The membrane was again washed 5 times with TBS and visualized using ECL™ Western Blotting Detection Reagents.
3.5 Binding of FAM20C to PERIOTIN
The 293 cells were plated onto 6 well culture plates at a density of 3 x 105 cells / well and transfected with mouse periostin with C-terminal HA tag (pCAGI-puro delta periostin (b-)-WT-HA; periostin -WT-HA) and FAM20C-WT-V5/His,
FAM20C-D478A-V5/His, or FAM20C-P328S-V5/His. After 24 hours of
transfection cell lysates were collected, immunoprecipitated (IP) with anti-V5 antibody and subjected to Western blotting (WB) analysis using anti-HA antibody to identify the binding. The same membrane was stripped by the stripping buffer (15g glycine, 1g SDS, 10ml Tween, and bring volume to 1L), and WB with anti-V5 antibody was performed to verify the expression of FAM20C-anti-V5/His proteins. An aliquot of the same cell lysates was subjected to WB analysis with anti-HA
antibody to verify the expression of PERIOSTIN-WT-HA.
3.6 Purification of FAM20C proteins from cell lysates
Clones that overexpress FAM20C that were previously generated in our lab were grown on ten 15 cm culture plates. The cell pellets of FAM20C-WT and FAM20C-D478A were collected after trypsin treatment and stored at -20 °C until use. Forty ml of Native Binding Buffer (1x Native purification buffer) with protease inhibitors was used to resuspend cells from one 15cm dish. Cells were frozen at -20 °C and thawed at room temperature for two cycles. Cells were then sheared by passing the preparation through 16-gauge needle four times. The cell lysates were centrifuged at 3,000 rpm for 15 min and the supernatant was transferred to new tubes. Ni-NTA agarose 400 µl was added to 40 ml of the clear supernatant and incubated overnight at 4 °C. After incubation, the lysates werepoured into the columns and washed with 500 ml of Native Wash Buffer. Proteins were then eluted into 15 1ml fractions by Native Elution Buffer. Forty µl samples from each fraction were taken,mixed with SDS sample buffer, applied to SDS-PAGE, and positive fractions were identified by Western blotting with anti-V5 AP antibody.
3.7 In vitro Binding assay
The mouse recombinant PERIOSTIN and WT-V5/His or FAM20C-D478A-V5/His proteins were incubated in PBS. The total protein per sample was
kept constant by adding bovine serum albumin (BSA). Samples were then immunoprecipitated (IP) with anti-V5 antibody followed by Western blot (WB) analysis with anti-PERIOSTIN (Abcam) antibody. The same membrane was stripped and WB with anti-V5 antibody was used to verify the expression of FAM20C-V5/His proteins.
3.8 Mapping of FAM20C-V5 binding domain to Periostin
Various deletion mutant forms of pCAGI-puro delta periostin (b-)-HA were a generous gift from Dr. Kudo’s laboratory (Japan). They were used to identify the interaction domain of periostin with Fam20C as previously described. The deletion forms; periostin -WT-HA, periostin delta CTR-HA lacking its C-terminal region, periostin delta EMI-HA lacking EMI domain, periostin delta EMI-CTR-HA lacking both EMI and CTR domains, and periostin EMI-HA (EMI domain only). Briefly, HEK 293 cells were transiently transfected with periostin -WT-HA,
periostin -dCTR-HA, periostin -dEMI-HA, periostin -dEMI-CTR-HA, or periostin -EMI-HA and FAM20C-WT-V5/His. After 24 hours of transfection, cell lysates were collected and the binding domain was identified by IP-WB analysis in the same manner as described above.
3.9 In vitro kinase assay
cold ATP (Thermo Fisher), 50 mM Tris-HCl pH 7.0, 10 mM MnCl2, 12.5 µg/ml of
recombinant POSTIN, 25 µg/ml of FAM20C-WT-V5/His, and 25 µg/ml of GST-FAM20A. The mixtures were incubated for 30 min at 30 °C, then stopped by adding SDS sample buffer and boiling for 5min. Reaction products were separated by 4~12% SDS-PAGE and detected by Western-blotting.
3.10 Phosphorylation analyses using Biotinylated phos-tag™
As complex of Biotin-pendant Zn2︎-Phos-tag and HRP-conjugated Streptavidin
HRP—10mg of BTL-104 was diluted with 0.13ml of methanol and 1.17ml of TBS-T and stored in the dark at 4°C. TBS-The 4:1 complex of biotin-pendant Zn2+-Phos- tag™ and HRP was mixed with 10ul of Phos-tag™ BTL-104 solution, 1µl of Pierce peroxide conjugate(Thermo), 20µl of 10 mM Zn(NO3) 2 , and 469µl of
TBS-T. The solution was allowed to stand for 30 min at room temperature. The mixed solution was put into a concentrator 30K filter unit Pierce and centrifuged for 20 min at 14,000µg to remove excess biotin-pendant Zn2+-Phos-tag. The remaining solution (<10 µl) in the reservoir was diluted with 30 ml of TBS-T solution and stored at 4 °C (Zn2+-Phos-tag-bound HRP solution).
Probing with Zn2+-Phos-tag-bound HRP—A protein-blotted PVDF membrane was soaked in a TBS-T solution for at least 1 h. The membrane was incubated with the Zn2+-Phos-tag-bound HRP solution (1 ml/5 cm2) in a plastic bag for 30
min and washed twice with a TBS-T solution at room temperature. Proteins were detected by the ECL method.
4. Results
PERIOSTIN was co-purified during the process of FAM20C protein
purification suggesting that PERIOSTIN may bind to FMA20C. To confirm this notion, IHC using anti-PERIOSTIN and anti-FAM20C antibodies was performed on serial sections of mouse maxillary tissues. Both FAM20C (Fig.1 b, c; indicated by black arrows) and PERIOSITIN (Fig.1 f, g; indicated by filled arrows) were identified in PDL. In sections, FAM20C was also present in dentin-enamel
junction (Fig.1 d; arrowheads) and alveolar bone (Fig.1 b, c; open arrows) where PERIOSTIN was not shown. No immunoreactivities were detected when non-immune Ig was used (Fig.1 i-p).
Binding of FAM20C to PERIOSTIN
To verify the interaction between FAM20C and POSTIN and investigate whether this interaction requires FAM20C’s kinase activity, an inactive kinase form of FAM20C (D478A) and a Raine-syndrome mutation form (P328S) in addition to FAM20C -WT were used for the binding assay. HEK 293 cells were transiently transfected with Periostin-WT-HA and WT-V5/His,
FAM20C-D478A-V5/His, or FAM20C-P328S-V5/His. Immunoprecipitation and Western blot analysis demonstrated that FAM20C -WT interacted with Periostin-WT (Fig. 2A upper panel, lane 3). Both inactive kinase forms of FAM20C, i.e. D478A and P328S, also bound to Periostin indicating that the interaction between Fam20c
and Periostin does not require FAM20C’s kinase activity (Fig. 2A, upper panel, lanes 4 and 5). The expression of each FAM20C form was similar (Fig. 2A, lower panel, lanes 3-5).
To determine whether the binding between FAM20C and PERIOSTIN is direct, recombinant PERIOSTIN protein produced by Sf21 baculovirus and FAM20C-WT-V5/His or FAM20C-D478A-V5/His protein purified from the HEK
293-conditioned media were immunoprecipitated (IP) with anti-V5 antibody. Western blot analysis with anti-PERIOSTIN antibody showed that FAM20C-D478A, but not WT interacted with PERIOSTIN (Fig. 2B, lower panel, lanes 4 and 5).
Mapping of FAM20C-binding domain in PERIOSTIN
Immunoprecipitation (IP)-Western blotting (WB) analysis using various forms of periositn-HA vectors and FAM20C-V5 was used to identify the binding domain between FAM20C and PERIOSTIN. In samples where both Periostin-HA and
Fam20c-V5 were co-expressed (Fig 3. lane 3, 5, 7, and 9), immunoreactive bands to anti-HA antibody were presented after IP with anti-V5 antibody but not with Periostin-dEMI-HA. The expression of Periostin-HA was confirmed by IP-Western blotting with anti-HA antibody (Fig. 3, middle panel, lanes 2~11). The
expression of FAM20C -WT was confirmed by Western blotting with anti-V5 antibody (Fig. 3, lower panel, lanes 3, 5, 7, 9 and11). The data demonstrated that
Periostin-HA, Periostin dCTR-HA, Periostin dEMI-HA, and Periostin dEMI-CTR-HA bound to Fam20c.Periostin-EMI-HA did not bind to Fam20c-V5 (Fig. 3, upper panel, lane11).If there were a band between FAM20C-WT and EMI, the
immunoreactive band would be detected at the position indicated by the open arrowhead. However this reactivity was not detected.The data indicate that
Periostin-Fas domain is likely the binding domain of Fam20c-V5 in HEK 293 cells.
Phosphorylation of PERIOSTIN by FAM20C
PERIOSTIN is a secretory protein predominantly expressed in collagen-rich fibrous connective tissue. It has several S-X-E motifs in the protein sequence, suggesting that FAM20C phosphorylates PERIOSTIN. Therefore, we
investigated whether FAM20C phosphorylates PERIOSTIN. FAM20A increases potentiates FAM20C kinase activity and phosphorylation ability. HEK 293 cell were transiently transfected with FAM20A-HA, FAM20C-WT-V5 and a kinase inactive form of FAM20C (D478A). Cell lysates were collected,
immunoprecipitated with anti-V5 antibody and protein A-sepharose beads were added. The in vitro kinase assay was performed with the collected protein
complex with anti-V5 antibody and recombinant PERIOSTIN protein produced by
Sf21 baculovirus. The level of POSTIN was confirmed by WB using anti-periostin. The expression of FAM20C was confirmed by WB using anti-V5
antibody. To analyze phosphorylation of PERIOSTIN, biotinylated phos-tag was used. The phos-tag method recombinant FAM20C (Rec. FAM20C; Fig. 4 lane 6) detected recombinant FAM20C (Rec. FAM20C; Fig. 4 lane 6) by WB with phos-tag, suggesting that FAM20C was phosphorylated. No expression of PERIOSTIN phosphorylation was detected using the pho-tag method. (Fig. 4 lane 1~5).
Secretion of PERIOSTIN
To determine whether FAM20C helps PERIOSTIN secretion, HEK 293 cells were transiently transfected with Periostin-WT-HA and Fam20c-WT-V5/His or
Fam20c-D478A-V5/His. After 12 hours, the conditioned medias were collected and analyzed by Immunoprecipitation (IP)-Western blots. Cell lysates were also collected and the expression of PERIOSTIN was confirmed by Western blotting with anti-HA antibody (Fig. 5, upper panel, lanes 2-4). The expression of
Fam20c-V5 was confirmed by Western blotting with anti-V5 antibody (Fig. 5, middle panel, lanes 3 and 4). The bands in the lower panel showed the
expression of PERIOSTIN. With Fam20c-WT-V5 is similar to PERIOSTIN only (Fig. 5 lane 2 and 3). Fam20c-D478A-V5 expression was weak compared to the previous two lanes (Fig 5. Lane 2, 3, and 4). The results indicate that there is no obvious relationship between PERIOSTIN secretion and FAM20C. Lanes 2, 3 and 4 showed thick bands where they were expected to display two bands with one at the same level as lane1 and one at a lower level. The overlapping thick bands may due to excessive exposure.
5. Discussion
Binding of FAM20C to PERIOSTIN
Our studies recently showed that PERIOSTIN is one of the direct binding partners of FAM20C. We have used the FAM20C-WT and FAM20C-D478A, and FAM20C- P238S proteins to differentiate dependent and
kinase-independent binding proteins. FAM20C-D478A and FAM20C-P238S are the mutants of FMA20C. FAM20C-D478A is an artificially created kinase-dead
mutant and FAM20C-P238S has decreased or impaired kinase activity (Kinoshita et al., 2014). In this study, it was shown that FAM20C interacted with
PERIOSTIN in cell culture using HEK293 cell and in vitro using purified proteins. In cells PERIOSTIN was transfected with a kinase inactive form of FAM20C (D478A) and a Raine syndrome mutation form (P328S) to determine if the kinase activity is necessary for the interaction between FAM20C and PERIOSTIN. The results demonstrated that FAM20C-WT interacted with PERIOSTIN. Both kinase inactive forms of FAM20C, i.e. D478A and P328S, also bound to PERIOSTIN, indicating that the interaction of kinase activity between FAM20C and
PERIOSTIN was not necessary (Fig. 2A). However, in vitro, the binding between WT with PERIOSTIN was below the detection level, though FAM20C-DA bound to PERIOSTIN (Fig. 2B). At this point, it is uncertain why PERIOSTIN directly binds to a kinase-inactive form, but not WT of FAM20C. A recent study mentioned that the kinase does not always stably associate with its
transient and reduces substrate detection (Ohyama et al., 2016). It is assumed that FAM20C-D478A lacks the kinase activity so that its substrates could not be phosphorylated and retained with FAM20C-D478A.
Mapping binding domain
Based on the FAM20C and PERIOSTIN binding experiment, we further identified the binding site of PERIOSTIN to determine the function of binding. Our binding assays used PERIOSTIN-HA, PERIOSTIN dCTR-HA,
PERIOSTIN dEMI-HA, PERIOSTIN dEMI-CTR-HA, and EMI-HA with FAM20C-WT. The results showed PERIOSTIN-HA, PERIOSTIN dCTR-HA, PERIOSTIN dEMI-HA, PERIOSTIN dEMI -CTR-HA bound to FAM20C but not to EMI-HA which indicated that the Fasciclin (Fas) domain in PERIOSTIN is necessary for binding of FAM20C (Fig.3). The S-X-E motif sites in PERIOSTIN might be phosphorylated by FAM20C. There are four S-X-E motif sites in mouse PERIOSTIN and three of these S-X-E motif sites are conserved in human PERIOSTIN (Fig.6).
FAM20C appears to be the Golgi casein kinase (G-CK) that phosphorylates secretory pathway proteins. The G-CK recognizes the consensus S-X-E/pS motif where X is any amino acid and E/pS can be Glu or phospho-serine (Ishikawa et al., 2012; Tagliabracci et al., 2012). Secretory calcium binding phosphoprotein (SCPP) members whose genes cluster on Chromosome 4 in humans, have multiple S-X-E/pS motifs and are phosphorylated by FMA20C (Kawasaki &
Weiss, 2003; Kinoshita et al., 2014). Analysis of the amino acid sequence of human, PERIOSTIN showed 3 probable FAM20C phosphorylation sites which are located in Fas I and Fas IV domain [Fig. 6A](Coutu et al, JBC, 2008). The
periostin isoform in mice has 4 potential phosphorylation sites which are in Fas I, Fas IV and CTR [Fig. 6B]. Since the binding sites are located in Fas domains, phosphorylation has high probability of occurring in Fas I and IV domains. Our next goal is to determine whether the binding reaction in Fas domains is related to phosphorylation.
Phosphorylation of PERIOSTIN by FAM20C
To determine whether PERIOSTIN is phosphorylated by FAM20C, FAM20A and FAM20C were co-expressed in cultured HEK293 cell with PERIOSTIN and analyzed by an in vitro kinase assay. To further investigate phosphorylation activity, Western-blot analysis with biotinylated phos-tag was used to detect whether PERIOSTIN be phosphorylated or not.
Protein phosphorylation, a fundamental mechanism that regulates many cellular function, was discovered in 1883 (Tagliabracci et al., 2012). Several
phosphoproteins have been implicated in regulating biomineralization, including Small Intergrin-Binding Ligand N-linked Glycoproteins (SIBLING) (Ishikawa et al., 2012). The common feature of SIBLING family members is the presence of S-X-E motifs in their amino acid sequences, which are often phosphorylated (Fisher, Torchia, Fohr, Young, & Fedarko, 2001; Qin, Baba, & Butler, 2004). FAM20C
phosphorylates serine residues within the S-X-E motifs of OPN and DMP1. Thus FAM20C is a protein kinase that phosphorylates extracellular proteins (Ishikawa et al., 2012; Tagliabracci et al., 2012).
PERIOSTIN, the ECM protein is highly expressed in the PDL [Fig.1.] and is a key factor required for periodontal integrity and hemostasis under normal
occlusal load (Padial-Molina, Volk, & Rios, 2013; Rios et al., 2008). It is
associated with cell proliferation, migration and activation of the survival signaling pathway PI3/AKT/mTOR (Padial-Molina et al., 2013). PERIOSTIN with several S-X-E motifs located within Fas domains is one of potential candidate substrates of FAM20C.
We did not detect phosphorylation of PERIOSTIN using biotinylated phos-tag. [Fig.4.]. Rec. FAM20C was used as a positive control which autophosphorylated the protein. Phosphorylation of Rec. FAM20C with biotinylated phos-tag was detected confirming the validity of this method (Fig.4, lane 6). This
under-detection might be caused by defeat of kinase assay or insufficient concentration of phosphorylated PERIOSTIN could not be expressed by phos-tag.
This study confirmed that the FAM20C and PERISOTIN binding location is within the FAS domain which contained S-X-E motifs. FAM20C is a secreted protein kinase that preferentially phosphorylates the peptides containing S-X-E motifs. Further studies are required to verify that PERIOSITN may be
phosphorylated by FAM20C and that phosphorylation may regulate cell functions such as biomineralization of extracellular matrix where PERIOSTIN is located.
Figure legends
Fig. 1. Localization of FAM20C and PERIOSTIN in mouse periodontium.
Serial sections of mouse maxillary regions were stained using anti-FAM20C (a-d) or anti-PERIOSTIN (e-h) antibody. Non-immune goat Ig was also used as a negative control for anti-FAM20C (i-l) and non-immune rabbit Ig as a negative control for anti-PERIOSTIN (m-p). Panels (b-d), (f-h), (j-l) or (n-p) show higher magnification views of the open boxed areas in (a), (e), (i) or (m), respectively. The white boxed areas in (b) and (f) are further magnified and shown at the lower right corner of (b) and (f). Immunolocalization for FAM20C (brown color) is
present in PDL (b, c; indicated by black arrows), dentin-enamel junction (d; arrowheads) and alveolar bone (c; indicated by open arrows). FAM20C is also observed in the PDL fibroblasts (b, c), dentinal tubules within odontoblasts (d; indicated by od) and osteoblasts (c; indicated by ob).
Note that PERIOSTIN is present (brown color) in PDL (f, g; filled arrows) with strong positive signals along the thick PDL collagen (f), but not observed in dentin/odontoblasts (h). No immunoreactivities were detected when non-immune Ig was used (i-p). Scale bar; 200 µm (a, e, i, m), 100 µm (b-d, f-h, j-l, n-p).
Fig. 2. Binding of FAM20C to PEIOSTIN. (A) FAM20C binds to PERIOSTIN (POSTIN) in cell cultures. HEK 293 cells were transiently transfected with
periostin-WT-HA and Fam20c-WT-V5/His, Fam20c -D478A-V5/His, or Fam20c
P328S-V5/His. The cell lysates were collected, and immunoprecipitation (IP)-Western blot (WB) analyses were performed. The binding of PERIOSTIN to
Fam20c -WT (upper panel, lane 3), -D478A (upper panel, lane 4), or -P328S (upper panel, lane 5) was observed, indicating that this interaction didn’t require FAM20C kinase activity. (B) Direct binding of FAM20C to PERIOSTIN. FAM20C and POSTIN proteins were incubated in vitro, immunoprecipitated with anti-V5 antibody, and subjected to WB analysis with anti-POSTIN antibody. FAM20C-D478A directly interacted with PERIOSTIN but no in FAM20C-WT (lane 2 and 3)
Fig. 3. Fasciclin domain in PERIOSTIN is the FAM20C-binding region. HEK 293 cells were transfected with Periostin-WT-HA, Periostin-dCTR-HA, Periostin -dEMI-HA, Periostin -dEMI-CTR-HA, or Periostin-EMI-HA and FAM20C-WT-V5/His. The “d” indicates lacking the following domain; for example, dCTR is a form of Periostin lacking the CTR region. The binding between POSTIN and FAM20C was investigated using cell lysates by immunoprecipitation (IP) - Western blotting (WB) analysis. The result showed that FAM20C interacted with
Periostin-WT-HA (upper panel, lane 3, indicated by an arrowhead), Periostin -dCTR-HA (upper panel, lane 5, indicated by an arrowhead), Periostin -dEMI-HA (upper panel, lane 7, indicated by an arrowhead), and Periostin -dEMI-CTR-HA (upper panel, lane 9, indicated by an arrowhead), but not with Periostin -EMI-HA (upper panel, lane 11, indicated by an open arrowhead). The expression of all
Periostin-HA constructs in the same cell lysate was confirmed by WB using anti-HA antibody (middle panel) and the level of immunoprecipitated FAM20C was confirmed by WB using anti-V5 antibody (lower panel). An asterisk indicates a non-specific band (upper and middle panels).
Figure 4
Fig. 4. Phosphorylation of PERIOSTIN by FAM20C. HEK 293 cells were
transiently transfected with Fam20a-HA, Fam20c-WT-V5/His, and Fam20c -D478A-V5/His. The cell lysates were collected, and kinase assays were performed with FAM20C and POSTIN proteins in vitro. Samples were then subjected to Western blot (WB) analysis. The result showed that only FAM20C be detected by phos-tag (upper panel lane 6). The level of POSTIN was
confirmed by WB using anti-periostin (middle panel), and the expression of FAM20C in the same cell lysate was confirmed by WB using anti-V5 antibody (lower panel). The phos-tag method was confirmed by Rec. FAM20C (upper panel lane 6).
Figure 5
Fig. 5. Secretion of PERIOSTIN. HEK 293 cells were transfected with periostin -WT-HA, Fam20c-WT-V5/His, and Fam20c--D478A-V5/His. Conditioned media were collected and immunoprecipitated (IP) with anti-HA followed by Western blot (WB) analysis. The data showed that there was no obvious difference between whether Fam20c--WT or Fam20c--DA (lower panel) means FAM20C cannot help PERIOSTIN for secretion. The level of PERIOSTIN was confirmed by WB using anti-HA (upper panel), and the expression of FAM20C was
Figure 6
A.
MIPFLPMFSL LLLLIVNPIN ANNHYDKILA HSRIRGRDQG PNVCALQQIL GTKKKYFSTC KNWYKKSICG QKTTVLYECC PGYMRMEGMK GCPAVLPIDH 100 VYGTLGIVGA TTTQRYSDAS KLREEIEGKG SFTYFAPSNE AWDNLDSDIR RGLESNVNVE LLNALHSHMI NKRMLTKDLK NGMIIPSMYN NLGLFINHYP 200 NGVVTVNCAR IIHGNQIATN GVVHVIDRVL TQIGTSIQDF IEAEDDLSSF RAAAITSDIL EALGRDGHFT LFAPTNEAFE KLPRGVLERI MGDKVASEAL 300 MKYHILNTLQ CSESIMGGAV FETLEGNTIE IGCDGDSITV NGIKMVNKKD IVTNNGVIHL IDQVLIPDSA KQVIELAGKQ QTTFTDLVAQ LGLASALRPD 400 GEYTLLAPVN NAFSDDTLSM DQRLLKLILQ NHILKVKVGL NELYNGQILE TIGGKQLRVF VYRTAVCIEN SCMEKGSKQG RNGAIHIFRE IIKPAEKSLH 500 EKLKQDKRFS TFLSLLEAAD LKELLTQPGD WTLFVPTNDA FKGMTSEEKE ILIRDKNALQ NIILYHLTPG VFIGKGFEPG VTNILKTTQG SKIFLKEVND 600 TLLVNELKSK ESDIMTTNGV IHVVDKLLYP ADTPVGNDQL LEILNKLIKY IQIKFVRGST FKEIPVTVYT TKIITKVVEP KIKVIEGSLQ PIIKTEGPTL 700 TKVKIEGEPE FRLIKEGETI TEVIHGEPII KKYTKIIDGV PVEITEKETR EERIITGPEI KYTRISTGGG ETEETLKKLL QEEVTKVTKF IEGGDGHLFE 800 DEEIKRLLQG DTPVRKLQAN KKVQGSRRRL REGRSQ
B.
MVPLLPLYAL LLLFLCDINP ANANSYYDKV LAHSRIRGRD QGPNVCALQQ ILGTKKKYFS SCKNWYQGAI CGKKTTVLYE CCPGYMRMEG MKGCPAVMPI 100 DHVYGTLGIV GATTTQHYSD VSKLREEIEG KGSYTYFAPS NEAWENLDSD IRRGLENNVN VELLNALHSH MVNKRMLTKD LKHGMVIPSM YNNLGLFINH 200 YPNGVVTVNC ARVIHGNQIA TNGVVHVIDR VLTQIGTSIQ DFLEAEDDLS SFRAAAITSD LLESLGRDGH FTLFAPTNEA FEKLPRGVLE RIMGDKVASE 300 ALMKYHILNT LQCSEAITGG AVFETMEGNT IEIGCEGDSI SINGIKMVNK KDIVTKNGVI HLIDEVLIPD SAKQVIELAG KQQTTFTDLV AQLGLASSLK 400 PDGEYTLLAP VNNAFSDDTL SMDQRLLKLI LQNHILKVKV GLSDLYNGQI LETIGGKQLR VFVYRTAICI ENSCMVRGSK QGRNGAIHIF REIIQPAEKS 500 LHDKLRQDKR FSIFLSLLEA ADLKDLLTQP GDWTLFAPTN DAFKGMTSEE RELLIGDKNA LQNIILYHLT PGVYIGKGFE PGVTNILKTT QGSKIYLKGV 600 NETLLVNELK SKESDIMTTN GVIHVVDKLL YPADIPVGND QLLELLNKLI KYIQIKFVRG STFKEIPMTV YRPAMTKIQI EGDPDFRLIK EGETVTEVIH 700 GEPVIKKYTK IIDGVPVEIT EKQTREERII TGPEIKYTRI STGGGETGET LQKFLQKEVS KVTKFIEGGD GHLFEDEEIK RLLQGDTPAK KIPANKRVQG 800 PRRRSREGRS Q 1-23: Signal peptide 42-89: EMI 111-232: Fasciclin 1 247-369: Fasciclin 2 384-496: Fasciclin 3 509-633: Fasciclin 4 634-811: CTR
Figure 6. Protein sequences of human periostin and mouse periostin
Isoform1. A. The human periostin has 3 probable S-X-E phosphorylation sites. B. There are five isoforms of the mouse periostin. The longest transcript mouse
periostin isoform 1 which has 4 probable phosphorylation sites.(Coutu et al., 2008).
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7. Vita
Personal Information
Ju-hsien Lin
Education
Henry M. Goldman School of Dental Medicine, Boston University
Masters of Science in Dentistry (MSD) and Certificate of Advanced Graduate Study (CAGS) in Oral Biology and Periodontology
July 2014-Present (Expected graduation is June, 2017)
Dentistry of Chung Shan Medical University Doctoral of Dental Surgery (D.D.S)
Academic Research
Boston University Henry M. Goldman School of Dental Medicine July 2014-July 2015
Department of Molecular & Cell Biology
Principal Investigator: Dr. Yoshiyuki Mochida, DDS, Ph.D.
Publication
FAM20A binds to and regulatesFAM20C localization. Nature Publishing Group, 6, 1–11. doi:10.1038/srep27784
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