Cell Tissue Res DOI 10.1007/s00441-014-1989-3

REGULAR ARTICLE

Characterisation of secretory calcium-binding phosphoprotein-proline-glutamine-rich 1: a novel basal lamina component expressed at cell-tooth interfaces Pierre Moffatt & Rima M. Wazen & Juliana Dos Santos Neves & Antonio Nanci

Received: 7 May 2014 / Accepted: 7 August 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Functional genomic screening of the rat enamel organ (EO) has led to the identification of a number of secreted proteins expressed during the maturation stage of amelogenesis, including amelotin (AMTN) and odontogenic ameloblast-associated (ODAM). In this study, we characterise the gene, protein and pattern of expression of a related protein called secretory calcium-binding phosphoprotein-proline-glutamine-rich 1 (SCPPPQ1). The Scpppq1 gene resides within the secretory calcium-binding phosphoprotein (Scpp) cluster. SCPPPQ1 is a highly conserved, 75-residue, secreted protein rich in proline, leucine, glutamine and phenylalanine. In silico data mining has revealed no correlation to any known sequences. Northern blotting of various rat tissues suggests that the expression of Scpppq1 is restricted to tooth and associated tissues. Immunohistochemical analyses show that the protein is expressed during the late maturation stage of amelogenesis and in the junctional epithelium where it localises to an Pierre Moffatt and Rima M. Wazen contributed equally to this work This study was supported by the Canadian Institutes of Health Research, Network for Oral and Bone Health Research and Shriners of North America. Juliana Dos Santos Neves was supported by CAPES, Government of Brazil. P. Moffatt Shriners Hospital for Children, Montréal, Québec, Canada P. Moffatt Department of Human Genetics, McGill University, Montréal, Québec, Canada R. M. Wazen : J. Dos Santos Neves : A. Nanci (*) Laboratory for the Study of Calcified Tissues and Biomaterials, Department of Stomatology, Faculty of Dentistry, Université de Montréal, Montréal, Québec, Canada e-mail: [email protected] A. Nanci Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, Québec, Canada

atypical basal lamina at the cell-tooth interface. This discrete localisation suggests that SCPPPQ1, together with AMTN and ODAM, participates in structuring the basal lamina and in mediating attachment of epithelia cells to mineralised tooth surfaces. Keywords Basal lamina . Enamel organ . Junctional epithelium . Secretory calcium-binding phosphoprotein-proline-glutamine-rich 1 . Rat . Mouse

Introduction The enamel organ (EO) is the epithelial structure responsible for creating the enamel layer during tooth development. Broad screening of the secretome of the EO has identified a number of non-annotated genes encoding for secreted proteins (Moffatt et al. 2006a). Two of them, called Amelotin (AMTN) and Odontogenic ameloblast-associated (ODAM; previously known as APIN), have unique biochemical characteristics (Moffatt et al. 2006a, 2006b, 2008). Based on their genomic location and architecture, genes for these proteins are classified as part of the secretory calcium-binding phosphoprotein (Scpp) gene cluster (Huq et al. 2005; Kawasaki and Weiss 2003, 2006). The Scpp gene family initially arose from secreted phosphoprotein acidic-rich like 1 (Sparcl1; Kawasaki et al. 2007). Genes from this cluster encode for proteins that stabilise calcium and phosphate ions in body fluids and/or guide CaPO4 deposition into receptive extracellular matrices such as in tooth and bone (Huq et al. 2005; Kawasaki et al. 2004; Kawasaki and Weiss 2003, 2008). Expression profiling has revealed that both AMTN and ODAM are expressed in the tooth during the maturation stage of amelogenesis and in the junctional epithelium (JE; Moffatt et al. 2006b, 2008). They localise at the cell-tooth interface and have been identified as novel components of the atypical

Cell Tissue Res

basal lamina (BL) present there (Dos Santos et al. 2012; Moffatt et al. 2006b, 2008; Nishio et al. 2010). Another nonannotated gene has been identified in the maturation stage of amelogenesis. In our original screens, this gene was referred to as EO-463 (Moffatt et al. 2006a) but is now known as secretory calcium-binding phosphoprotein-prolineglutamine-rich 1 (Scpppq1). It is also part of the Scpp gene cluster (Kawasaki et al. 2004; Kawasaki and Weiss 2003) and is evolutionarily related to Odam and Amtn. Relatively little else is known about SCPPPQ1; in particular, the nature of this protein, its tissue distribution and histological localisation remain to be determined. Therefore, the objective of this study was to provide a detailed characterisation of the Scpppq1 gene, transcripts and protein, its developmental pattern of expression and its precise localisation.

Materials and methods Cell culture The rat osteosarcoma UMR106 and ROS17/2.8 cell lines (ATCC, Manassas, Va., USA) and HEK293 (human embryonic kidney) cells were grown in DMEM (Life Technologies, Mississauga, ON, Canada) supplemented with 10 % (v/v) fetal bovine serum. Culture media were replenished every 2–3 days. Northern blot analysis EOs were dissected from freeze-dried upper and lower incisors of 100-g male Wistar rats, as previously described (Smith and Nanci 1989). Total RNA was prepared from multiple adult rat tissues, from cultured cell lines, from entire EOs and from EOs that had been partitioned into secretory (EO-S) and maturation (EO-M) stages. Total RNA was extracted by using TRIzol (Life Technologies) and electrophoresed on a MOPSbuffered 1.2 % (w/v) agarose gel containing 1.2 % (v/v) formaldehyde. The integrity and loading of the RNA samples were verified by ethidium bromide staining, before transfer on to GE Magna nylon membranes (Fisher Scientific, Whitby, ON, Canada) with 20× SSC (1× SSC is 0.15 M NaCl and 0.015 M sodium citrate). The blot was UV cross-linked and then prehybridized for 2 h in Church buffer (250 mM sodium phosphate, pH 7.2, 1 mM EDTA, 1 % [w/v] bovine serum albumin and 7 % [w/v] SDS; Church and Gilbert 1984) at 65 °C and hybridized under the same conditions overnight with a Scpppq1 cDNA fragment labelled by random-priming with High Prime (Roche Applied Science, Laval, QC, Canada) and [α-32P]dCTP (GE Healthcare, Baie d’Urfé, QC, Canada). The blot was washed with 0.2× SSC/0.1 % SDS at 65 °C and autoradiographed.

Cloning of rat and mouse full-length Scpppq1 cDNA The rat cDNA encoding Scpppq1 was amplified by 3′ rapid amplication of cDNA ends (RACE) reverse transcription plus the polymerase chain reaction (RT-PCR) on EO total RNA as described previously (Moffatt et al. 2006b, 2008). Briefly, single-stranded cDNA was prepared by using a dT primer (5′-GAGATGAATTCCTCGAGCTTTTTTTTTTTTTTT-3′) and SuperscriptIII (Life Technologies) at 42 °C for 50 min. After RNAseH1 treatment, the cDNAs were purified on a Qiaquick column (QIAGEN, Mississauga, ON, Canada) and used for PCR amplification by using Titan polymerase (Roche) and SCPPPQ1-specific forward primer (5′-ACCT CCAAGCTCTCCCTTCAG-3′) and a reverse primer complementary to the dT oligo (5′-GAGATGAATTCCTCGAGC-3′). The mouse Scpppq1 cDNA was amplified directly from total RNA extracted from a 1-month-old mouse mandible by using the gene-specific primers (5′-GCTCTGCCAATCCCTCTTG3′ and 5′-GCTGTAGATAGTAGATTTGCC-3′) and the Superscript III One-Step RT–PCR system with Platinum Taq (Life Technologies). The PCR products were cloned into pBluescriptII and sequenced. Sequences have been deposited at Genbank with accession numbers HM015624 (rat) and HM015622 and HM015623 (mouse). Scpppq1 gene expression by standard and real-time quantitative RT-PCR Total RNA extracted from adult mouse EO-S, EO-M, gingiva, liver and kidney were analysed and reactions were set up with 250 ng total RNA and analysed with the Superscript III OneStep RT-PCR system with Platinum Taq (Life Technologies). The Scpppq1 gene-specific forward (5′-GCTCTGCCAATC CCTCTTG-3′) and reverse (5′-GCTGTAGATAGTAGATTT GCC-3′) primers were designed with regard to the junction between exons 2 and 3 and within exon 9, respectively. Actinspecific primers were performed to monitor the integrity and uniformity of the RNAs as previously described (Moffatt et al. 2006b). The RT-PCR conditions were as follows: 55 °C/ 30 min, 94 °C/2 min, followed by cycling at 94 °C/30 s, 55 °C/30 s, 68 °C/30 s and a final elongation step at 68 °C for 5 min. PCRs were stopped after 25, 30 and 35 cycles, resolved on 2 % agarose gels and stained with ethidium bromide. PCR product sizes were 388 and 348 bp for Scpppq1 and β-actin, respectively. For real-time quantitative RT-PCR (qPCR), cDNA were prepared by using the High Capacity cDNA synthesis kit (Applied Biosystems, Life Technologies). Real-time qPCR was performed on an Applied Biosystems 7500 PCR machine in a 25-μl reaction volume with the 2× Universal PCR Master Mix and the following Taqman probes (Applied Biosystems): amelogenin (Amel; Mm00711642_m1), ameloblastin (Ambn; Mm00477485_m1), Scpppq1 (Mm03990687_m1), follicular

Cell Tissue Res

dendritic cell secreted protein (Fdc-sp; Mm03951982_m1) and β-actin (435933E). All data were normalised to β-actin and the values were expressed as 2e(−ΔCt).

quantities of aprotinin. The recombinant 6His-SCPPPQ1 protein was mixed with complete Freund’s adjuvant, injected into rabbits. Whole serum was collected according to standard protocols (Open Biosystems, Huntsville, Al., USA).

Generation of a SCPPPQ1 antibody The cDNA fragment encoding rat SCPPPQ1 (residues 16–90) was amplified on the expression plasmid detailed above with the following primers: 5′-ATAGGATCCCTGCCAATCCCT CTTGGAG-3′ and 5′-GGAAGCTTATCTCCCAAGGAA GCCCTG-3′. The PCR product was digested with BamHI and HindIII (sites underlined in primer sequences), purified on agarose gel and cloned at the BamHI-HindIII sites of the pQE30 bacterial expression plasmid (QIAGEN). The resulting plasmid was expected to produce an 87-residuelong recombinant protein with an N-terminal 6-histidine tag (MRGSHHHHHHGS) fused to rat mature SCPPPQ1 lacking its signal peptide. The plasmid was transformed into the SG13009 [pREP4] bacterial strain (QIAGEN). A 500-ml culture of a positive clone was grown to an OD600nm of 0.7 and further grown for 4 h in the presence of 1 mM isopropyl thiogalactoside to induce the production of 6His-SCPPPQ1. Bacteria were centrifuged, washed twice with cold phosphatebuffered saline (PBS) and resuspended with lysis buffer A (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0). The bacterial suspension was sonicated on ice and centrifuged at 10,000 rpm for 30 min at 4 °C. The insoluble pellet was washed once with lysis buffer A and re-centrifuged. The pellet was extracted with lysis buffer B (100 mM NaH2PO4, 10 mM TRIS–HCl, 8 M urea, pH 8.0) for 15 min at room temperature with vigorous mixing. The lysate was centrifuged at 10,000 rpm for 30 min at 4 °C and the supernatant was collected and mixed for 1 h at room temperature with 1 ml pre-equilibrated 50 % Ni-NTA-sepharose slurry (QIAGEN). The mixture was loaded into a disposable chromatography column (Pierce-Thermo Scientific, Rockford, Il, USA) and the flow through was collected. The resin was washed with 12 ml lysis buffer B pH 6.4 and eluted sequentially with 4 ml each of lysis buffer B at pH 5.9 and at pH 4.5. Elution fractions of 0.5 ml were collected on ice. The production and purification of 6His-rSCPPPQ1 was monitored by loading equivalent amounts on 16 % SDS-polyacrylamide gel electrophoresis (SDS-PAGE) TRIS-Tricine gels and staining with Coomassie blue or Western blotting with the monoclonal anti-6His antibody clone HIS-1 (Sigma-Aldrich, Oakville, ON, Canada). Purified fractions were pooled and dialyzed in 3.5-kDa molecular weight cut-off Slide-a-lyzer (Pierce) for two consecutive 24 h periods against 2 l PBS containing 2.5 M urea (pH 7.2). The protein concentration of the final 6HisSCPPPQ1 preparation was quantified by using the Bradford reagent (Bio-Rad Laboratories, Mississauga, ON, Canada) and purity was estimated by running serial dilutions on 16 % SDS-PAGE TRIS-Tricine gels, together with known

Transfection and immunofluorescence labelling for SCPPPQ1 The rat Scpppq1 cDNA was subcloned into the CMV-based expression plasmid pCDNA3.1. HEK293 cells were seeded into 6-well plates at 20,000 cells/cm2. The next day, cells were transfected with 1 μg of the CMV-Scpppq1 plasmid or with the empty pCDNA3.1 as a control by using Fugene6 (Roche) at a ratio of 3:1. Cells were processed 24 h later for immunofluorescence staining (all steps were performed at room temperature). Briefly, cells were washed with PBS, fixed with paraformaldehyde (3 % w/v in PBS) for 10 min, permeabilised with Triton X-100 (0.1 % v/v in PBS) for 2 min and washed with PBS. Cells were incubated successively for 1 h each in skim milk (5 % w/v in PBS), diluted antiSCPPPQ1 (1/250) and Alexa-Fluor-594-conjugated rabbit secondary antibody (1/1000). After being washed with PBS, cells were mounted in ProlongGold with 4,6-diamidino-2phenylindole (DAPI) and imaged under epifluorescence with a Canon DP70 digital camera. Tissue processing for immunolocalisation of SCPPPQ1 in tissues All animal procedures were approved by the Comité de déontologie de l’expérimentation sur les animaux of Université de Montréal. The expression of SCPPPQ1 in mouse embryos, during postnatal rat tooth development and in young mice was investigated. Formation of the primary JE was followed in erupting maxillary molars of male Wistar rats (Charles River Canada, St Constant, QC, Canada) of 1 weeks (body weight 25±0.5 g), 2 weeks (body weight 40±0.5 g), 3 weeks (50±0.5 g) and 8 weeks of age (250±0.5 g). Mouse embryos were collected at day 17–18 from C57BL/6 pregnant females that were staged by detection of vaginal plugs (set as day 0.5). C57BL6 mice of 8 weeks of age (Charles River Canada) were also studied. Each age group comprised three animals. Animals were anaesthetised with 20 % chloral hydrate solution (0.4 mg/g body weight; Fisher Scientific) and ketamine hydrochloride (10 mg/kg). Mouse embryos were dissected, rinsed with PBS and fixed with 4 % paraformaldehyde (Acros organics, Morris Plains, N.J., USA) and 0.1 % glutaraldehyde (Electron Microscopy Sciences, Fort Washington, Pa., USA) overnight at 4 °C. Young adult mice and rats were sacrificed by perfusion through the left ventricle with Ringer’s lactate (Hospira, Montreal, QC, Canada) for 30 s, followed by a fixative solution consisting of 4 % paraformaldehyde (Acros

Cell Tissue Res

organics) and 0.1 % glutaraldehyde (Electron Microscopy Sciences) in 0.1 M PBS (pH 7.2) for 20 min. Hemimandibles and hemimaxillae were dissected and specimens were immersed in the same fixative solution overnight at 4 °C. Specimens were decalcified with 4.13 % disodium ethylenediamine tetraacetic acid (Fisher Scientific) for 21 days and then were washed for 24 h in 0.1 M PBS buffer, pH 7.2. Some decalcified specimens were processed for paraffin embedding. Sections of 5 μm in thickness were prepared with a Leica RM2155 microtome (Leica Microsystems Canada, Richmond Hill, ON, Canada) and mounted on Superfrost/ Plus slides (Fisher Scientific) for immunoperoxidase staining. After decalcification and washes, some incisor EO segments containing the early to mid- and late-maturation stages of amelogenesis from young adult mice were prepared by using a molar reference line (Smith and Nanci 1989). Specimens were post-fixed in potassium-ferrocyanidereduced osmium tetroxide (Neiss 1984) and then processed for embedding in LR White resin (London Resin, Berkshire, UK). Sections of 1 μm in thickness were cut with glass knives on a Reichert Jung Ultracut E ultramicrotome and stained with toluidine blue. Ultrathin sections of 80–100 nm in thickness were cut with a diamond knife, collected on Formvar-carboncoated 200-mesh nickel grids and processed for postembedding colloidal gold labelling. Immunohistochemistry Sections were deparaffinised with Citrisolv (Fisher Scientific), rehydrated through a descending ethanol series and washed in distilled water. In order to avoid non-specific binding, sections were blocked with 0.01 M PBS (pH 7.2) containing 5 % skim milk for 20 min. They were then incubated for 3 h at room temperature with rabbit antibody raised against rat SCPPPQ1 (1:2000, crude antiserum) followed by treatment with the DakoEnvision + System, horseradish-peroxidase-labelled polymer anti-rabbit kit (Dako, Glostrup, Denmark), as recommended by the manufacturer. Visualisation of the staining was performed with 3,3′-diaminobenzidine and sections were then counterstained with 0.5 % methyl green. As negative controls, some sections were incubated with PBS instead of primary antibodies. Sections were examined under an AxioImager M2 microscope (Carl Zeiss, Oberkochen, Germany). Postembedding colloidal gold immunocytochemistry Ultrathin sections of osmicated samples were first treated with an aqueous solution of 5 % sodium metaperiodate (Bendayan and Zollinger 1983) for 45 min and washed with distilled water. Grids were then floated on a drop of 1 % ovalbumin in PBS for 15 min for blocking unspecific binding and then transferred onto a drop of SCPPPQ1 antibody for 1 h (1:500). Following incubation with primary antibody, the grids were

rinsed with PBS and placed again on the blocking solution for 15 min. The sites of antibody-antigen binding were then revealed by floating the grids on a drop of the protein Agold complex for 30 min (prepared in-house; Bendayan 1995). Finally, the grids were washed with PBS followed by distilled water. All steps were carried out at room temperature. Controls consisted in incubations with protein A-gold alone. Sections were then stained with uranyl acetate and lead citrate and examined in an FEI Tecnai 12 (Eindhoven, The Netherlands) transmission electron microscope operated at 80 kV.

Results Identification and cloning of the full-length cDNA of Scpppq1 A rat Scpppq1 cDNA fragment was originally retrieved as part of a signal-trap screen performed to identify genes encoding secreted or membrane-bound proteins in a rat EO cDNA library (Moffatt et al. 2006a). The initial 214-bp-long clone, called EO-463, contained 105 bp of the 5′ untranslated region (UTR) followed by an open reading frame of 36 residues with a predicted cleavable signal peptide according to SignalP4.0 (Petersen et al. 2011). At that time, database mining with this EO-463 cDNA fragment did not match any previously identified sequences but hit on four different segments of the rat chromosome 14 in which flanking nucleotides conformed to a GT/AG intron/exon structure. Amplification by 3′RACE allowed the cloning of the full-length 585-bp cDNA for the rat and the precise mapping of the genomic location and architecture of the gene. While this work was in preparation, Kawasaki (2009) identified the same gene by in silico mining and named it Scpppq1 because of its evolutionary relationship and physical localisation in the Scpp gene cluster. To avoid any confusion regarding gene nomenclature, we have thus decided to employ Scpppq1 throughout the text. Existing sequences related to Scpppq1 are still scarce in the expressed sequence tag (EST) databases and have only been formally identified for the prairie deer mouse (GenBank GH514299 and GH514300). All other EST annotated at Unigene for mouse (Mm.366664) and rat (Rn.226724) hit on contiguous stretches of genomic DNA at the Scpppq1 gene locus suggesting that they are not genuine transcript sequences. The only other cloned Scpppq1 sequences have been obtained from the anole lizard and the mouse (Kawasaki et al. 2011). The Scpppq1 gene is located on rat chromosome 14 within the SCPP cluster and its immediate neighbours are Sparcl1 and Nudt9 (nudix [nucleoside diphosphate linked moiety X]type motif 9; Fig. 1a, b). Rat Scpppq1 contains 10 exons covering approximately 9 kb and is transcribed in the same

Cell Tissue Res

orientation as the adjacent Sparcl1 gene (Fig. 1c, d). Fulllength cDNA cloning for the mouse Scpppq1 has identified two transcripts differing in length by 45 bp (Fig. 1f). This change would only affect the 5′UTR length and is caused by an alternate splice acceptor site located at the beginning of exon 2. The Scpppq1 cDNA from other species were deduced after Blast searches with either the rat or human exonic sequences on all available genomic sequences. The deduced protein

alignment is presented in Fig. 2. In all mammalian genomes interrogated, the Scpppq1 gene structure presents with the same characteristics as those found in the rat. In some species, the deduced SCPPPQ1 protein sequences are partially at the C-terminus, because of incomplete genomic sequencing data or difficulties associated with identification of the last exons 8 and 9, which encode for the last five residues. Another (nontested) possibility is that, in apes, non-canonical splice acceptor sites (GG instead of AG) in intron 7 might cause skipping

Fig. 1 Representation of the location and structure of the rat Scpppq1 gene, its full-length cDNA and deduced protein sequence. a The rat ScpppqQ1 gene is located on chromosome (chr.) 14 flanked by the SPARC-like protein 1 (Sparcl1) and Nudix (nucleoside diphosphate linked moiety X-type motif 9; Nudt9) genes. b The 450-kb locus is rich in genes encoding secreted proteins such as osteopontin (Opn), matrix extracellular phosphoglycoprotein (Mepe), bone sialoprotein (Bsp), dentin matrix acidic phosphoprotein 1 (Dmp1) and dentin sialophosphoprotein (Dspp). c, d The Scpppq1 gene is composed of 10 exons covering 8.77 kb (ORF open

reading frame, UTR untranslated regions). e Full-length cDNA for Scpppq1 and deduced protein sequence. Normal and italicised numbers left refer to the nucleotide and protein sequences, respectively. The 5′UTR and 3′UTR are in lower-case and the coding region is in upper-case. The start and stop codons are boxed. Nucleotides corresponding to different exons are depicted by alternating normal/bold. The signal peptide is underlined. f A second transcript differing in length by 45 bp has been identified on the full-length cDNA for mouse Scpppq1

Cell Tissue Res

Fig. 2 Alignment of the SCPPPQ1 protein sequence from various mammalian species. a Numbering at the end of each sequence refers to the total number of residues in each protein. Identical residues and blocks of similar, weakly similar and non-similar residues are highlighted in black, grey, yellow and blue, respectively. The coding exons numbered E2 to E9 are indicated top and the signal peptide (SP) sequence is indicated bottom by the box. b Signal peptide prediction for rat SCPPPQ1 by SignalP4.0 suggests a point of cleavage (arrow) between amino acids 15 and 16. The

graphical output shows the three different scores (C-score raw cleavage site score, S-score signal peptide score, Y-score combined cleavage site score) for each position in the sequence. c Ribbon-like representation of the three-dimensional structure for rat SCPPPQ1 as predicted by Phyre2 software. The N-terminus (starting at L16) and the C-terminus are indicated. Alpha helices are predicted over residues 29–33 and 71–86 and a beta-strand over residues 67–68, whereas the remainder of the polypeptide adopts a random coil

of exons 8 and 9. Alignment of SCPPPQ1 protein sequences shows cross-species conservation sharing 68 % similarity between rat and human, 63 % between mouse and human and 94 % between rat and mouse (Fig. 2a). The SCPPPQ1 protein sequences are composed of between 87 and 95 amino acids, including a predicted cleavable signal peptide of 15 residues (Fig. 2b). For all species, the mature protein (without the signal peptide) has a computed pI of close to 6 and is rich in proline (~20 %), leucine (~17 %), glutamine (~12 %) and phenylalanine (~10 %). The amino acid composition predicts that SCPPPQ1 possesses mostly a random coil structure and is relatively hydrophobic in nature (residues AILFWV constitute 42 % by weight; Fig. 2a, c). Prediction of secondary structures by using various algorithm servers such as Phyre (Kelley and Sternberg 2009) and I-TASSER (Zhang 2008) has identified

two alpha helices and two extended strands (Fig. 2a, c). A putative serine phosphorylation motif (SSSE) located at position 26–29 is conserved among most species, with the first two serines potentially being phosphorylation sites (according to NetPhos 2.0 Server; Blom et al. 1999). However, no other sequence motifs resembling any other proteins have been found. Expression profile by Northern blot and by standard and qRT-PCR In order to define the expression pattern of rat Scpppq1, Northern blotting analyses were performed on various tissue RNAs (Fig. 3). The expression of Scpppq1 was detected as a short (~500 nt) transcript in RNA samples from EOs,

Cell Tissue Res

Fig. 3 Expression profiling of Scpppq1 in rat tissues and cells. Total RNA was extracted from adult tissues and/or cell lines of rat. RNA was also isolated from enamel organ (EO) at the secretory (EO-S) or maturation (EO-M) stage of amelogenesis. RNA was loaded at 15 μg/lane (except for enamel organs at 5 μg), separated on agarose gels and

processed for hybridisation with a specific Scpppq1 cDNA probe labelled with α32P-dCTP (top). The integrity of RNA was monitored after staining the blots with methylene blue (bottom). Positions of 28S and 18S ribosomal RNA are indicated

mandible and gingiva. A signal was also visible at the 18S marker but this was considered non-specific hybridisation. All other tissues tested were negative. The site of expression of the Scpppq1 gene was further redefined on RNA samples extracted from the EO-S and EO-M stages of amelogenesis. Northern blot and RT-PCR analyses revealed that the expression of Scpppq1 in EOs was restricted to the maturation stage (Figs. 3 and 4a). The strongest signal was found in the late stage of maturation in comparison with the early to midmaturation of amelogenesis (Fig. 4). Relative levels of expression of Scpppq1 in the late maturation stage (20×) are much higher than in the early to mid-stages (Fig. 4b). As expected, the overall expression of amelogenin (Amel) and ameloblastin (Ambn) transcripts decreased as development progressed to the maturation stage. The follicular dendritic cell secreted protein (Fdc-sp) transcript, another member of the SCPP family (Kawasaki 2009), was also analysed and found to be highly expressed mostly in the gingiva.

slightly faster than the expected 9.7 kDa, suggesting that this behaviour was intrinsic to the properties of the polypeptide primary sequence. Hence, upon dialysis of the final protein preparation, urea had to be included, because SCPPPQ1 precipitated out of solution. Quantification and purity of our final preparation were estimated by SDS-PAGE and Coomassie staining. The recombinant SCPPPQ1 protein was found to be >95 % pure and migrated again slightly faster than the aprotinin (6.5 kDa) standards (Fig. 5c). When this protein was used as an immunogen for the production of antibodies, sera collected at day 35, day 58 or after exsanguination revealed no significant difference in immunolabelling intensity (data not shown).

Production of bacterial 6His-tagged SCPPPQ1 and generation of a polyclonal antibody A polyclonal antibody against a bacterially produced rat SCPPPQ1 was generated in order to carry out immunolocalisation analyses. The recombinant SCPPPQ1 protein was tagged at its N-terminal with a 6His-tag (Fig. 5a). The protein was purified by Ni-affinity chromatography from the insoluble bacterial material by using urea extraction (Fig. 5b, top); this material was also immunoreactive with the anti-6His antibody (Fig. 5b, bottom). The Coomassie-blue-stained SDS-PAGE gel revealed a band at around 6.5 kDa (Fig. 5b). The recombinant protein migrated

Immunofluorescence detection of rat SCPPPQ1 in transfected HEK293 cells The antiserum was first tested in HEK293 overexpressing the rat SCPPPQ1 after transient transfection. Immunofluorescence staining with the anti-SCPPPQ1 antiserum revealed strong crescent-like labelling over structures abutting the nucleus, resembling the Golgi signal. This suggested that the SCPPPQ1 protein was targeted to the secretory pathway (Fig. 5d). Cells transfected with empty plasmid showed no specific signal (Fig. 5e). Immunolocalisation of SCPPPQ1 during tooth development and primary JE formation In the continuously erupting lower incisor in young adult rats, no labelling was apparent in presecretory (Fig. 6b), secretory (Fig. 6c) or post-secretory (Fig. 6d) transition stage

Cell Tissue Res

Fig. 4 Reverse transcription plus the polymerase chain reaction (RTPCR) and real-time RT-PCR expression analyses of mouse enamel organ (EO). Total RNA was extracted from adult mouse EOs, gingiva, liver and kidney. a Aliquots containing 250 ng were used in one-step RT-PCRs and PCR products were resolved on 2 % agarose gels stained with ethidium bromide. Scpppq1 (band at 390 bp) and actin (band at 348 bp) primers were used in parallel reactions. PCRs were stopped at 25, 30 and 35 cycles. The strongest signal for Scpppq1 was found in the late-maturation stage (EO-M late) of amelogenesis as compared with the secretory (EO-S) and early maturation (EO-M early) stages. No signal was noted in the liver. A 100-bp DNA ladder was used as markers. Molecular size (bp) is given left. b RNA was reverse-transcribed with random primers by using

the High Capacity cDNA reverse transcription kit. The cDNA was used for real-time PCR analysis with gene-specific Taqman probes for amelogenin (Amel, Mm00711642_m1), ameloblastin (Ambn, Mm00477485_m1), follicular dendritic cell secreted protein (Fdc-sp, Mm03951982_m1) and Scpppq1 (Mm03990687_m1). All reactions were performed in triplicate and normalised to the levels of beta-actin (435933E). Results were expressed as 2e(−ΔCt) and represent the mean ± range from two independent samples. A much higher (20×) relative level of expression for Scpppq1 was found in the EO-M late stage than in the EO-M early stage. As expected, the overall expression of Amel and Ambn transcripts decreased as development progressed to the maturation stage. The Fdc-sp transcript was highly expressed mostly in the gingiva

ameloblasts. In addition, no immunoreactivity for SCPPPQ1 was observed over the various cell layers of EO (including ameloblasts) forming and maturing enamel matrix, dentin, odontoblasts, pulp or bone (Fig. 6a). In the later part of the mid-stage of maturation, only the faint expression of SCPPPQ1 was detected in ameloblasts and the interface between their apical surface and enamel (Fig. 6e). The Golgi-like and interfacial immunoreactions became more intense and readily evident as maturation progressed towards the gingival margin (Fig. 6f). Immunogold labelling confirmed these reactivities and revealed that the interfacial labelling essentially reflected the presence of SCPPPQ1 over the BL (Fig. 6g, h). Immunohistochemical localisation of SCPPPQ1 in sections of 17.5-day-old mouse embryos revealed no labelling in dental and non-dental tissues (data not shown). Consistent with findings in the continuously erupting incisor, the developing second and third molar crowns of 1-week-old rats in which amelogenesis is still in the secretory stage, no immunoreactivity for SCPPPQ1 was observed (Fig. 7b). In the first molar, amelogenesis was more advanced and entered the maturation stage. Highly conspicuous staining was now seen over the EO (Fig. 7c, d), including at the ameloblast-enamel interface along the

lateral aspects of the molars (Fig. 7f, h). When the enamel matrix was completely EDTA-soluble, the exposed dentin surface at the dentino-enamel junction was immunoreactive but this was not observed in the incisor (Fig. 7f). In these teeth, in the enamel-free areas (Bosshardt and Nanci 1997), the EO was intensely reactive (Fig. 7d). When the tooth was close to erupting, the oral epithelium only immediately over the erupting path and clusters of cells between the epithelium and the tooth also expressed SCPPPQ1 (Fig. 7d–g). As the tooth pierced the oral epithelium, the EO situated incisally was destroyed but the cervical portions fused with the oral epithelium to form the primary JE. Cells of the primary forming JE were labelled for SCPPPQ1 (Fig. 7h). At 8 weeks of age, when the JE was fully established, the labelling mainly appeared as a thin and defined linear reaction at the interface between the JE cells and enamel space (Fig. 7i). Occasionally, some cellular labelling in the JE was seen (data not shown). Immunocytochemistry with colloidal gold confirmed that the labelling for SCPPPQ1 was associated with the internal BL of the JE (Fig. 7j). In some sections, the signal for SCPPPQ1 was also noted over the epithelium of the nasal cavities (data not shown).

Cell Tissue Res Fig. 5 Production of a recombinant bacterial 6His-SCPPPQ1 and purification by nickel affinity chromatography. a A cDNA fragment encoding rat SCPPPQ1 (without residues 1–15) was subcloned in fusion with the 6-histidine tag of the pQE30 bacterial expression plasmid. b The soluble 6His-rSCPPPQ1 protein was purified through a NiNTA-agarose column and eluted with imidazole. The presence of 6His-rSCPPPQ1 was monitored at each step on 16 % SDS polyacrylamide gel eletrophoresis (SDS-PAGE) Tris-Tricine gels and stained with Coomassie blue (top) and by immunoblotting (I.B., bottom) with anti-6His antibody. The final preparation contained the predominant fulllength 6His-rSCPPPQ1 (arrow ~ Mr 6.5 kDa). c The protein purity of the final preparation of 6His-SCPPPQ1 was estimated by running serial dilutions on 16 % SDS-PAGE Tris-Tricine gels, alongside known quantities of aprotinin. Detection was achieved with Coomassie blue. d Immunolabelling for rat SCPPPQ1 in HEK293 transfected cells revealed a perinuclear reaction. e Control incubation consisting in transfection of cells with an empty vector (MOCK) gave no labelling. Nuclei are counterstained with DAPI (blue)

Discussion Using a signal-trap strategy to identify the secretome of the EO (secreted and membrane-associated proteins), we have previously identified several novel non-annotated genes including Scpppq1 (Moffatt et al. 2006a). In subsequent studies, we characterised the proteins of two of these genes, namely AMTN (Moffatt et al. 2006b) and ODAM (Moffatt et al. 2008). This paper describes the complete gene sequence of Scpppq1, the protein that it encodes and its pattern of expression. Northern blotting and standard and qRT-PCR revealed

that, in rat tissues, the expression of Scpppq1 is essentially restricted to the maturation stage of amelogenesis and to the gingiva surrounding the teeth. Immunohistochemical analysis confirmed the expression of SCPPPQ1 at these sites and further revealed that this small secreted protein is localised at the interface between the enamel and the apical surface of late maturation stage ameloblasts and between the JE and erupted tooth surfaces. No or little signal is found over the maturing enamel layer suggesting that it does not accumulate to any significant extent within this calcifying layer, consistent with the finding that the organic matrix is actively removed during

Cell Tissue Res

Cell Tissue Res

ƒFig.

6 Immunohistochemical labelling for SCPPPQ1 in the rat mandibular incisor. a Composite image showing SCPPPQ1 labelling in the rat lower incisor. b–f Higher magnifications of the relevant boxed areas in a. No labelling for SCPPPQ1 is found in the presecretory (b), secretory (c) or post-secretory (d) transition of amelogenesis. e In the early to mid-maturation stage, Golgi-like staining in the ameloblasts is observed and some regions along the cell-tooth interface are stained. f In the late stage of maturation, a great increase in the expression of SCPPPQ1 is noted. g, h Transmission electron micrographs of immunogold labelling showing the weak expression of SCPPPQ1 at the early to mid-stage of maturation (g) and its abundant presence in the later stage (h)

this stage (Smith and Nanci 1996). Alignment of the SCPPPQ1 sequence has revealed its high homology among species (Kawasaki et al. 2007, 2011; Kawasaki and Weiss 2008). By virtue of its genomic location in the Scpp gene cluster and architecture, its restricted expression and its histological distribution, SCPPPQ1 is reminiscent of ODAM and AMTN (Moffatt et al. 2006b, 2008). Phylogenetically, Scpppq1 originated from Odam and itself gave rise to the milk Ca-sensitive caseins supporting the notion that Scpp genes are involved in calcium phosphate biology (Kawasaki et al. 2011). SCPPPQ1 is a small molecular weight protein with a rather uncharacteristic sequence that exhibits no conserved domains or motifs with other members of the Scpp cluster thereby reinforcing the notion that it has diverged following its evolution by tandem duplication (Kawasaki 2011; Kawasaki et al. 2011). The absence of identifiable known motifs or domains on SCPPPQ1 makes it difficult at present to infer any potential function. Interestingly, it shares a putative phosphorylation site with AMTN. The difference in molecular weight between the actual Western blot band size (6.5 kDa) and the predicted molecular weight (9.7 kDa) is notable and might be explained by the highly hydrophobic nature of SCPPPQ1. The epithelial attachment complex at the cell-tooth interface in the maturation stage of amelogenesis and in the JE consists in an atypical BL to which cells conventionally attach by hemidesmosomes. However, the adhesion mechanism of this BL to the tooth surface is still not understood. Characterisation of its structural components has been particularly challenging; the inner BL of the JE has been demonstrated to contain laminin-332 but other typical components such as γ1-chaincontaining laminins and type IV and VII collagens have not been convincingly shown and believed to be absent (Hormia et al. 2001). Our previous studies identified ODAM and AMTN as two novel constituents of this BL (Dos Santos et al. 2012; Moffatt et al. 2006b, 2008) and the present study identified SCPPPQ1 as an additional component. Typical BLs bind to the connective tissue via collagen type VII (Erickson and Couchman 2000; McMillan et al. 2003; Yurchenco et al. 2004) but this is not present at the tooth surface, which essentially consists of calcium phosphate mineral. The epithelial cells have adapted to this situation by possibly exploiting proteins from the Scpp gene cluster with an affinity for mineral. All three

proteins, namely ODAM, AMTN and SCPPPQ1, have evolved as hypermineralisation proteins and represent logical candidates to mediate attachment to the mineralised tooth surface (Kawasaki 2009, 2011; Kawasaki et al. 2009). The available data, however, do not allow to determine as to whether any of them actually binds to these surfaces. All three proteins probably interact among themselves and with other components of the BL such as laminin-332 to constitute a macromolecular interfacial layer binding on one side to the epithelial cells and on the other to mineralised surfaces. The yeast-two hybrid approach has shown that ODAM and AMTN can interact (Holcroft and Ganss 2011) and the co-localisation of ODAM, AMTN and SCPPPQ1 in the BL certainly supports such a possibility. Dos Santos et al. (2012) reported that ODAM in the BL appears to concentrate closer to the cell surface, whereas AMTN seems to be distributed closer to the enamel surface during early to mid-maturation. However, in the late maturation stage, both molecules show a similar pattern of distribution suggesting that the BL is restructured and/or that it has a “loose” organisation that allows the accommodation of additional small-molecular-weight components, such as we report here for SCPPPQ1. The necessity for additional components might be related to the changing nature of the mineralising enamel layer, which transforms from a partially mineralised material to an almost pure mineral (Schmitz et al. 2014; Smith and Nanci 1989). Another interesting observation is that both ODAM (Dos Santos et al. 2012; Moffatt et al. 2008; Nishio et al. 2010) and SCPPPQ1 exhibit intense Golgi reactions in ameloblasts, whereas cellular labelling for AMTN (Dos Santos et al. 2012; Moffatt et al. 2006b; Nishio et al. 2010) is extremely weak and seen only at the very beginning of maturation. The sustained Golgi labelling for ODAM and SCPPPQ1 suggests that ODAM and SCPPPQ1 undergo renewal throughout maturation. In contrast, AMTN seems to be produced in amounts detectable by our immunolabelling procedures only during a narrow time frame and to remain stable over time. In a wellestablished JE, under normal conditions, both AMTN (Moffatt et al. 2006b; Nishio et al. 2013) and SCPPPQ1 appear as a discrete line at the cell-tooth interface, whereas ODAM is also found among cells of the JE. In a recent study, Sawada et al. (2014) reported that Amtn RNA is not found in monkey JE supporting the hypothesis that certain proteins of the BL in the epithelial attachment are produced phasically as a response to normal turnover or to insults that require repair to restore integrity of the JE. Indeed, the gamma 2 component of laminin-332, which is a major constituent of the BL, seems to have a turnover of approximately 3 weeks (Adair-Kirk et al. 2012; Kim et al. 2012). In conclusion, our data demonstrate that SCPPPQ1 is a novel component of the BL associated with maturation stage, ameloblasts and JE. Its exact function, the way that it interacts

Cell Tissue Res

Fig. 7 Immunolocalisation of SCPPPQ1 during rat molar development. a Composite image of the immunohistochemical localisation of SCPPPQ1 during tooth eruption showing the three molars at various stages of development. The first molar has pierced the oral epithelium and the primary junctional epithelium (JE) has started to form. When the second molar approaches the oral epithelium, a thin layer of connective tissue still separates the enamel organ from the oral epithelium. The development of enamel in the third molar is still underway at this stage. b At 1 week of age, enamel formation is still at an early secretory stage of amelogenesis and no labelling for SCPPPQ1 is found. c–e Although the enamel has not yet completely formed over the tooth crown (c),

SCPPPQ1 is detected in the enamel-free area (d) and in epithelial cell clusters of the outer enamel epithelium (e). d, e Higher magnification of the boxed areas in c. f–h As the tooth erupts, ameloblasts and an adjacent cell layer form the reduced enamel organ. Expression of SCPPPQ1 is detected in the latter cell layer (arrowhead) and in the overlying oral epithelium. i In adult rats, the expression of SCPPPQ1 is detected at the junctional epithelium cell-tooth interface in which the atypical basal lamina is found (arrowheads). j Transmission electron micrograph of immunogold labelling showing the presence of SCPPPQ1 in the atypical basal lamina of the junctional epithelium

Cell Tissue Res

with other constituents of the BL and whether it can bind to mineral remain to be determined. The temporospatial distribution of SCPPPQ1 suggests that it contributes directly or indirectly to the binding mechanism of epithelial cells to tooth surfaces and also possibly to late enamel maturation events. Similar to epidermal bullosas, mutations in any of these molecules could affect the integrity of the BL leading to problems with enamel maturation or poor attachment of the JE to the tooth surface. A better understading of this system is thus mandatory. Acknowledgments We extend our thanks to Mrs. Cynthia Török and Katia Julissa Ponce for technical assistance.

References Adair-Kirk TL, Griffin GL, Meyer MJ, Kelley DG, Miner JH, Keene DR, Marinkovich MP, Ruppert JM, Uitto J, Senior RM (2012) Keratinocyte-targeted expression of human laminin gamma2 rescues skin blistering and early lethality of laminin gamma2 deficient mice. PLoS ONE 7:e45546 Bendayan M (1995) Colloidal gold post-embedding immunocytochemistry. Prog Histochem Cytochem 29:1–159 Bendayan M, Zollinger M (1983) Ultrastructural localization of antigenic sites on osmium-fixed tissues applying the protein A-gold technique. J Histochem Cytochem 31:101–109 Blom N, Gammeltoft S, Brunak S (1999) Sequence and structure-based prediction of eukaryotic protein phosphorylation sites. J Mol Biol 294:1351–1362 Bosshardt DD, Nanci A (1997) Immunodetection of enamel- and cementum-related (bone) proteins at the enamel-free area and cervical portion of the tooth in rat molars. J Bone Miner Res 12:367–379 Church GM, Gilbert W (1984) Genomic sequencing. Proc Natl Acad Sci U S A 81:1991–1995 Dos Santos NJ, Wazen RM, Kuroda S, Francis ZS, Moffatt P, Nanci A (2012) Odontogenic ameloblast-associated and amelotin are novel basal lamina components. Histochem Cell Biol 137:329–338 Erickson AC, Couchman JR (2000) Still more complexity in mammalian basement membranes. J Histochem Cytochem 48:1291–1306 Holcroft J, Ganss B (2011) Identification of amelotin- and ODAMinteracting enamel matrix proteins using the yeast two-hybrid system. Eur J Oral Sci 119 (Suppl 1):301–306 Hormia M, Owaribe K, Virtanen I (2001) The dento-epithelial junction: cell adhesion by type I hemidesmosomes in the absence of a true basal lamina. J Periodontol 72:788–797 Huq NL, Cross KJ, Ung M, Reynolds EC (2005) A review of protein structure and gene organisation for proteins associated with mineralised tissue and calcium phosphate stabilisation encoded on human chromosome 4. Arch Oral Biol 50:599–609 Kawasaki K (2009) The SCPP gene repertoire in bony vertebrates and graded differences in mineralized tissues. Dev Genes Evol 219:147– 157 Kawasaki K (2011) The SCPP gene family and the complexity of hard tissues in vertebrates. Cells Tissues Organs 194:108–112 Kawasaki K, Weiss KM (2003) Mineralized tissue and vertebrate evolution: the secretory calcium-binding phosphoprotein gene cluster. Proc Natl Acad Sci U S A 100:4060–4065 Kawasaki K, Weiss KM (2006) Evolutionary genetics of vertebrate tissue mineralization: the origin and evolution of the secretory calcium-

binding phosphoprotein family. J Exp Zool B Mol Dev Evol 306: 295–316 Kawasaki K, Weiss KM (2008) SCPP gene evolution and the dental mineralization continuum. J Dent Res 87:520–531 Kawasaki K, Suzuki T, Weiss KM (2004) Genetic basis for the evolution of vertebrate mineralized tissue. Proc Natl Acad Sci U S A 101: 11356–11361 Kawasaki K, Buchanan AV, Weiss KM (2007) Gene duplication and the evolution of vertebrate skeletal mineralization. Cells Tissues Organs 186:7–24 Kawasaki K, Buchanan AV, Weiss KM (2009) Biomineralization in humans: making the hard choices in life. Annu Rev Genet 43: 119–142 Kawasaki K, Lafont AG, Sire JY (2011) The evolution of milk casein genes from tooth genes before the origin of mammals. Mol Biol Evol 28:2053–2061 Kelley LA, Sternberg MJ (2009) Protein structure prediction on the Web: a case study using the Phyre server. Nat Protoc 4:363–371 Kim ST, Adair-Kirk TL, Senior RM, Miner JH (2012) Functional consequences of cell type-restricted expression of laminin alpha5 in mouse placental labyrinth and kidney glomerular capillaries. PLoS ONE 7:e41348 McMillan JR, Akiyama M, Shimizu H (2003) Epidermal basement membrane zone components: ultrastructural distribution and molecular interactions. J Dermatol Sci 31:169–177 Moffatt P, Smith CE, Sooknanan R, St-Arnaud R, Nanci A (2006a) Identification of secreted and membrane proteins in the rat incisor enamel organ using a signal-trap screening approach. Eur J Oral Sci 114 (Suppl 1):139–146 Moffatt P, Smith CE, St-Arnaud R, Simmons D, Wright JT, Nanci A (2006b) Cloning of rat amelotin and localization of the protein to the basal lamina of maturation stage ameloblasts and junctional epithelium. Biochem J 399:37–46 Moffatt P, Smith CE, St Arnaud R, Nanci A (2008) Characterization of Apin, a secreted protein highly expressed in tooth-associated epithelia. J Cell Biochem 103:941–956 Neiss WF (1984) Electron staining of the cell surface coat by osmiumlow ferrocyanide. Histochemistry 80:231–242 Nishio C, Wazen R, Kuroda S, Moffatt P, Nanci A (2010) Expression pattern of odontogenic ameloblast-associated and amelotin during formation and regeneration of the junctional epithelium. Eur Cell Mater 20:393–402 Nishio C, Wazen R, Moffatt P, Nanci A (2013) Expression of odontogenic ameloblast-associated and amelotin proteins in the junctional epithelium. Periodontol 2000 63:59–66 Petersen TN, Brunak S, Heijne G von, Nielsen H (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 8:785–786 Sawada T, Yamazaki T, Shibayama K, Kumazawa K, Yamaguchi Y, Ohshima M (2014) Expression and localization of laminin 5, laminin 10, type IV collagen, and amelotin in adult murine gingiva. J Mol Histol 45:293–302 Schmitz JE, Teepe JD, Hu Y, Smith CE, Fajardo RJ, Chun YH (2014) Estimating mineral changes in enamel formation by ashing/BSE and MicroCT. J Dent Res 93:256–262 Smith CE, Nanci A (1989) A method for sampling the stages of amelogenesis on mandibular rat incisors using the molars as a reference for dissection. Anat Rec 225:257–266 Smith CE, Nanci A (1996) The protein dynamics of amelogenesis. Anat Rec 245:186–207 Yurchenco PD, Amenta PS, Patton BL (2004) Basement membrane assembly, stability and activities observed through a developmental lens. Matrix Biol 22:521–538 Zhang Y (2008) I-TASSER server for protein 3D structure prediction. BMC Bioinformatics 9:40