Villarreal - Ramirez 2009 Biochemical - and - Biophysical - Research - Communications

Villarreal - Ramirez 2009 Biochemical - and - Biophysical - Research - Communications

(Parte 1 de 3)

Characterization of recombinant human cementum protein 1 (hrCEMP1): Primary role in biomineralization

Eduardo Villarreal-Ramíreza, Abel Morenob, Jaime Mas-Olivac, Juan Luis Chávez-Pachecoa,

A. Sampath Narayanan d, Ivet Gil-Chavarría a, Margarita Zeichner-David e, Higinio Arzate a,*Laboratorio de Biología Periodontal y Tejidos Mineralizados, Facultad de Odontología, UNAM, México D.F. 04510, MexicoInstituto de Química, UNAM, México D.F. 04510, MexicoInstituto de Fisiología Celular, UNAM, México D.F. 04510, MexicoDepartment of Pathology, School of Medicine, University of Washington, Seattle 98195, USACenter for Craniofacial Molecular Biology, University of Southern California, USA article info

Article history: Received 2 April 2009 Available online 23 April 2009

Keywords: Cementum Biomineralization Cementum protein 1 Mineralized tissues Hydroxyapatite Octacalcium phosphate Periodontal regeneration abstra ct

Cementum protein 1 (CEMP1) has been recently cloned, and in vitro experiments have shown functions as regulator of cementoblast behavior and inducer of differentiation of non-osteogenic cells toward a cementoblastic/osteoblastic phenotype. In this study, we have produced a full-length human recombi- nant CEMP1 protein in a human gingival fibroblast cell line. The purified protein (hrCEMP1) has a Mr 50,0. Characterization of hrCEMP1 indicates that its secondary structure is mainly composed of b-sheet

(5%), where random coil and alpha helix conformations correspond to 35% and 10%, respectively. It was found that hrCEMP1 is N-glycosylated, phosphorylated and possesses strong affinity for hydroxyapatite. Even more important, our results show that hrCEMP1 plays a role during the biomineralization process by promoting octacalcium phosphate (OCP) crystal nucleation. These features make CEMP1 a very good candidate for biotechnological applications in order to achieve cementum and/or bone regeneration. 2009 Elsevier Inc. All rights reserved.


Cementum is a unique avascular mineralized connective tissue that covers the root surface of teeth and provides the interface through which the root surface is anchored to collagen Sharpey’s fibers of the periodontal ligament. Nevertheless, the complex processes thatregulatecementogenesis andnormalcementummetabolism remain unclear to date. Recent evidence indicates that cementum formation is critical for appropriate maturation of the periodontium [1]. Recently we have isolated and characterized a human cementum protein which we named Cementum Protein 1 (CEMP1), (GenBank Accession No. NP_001041677; HGNC: ID 32553) [2]. Antibodies against this protein recognize the cementoid layer and adjacent cementoblasticcell layer, cementocytes, progenitor cells located near the blood vessels in the periodontal ligament, cells located in the endosteal spaces of human alveolar bone, dental follicle-derived cells and human periodontal ligament cells [2–4]. CEMP1mRNAishighlyexpressedincementoblasts,subpopulations and progenitor cells of the human periodontal ligament [5]. In vitro experiments showed that CEMP1 promotes cell attachment, differ- entiation [6,7], and deposition rate, composition, and morphology of hydroxyapatite crystals formed by human cementoblast cells [7]. Since CEMP1 is synthesized by cementoblast cells and, a restricted periodontal ligament cell subpopulations (cementoblast precursors),itissuggestedthatthismoleculeisacementum-specific biological marker and it might play a role as regulator of cell differentiation. Furthermore, CEMP1 transfection into non-osteogenic cells such as adult human gingival fibroblasts results in differentiation of these cells into a ‘‘mineralizing” cell phenotype [8]. Although thephysiologicalfunctionofCEMP1isnotcompletelyunderstood,it is our hypothesis that this molecule plays an important role during the cementogenesis process and also as an inducer of the formation ofmineralizingnodulesandcalciumdepositionduringhydroxyapatiteformation.Therefore,theaimofthepresentstudywastocharacterizethephysic-chemicalcharacteristicsofhrCEMP1expressedina humangingivalfibroblastcellline anddeterminepost-translational modifications and their influence on CEMP1’s functional properties during the mineralization process.

Materials and methods

Expression and purification of CEMP1. The open reading frame of CEMP1 (GenBank Accession No. NP_001041677), was subcloned

0006-291X/$ - see front matter 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.04.072

* Corresponding author. Fax: +52 5556225563. E-mail address: (H. Arzate).

Biochemical and Biophysical Research Communications 384 (2009) 49–54 Contents lists available at ScienceDirect

Biochemical and Biophysical Research Communications journal homepage: w.else into the pENTR/SD/D vector (Invitrogen, Carlsbad, CA) and the resultant pENTR/SD/D-CEMP1 cDNA construct ligated into a pcDNA40(+) vector pcDNA40-CEMP1(+). Human gingival fibroblasts (HGF) were isolated and grown as previously described [3]. The plasmid pcDNA40-CEMP1(+) was transfected into human gingival fibroblasts cells as described elsewhere [8].

Recombinant human CEMP1 protein collected from conditioned media of HGF expressing CEMP1 was purified by Ni2+ affinity chromatography (HiTrap Chelating HP column, Invitrogen, Carlsbad, CA). Determination of protein purity was performed by 12% SDS– PAGE.

Western blot analysis. Recombinant human CEMP1 (10 lg) was separated by 12% SDS–PAGE and electroblotted onto Inmobilon-P (PVDF) nitrocellulose membrane (Millipore Corp., Bedford, MA). Anti-hrCEMP1 and anti-6XHis (C-term) polyclonal antibodies were used to specifically identify the CEMP1 gene product and the fused histidines. Peroxidase-conjugated goat anti-rabbit IgG was used and secondary antibody and detection was performed as previously described [2].

Hydroxyapatite affinity chromatography. To determine if hrCEMP1 has affinity to hydroxyapatite, an Econo-Pac CHT-I cartridge (1 mL) (Bio Rad, Hercules, CA) was used. The column was equilibrated with 10 mM sodium phosphate, pH 7.2. Fifty micrograms of purified hrCEMP1 was loaded and unbound proteins removed with a solution containing 10 mM sodium phosphate, pH 7.2. Bound proteins were eluted with a solution containing 0.05, 0.1, 0.2, 0.3, 0.4 and 0.5 M sodium phosphate, pH 7.2. Fractions were subjected to 12% SDS–PAGE and Western blotting.

Circular dichroism spectroscopy. hrCEMP1 protein was dissolved in PBS, pH 7.4, at 200 lg/mL. The concentration was calculated from the absorption at 280 nm using an extinction coefficient of 28,125 M 1 cm 1 and deduced from the amino acid sequence [9]. CD spectra were recorded in thermostatted (25 C) quartz cells of 1-m optical path length within a wavelength range of 190– 260 nm using a AVIV62DS spectropolarimeter. The molar ellipticity (h) expressed in degrees.cm2 dmol 1 was calculated on the basis of a mean residue of Mr 50,0. Five spectra were accumulated to improve the signal to noise ratio. A baseline with buffer (PBS, pH 7.4) was recorded separately and subtracted from each spectrum. The program CONTIN was used to calculate secondary structure content [10].

Dynamic light scattering (DLS). Light scattering experiments were performed using a Zetasizer Nano S (Malvern Instruments, Ltd., UK) molecular sizing instrument which employs a 4 mw, 633 nm semiconductor laser as light source and NIBS technology (Malvern Instruments, Ltd., UK) [1]. During experiments the temperature was held at 25 (0.1 C) via a Peltier unit. Data analysis was performed using the Zetasizer Nano S DTS software package (Malvern Instruments, Ltd., UK).

Presence of cysteine disulfide bonds. Human recombinant CEMP1 at a 2.5 mM concentration was dissolved with 6 M guanidine–HCl containing 200 mM DTT. The protein was reduced at 37 C overnight, and boiled for 5 min before the protein was loaded into a gel filtration column (1.5 10 cm Sephadex G-10, Pharmacia, Uppsala, SW) equilibrated with 300 mM acetic acid. The reduction state of hrCEMP1 was assessed by quantitation of thiols using an assay for dithiodipyridine. Briefly, hrCEMP1 was incubated with

6 M guanidine–HCl, 10 mM EDTA, 120 mM Na2HPO4, pH 6.6, and DTPD (4,40-dithiodipyridine) added to a final concentration of

500 nM. Samples were incubated for 30 min at 25 C and the A324 was monitored to estimate the number of cysteine residues present.

Glycosylation analysis. Carbohydrates contained in hrCEMP1 were determined using the ECL glycoprotein detection system (Amersham Biosciences, UK). Briefly, samples were separated by 12% SDS–PAGE and electro-transferred as described above. Oxida- tion was carried out in the dark with 10 mM sodium metaperiodate dissolved in 100 mM acetate buffer, pH 5.5. Samples were treated with biotin hydrazide to incorporate biotin into the oxidized carbohydrate and biotin was detected by the horseradish peroxidase-conjugated streptavidin system using enhanced chemiluminescence (ECL, Millipore Corp., Bedford, MA).

Release of N-linked oligosaccharides. N-Glycans were released from 200 lgo f hrCEMP1 by enzymatic cleavage using peptide N- glycosidase F (Calbiochem Glycoprotein Deglycosylation kit, Merck Biosciences Ltd., Nottingham, UK). The protein was resuspended with 10 lL of 250 mM sodium phosphate buffer, pH 7.0 and 2.5 lL of denaturation solution (2% w/v SDS, 1 M b-mercaptoethanol). The mixture was heated at 100 C for 5 min. One unit of PNGasa F was added and incubated for 24 h at 37 C. The N- linked glycosylation pattern of hrCEMP1 was resolved by 12% SDS–PAGE. hrCEMP1 phosphorylation. According to ‘in silico’ analysis, hrCEMP1 possesses multiple potential phosphorylation sites. Eighteen phosphorylation sites (10 serine, 8 threonine) were predicted by the NetPhos 2.0 program [2]. Tyrosine is not present in the hrCEMP1 amino acid sequence. To determine if serine and threonine phosphorylation is present in hrCEMP1, hrCEMP1 secreted to the media and purified by Ni2+ affinity chromatography was used. Human recombinant CEMP1 was subjected to 12% SDS–PAGE and electrotransfered as described above. Membranes were blocked as described, and incubated with primary polyclonal antibodies against phosphothreonine and phosphoserine (Zymed, San Francisco, CA, USA). After washing, membranes were incubated with the horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody. Membranes were washed and detection of secondary antibody performed as previously described. hrCEMP1 effect on apatite formation. To determine if hrCEMP1 promotes apatite formation, a capillary counterdiffusion system was used [13–15]. Briefly, 1% (w/v) agarose gel containing 20 lg/ mL of hrCEMP1 was poured into the capillaries (0.5 m diameter and 30 m long). The ends of the capillaries were injected with

100 mM CaCl2 and 100 mM NaH2PO4. All experiments were carried out at37 C. After 7 days, the crystals were recovered by dissolving the gel in hot milli Q water and air-dried.

Energy-dispersive X-ray micro-analysis (EDX). The composition of crystals formed by induction of hrCEMP1 into the capillaries was analyzed using a Jeol 5600 scanning electron microscope fitted with a detector of energy dispersive X-ray microanalysis microprobe. All analyses were performed at 20 kV for 300 s [16]. Crystals were analyzed in low vacuum and the calcium/phosphate (Ca/P) ratio was calculated from the intensity of the peaks present in the EDX pattern. After determining the composition of the crystals, they were covered with a thin gold film, 100 nm thick, to avoid electron disturbances that could interfere with the SEM images.

Electron diffraction pattern by transmission electron microscopy.

Crystals were mounted on carbon-coated 150-mesh gold grids and examined for diffraction techniques. D-spacings of diffraction patterns were calibrated against those used as gold standard with identical diffraction conditions. The mineral phase was analyzed by means of a JEOL 100 CX analytical transmission microscope employing 100 kV.

Results and discussion Isolation of human CEMP1 by recombinant expression

Previously, we have expressed hrCEMP1 in a prokaryotic expression system; however, this system is not able to express the full-length recombinant CEMP1 [2]. In this study, human recombinant CEMP1 was expressed in human-derived gingival fibroblasts as a secreted 6XHis fusion protein and latter purified

50 E. Villarreal-Ramírez et al./Biochemical and Biophysical Research Communications 384 (2009) 49–54 by affinity chromatography using a Ni2+ column. The yield of recombinant CEMP1 per liter of serum-free media was about 1 mg. After SDS–PAGE and coomassie blue staining, the protein pre- sented a Mr 50,0 (Fig. 1A, lane 2). The identity of the human recombinant protein was determined by Western blot analysis using a specific polyclonal antibody against the 6XHis tag (Fig. 1A, lane 3) and a specific polyclonal antibody against hrCEMP1 (Fig. 1A, lane

4). Both antibodies recognized a single protein species of Mr 50,0 which is almost twice the theoretical molecular mass de- duced from the cDNA sequence (25.9 kDa) [2]. This data shows that we are able to produce the full-length recombinant human CEMP1 in high yields using a human gingival fibroblasts-derived cell line.

hrCEMP1 secondary structure

Circular dichroism of hrCEMP1 showed that the spectra bands present its maximum value at 218 nm. This determines that the secondary structure present in hrCEMP1 is mainly composed of b-sheet. CD spectra analysis revealed 10% a-helix, 32.4% b-antiparallel, 5.8% b-parallel, 16.7% b-turn and 35% random coil (Fig. 1B). This result was consistent even when different concentrations of trifluoride ethanol (20% and 40%) were used in order to determine if a change in the structure could be induced. Recently, it has been show that proteins with high percentages of random coil structure are multifunctional and allow proteins to have diverse binding properties such as SIBLING and HMGI(Y) [16,17]. This feature might explain why hrCEMP1 regulates crystal growth and composition of hydroxyapatite crystals [7]. CEMP1 also induces the expression of proteins related to mineralization and promotes in vitro osteoblastic and/or cementoblastic cell differentiation of HGF [8].

Dynamic light scattering and cysteine disulfide bonds

Dynamic light scattering analysis revealed that hrCEMP1 aggregates mainly as 6.50 nm particles. Such aggregates are contributed by one type of molecule with a Mr 50,0 (Fig. 1C). Our results showed that hrCEMP1 did not react with sulfhydryl groups. Therefore, all cysteine residues present in hrCEMP1 might be linked to disulfide bridges. Disulfide bridges generally play a role stabilizing protein structure [18–20]. From our results we infer that disulfide bridges contribute to hrCEMP1‘s secondary structure stabilization.

hrCEMP1 glycosylation and N-linked oligosaccharides

According to in silico analysis (NetNGlyc, neural net bioinformatic program) [12], hrCEMP1 possesses two N-glycosylation sites Asn-X-Ser/Thr in amino acids 20 and 25. After digestion of the protein with PNGase F, our results demonstrate that hrCEMP1 is a glycoprotein with a Mr 50,0 and shifted from Mr 50,0

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