Characterization of Osteocalcin (BGP) and Matrix Gla Protein (MGP) Fish Specific Antibodies: Validation for Immunodetection Studies in Lower Vertebrates

Abstract

Abstract. In fish species the basic mechanisms of bone development and bone remodeling are not fully understood. The classification of bone tissue in teleosts as cellular or acellular and the presence of transitional states between bone and cartilage and the finding of different types of cartilage in teleosts not previously recognized in higher vertebrates emphasizes the need for a study on the accumulation of the Gla-containing proteins MGP and BGP at the cellular level. In the present study, polyclonal antibodies developed against BGP and MGP from A. regius (a local marine teleost fish) and against MGP from G. galeus (a Pacific Ocean shark), were tested by Western blot for their specificity against BGP and MGP from several other species of teleost fish and shark. For this purpose we extracted and purified both proteins from various marine and freshwater teleosts, identified them by N-terminal amino acid sequence analysis and confirmed the presence of gamma-carboxylation in the proteins with the use of a stain specific for Gla residues. Each antibody recognized either BGP or MGP with no cross-reaction between proteins detected. All purified fish BGPs and MGPs tested were shown to be specifically recognized, thus validating the use of these antibodies for further studies.Matrix Gla Protein (MGP) and Bone Gla Protein (BGP, osteocalcin) belong to the family of vitamin K-dependent (VKD), c-carboxyglutamic acid (Gla)-containing proteins that have been unequivocally associated with bone formation and mineralization [1][2][3][4], and more recently, with vascular calcification [5][6][7][8][9][10].MGP is a 10-15 kDa-secreted protein, containing 4-5 residues (depending on the species) of the Ca 2+ binding Gla residue [11][12][13][14][15] while BGP is a small secreted protein with approximately 6 kDa molecular weight and includes three Gla residues. Although there is little information about the regulation of expression of these proteins in teleosts, BGP in teleost fish has been shown to be associated with bone-like mineralized tissues present in branchial arches, jaw, vertebra and scales [15,16] while MGP was only recently found to accumulate, mainly in the extracellular matrix of calcified cartilage [15]. In this previous work, the MGP gene was found to be predominantly expressed in chondrocytes from branchial arches, with no expression detected in the different bone-like mineralized tissues analyzed while BGP mRNA was mainly located in bony tissues associated with osteoblast-like cells, as expected [15]. As previously seen in mammals and Xenopus, MGP mRNA was also found to be present in teleost soft tissues, predominantly in heart and kidney [15], with the expression of MGP in heart tissue mainly associated with two specific cell types, smooth muscle and endothelial cells [15].We have recently developed specific polyclonal antibodies against BGP and MGP from the teleost fish A. regius [15] as well as against MGP purified from soupfin shark (Galeorhinus galeus) (our unpublished results). The purpose of the present work ...

Matrix Gla protein in turbot (Scophthalmus maximus): Gene expression analysis and identification of sites of protein accumulation

“…DBS staining permitted the identification of a Gla containing protein in sample extracts (Fig. 5.1) corresponding to a single immunoreactive band observed in western blotting with anti-ArMgp with a migration behaviour (14-18 kDa) similar to that previously observed for other fish Mgp entities (Simes et al, 2004). Similarly, SDS-PAGE electrophoresis of guanidine-HCL extracts obtained from heart, followed by blotting onto nitrocellulose membranes and incubation with anti-ArMgp polyclonal antiserum, revealed the Fig.…”

“…The resulting tissue powder was extracted using a 10-fold excess (w/v) of 6 M guanidineHCl solution with vigorous stirring at room temperature for 24 h followed by a 10 min centrifugation at 10,000 rpm, at room temperature. The tissue pellet was subjected to a similar second extraction procedure during 24 h. Supernatants from both extractions were mixed, dialyzed (SpectraPor 3; Spectrum, Gardena, CA, USA) against 50 mM HCl at 4°C, cleared by centrifugation at (10,000 rpm, 10 min, 4°C) and total supernatant protein content was as previously described (Simes et al, 2004). The presence of Mgp in both dialyzed guanidine extracts was analysed by western blot.…”

“…Western-blot analysis for both validation of ArMgp antibody (developed against Argyrosomus regius, meagre, Mgp) and detection of Mgp in heart samples was performed as described (Simes et al, 2004) using 1:250 dilution of ArMgp primary polyclonal antibody. Immunoreactive protein bands were detected using alkaline phosphatase-labelled goat anti-rabbit IgG antibody (Gibco-BRL, Paisley, UK) diluted 1:20,000 in TBST and visualized using NBT/BCIP substrate solution (Sigma).…”

“…MWM, SeeBlue pre-stained molecular weight markers (Invitrogen); 2 -western-blot analysis of dialyzed turbot heart guanidine-HCl 6 M extract. Validated anti-ArMgp polyclonal antiserum (Simes et al, 2004) was used to detect the presence of Mgp in the first (heart extract-1) and second (heart extract-2) dialyzed guanidine extraction solution by western blot as described in the Materials and Methods. MWM, SeeBlue pre-stained molecular weight markers (Invitrogen).…”

Normal and histopathological organization of the opercular bone and vertebrae in gilthead sea bream Sparus aurata

“…Anti meagre-OC and -MGP antibodies presented a specific cross reactivity for gilthead sea bream-OC and -MGP (Simes et al 2004). Immunohistochemical staining was performed with peroxidase-conjugated antirabbit IgG (Sigma) as secondary antibody, using the methodology proposed by Simes et al (2003Simes et al ( , 2004.…”