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 (MGP) belongs to the family of vitamin K dependent, Gla containing proteins and, in mammals, birds and Xenopus, its mRNA has been previously detected in bone, cartilage and soft tissue extracts, while the accumulation of the protein was found mainly in calcified tissues. More recently, the MGP gene expression was also studied in marine teleost fish where it was found to be associated with chondrocytes, smooth muscle and endothelial cells. To date no information is available on the sites of MGP expression or accumulation in cartilaginous fishes that diverged from osteichthyans, a group that includes mammals, over 400 million years ago. The main objectives of this work were to study the sites of MGP gene expression and protein accumulation by means of in situ hybridization and immunohistochemistry. MGP mRNA and protein were localized as expected not only in cartilage from branchial arches and vertebra but also in the endothelia of the vascular system as well as in the tubular renal endothelium. The accumulation of MGP in non mineralized soft tissues was unexpected and suggests differences in localization or regulation of this protein in shark soft tissues compared to tetrapods and teleosts. Our results also corroborate the hypothesis that in Prionace glauca, as previously shown in mammals, the MGP protein probably also acts as a calcification inhibitor, protecting soft tissues from abnormal and ectopic calcification.
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