Matrix Gla Protein in Xenopus laevis: Molecular Cloning, Tissue Distribution, and Evolutionary Considerations

Abstract

Matrix Gla protein (MGP) belongs to the family of vitamin K-dependent, Gla-containing proteins and in higher vertebrates, is found in the extracellular matrix of mineralized tissues and soft tissues. MGP synthesis is highly regulated at the transcription and posttranscription levels and is now known to be involved in the regulation of extracellular matrix calcification and maintenance of cartilage and soft tissue integrity during growth and development. However, its mode of action at the molecular level remains unknown. Because there is a large degree of conservation between amino acid sequences of shark and human MGP, the function of MGP probably has been conserved throughout evolution. Given the complexity of the mammalian system, the study of MGP in a lower vertebrate might be advantageous to relate the onset of MGP expression with specific events during development. Toward this goal, MGP was purified from Xenopus long bones and its N-terminal amino acid sequence was determined and used to clone the Xenopus MGP complementary DNA (cDNA) by a mixture of reverse-transcription (RT)-and 5-rapid amplification of cDNA ends (RACE)-polymerase chain reaction (PCR). MGP messenger RNA (mRNA) was present in all tissues analyzed although predominantly expressed in Xenopus bone and heart and its presence was detected early in development at the onset of chondrocranium development and long before the appearance of the first calcified structures and metamorphosis. These results show that in this system, as in mammals, MGP may be required to delay or prevent mineralization of cartilage and soft tissues during the early stages of development and indicate that Xenopus is an adequate model organism to further study MGP function during growth and development. (J Bone Miner Res 2001;16:1611-1621)

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

“…In contrast with mammals, two major histological types of bone can be found in teleost fish (cellular and acellular bone) [27,29]. Despite these structural differences, our results indicate that in fish, as in mammals [26], fish bone-like mineralized tissue is the major site of accumulation for BGP [14,15,17] , S. aurata, H. didactylus and D. rerio), as well as from X. laevis and B. taurus, showed clear differences. While X. laevis, H. didactylus and A. regius BGPs had a similar behavior and migrated more like the B. taurus protein, the S. aurata and D. rerio BGPs migrated as smaller entities (Fig.…”

“…Vertebra from Sparus aurata, Solea senegalensis and Prionace glauca, branchial arches from Halobatrachus didactylus and the entire skeleton of Xenopus laevis and Danio rerio were cleaned from adhering soft tissues and the Gla-containing proteins were extracted based on previously described procedures [14,15,17]. Briefly, the mineralized material was ground in a mortar with liquid nitrogen to less than 1 mm in diameter, Correspondence to: M. L. Cancela; E-mail: lcancela@ualg.pt extensively washed with 6 M guanidine HCl and water and dried with acetone.…”

“…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].…”

“…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].…”

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

“…However, the evolutionary conservation of many invariant residues identified from the alignment of all vertebrate Mgp sequences, suggests that they are required for maintenance of a correct protein structure and/or to preserve a critical function. Among the unique characteristics that distinguish Mgp from other vitamin Kdependent proteins is the lack of a pro-peptide, with the gammacarboxylase recognition sequence encoded inside the mature protein (Cancela et al, 2001;Price et al, 1987), demonstrating that gammacarboxylation and secretion of this VKD protein are not directly related to the proteolytic cleavage of a pro-peptide domain (Price et al, 1987). An ANxF putative proteolytic cleavage site (reviewed in Laizé et al, 2005) and three highly conserved serine phosphorylation sites near the N-terminus of all known Mgps (Laizé et al, 2005;Price et al, 1994;Simes et al, 2003) are some of the potential targets for post-translational events that may alter the activity and/or properties of Mgp.…”