ECTS2014 Poster Presentations Other diseases of bone and mineral metabolism (44 abstracts)
1Centre of Marine Sciences (CCMAR), Faro, Portugal; 2European Board of Orthopedics and TraumatologyAlgarve Medical Centre, Faro, Portugal; 3VitaK, Maastricht University, Maastricht, The Netherlands; 4GenoGla Diagnostics, Faro, Portugal.
Gla-rich protein (GRP) is a vitamin K-dependent protein, characterized by a high density of γ-carboxylated Glu residues and high calcium binding affinity. It was shown to accumulate in mouse and sturgeon cartilage and in sites of skin and vascular calcification in humans. Four alternatively spliced transcripts of the GRP gene (GRPF1, F2, F3 and F4) were described in mouse chondrocytes and zebrafish. Osteoarthritis (OA) is a common degenerative joint disease, and reported to be associated with basic calcium phosphate crystals deposition either in the articular cartilage, synovial fluid or synovial membrane, which lead us to investigate the relation between calcium mineral deposition and GRP expression/accumulation during OA.
Comparative analysis of GRP patterning at transcriptional and translational levels was performed between controls and OA patients. We have first identified novel alternative splice variants in humans by RT-PCR and the respective protein isoforms are characterized by the loss of full γ-carboxylation and secretion functional motifs. These findings led us to produce an overexpressing human cell system to further understand the GRP secretory and γ-carboxylation potentials. Using newly developed and validated GRP conformation-specific antibodies we determined the differential accumulation pattern of human γ-carboxylated GRP (cGRP) and under-carboxylated GRP (ucGRP) in healthy and OA tissues by immunohistochemistry. Furthermore we used a cell-free in vitro assay to evaluate the calcium/phosphate (Ca/P) mineral-binding capacity of cGRP and ucGRP protein forms.
Our results show that GRPF1 appears to be the predominant splice variant expressed in mouse and human adult tissues, particularly in OA cartilage, while the overexpressed protein appears to be γ-carboxylated. Immunohistochemistry results using the conformational-specific antibodies show a preferred cGRP accumulation in controls, whereas ucGRP was the predominant form in OA-affected tissues, co-localizing at sites of ectopic calcifications in OA. Overall our results indicate an association of under-carboxylated GRP with OA.