GENETIC EXPRESSION OF NONCOLLAGENOUS BONE PROTEINS

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Osteonectin (ON) and bone gla protein (BGP) are proteins predominantly found in the extracellular matrix of bone and dentin where they are thought to play a role in mineralization. A third protein, originally identified as an Saos-2 antigen (SAOS-P80), is currently unknown structure and function and appears to be specific to osteoblastic cells. ON is the species homologue of mouse SPARC found extracellularly in parietal endoderm and bovine 43K protein first identified in endothelial cell cultures. The latter proteins are believed to be involved in cell migration and basement membrane extracellular matrix synthesis and modeling. Recently, it was discovered that human platelets contain a significant amount of ON and that it is releasable by thrombin activation. Platelet ON may play an important role in maintaining hemostasis and the vascular bed, and in bringing about repair following tissue injury and pathogenesis. The amino acid sequence for platelet ON will be determined from synthesis, cloning, and DNA sequencing of cDNAs encoding ON in human erythroleukemic (HEL) and normal megakaryocytic cells. Epitope mapping of antibodies to bone and platelet ONs will be performed with a lambdagt11 deletion/expression system. The 5' end of the human gene for ON will also be isolated and characterized as to genetic regulatory elements. The region upstream of the 5' transcriptional start site as well as portions of a large intron in the 5' non-translated region of the cDNA will be sequenced and put into a pSV2-CAT/mammalian cell expression system to evaluate regulatory elements of the gene. Saos- 2/lambdagt11 expression libraries will be screened with monoclonal antibodies for SAOS-P80 cDNAs. Positive phage will be characterized by enzyme mapping and DNA sequencing and the amino acid sequence thereby derived. SAOS-P80 cDNA will be used as a probe to isolate the human SAOS- P80 gene from a human genomic/lambdaEMBL3 library. The gene will be characterized in a manner similar to that proposed for ON and compared to those of other osteoblastic proteins including ON, BGP, alkaline phosphatase, and type I collagen. cDNAs from these studies will be used as probes for in situ hybridization and to study mRNA levels in a variety of cells undergoing osteoblastic differentiation and development or associated with bone and connective tissue disease.