Structure of human osteonectin based upon analysis of cDNA and genomic sequences

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Abstract
Overlapping human have bone osteonectin DNA were obtained by screening two independent human SaOS-2 .lambda.gt11 libraries using antibovine osteonectin monoclonal antibodies. One clone contains a 0.54-kb insert and the other a 1.9-kb insert. Insertion fragments from .lambda. clones were liberated by restriction digestion and subcloned into pUC19 for sequencing. Digestion of the 1.9-kb insert with EcoRI released 0.4- and 1.5-kb fragments. Sequencing analysis revealed that the 0.54- and 0.4-kb fragments are identical except for 150 nucleotides missing at the 5'' region of the 0.4-kb fragment. The composite nucleotide sequence of human osteonectin has a total length of 2091 nucleotides and is comprised of 50 nucleotides of 5''-noncoding sequence, a coding segment for 303 amino aicds, a termination codon, and 1114 nucleotides of 3''-noncoding sequence. The primary transcript codes for 286 amino acids of mature protein and a 17-residue amino-terminal hydrophobic signal peptide. Outstanding properties inferred from the primary structure are putative Ca2+ binding domains located in the glutamic acid rich NH2 terminus (residues 1-52) and two "EF"-hand domains in the C-terminal half of the protein (residues 165-176 and 257-286). The mature protein also contains a cysteine-rich, highly hydrophilic region homologous to the ovomucoid serine protease inhibitors (residues 76-132). Overlapping human genomic clones in .lambda.EMBL3 for osteonectin have been isolated and characterized. Intron/exon junction sequencing of the human osteonectin gene shown the presence of 10 exons and 9 introns. The mature protein is encoded by nine exons separated by eight introns. Exonic sequences are in complete agreement with that of cDNA from the SaOS-2 cell line. The position of introns is in good agreement with predicted protein domains. Comparison at both the nucleic acid and translated amino acid level demonstrates human bone osteonectin to be identical with human placental SPARC and very similar to bovine osteonectin and mouse SPARC. These findings clearly establish that the nascent translation product for bone osteonectin and SPARC from nonmineralized tissue are identical.