Molecular cloning and sequence analysis of cDNA for a 59 kD bone sialoprotein of the rat: demonstration that it is a counterpart of human alpha 2-HS glycoprotein and bovine fetuin.

A complementary DNA (cDNA) for the 59 kD bone sialoprotein, which is supposed to be the rat counterpart of human alpha 2-HS glycoprotein (alpha 2-HSG) and is synthesized by both hepatocytes and osteoblasts, has been cloned from a rat liver cDNA library. Polyclonal rabbit antibodies to rat 59 kD bone sialoprotein were used to identify and isolate the cDNA. The amino acid sequence of 59 kD bone sialoprotein deduced from the cDNA revealed that the entire protein consisted of 352 amino acid residues, including a signal peptide of 18 amino acid residues, and contained three possible N-glycosylation sites. On Northern blot analysis of rat liver, an mRNA of about 1.5 kilobases was detected. An mRNA of 59 kD bone sialoprotein was also detectable in rat bone but not in other tissues, such as kidney, brain, and lung. A computer search of protein and nucleic acid data bases revealed that 68.2, 63.2, and 97.4% amino acid residues of 59 kD bone sialoprotein were identical with those of human alpha 2-HSG, bovine fetuin, and rat phosphorylated N-glycoprotein (pp63), respectively. The positions of cysteine residues in 59 kD bone sialoprotein also completely matched those in human alpha 2-HSG and bovine fetuin, indicating that the sialoprotein is the rat counterpart of human alpha 2-HSG and bovine fetuin. In addition, comparison of the nucleotide sequence of cDNA for rat fetuin/alpha 2-HSG with that for pp63 recently corrected showed only two differences in nucleotides in the entire protein coding regions of the two proteins, and immunoreactive rat fetuin/alpha 2-HSG in the conditioned medium of adult rat hepatocytes in primary culture was found to be phosphorylated. Thus, because rat fetuin/alpha 2-HSG isolated from bone and synthesized by osteoblasts in culture does not contain phosphorus, it seems to be pp63 dephosphorylated during circulation or in the bone matrix.