The Isolation and Characterization of Glycosylated Phosphoproteins from Herring Fish Bones

Abstract

Past studies of bone extracellular matrix phosphoproteins such as osteopontin and bone sialoprotein have yielded important biological information regarding their role in calcification and the regulation of cellular activity. Most of these studies have been limited to proteins extracted from mammalian and avian vertebrates and nonvertebrates. The present work describes the isolation and purification of two major highly glycosylated and phosphorylated extracellular matrix proteins of 70 and 22 kDa from herring fish bones. The 70-kDa phosphoprotein has some characteristics of osteopontin with respect to amino acid composition and susceptibility to thrombin cleavage. Unlike osteopontin, however, it was found to contain high levels of sialic acid similar to bone sialoprotein. The 22-kDa protein has very different properties such as very high content of phosphoserine (ϳ270 Ser(P) residues/1000 amino acid residues), Ala, and Asx residues. The N-terminal amino acid sequence analysis of both the 70-kDa (NPIMA(M)ETTS(M)DSKVNPLL) and the 22-kDa (NQDMAMEASSDPEAA) fish phosphoproteins indicate that these unique amino acid sequences are unlike any published in protein databases. An enzyme-linked immunosorbent assay revealed that the 70-kDa phosphoprotein was present principally in bone and in calcified scales, whereas the 22-kDa phosphoprotein was detected only in bone. Immunohistological analysis revealed diffusely positive immunostaining for both the 70-and 22-kDa phosphoproteins throughout the matrix of the bone. Overall, this work adds additional support to the concept that the mechanism of biological calcification has common evolutionary and fundamental bases throughout vertebrate species.The noncollagenous phosphoproteins of bone extracellular matrix (ECM) 2 have been of major scientific interest for more than three decades and continue to be the subject of a number of studies. This is due to observations and experimental evidence that they are involved in a number of biological events such as regulation of biomineralization and cellular activity/ behavior in normally mineralizing tissues including bone (1-7), cartilage (8), and dentin (9). ECM phosphoproteins have been also found in the pathologically mineralizing tissues such as atherosclerotic plaque (10), kidney stones (11), dental calculus (12), and breast tumors (13). Two most abundant and well known are bone sialoprotein (BSP) and osteopontin (OPN), which are synthesized by osteoblasts during bone formation (1-7, 14, 15). In addition to their involvement in biomineralization and its regulation (6, 7, 16 -19), bone ECM phosphoproteins are implicated in modulating cellular function and the behavior of bone cells such as osteoblasts (20 -22), osteoclasts (23-27), and other cell types such as tumor and immune (28, 29) where they promote cell adhesion, motility, and transmembrane signaling. The involvement of both BSP and OPN in such biological functions has been predominantly linked to the presence of an integrin receptor-binding tripeptide Arg-Gly-Asp sequence in t...

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