The isolation and characterization of glycosylated phosphoproteins from herring fish bones.
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 (∼270 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.
Zhou
Hai-Yan
HY
Laboratory for the Study of Skeletal Disorders and Rehabilitation, Department of Orthopedic Surgery, Harvard Medical School and Children's Hospital Boston, Boston, Massachusetts 02115-5737, USA.
Salih
Erdjan
E
Glimcher
Melvin J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D001842:Bone and Bones / Q000378:metabolism*; D002241:Carbohydrates / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D029941:Fish Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D005399:Fishes / Q000378:metabolism*; D006031:Glycosylation; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry; D010710:Phosphates / Q000032:analysis; D010750:Phosphoproteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D020539:Sequence Analysis, Protein; D053719:Tandem Mass Spectrometry
D002241:Carbohydrates; D029941:Fish Proteins; D010446:Peptide Fragments; D010710:Phosphates; D010750:Phosphoproteins