Isolation and Partial Characterization of a Glycoprotein from Bovine Cortical Bone

Abstract

1.A fraction (G2) which contained 300/, of the total non-collagenous proteins was prepared from neutral EDTA extracts of bovine cortical bone. Three hitherto undescribed proteins (G2B-glycoprotein, GZC-glycoprotein, G~C F ) were identified in this fraction together with collagen, serum albumin, immunoglobulin G and transferrin.2 . The G2B-glycoprotein was prepared from the G2 fraction and shown to be homogenous by electrophoretic and immunochemical criteria.3. The G2B-glycoprotein was demonstrated to be present in bovine plasma at a concentration of approximately 20 mg/lOO ml. It was found that in the collagenase digest of decalcified bone matrix the ratio of the GZB-glycoprotein concentration to the serum albumin or immunoglobin G concentration was upto 250 times higher than the ratio in plasma.hexosamines and 14O/, neutral sugars, and had an apparent molecular weight of around 50000. 4.The G2B-glycoprotein contained approximately 7S0/, protein, 4O/, sialic acids, 3Analyses of mature cortical bone demonstrate that collagen constitutes OOo/, by weight of the organic matrix [l]. This has led to the frequent assumption that the early stages of calcification must occur in a highly collagenous matrix. It has been shown, however, that in the osteoid border where the first mineralized deposits are formed, non-collagenous protein constitute between 30°/, and 50°/, (w/w) of the total protein, and that during the course of mineralization the non-collagenous protein content decreases while the amount of collagen remains constant [2,3]. A similar decrease in the amount of protein polysaccharides during mineralization has been demonstrated [4].Plasma proteins [5,6], peptides [7], proteoglycans and glycoproteins [S -111 have been identified as constituents of the non-collagenous protein pool present in compact bone. The proteoglycans and acidic glycoproteins of bovine and rabbit bone have been isolated and characterized in some detail but the presence of considerable amounts of other glycoproteins has also been demonstrated [ll, 121. The present studies were directed toward the isolation and characterization of components from the major glycoprotein fraction prepared from the EDTA extract of bovine cortical bone. Abbreviation. IgG, immunoglobulin G.Enzyme. Collagenase (EC 3.4.4.19). MATERIALS AND METHODS Preparation of Bone FractionsAdult bovine cortical bone was powdered and extracted with sodium-EDTA solutions a t pH 7.5 [13] and the EDTA extract fractionated according to the method of Herring [S], represented schematically in Scheme 1. Recoveries of protein, hydroxyproline and sialic acid were determined a t each step.A sample (240 mg) of the G2 fraction was separated into several components by chromatography on a column (1 x I0 cm) of DEAE-cellulose (DE32, W & R. Balston, Kent, England) which had been equilibrated with 0.05M Tris-HC1 buffer pH 7.2. The column was eluted with this equilibration buffer (100 ml), followed by a linear salt gradient of 0 to 0.4 M NaCl in 0.05 M Tris-HC1 buffer pH 7.2 (total volume 200 ml). ...

Serum concentration of human alpha 2 HS glycoprotein during the inflammatory process: evidence that alpha 2 HS glycoprotein is a negative acute-phase reactant.

“…In our curreint work, these spontaneous fragments could be obtained by serum proteases, because trypsin in vitro produced alpha2 HS fragments of a size similar to those spontaneously obtained. Alpha2 HS fragility could be caused by the fact that it is the serum protein that contains the greatest amount of hydrophobic residues (27). The high amount of proline amino acids in alpha2 HS, which prevents formation of alpha helix, could cause an increased sensitivity to proteolytic enzymes.…”

“…As is well-and fetal bone, and is located in areas of mineralizaknown, kinins play a pharmacological role in the in-tion. Triffitt et al (25,26) have shown that a rabbit flammatory process (23 (27) was shown to be present in the similar physico-chemical properties, it was thought that collagenase digest of decalcified bone matrix at a much they fulfilled identical physiological functions (27,28 FIGuRE 5 Ouchterlony analysis with an anti-alpha2 HS immune serum of (1) These results, added to those indicating that alpha2 HS was precipitated by calcium phosphate complexes (28) of their correlation with the two first principal components (group I). It would be of interest to assess serum levels of these two proteins in other types of illnesses.…”

Mineralization of Decalcified Bone Occurs Under Cell Culture Conditions and Requires Bovine Serum But Not Cells

“…We speculate that nucleator(s) of bone matrix mineralization are secreted by osteoblasts in the BMU, and that due to the vascular nature of these structures, some of the nucleator unavoidably escapes to blood. The escape of the nucleator from the BMU to serum is supported by the appearance in serum of other proteins synthesized in the BMU [e.g., alkaline phosphatase and bone Gla protein (osteocalcin)] and by the massive accumulation of some serum proteins in the extracellular bone matrix (e.g., fetuin, also called a-2HS glycoprotein, which is one of the most abundant noncollagenous proteins in rat, human, and bovine bone [25][26][27]). …”

Mineralization by Inhibitor Exclusion

“…Because fetuin is the subject of this study, it is useful to review briefly its occurrence and calcification-inhibitory activity. Fetuin is a 48-kDa glycoprotein that is synthesized in the liver and is found at high concentrations in mammalian serum (15,16) and bone (17)(18)(19)(20)(21)(22). The serum fetuin concentration in adult mammals ranges from 0.5 to 1.5 mg/ml, whereas the serum fetuin concentration in the fetus and neonate is typically far higher (16).…”