Characterization of fetal porcine bone sialoproteins, secreted phosphoprotein I (SPPI, osteopontin), bone sialoprotein, and a 23-kDa glycoprotein. Demonstration that the 23-kDa glycoprotein is derived from the carboxyl terminus of SPPI

Demineralizing extracts of porcine bone contain two large 66-80-kDa sialoproteins and smaller 20- and 23-kDa glycoproteins with similar chemical properties. Each protein was characterized following extraction from fetal calvariae and purification under dissociative conditions using Sepharose CL-6B, followed by fast protein liquid chromatography fractionation on hydroxyapatite and Mono Q resins. Unlike the large sialoproteins, the 20- and 23-kDa glycoproteins did not contain sialic acid. Nevertheless, affinity-purified antibodies raised against the 23-kDa protein recognized both the 20-kDa protein and a 67-kDa sialoprotein on immunoblots. These antibodies also immunoprecipitated a 60-kDa [35S]methionine-labeled protein produced by cell-free synthesis of calvarial bone mRNA, indicating that the smaller proteins were derived from the 67-kDa protein. The two sialoproteins were shown by primary sequence analysis to be secreted phosphoprotein I (SPPI, osteopontin, bone sialoprotein I) and bone sialoprotein (BSP, bone sialoprotein II). The SPPI was also characterized by its susceptibility to thrombin which produced a 23-kDa fragment, similar to the glycoprotein isolated, and a 30-kDa fragment. Amino-terminal sequence analysis of the 23- and 20-kDa proteins revealed that these proteins were derived from the carboxyl-terminal half of the SPPI molecule, the proteins showing 58% identity with human and rat, and 50% identity with mouse, SPPI sequences. Both the 23- and 20-kDa proteins appeared to be generated by the activity of an endogenous trypsin-like protease that cleaves at Arg-Ser (residues 155-156) and Lys-Ala (residues 182-183) bonds. Radiolabeling studies performed in vitro showed that the 23-kDa fragment was detectable in mineralized tissue within 4 h. The fragment was phosphorylated but, unlike SPPI, was not sulfated. The rapid generation of the 23-kDa glycoprotein and its presence in different bone tissues at different developmental stages indicate that the fragmentation of SPPI is important in bone formation and remodeling.     

Osteoblast-related gene expression of bone marrow cells during the osteoblastic differentiation induced by type I collagen

Bone marrow contains multipotent cells that differentiate into fibroblasts, adipocytes, and osteoblasts. Recently we found that type I collagen matrix induced the osteoblastic differentiation of bone marrow cells. Three weeks after cells were cultured with type I collagen, they formed mineralized tissues. In this study, we investigated the expression of osteoblast-related genes (alkaline phosphatase, osteocalcin, bone sialoprotein, osteopontin, and cbfa-1) during the osteoblastic differentiation. The expression of alkaline phosphatase and osteopontin genes increased time-dependently during the osteoblastic differentiation. Osteocalcin and bone sialoprotein genes were expressed in cells that formed mineralized tissues, and both were expressed only after cells reached the mineralized tissue-formation stage. On the other hand, the cbfa-1 gene was expressed from the early differentiation stage. The Asp-Gly-Glu-Ala (DGEA) amino acid domain of type I collagen interacts with the alpha2beta1 integrin receptor on the cell membrane and mediates extracellular signals into cells. When the collagen-integrin interaction was interrupted by the addition of DGEA peptide to the culture, the expression of osteoblastic phenotypes of bone marrow cells was inhibited. These findings imply that the collagen-alpha2beta1 integrin interaction is an important signal for the osteoblastic differentiation of bone marrow cells.     

Purification of the rat hepatic alpha 2-macroglobulin receptor as an approximately 440-kDa single chain protein

alpha 2-Macroglobulin-trypsin complex (alpha 2M.T) and alpha 2M-methylamine bind in a Ca2+-dependent way to a 400- to 500-kDa receptor in rat and human liver membranes (Gliemann, J., Davidsen, O., and Moestrup, S. K. (1989) Biochim. Biophys. Acta 980, 326-332). Here we report the preparation of alpha 2M receptors from rat liver membranes solubilized in 3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulfonic acid (CHAPS) dihydrate and incubated with Sepharose-immobilized alpha 2M-methylamine. The receptor preparation eluted with EDTA (pH 6.0) contained a protein larger than the 360-kDa alpha 2M (nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and some minor contaminants. The reduced large protein was about 440 kDa using reduced laminin (heavy chain: 400 kDa) as a standard. About 10 micrograms of receptor protein was obtained from 100 mg of liver membranes. The receptor preparation immobilized on nitrocellulose sheets bound 125I-alpha 2M.T, and the binding activity co-eluted with the 440-kDa protein. 125I-Labeled rat alpha 1-inhibitor-3 (alpha 1I3), a 200-kDa analogue of the alpha 2M subunit which binds to the alpha 2M receptors, was cross-linked to the 440-kDa protein. The receptor preparation was iodinated, and the 125I-labeled 440-kDa protein was isolated. It showed Ca2+-dependent saturable binding to alpha 2M-methylamine. In conclusion, we have purified the major hepatic alpha 2M receptor as an approximately 440-kDa single chain protein.     

The primary structure of a cell-binding bone sialoprotein

We have determined the amino acid sequence of rat bone sialoprotein (BSP). The sequence deduced from a 1974-base pair cDNA encodes a protein of 320 residues, including a 16-residues long signal peptide. The mature BSP has a molecular mass of 33,600 and contains predominantly glutamic acid and glycine residues, which constitute 32% of all residues. The glutamic acid residues are typically distributed in clusters of up to 10 consecutive residues. The tissue distribution of BSP mRNA suggests that the protein may be a unique product of cells in bone tissue. BSP contains an Arg-Gly-Asp sequence, which presumably is responsible for its cell binding properties (Oldberg, A., Franzén, A., Heineg?rd, D., Pierschbacher, M., and Ruoslahti, E. (1988) J. Biol. Chem. 263, 19433-19436).     

骨唾液酸蛋白(BSP)的结构与功能

骨唾液酸蛋白 (Bonesialoprotein ,简称BSP)是细胞外基质中的一种糖蛋白 ,主要分布在矿化组织中 ,参与骨代谢 ,但近年研究发现BSP在癌细胞的骨转移中发挥作用并对血管生成有促进作用。本文对BSP的结构与功能及研究状况作一介绍。     

Posttranslational modifications to human bone sialoprotein determined by mass spectrometry.

Bone sialoprotein (BSP) is an acidic 301 amino acid protein expressed by osteoblasts and at a low level by hypertrophic chondrocytes. Its expression is highest during early stages of bone formation, and it is particularly abundant in the cells lining the surface of newly formed trabeculae. BSP contains numerous substituents which are anionic in nature and apparently essential for the function of the protein. Thus, the proposed role of BSP in hydroxyapatite nucleation and growth may depend on such modifying groups. The posttranslational modifications include several acidic oligosaccharides as well as phosphate and sulfate groups. This work combines matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry with selective enzyme treatment of BSP to provide new information on the precise distribution and structure of oligosaccharides, sulfate, and phosphate groups in BSP isolated from human bone. The results provide a high level of detail in the location of these modifying groups toward the end of providing a basis for further understanding the function of BSP in bone nucleation.     

Immunohistochemical localization of bone sialoprotein in foetal porcine bone tissues: comparisons with secreted phosphoprotein 1 (SPP-1, osteopontin) and SPARC (osteonectin)

Summary Bone sialoprotein (BSP) is a prominent component of bone tissues that is expressed by differentiated osteoblastic cells. Affinity-purified antibodies to BSP were prepared and used in combination with biotin-conjugated peroxidase-labeled second antibodies to demonstrate the distribution of this protein in sections of demineralized foetal porcine tibia and calvarial bone. Staining for BSP was observed in the matrix of mineralized bone and also in the mineralized cartilage and associated cells of the epiphysis, but was not observed in the hypertrophic zone nor in any of the soft tissues including the periosteum. In comparison, SPP-1 (osteopontin) and SPARC (osteonectin), which are also major proteins in porcine bone, were observed in the cartilage as well as in the mineralized bone matrix, In addition, SPARC was also present in soft connective tissues. Although SPP-1 distribution was more restricted than SPARC, hypertrophic chondrocytes, periosteal cells and some stromal cells in the bone marrow spaces were stained in addition to osteoblastic cells. The variations in the distribution and cellular expression of BSP, SPARC and SPP-1 in bone and mineralizing cartilage indicate these proteins perform different functions in the formation and remodelling of mineralized connective tissues.     

A rat osteogenic cell line (UMR 106-01) synthesizes a highly sulfated form of bone sialoprotein

The rat osteosarcoma cell line (UMR 106-01) synthesizes and secretes relatively large amounts of a sulfated glycoprotein into its culture medium (approximately 240 ng/10(6) cells/day). This glycoprotein was purified, and amino-terminal sequence analysis identified it as bone sialoprotein (BSP). [35S]Sulfate, [3H]glucosamine, and [3H]tyrosine were used as metabolic precursors to label the BSP. Sulfate esters were found on N- and O-linked oligosaccharides and on tyrosine residues, with about half of the total tyrosines in the BSP being sulfated. The proportion of 35S activity in tyrosine-O-sulfate (approximately 70%) was greater than that in N-linked (approximately 20%) and O-linked (approximately 10%) oligosaccharides. From the deduced amino acid sequence for rat BSP (Oldberg, A., Franzén, A., and Heineg?rd, D. (1988) J. Biol. Chem. 263, 19430-19432), the results indicate that on average approximately 12 tyrosine residues, approximately 3 N-linked, and approximately 2 O-linked oligosaccharides are sulfated/molecule. The carboxyl-terminal quarter of the BSP probably contains most, if not all, of the sulfated tyrosine residues because this region of the polypeptide contains the necessary requirements for tyrosine sulfation. Oligosaccharide analyses indicated that for every N-linked oligosaccharide on the BSP, there are also approximately 2 hexa-, approximately 5 tetra-, and approximately 2 trisaccharides O-linked to serine and threonine residues. On average, the BSP synthesized by UMR 106-01 cells would contain a total of approximately 3 N-linked and approximately 25 of the above O-linked oligosaccharides. This large number of oligosaccharides is in agreement with the known carbohydrate content (approximately 50%) of the BSP.     

A histomorphometric, structural, and immunocytochemical study of the effects of diet-induced hypocalcemia on bone in growing rats.

Despite several studies on the effect of calcium deficiency on bone status, there is relatively little information on the ensuing histological alterations. To investigate bone changes during chronic hypocalcemia, weanling rats were kept on a calcium-free diet and deionized water for 28 days while control animals were fed normal chow. The epiphyseal-metaphyseal region of the tibiae were processed for histomorphometric, histochemical, and structural analyses. The distribution of bone sialoprotein (BSP), osteocalcin (OC), and osteopontin (OPN), three noncollagenous bone matrix proteins implicated in cell-matrix interactions and regulation of mineral deposition, was examined using postembedding colloidal gold immunocytochemistry. The experimental regimen resulted in serum calcium levels almost half those of control rats. Trabecular bone volume showed no change but osteoid exhibited a significant increase in all its variables. There were a multitude of mineralization foci in the widened osteoid seam, and intact matrix vesicles were observed in the forming bone. Many of the osteoblasts apposed to osteoid were tartrate-resistant acid phosphatase (TRAP)- and alkaline phosphatase-positive, whereas controls showed few such TRAP-reactive cells. Osteoclasts in hypocalcemic rats generally exhibited poorly developed ruffled borders and were inconsistently apposed to bony surfaces showing a lamina limitans. Sometimes osteoclasts were in contact with osteoid, suggesting that they may resorb uncalcified matrix. Cement lines at the bone-calcified cartilage interface in some cases were thickened but generally did not appear affected at bone-bone interfaces. As in controls, electron-dense portions of the mineralized matrix showed labeling for BSP, OC, and OPN but, in contrast, there was an abundance of immunoreactive mineralization foci in osteoid of hypocalcemic rats. These data suggest that chronic hypocalcemia affects both bone formation and resorption.     

An immunohistochemical study on hard tissue formation in a subcutaneously transplanted rat molar

While dental pulp undergoes calcification following tooth replantation or transplantation, we actually know little about these mechanisms. We therefore conducted histological and immunohistochemical evaluations of mineralized tissue that formed in the pulp of rat maxillary molar transplanted into abdominal subcutaneous tissue. One, 2, 3, and 4 weeks post-transplantation, the teeth were investigated immunohistochemically using antibodies to osteocalcin (OCN), osteopontin (OPN), bone sialoprotein (BSP), dentin sialoprotein (DSP), and tissue non-specific alkaline phosphatase (TNAP). In the 1st week after transplantation, cell-rich hard tissue was formed at the root apex. At 2 weeks, formations of hard tissue, with few cells in the root canals and bone-like tissue in the coronal pulp chamber, were noted. After 3 and 4 weeks, the amounts of these hard tissues were increased. The immunolocalization of OCN, OPN, and BSP was seen strongly in coronal and apical hard tissues, but weakly in the root hard tissue. Conversely, DSP localized in the root hard tissue, but not in other newly formed hard tissues. At 1 week, TNAP localized along the periphery of the apical hard tissue and the lower surfaces of root predentin. These results demonstrate that the newly formed hard tissues in the pulp cavity of subcutaneously transplanted molars could be classified into three types, suggesting that these might be formed by type-specific cells.