Structure and properties of protein of an osteopontin

Recently the increasing attention of explorers is involved with the squirrels-cytokines involved in processes of a bone formation and a dentogenesis, and also reconstruction of a denta and an osteal tissue. Molecular mechanisms of action of the given proteins, regulation of an expression of their genes, allocation in various types of tissues are studied, materials - inhibitors or, on the contrary, activators of their biological potency etc. are found.

Cause of such steadfast attention is not only necessity of reception of fundamental knowledge of structural bases of their biological potency, but also a role of growth factors for development of variety of diseases of the human. Adhesive squirrels and squirrels-CYTOKINES of an osteal tissue are involved in a pathogenesis of an osteoporosis, an osteoperosis, development of primary and secondary osteosarcomas of a various parentage, and also correcting of mechanical damages of a bone and a denta, etc.

One of the most widespread pathologies of an osteal tissue is the osteoporosis - the serious disease, leading demineralizations and bone destructions. Only in the USA for today it suffer more than 10 million humans that predetermines requirement for an effective medical product for therapy of an osteoporosis and prevention of the mechanical damages of a bone bound to it.

Within last several years the method of treatment of an osteoporosis by means of moderate doses of preparations of a parathyroid hormone has been developed and successfully tested. As have shown clinical tests, the hormonetherapy accelerates formation of a new osteal tissue, and also enlarges its density. At the same time, the researches carried out on modelling animals have allowed to establish, that a bone formation - activating action of a parathyroid hormone in many respects is caused by activity of an osteopontin (OP) - one of adhesive proteins of the osteal matrix involved in process of a resorption of a bone through simultaneous interaction with an osteal surface and osteoclasts.

The osteal tissue and denta of animals and the human contains a number of phosphorylated sialoproteins. Them concern: BSP (an osteal sialoprotein), BAG-75 (acidic osteal glikoprotein-75), DMP1 (a protein of a matrix of a denta-1), DSP (the denta sialoprotein), OP, etc. has been open and studied by the First of them a fragment of an osteal sialoprotein (BSP). Later, after introduction of an inhibitor of proteinases in an extract of a tissue of a bone, properties of full-size protein have been described. The subsequent cloning and nucleotide sequence definition cDNA BSP has allowed to define translation on the found open reading frame full amino-acid sequence. Use of the working methods fulfilled at studying BSP with an osteal tissue has given further possibility to secure and characterise the big number of the osteal proteins possessing adhesive and cytokinetic properties. One of such proteins also is the osteopontin - OP.

Osteopontin of the human has been secured and identified for the first time in 1985 After has been cloned cDNA OP mice and its nucleotide sequence, amino-acid sequence of protein, and subsequently and the organisation of a gene full an exon-intronnaja are defined. Full-size gene OP of the human cloned, using as hybridization probes labelled fragments cDNA OP mice. Comparison of the structural and functional organisation of two genes has taped a number of the general properties. In particular, both genes include seven exons in the structure, and their regulatory ranges contain a number of similar Consensus sequences. At the same time, the third intron of gene OP of the human contains an insert in length of 1,8 thousand pairs the establishments. The further studying of regulation of an expression of genes OP of the mouse and the human, no less than a path of their splicing mRNA in different lines of cells and types of tissues will allow to define further functional value of features of the organisation of genes OP from different sources.

OP - the secretory sialoprotein, which pro-peptide is formed by 314 amino-acid residual from which on a lobe of in the lead sequence it is necessary 16 and. Island OP has appeared a little less acidic, than BSP, but its amino-acid sequence as it has been established includes some blocks from the residual dicarboxylic amino acids, and one total length nine residual (see the drawing). Protein O-glikozilirovan also contains a number of the phosphorylated residual of a serine. Comparison of amino-acid sequence OP with primary structures of other osteal proteins has not allowed to find extensive fields of appreciable homology. At the same time, amino-acid sequence OP contains a Consensus RGD-field in the structure. The given structure answers in a number of proteins - BSP, etc., for interaction with being on a surface of cells receptors of family of integrins, and for the first time has been established in responsible for linkage of cells the domain of protein of a fibronectin. It is interesting to notice also, that as it was revealed in the course of abjection, OP is capable to contact a hydroxuapatite strongly enough. It can speak its functions in a mineralised osteal tissue.

It is necessary to underline, that though for the last years a number of proteins of an osteal matrix has been secured and characterised, now there are only assumptions of a possible functional role of some of them. Any of these proteins is not unique from the point of view of localisation in an osteal tissue. All of them also are present and at other types of tissues, thus OP is a little more circumscribed in localisation in comparison with the others to squirrels. So, level mRNA OP, high in an osteal tissue, also essential and in nephroses. The given protein as it has been shown, often is a part of renal stones and, very possibly, influences their formation., In particular, presence OP at calcium-phosphatic renal stones has been shown. Communication between the low maintenance of the given protein in an urine of patients and formation of stones of the oxalic nature is besides, found. Appreciable level of accumulation OP has been found in a placenta and brain tissues. Gene OP expresses also in many cellular lines where finds various types of an expression. Constitutive expression OP is found in cells from an osteal tissue, nephroses of a placenta, the excitatory cells, macrophages. At the same time inducible the type was observed in T-limfotsitah, false skin cells. Expression intensifying descends under the influence of various agents: 1,25 dihydroxyvitamin D3, a major factor of growth of fibroblasts (bFGF), the factor of a necrosis of a tumour (TNF), interlejkina-1 (IL-1), lipopolysaccharides, g-interferon (g-IFN). Besides it, the expression of gene OP descends at a neoplastic condition of a cell. Thus, regulation of an expression of gene OP is under the complex monitoring system which can differ at cells of different types.

In particular, for the purpose of studying of role OP in processes bone formation, mRNA initial research of influence on level of accumulation OP has been carried out osteoblasts from an osteosarcoma (line ROS 17/2.8) the various factors influencing regulation of growth and restoration of a bone. It has been found, that 1,25-digidroksivitamin-D3 several times raises both accumulation level mRNA, and secretion of mature protein from cells. These data show, that synthesis OP is positively regulated by vitamin D3, i.e. the factor inducing mobilisation of calcium from a bone. According to authors of the monography, it, most likely, is bound to an induction of activity of osteoclasts. It is interesting, that on literary data a gene of other osteal sialoprotein - BSP, it is subject to negative regulation by vitamin D3, but it is positively regulated by glucocorticoids.

Whereas protein incorporates Consensus RGD-sequence, and also has ability to contact a hydroxuapatite, it was possible to assume, that OP is involved in linkage of osteoclasts on a mineralised surface of a bone. Really, later linkage of osteoclasts with OP, put on a surface from glass and plastic has been shown. In a number of the subsequent experiments also it was revealed, that linkage of the isolated osteoclasts with OP can be inhibited by synthetic peptide RGD, but not control RGE, and also monoclones to a fragment of the amino-acid sequence OP containing a RGD-Consensus site. The functional role of RGD-structure has been in addition confirmed by building by site-directed mutagenesis of forms OP of the human with amino-acid changings. The obtained data have induced some explorers to try to define a type and a subunit composition involved in interaction with OP an integrin of osteoclasts. The carried out researches have shown, that only antibodies to an avb3-integrin inhibited linkage of cells with OP. These data show, that linkage of the isolated osteoclasts with OP, apparently, is mediated avb3-integrinom. The given type of integrins is a receptor of a vitronectin and as it has been earlier shown, is localised on a membrane the colossal cells of the human isolated an osteoclast-like. Probably, that the microenvironment on a surface of osteoclasts can induce specific conformstion of an avb3-integrin which allows it to be bound only with OP, i.e. specificity of cellular linkage can be highly selective.

It is interesting, that in proximal range OP behind RGD-sequence there is a site of scission by Thrombinum.

As have shown the researches carried out for the last years, descending in vivo scission OP by Thrombinum has essential physiological value.

In particular, the obtained data have allowed to show convincingly, that unlike native OP, its N-terminal fragment which contains the RGD-domain, sustains adhesion of lines of cells of a melanoma. It assumes, that some adhesive properties OP is supervised through scission by Thrombinum. Also it was revealed, that OP contains the latent sites of linkage which can co-operate with distinct from avb3 types of receptors. Subsequently new receptors integrin a number (avb1, avb5, a4b1, a5b1, a8b1, a9b1) and other kinds (CD44v6, CD44v7), responsible interactions OP with a surface of different types of cells have been identified.