Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
1999-11-18
2002-09-17
Nashed, Nashaat T. (Department: 1652)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C004S111100, C004S218000, C004S219000
Reexamination Certificate
active
06451548
ABSTRACT:
The invention relates to the use of activated recombinant TRAP (tartrate-resistant and purple acid phosphatases) for screening for specific inhibitor of TRAP activity useful in the treatment of diseases or degenerative conditions resulting in increased bone resorption, such as tissue damages, bone metabolic disorders, osteoporosis.
TRAP can be activated by proteolytic activation of TRAP e.g. by cleaving with a protease, papain-like enzyme.
BACKGROUND
Normal bone function requires a turnover of bone. Bone is constantly being rebuilt by cycles of resorption and formation which means that formation is closely linked to resorption (a phenomenon referred to as coupling).
TRAP is an enzyme expressed predominantly in bone resorbing cells (osteoclasts). Investigations in TRAP knockout mice show that the resorption process is disrupted so that, with increasing age, TRAP knockout mice become osteopetrotic, i.e. have an increased bone mineral content and more dense bone is formed. Osteoclasts prepared from these animals are functional and do resorb bone but to a lesser extent than wild type mouse osteoclasts.
Phosphatases are enzymes that remove organic phosphates from proteins. The mammalian Purple Acid Phosphatases (PAPs), a group of enzymes to which Tartrate Resistant and purple Acid Phosphatase (TRAP) belongs, are characterized by a binuclear iron center at the active site. Purple acid phosphatases (PAPs) are acid metallohydrolases that contain a binuclear Fe
3+
M
2+
center in their active site, where M=Fe or Zn [1-3]. In mammals, these enzymes are also referred to as tartrate-resistant acid phosphatases (TRAPs) (EC 3.1.3.2) or type 5 acid phosphatases [4]. TRAPs are iron-containing, monomeric glycoproteins with molecular weights of around 35,000 Da [5]. The deduced amino acid sequences of human, rat and mouse TRAPs show a high degree of homology with the mammalian members of the PAP family, e.g uteroferrin (Uf) and bovine spleen PAP [6-9]. Recently, EPR spectroscopic analysis of rat recombinant TRAP [10] has provided compelling evidence that this enzyme also belongs to the PAP family.
Mammalian PAPs contain a FeFe center, while a plant PAP from red kidney beans (KBPAP) instead has a FeZn center [11]. Moreover, the mammalian protein phosphatases calcineurin (type 2B) [12] and protein phosphatase type 1 (PP-1) [13] both contain a di-nuclear metal centre and also reveal a striking similarity to the plant PAP enzyme in the coordination environment of the active site, except for the absence of the tyrosine ligand. PP-1 and calcineurin are serine/threonine protein phosphatases, suggesting that also PAPs may function as protein phosphatases. It has been shown that PAP enzymes exhibit a rather broad specificity as these enzymes can dephosphorylate both serine- and tyrosine-bound phosphate moieties in phosphoproteins [10, 14-19].
The binuclear iron center, low pH optimum (≈5), high isoelectric point (≈9) and insensitivity to inhibition by L(+) tartrate are features of TRAP that may be involved in the apparent substrate specificity at the low pH in the osteoclastic resorption area. The TRAP enzyme is a cationic glycoprotein with a molecular mass of 35 kD and a monomeric 325 amino acid peptide structure. The peptide sequence of rat bone TRAP displays 89-94% homology to TRAP enzyme of the human placenta, bovine spleen, and uteroferrin. TRAP hydrolyzes aryl phosphates, nucleoside di- and triphosphates, pyrophosphate and phosphoproteins. Its physiological role remains unclear but TRAP may mediate dephosphorylation of bone matrix proteins such as osteopontin and bone sialoprotein.
Dephosphorylation of bone matrix proteins enables osteoclasts to migrate over the bone surface and TRAP is therefore likely to be involved in the attachment of osteoclasts to the bone surface.
In humans and rats, PAP enzymes are highly expressed in certain cells of the monocyte-macrophage lineage, such as the bone-resorbing osteoclasts and certain activated macrophages in spleen, liver and lung [20-23], and TRAP has since long been used as a histochemical marker for these cells. Given the broad substrate specificity of PAP enzymes, it is conceivable that other factors, such as local availability and proper compartmentalisation of PAPs with their potential substrates, are other important factors in determining the physiological action of PAPs in biological systems.
The cDNA sequences of TRAP/PAP enzymes from different species and organs all indicate that these enzymes are translated as a single polypeptide of around 35 kDa [7-9, 24]. This contrasts with the predominantly two subunit structure, consisting of a 20-23 kDa N-terminal domain linked through a disulphide bond to a 15-17 kDa C-terminal domain, observed in purified enzyme preparations from a variety of sources including human and rat bone [25, 26], giant cell tumors [27] and normal and pathological spleen [28-30]. In contrast, uteroferrin purified from endometrial secretions are mostly in the single subunit form [28, 31] as are the recombinant PAPs generated by overexpression using the Baculovirus system [10, 17, 32]. Orlando et al [29] managed to separate the monomeric and two-subunit variants of PAP from bovine spleen, and demonstrated a markedly higher specific enzyme activity associated with the two subunit form. Moreover, digestion of the single subunit form with the serine proteases trypsin or chymotrypsin generated the 23 kIDa and 15 kDa disulfide-linked fragments characteristic of the two subunit form together with a significant enhancement of enzyme activity. Similar nicking and activation of the non-cleaved purified recombinant human and mouse PAPs were noted upon prolonged storage [17].
Inhibitors of TRAP are known, such as PGE2 [Quinn et al Calcif. Tissue Int (1997), 60 (1) 63-70], which has an influence on the formation of osteoclasts and thus reduce the amount of TRAP, hemin (ferric protoporphyrin) [Reddy et al, Blood (1996), 88 (6) 2288-2297], which regulates the TRAP on a gene level i.e. a lowering of the expression of TRAP and calcitonin which inhibits the release of TRAP. Calcitonin, has an effect against osteoclasts and is used as medicament against osteoporosis.
These known inhibitors are not specific (no direct inhibitor) and synthetic inhibitors for the protein and the enzyme activity can therefore not be used in vivo.
Current drugs on the market for treatment of osteoporosis turn off bone resorption. We now have found a possibility to modulate and lower the bone resorption rate, but not to turn it off completely.
THE INVENTION
The overall goal of this invention is to develop drugs for the treatment of osteoporosis. We have compared certain structural and enzyme kinetic properties of recombinant rat TRAP (single-subunit) with those of the native TRAP/TRAP enzyme (two-sub-unit), and examined the effects of cleaving the monomeric recombinant TRAP with the serine proteinase trypsin or the cysteine proteinase papain. Cysteine proteinases were chosen because enzymes belonging to this family appear to serve important roles in resorptive and degradative processes in cells of the monocyte-macrophage lineage [33-35].
The results show that the monomeric form of TRAP represents a latent proenzyme with low enzymatic activity towards both tyrosine- and serine-containing phosphosubstrates. We have found that members of the cystein proteinase family play an important role in degradative processes involving the TRAP enzymes by converting the TRAPs to enzymatically active and micro environmentally regulated species.
To our surprise, we found that cleavage with special proteases, such as papain, but not trypsin, significantly activate the enzyme and confer similar properties with regard to enzymatic parameters such as pH-dependence and sensitivity to reducing agents, as well as in size of the subunits and the site of initial
Andersson Göran
Ek-Rylander Barbro
Oellig Cornelia
Biovitrum AB
Fronda Christian L.
Nashed Nashaat T.
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