Characterization of a human angiotensinogen cleaved in its reactive center loop by a proteolytic activity from Chinese hamster ovary cells

Angiotensinogen, the renin (E.C. 3.4.23.15) substrate, belongs to the serpins superfamily and has been classified as a noninhibitory serpin. Using mass spectroscopy, angiotensinogen purified from Chinese hamster ovary cell supernatant shows a broad spectrum. The absence of protease inhibitors throughout the purification leads to an angiotensinogen cleaved within the reactive center loop. This cleavage does not affect the Ang I generation because kinetic parameters are similar to the values of the full-length angiotensinogen. Although cleavage is complete, the cleaved angiotensinogen migrates after deglycosylation on SDS-polyacrylamide gel electrophoresis as a doublet differing by 4 kDa. To test whether the circulating angiotensinogen is cleaved in the reactive center loop, it was purified from a pool of human plasma and was shown to be uncleaved. Its migration was obviously slower than of cleaved angiotensinogen but also consisted of two bands pointing to a so far unexplained residual heterogeneity. We then compared the heat-induced polymerization of full-length- and reactive center loop-cleaved angiotensinogens. Both monomers were able to aggregate, revealing a particular behavior of angiotensinogen distinct from that of reactive center loop-cleaved serpins. Lacking the three-dimensional structure of angiotensinogen, we propose and discuss a structural model of the serpin fold within the renin substrate.     

Sufficiency of the reactive site loop of maspin for induction of cell-matrix adhesion and inhibition of cell invasion. Conversion of ovalbumin to a maspin-like molecule

Maspin, an ov-serpin, inhibits tumor invasion and induces cell adhesion to extracellular matrix molecules. Here, we use maspin/ovalbumin chimeric proteins and the maspin reactive site loop (RSL) peptide to characterize the role of the RSL in maspin-mediated functions. Replacement of the RSL plus the C-terminal region or the RSL alone of maspin with that of ovalbumin resulted in the loss of the stimulatory effect on adhesion of corneal stromal cells to type I collagen, fibronectin, and laminin and of mammary carcinoma MDA-MB-231 cells to fibronectin. Maspin with ovalbumin as the C-terminal region retained activity, suggesting the maspin C-terminal polypeptide is not required. An R340Q mutant retained full maspin activity; however, an R340A mutant lost activity. This indicates the arginine side chain at the putative P1 site forms a hydrogen bond and not an ionic bond. The RSL peptide (P10-P5', amino acids 330-345) alone induced cell-matrix adhesion of mammary carcinoma cells and corneal stromal cells and inhibited invasion of the carcinoma cells. Substitution of the RSL of ovalbumin with that of maspin converted inactive ovalbumin into a fully active molecule. Maspin bound specifically to the surface of the mammary carcinoma cells with a kd of 367 +/- 67 nM and 32.0 +/- 2.2 x 10(6) binding sites/cell. The maspin RSL peptide inhibited binding, suggesting the RSL is involved in maspin binding to cells. Sufficiency of the maspin RSL for activity suggests the mechanism by which maspin regulates cell-matrix adhesion and tumor cell invasion does not involve the serpin mechanism of protease inhibition.     

S-ovalbumin, an ovalbumin conformer with properties analogous to those of loop-inserted serpins.

Most serpins are inhibitors of serine proteinases and are thought to undergo a conformational change upon complex formation with proteinase that involves partial insertion of the reactive center loop into a beta-sheet of the inhibitor. Ovalbumin, although a serpin, is not an inhibitor of serine proteinases. It has been proposed that this deficiency arises from the presence of a charged residue, arginine, at a critical point (P14) in the reactive center region, which prevents loop insertion into the beta-sheet and thereby precludes inhibitory properties. To test whether loop insertion is prevented in ovalbumin we have examined the properties of two forms of ovalbumin: the native protein and S-ovalbumin, a form that forms spontaneously from native ovalbumin and has increased stability. Calorimetric measurements showed that S-ovalbumin was more stable than ovalbumin by about 3 kcal mol-1. CD spectra, which indicated that S-ovalbumin had less alpha-helix than native ovalbumin, and 1H NMR spectra, which indicated very similar overall structures, suggest limited conformational differences between the two forms. From comparison of the susceptibility of the reactive center region of each protein to proteolysis by porcine pancreatic elastase and by subtilisin Carlsberg, we concluded that the limited native-to-S conformational change specifically affected the reactive center region. These data are consistent with a structure for S-ovalbumin in which part of the reactive center loop has inserted into beta-sheet A to give a more stable structure, analogously to other serpins. However, the rate of loop insertion appears to be very much lower than for inhibitory serpins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Absence of large-scale conformational change upon limited proteolysis of ovalbumin, the prototypic serpin

1H and 31P NMR spectroscopies have been used to examine the effects of limited proteolysis with subtilisin Carlsberg on the global conformation of ovalbumin and on the local environment of phosphoserine 344, a residue two positions removed from the site of proteolysis. Such limited proteolysis has been shown to result in excision of a hexapeptide from the region of the protein that, in other serine protease inhibitors (serpins), contains the reactive center. Based on the structure of the related serpin alpha 1-antitrypsin, it has been predicted that phosphoserine 344 should undergo a large change in environment upon proteolysis of ovalbumin (L?bermann, H., Tokuoka, R., Deisenhofer, J., and Huber, R. (1984) J. Mol. Biol. 177, 531-550). Proteolysis of ovalbumin produces a small upfield shift (0.15 ppm) of the 31P resonance of phosphoserine 344. In addition, the pKa of phosphoserine 344 is raised by 0.1 pH unit. At pH 8.5, phosphoserine 344 in cleaved ovalbumin (plakalbumin) is as accessible to hydrolysis by Escherichia coli alkaline phosphatase as it is in native ovalbumin. 1H NMR shows that dephosphorylation of serine 344 has an imperceptible effect on the protein's conformation. Similarly, little effect on conformation is seen by 1H NMR upon proteolysis of ovalbumin. These findings suggest that ovalbumin does not undergo a marked conformational change analogous to that inferred for the related members of the serpin superfamily, alpha 1-antitrypsin and antithrombin III, nor do the residues close to the site of proteolysis appear to change environment from that of an exposed loop to a buried strand of beta-sheet. These findings are not consistent with the hypothesis of Carrell and Owen ((1985) Nature 317, 730-732) for the role of the exposed loop in serpins of directly facilitating conformational change upon cleavage of the loop. Instead, it is proposed that cleavage of the exposed loop alters the solvent accessibility of residues formerly covered by the loop and that this provides the thermodynamic impetus for conformational change, perhaps by disruption of a salt bridge crucial to the integrity of the native structure.     

DNA methylation: correlation with DNase I sensitivity of chicken ovalbumin and conalbumin chromatin.

To analyse the relationship between DNA undermethylation at some sites in the ovalbumin and conalbumin gene regions (1) and the expression of these genes in chick oviduct, digestions with HhaI, which differentiates between methylated and unmethylated HhaI restriction sites, was performed on DNA isolated from chicken erythrocyte or oviduct chromatin treated with DNase I which degrades preferentially "active" chromatin. This was followed by analysis with ovalbumin- and conalbumin-specific hybridization probes. We conclude that the residual DNA methylation found at some sites of the ovalbumin and conalbumin gene regions is derived from the fraction of cells in which the chromatin of these genes is not in an "active" form. On the other hand, the ovalbumin and conalbumin sites which are partially unmethylated in erythrocyte DNA correspond to chromatin regions which are not DNase I-senitive. We have also detected a site about 1 kb downstream from the 3' end of the conalbumin gene that is hypersensitive to DNase I in all tissues tested.     

Efficient liver-specific deletion of a floxed human angiotensinogen transgene by adenoviral delivery of Cre recombinase in vivo.

Tissue-specific ablation of gene function is possible in vivo by the Cre-loxP recombinase system. We generated transgenic mice containing a human angiotensinogen gene flanked by loxP sites (hAGT(flox)). To examine the physiologic consequences of tissue-specific loss of angiotensinogen gene function in vivo, we constructed an adenovirus expressing Cre recombinase. Studies were performed in several independent lines of hAGT(flox) mice before and after intravenous administration of either Adcre or AdbetaGal as a control. Systemic administration of Adcre caused a significant decrease in circulating human angiotensinogen and markedly blunted the pressor response to administration of purified recombinant human renin. Southern blot analysis of genomic DNA from various organs revealed that the Cre-mediated deletion was liver-specific. Further analysis revealed the absence of full-length human angiotensinogen mRNA and protein in the liver but not the kidney of Adcre mice, consistent with the liver being the target for adenoviruses administered intravenously. These studies demonstrate that extra-hepatic sources of angiotensinogen do not contribute significantly to the circulating pool of angiotensinogen and provide proof-of-principle that the Cre-loxP system can be used effectively to examine the contribution of the systemic and tissue renin-angiotensin system to normal and pathological regulation of blood pressure.     

Location of two of the introns in the antithrombin-III gene

At least two of the introns in the antithrombin-III (AT-III) gene are located in positions different from those of the other three proteins in this superfamily for which the gene structures are known, namely, ovalbumin, alpha 1-antitrypsin and angiotensinogen. In another part of the 3'-portion of the AT-III gene there is no intron where each of the other three gene structures has one.     

Chicken Ovalbumin Promoter Is Demethylated upon Expression in the Regions Specifically Involved in Estrogen-Responsiveness

Here we report the methylation status of the chicken ovalbumin promoter. Genomic DNA of oviduct from immature chickens and laying hens was analyzed through bisulfite genomic sequencing. In the ovalbumin control locus up to the 6 kb upstream region, CpG sites were methylated in immature chickens, except for several sites, and almost all CpGs residing in DNase I hypersensitive sites I, II, and III, but not IV, were selectively unmethylated in ovalbumin expressing chickens. Chromatin immunoprecipitation assays showed that the ovalbumin control region was associated with acetylated histone H3 but not with dimethylated histone H3 at Lys 27. These results demonstrate that DNA demethylation was restricted to short DNA regions of DNase I hypersensitive sites, especially to those which participated in estrogen-responsiveness, even when cells expressed extremely high levels of ovalbumin and these sites were associated with acetylated histones.     

Loop-inserted and thermostabilized structure of P1-P1' cleaved ovalbumin mutant R339T.

Ovalbumin is a member of a superfamily of serine proteinase inhibitors, known as the serpins. It is, however, non-inhibitory towards serine proteinases, and lacks the loop insertion mechanism common to the serpins due to unknown structural factors. Mutant ovalbumin, R339T, in which the P14 hinge residue is replaced, was produced and analyzed for its thermostability and three-dimensional structure. Differential scanning calorimetry revealed that the mutant ovalbumin, but not the wild-type protein, undergoes a marked thermostabilization (DeltaT(m)=15.8 degrees C) following the P1-P1' cleavage. Furthermore, the crystal structure, solved at 2.3 A resolution, clearly proved that the P1-P1' cleaved form assumes the fully loop-inserted conformation as seen in serpin that possess inhibitory activity. We therefore conclude that ovalbumin acquires the structural transition mechanism into the loop-inserted, thermostabilized form by the single hinge mutation. The mutant protein does not, however, possess inhibitory activity. The solved structure displays the occurrence of specific interactions that may prevent the smooth motion, relative to sheet A, of helices E and F and of the loop that follows helix F. These observations provide crucial insights into the question why R339T is still non-inhibitory.     

Chicken ovalbumin promoter is demethylated upon expression in the regions specifically involved in estrogen-responsiveness

Here we report the methylation status of the chicken ovalbumin promoter. Genomic DNA of oviduct from immature chickens and laying hens was analyzed through bisulfite genomic sequencing. In the ovalbumin control locus up to the 6 kb upstream region, CpG sites were methylated in immature chickens, except for several sites, and almost all CpGs residing in DNase I hypersensitive sites I, II, and III, but not IV, were selectively unmethylated in ovalbumin expressing chickens. Chromatin immunoprecipitation assays showed that the ovalbumin control region was associated with acetylated histone H3 but not with dimethylated histone H3 at Lys 27. These results demonstrate that DNA demethylation was restricted to short DNA regions of DNase I hypersensitive sites, especially to those which participated in estrogen-responsiveness, even when cells expressed extremely high levels of ovalbumin and these sites were associated with acetylated histones.     

Dynamic mechanism for the serpin loop insertion as revealed by quantitative kinetics

The serpin conformational change by insertion of the reactive center loop into beta-sheet A plays a central role in multiple physiological consequences such as serine proteinase inhibition, latency and serpinopathic polymerization. To study the dynamic mechanism for the loop insertion, a novel kinetic method was established utilizing the ovalbumin mutant R339T/A352R; the loop insertion progressed after the cleavage of P1-P1' (Arg352-Ser353) by trypsin was quenched at pH 8 and 0.5 degrees C, and different conformers were quantified by separation using ion-exchange HPLC. The apparent first-order rate constant k(app) determined for various R339T/A352R derivatives differing in conformational stability was greatly increased by lowering the pH. The pH-dependence of k(app) indicated that the protonation of side-chain(s) with a pK(a) value of around 4.6 is a pre-requisite for the loop insertion. The theoretical rate constant k for the protonated form calculated from k(app) was highly variable, depending on the ovalbumin derivative; structural modifications that give increased mobility to helix F and the sheet-A half (s3A/s2A/s1A) resulted in a striking increase in the loop insertion rate constant k. The k values were determined at different temperatures for all the ovalbumin derivatives, and DeltaH(double dagger) and DeltaS(double dagger) values for the loop insertion reaction were determined according to the transition theory. The formation of the transition state was highly endothermic with minor entropy gain, requiring a DeltaG(double dagger) larger than 18 kcal/mol, which can offset the hydrogen-bond cleavages between s3A and s5A. These results are consistent with the transition state with an opened sheet A and altered orientation of helix F.     

Molecular evolution of serpins: homologous structure of the human alpha 1-antichymotrypsin and alpha 1-antitrypsin genes

alpha 1-Antichymotrypsin belongs to a supergene family that includes alpha 1-antitrypsin, antithrombin III, ovalbumin, and angiotensinogen. The human chromosomal alpha 1-antichymotrypsin gene has been cloned and its molecular structure established. The gene is approximately 12 kb in length and contains five exons and four introns. The locations of the introns within the alpha 1-antichymotrypsin gene are identical with those of the human alpha 1-antitrypsin and angiotensinogen genes. Other members of this supergene family contain introns located at nonhomologous positions of the genes. The homologous organization of the alpha 1-antichymotrypsin and alpha 1-antitrypsin genes corresponds with the high degree of homology between their protein sequences and suggests that these loci arose by recent gene duplication. A model is presented for the evolution of both the genomic structure and the protein sequences of the serine protease inhibitor superfamily.     

The immunocytochemical localization of angiotensinogen in the rat ovary

Summary The present study examined the presence and cellular distribution of angiotensinogen, the precursor to the angiotensin peptides, in the ovary of the normal cycling rat by immunocytochemistry. Angiotensinogen staining was present in the granulosa cells of maturing follicles and to a lesser extent in those undergoing atresia. Staining was not seen in the granulosa cells of primordial or early primary follicles. In maturing follicles intense staining for angiotensinogen was confined to the antral cell layers, cells of the cumulus oophorus and in the follicular fluid. Strong immunostaining was also seen in the germinal epithelium covering the ovary. Lighter angiotensinogen staining was observed in some parts of the cortical and medullary stroma and occasionally in corpora lutea. No variation in the intensity or pattern of angiotensinogen staining was observed throughout the estrous cycle. Comparison of the distribution of angiotensinogen with the previously described localization of renin, AII, angiotensin converting enzyme and AII receptors, suggests that there are a number of intra-ovarian sites at which AII could be produced.     

Thermostabilization of ovalbumin by an alkaline treatment: examination for the possible implications of an altered serpin loop structure

Ovalbumin, a member of the serpin superfamily, is transformed via an intermediate state into a non-cleaved, thermostabilized form (S-ovalbumin) during either the storage of unfertilized eggs or development of fertilized eggs; essentially the same thermostabilization also occurs upon in vitro incubation of isolated ovalubumin under alkaline conditions. To investigate the implications of a partial insertion of the alpha-helical serpin loop into beta-sheet A that has been proposed as a conformational mechanism for S-ovalbumin production, we examined the thermostabilization process of ovalbumin with different loop structures. When the thermostabilization processes were compared for the intact, P1-P1'-cleaved and P1-P1'/P8-P7-cleaved forms of egg white ovalbumin, both the rates for the conversion from the native to intermediate and from the intermediate to S-ovalbumin were almost indistinguishable among the three protein forms. Furthermore, the fully loop-inserted form of recombinant ovalbumin mutant R339T that had been thermostabilized by P1-P1' cleavage with Tm values from 72 to 88 degrees C was further thermostabilized by an alkaline treatment, yielding a final product (loop inserted S-ovalbumin) with a Tm value of 93 degrees C. No significant difference was found between native ovalbumin and S-ovalbumin in respect of the rate of proteolytic cleavage of the loop by elastase and subtilisin. These data strongly suggest that S-ovalbumin is produced by a mechanism other than that of the partial loop insertion model.     

DNA polymorphisms and linkage disequilibrium in the angiotensinogen gene

A number of recent studies have implicated the angiotensinogen gene in the aetiology of essential hypertension in Caucasian, Japanese and African Caribbean subjects. We have genotyped 153 healthy white Caucasian subjects at a dinucleotide repeat polymorphism and seven diallelic sites in the coding or flanking regions of the angiotensinogen gene, including one polymorphism not previously studied. We have also documented patterns of linkage disequilibrium between polymorphisms. There is evidence of variation in the frequency of several mutations when compared with published results from other Caucasian control populations, possibly due to cryptic ethnic differences between these groups. This should be considered in the design and interpretation of studies of the angiotensinogen gene. Received: 10 November 1995 / Revised: 25 March 1996     

Secondary structure of ovalbumin messenger RNA.

The secondary structure of highly purified ovalbumin mRNA was studied by automated thermal denaturation techniques and the data were subjected to computer processing. Comparative studies with 20 natural and synthetic model nucleic acids suggested that the secondary structure of ovalbumin mRNA possesses the following features: the extent of base pairing of ovalbumin mRNA is similar to that found in tRNAs or ribosomal RNAs; the secondary structure of ovalbumin mRNA is more thermolabile than any of the model compounds tested, including the copolymer poly(A-U); ovalbumin mRNA does not have extensive G-C rich stems as found in tRNAs or ribosomal RNAs; the base composition of the double-stranded regions reveals 54% G-C residues which was significantly higher than that noted in the whole molecule (approximately 41.5% G-C). The presence of 46% A-U pairs in short stems of about five base pairs would have a very large destabilizing effect on the secondary structure of ovalbumin mRNA. However, at 0.175 M monovalent cations and 36 degrees C most of the secondary structure of ovalbumin mRNA is preserved. These data suggest that the double-stranded regions in ovalbumin mRNA are of sufficient length to provide the necessary stability for maintaining the open loop regions in an appropriate conformation which may be required for the biological function of ovalbumin mRNA. Furthermore, the lability of the double-stranded regions in ovalbumin mRNA may also be important for the biological function of this mRNA.     

A significant lag in the induction of ovalbumin messenger RNA by steroid hormones: a receptor translocation hypothesis.

Although ovalbumin and conalbumin mRNA accumulate in the same tubular gland cells of the chick oviduct in response to estrogen or progesterone treatment, the kinetics of induction are markedly different. Conalbumin mRNA begins to accumulate within 30 min after estrogen administration, whereas there is a lag of approximately 3 hr before ovalbumin mRNA begins to accumulate, as measured by three independent assays. The kinetics of estrogen-receptor binding to chromatin indicate that these sites are saturated within 15 min of estrogen administration to the chicks, demonstrating that the lag is not due to slow uptake of the steroid. Suboptimal doses of estrogen produce the same lag, but the resultant rate of ovalbumin mRNA accumulation is lower than with an optimal dose. Partial induction of ovalbumin mRNA by a low dose of estrogen does not shorten the lag with an optimal dose. With progesteone, there is a lag of about 2 hr before either ovalbumin or conalbumin mRNA begins to accumulate. Treatment of chicks with hydroxyurea shortens the lag for ovalbumin induction with either hormone. Inhibition of protein synthesis with emetine does not prevent the accumulation of either ovalbumin or conalbumin mRNA. With cycloheximide, however, ovalbumin mRNA accumulation can be prevented. The existence of a lag suggests that there are intermediate steps between the binding of steroid receptors to chromatin and the induction of ovalbumin mRNA. There are basically two models to explain these delays in response: one involving the accumulation of an essential intermediate, and the other involving a rate-limiting translocation of steroid receptors from initial nonproductive chromatin-binding sites to productive sites. Several aspects of the kinetics of ovalbumin mRNA induction are more consistent with the latter model.     

Ovalbumin is an elastase substrate

Ovalbumin is partially homologous in sequence with the proteinase inhibitors alpha 1-proteinase inhibitor and anti-thrombin III. The region of sequence in ovalbumin which corresponds to the reactive sites of these proteinase inhibitors is susceptible to attack by subtilisin, elastase, thermolysin, bromelain, and Bacillus cereus protease. The esterase activity of elastase is not inhibited by ovalbumin, but ovalbumin is efficiently cleaved by elastase. In contrast with these proteases, trypsin does not cleave ovalbumin.     

DNA methylation: organ specific variations in the methylation pattern within and around ovalbumin and other chicken genes.

The restriction enzymes HhaI and HpaII, whose activity is inhibited by cytosine methylation within their recognition sites, have been utilised as probes to study methylation in the vicinity of the ovalbumin gene in DNA from various chicken tissues. This was complemented by a preliminary study of methylation in the regions of chicken ovotransferrin (conalbumin), ovomucoid and beta-globin genes. From our data we conclude that HaI or HpaII sites can be divided in 3 classes according to their pattern of methylation in different tissues. In the first class of sites (mV class) the extent of methylation varies in different tissues. The patterns obtained show that methylation at the sites located within and around the 3 genes which code for egg white proteins is in general lowest in oviduct of laying hen, where these genes are expressed. However some sites are not methylated (m- class) and others are 95 to 100% resistant (m+ class) to digestion by HhaI or HpaII in the DNAs of all the tissues which were tested. Our study has also revealed a remarkable number of allelic variants for the presence of HhaI or HpaII sites in the region of the ovalbumin gene.