Physiological non-equivalence of the two isoforms of angiotensin-converting enzyme.

The structurally related somatic and germinal isoforms of angiotensin-converting enzyme (ACE) contain the same catalytic active center and are encoded by the same gene, whose disruption causes renal atrophy, hypotension, and male sterility. The reason for the evolutionary conservation of both isozymes is an enigma, because, in vitro, they have very similar enzymatic properties. Despite the common enzymatic properties, discrete expression of both isoforms is maintained in alternate cell types. We have previously shown that sperm-specific expression of transgenic germinal ACE in Ace -/- male mice restores fertility without curing their other abnormalities (Ramaraj, P., Kessler, S. P., Colmenares, C. & Sen, G. C. (1998) J. Clin. Invest. 102, 371-378). In this report we tested the biological equivalence of somatic ACE and germinal ACE utilizing an in vivo isozymic substitution approach. Here we report that restoration of male fertility was not achieved by the transgenic expression of enzymatically active, somatic ACE in the sperm of Ace -/- mice. Therefore, the requisite physiological functions of the two tissue-specific isozymes of ACE are not interchangeable.

Selective restoration of male fertility in mice lacking angiotensin-converting enzymes by sperm-specific expression of the testicular isozyme.

Although angiotensin-converting enzyme (ACE) has been studied primarily in the context of its role in blood pressure regulation, this widely distributed enzyme has many other physiological functions. The ACE gene encodes two isozymes. The somatic isozyme is expressed in many tissues, including vascular endothelial cells, renal epithelial cells, and testicular Leydig cells, whereas the testicular or germinal angiotensin-converting enzyme is expressed only in sperm. The ACE gene knockout mice lack both isozymes and they exhibit low blood pressure, kidney dysfunctions, and male infertility. Here, we report the use of a sperm-specific promoter and interbreeding of transgenic and gene knockout mice for generating a mouse strain that expressed ACE only in sperm. The experimental mice maintained the kidney defects of ACE-/- mice, but unlike the knockout strain, the males were fertile. Thus, we established that the role of ACE in male fertility is completely dependent on its exclusive expression in sperm. Our study clearly demonstrated how transgenic and knockout techniques can be combined for ascribing a specific physiological function to the expression of a multifunctional protein in a given tissue.

A cyclic AMP response element in the angiotensin-converting enzyme gene and the transcription factor CREM are required for transcription of the mRNA for the testicular isozyme.

The angiotensin-converting enzyme (ACE) gene produces two mRNA species from tissue-specific promoters. The transcription start site of the mRNA for the smaller testicular isozyme (ACET) is located within an intron of the larger transcription unit that encodes the pulmonary isozyme (ACEP).We have previously demonstrated that a 298-base pair DNA fragment, 5' to the rabbit ACET mRNA transcription initiation site, can activate the testicular expression of a transgenic reporter gene. In the current study, using the same transgenic reporter system, we identified a putative cyclic AMP response element present within this DNA fragment to be absolutely essential for transcriptional activation. Moreover, we observed that ACET mRNA was not expressed in the testes of mice homozygous for a null mutation in the transcription factor CREM. However, in the same mice, ACEP mRNA was abundantly expressed in the lung. Our observations indicate that ACET mRNA expression in the testes is regulated by the putative cyclic AMP response element present 5' to the transcription start site and the corresponding transcription factor CREM.

Analysis of the negative transcriptional regulatory element in the angiotensin-converting enzyme gene.

We have characterized the sequence requirements and the protein binding properties of the previously identified transcriptional negative element present in the rabbit angiotensin-converting enzyme (ACE) gene. DNase footprinting experiments revealed that within the negative element (-715 to -610) several regions interact with proteins present in the nuclear extracts of ACE-expressing and -nonexpressing cell lines. Transfection analysis using the heterologous beta-actin promoter and mutated negative elements demonstrated that the SP1 site, the collagen-silencer-like sequence, and the inverted repeat elements are dispensable for their functioning. Deletion of the region between -692 to -668, however, completely eliminated the activity of the negative element, and mutation of the synapsin-silencer-like sequence present within this region vastly reduced it. This region (-692 to -668) by itself, when present in two copies, could effectively repress the activity of the beta-actin promoter. The same point mutations in the silencer element that destroyed its action on the beta-actin promoter greatly increased the transcriptional efficiency of the native ACE promoter. Electrophoretic mobility shift assay using the -692 to -668 ACE silencer sequence demonstrated the formation of a DNA/protein complex. UV cross-linking of the components of this complex revealed the presence of one prominent protein of approximately 21.5 kDa. This protein may be responsible for mediating the transcriptional-repressing activity of the ACE negative element. Homology between the ACE silencer and neuronal silencer consensus sequence, together with the promoter- and tissue-independent function of the the ACE silencer, suggests this element may bind a member of a large family of common negative regulatory transcription factors.

The cyclic AMP response elements of the genes for angiotensin converting enzyme and phosphoenolpyruvate carboxykinase (GTP) can mediate transcriptional activation by CREM tau and CREM alpha.

The potential of the CREM family of proteins to activate transcription of the genes encoding the testis-specific isozyme of angiotensin converting enzyme (ACET) and the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (GTP) (PEPCK) (EC 4.1.1.32) were investigated. Both CREM tau and CREM alpha bind efficiently to the putative cyclic AMP response element (CRE) present in the ACET gene (CRET) and to the CRE in the PEPCK gene. In HepG2 cells, the CRE was required for the strong stimulation by CREM tau of the expression of a chimeric PEPCK (-210 to +73)-chloramphenicol acetyl transferase (CAT) gene. The CRE could be mutated to the CRET sequence without losing the stimulatory effects of CREM tau. However, a similar chimeric gene driven by the regulatory region of the ACET gene, which contains the CRET site, could only be stimulated by CREM tau when its imperfect TATA element was mutated to an authentic TATA. Surprisingly, CREM alpha, an alleged inhibitor of CRE-mediated transcription, stimulated the expression of both PEPCK-CAT and ACET-CAT genes in HepG2 cells, a process which required the presence of the CRE and the CRET sites, respectively. In contrast, when the same CRE elements were used to drive the transcription of a chimeric gene containing the thymidine kinase promoter linked to the CAT structural gene, CREM alpha inhibited its expression in HepG2 and JEG3 cells. The expression of the same chimeric gene, however, was stimulated by CREM alpha in F9 embryonal carcinoma cells. These results demonstrated that the nature of the transcriptional effects of CREM isoforms on CRE-mediated transcription depends on the specific gene, the specific cell type and the promoter context of the CRE site.

Transcriptional repression of heterologous constitutive and inducible promoters by the negative element of the rabbit angiotensin-converting enzyme gene.

We previously identified a transcriptional negative element (NE) present in the rabbit angiotensin-converting enzyme (ACE) gene. Here, we report that the NE can also repress transcription driven by the strong constitutive promoters of the human beta-actin gene and SV40 in both ACE-expressing and nonexpressing cell lines. The extent of repression was influenced by the relative positions of the NE and the SV40 promoter and enhancer. The NE could also repress transcription driven by interleukin-1 and interferon-alpha-inducible promoters. Finally, transcription from a TATA-less promoter was equally repressed by the NE. Taken together, these results suggest that the NE of the rabbit ACE gene can function as a universal transcriptional silencer element.

Identification of positive and negative transcriptional regulatory elements of the rabbit angiotensin-converting enzyme gene.

The two tissue-specific mRNAs encoding the isozymes of rabbit angiotensin-converting enzyme (ACE) are generated from the same gene by alternative choice of two transcription initiation sites 5.7 kb apart. In the current study, we have characterized the regulatory sites controlling the transcription of the larger pulmonary isozyme mRNA. For this purpose, reporter genes driven by varying lengths of upstream region of the ACE gene were transfected into ACE-producing cells. Our results demonstrated that the transcription of this gene is primarily driven by positive elements within the first 274 bp DNA upstream of the transcription initiation site. The reporter gene driven by this region was expressed in two ACE-producing cells but not in two ACE-non-producing cells thereby establishing its tissue specificity. Our experiments also revealed the existence of a strong negative element located between -692 and -610 positions. This element suppressed the expression of the reporter gene in a dose-dependent and position and orientation-independent fashion thus suggesting that it is a true silencer element. It could also repress the expression of a reporter gene driven by the heterologous strong promoter of the beta-actin gene. The repressing effects of the negative element could be partially overcome by cotransfecting the isolated negative element along with the reporter gene containing the negative element. This result was possibly due to the functional removal of a limiting trans-acting factor which binds to this element. Electrophoretic mobility shift assays revealed that the negative element can form several complexes with proteins present in the nuclear extract of an ACE-producing cell line. At least part of the negative element is strongly conserved in the upstream regions of the human and mouse ACE genes.

Pseudomonas aeruginosa exotoxin A interaction with eucaryotic elongation factor 2. Role of the His426 residue.

Pseudomonas aeruginosa exotoxin A (ETA) catalyzes the transfer of the ADP-ribose moiety of NAD+ onto eucaryotic elongation factor 2 (EF-2). To study the ETA site of interaction with EF-2, an immobilized EF-2 binding assay was developed. This assay demonstrates that ETA, in the presence of NAD+, binds to immobilized EF-2. Additionally, diphtheria toxin was also found to bind to the immobilized EF-2 in the presence of NAD+. Comparative analysis was performed with a mutated form of ETA (CRM 66) in which a histidine residue at position 426 has been replaced with a tyrosine residue. This immunologically cross-reactive, ADP-ribosyl transferase-deficient toxin does not bind to immobilized EF-2, thus explaining its lack of ADPRT activity. ETA bound to immobilized EF-2 cannot bind the monoclonal antibody TC-1 which specifically recognizes the ETA epitope containing His426. Immunoprecipitation of native ETA by mAb TC-1 is only achieved by incubating ETA in the presence of NAD+. Diethyl pyrocarbonate modification of the His426 residue blocks ETA binding to EF-2 and prevents the binding of the TC-1 antibody. Analogs of NAD+ containing a reduced nicotinamide ring or modified adenine moieties cannot substitute for NAD+ in the immobilized binding assay. Collectively, these data support our proposal that the site of ETA interaction with EF-2 includes His426 and that a molecule of NAD+ is required for stable interaction.

Immunochemical analysis of Pseudomonas aeruginosa exotoxin A. Analysis of the His426 determinant.

This study describes a combined immunochemical and genetic approach defining a site on Pseudomonas aeruginosa exotoxin A (ETA) which is critical to the ADP-ribosyltransferase (ADPRT) activity of the toxin. The sequential epitope of a monoclonal antibody (TO-1) which binds to domain III (residues 405-613), containing the ADPRT activity of ETA, has been defined using a series of synthetic peptides. This epitope spans residues 422-432 which composes the major alpha-helical segment of domain III and includes His426 which has previously been shown to be essential for ADPRT activity (Wozniak, D.J., Hsu, L.-Y., and Galloway, D. R. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8880-8884). The critical His426 residue which projects into a major cleft becomes exposed when the ETA protein is in an ADPRT-active configuration. Since the TC-1 mAb does not block the binding of NAD+, it is possible that the alpha-helix site containing the TC-1 epitope and the His426 residue is associated with the interaction between ETA and its elongation factor 2 substrate.

Biochemical analysis of CRM 66. A nonfunctional Pseudomonas aeruginosa exotoxin A.

Pseudomonas aeruginosa exotoxin A (ETA) is an ADP-ribosyltransferase which inactivates protein synthesis by covalently attaching the ADP-ribose portion of NAD+ onto eucaryotic elongation factor 2 (EF-2). A direct biochemical comparison has been made between ETA and a nonenzymatically active mutant toxin (CRM 66) using highly purified preparations of each protein. The loss of ADP-ribosyltransferase activity and subsequent cytotoxicity have been correlated with the presence of a tyrosine residue in place of a histidine at position 426 in CRM 66. In the native conformation, CRM 66 demonstrated a limited ability (by a factor or at least 100,000) to modify EF-2 covalently and lacked in vitro and in vivo cytotoxicity, yet CRM 66 appeared to be normal with respect to NAD+ binding. Upon activation with urea and dithiothreitol, CRM 66 lost ADP-ribosyltransferase activity entirely yet CRM 66 retained the ability to bind NAD+. Replacement of Tyr-426 with histidine in CRM 66 completely restored cytotoxicity and ADP-ribosyltransferase activity. These results support previous findings from this laboratory (Wozniak, D. J., Hsu, L.-Y., and Galloway, D. R. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8880-8884) which suggest that the His-426 residue of ETA is not involved in NAD+ binding but appears to be associated with the interaction between ETA and EF-2.