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ARTICLES: Karin Kohlstedt, Cynthia Gershome, Matthias Friedrich, Werner Muller-Esterl, François Alhenc-Gelas, Rudi Busse, and Ingrid Fleming Angiotensin-Converting Enzyme (ACE) Dimerization Is the Initial Step in the ACE Inhibitor-Induced ACE Signaling Cascade in Endothelial Cells Mol Pharmacol 2006; 69: 1725-1732 [Abstract] [Full text] [PDF]

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The Missing Link: ACE Dimerization and Shedding

Sergei M. Danilov, Olga A. Kost and Edward E. Sturrock   (5 July 2006)

Authors' Response

Ingrid Fleming, Karin Kohlstedt   (5 July 2006)

The Missing Link: ACE Dimerization and Shedding 5 July 2006
Sergei M. Danilov University of Illinois at Chicago, Olga A. Kost and Edward E. Sturrock Send letter to journal: Re: The Missing Link: ACE Dimerization and Shedding danilov{at}uic.edu Sergei M. Danilov, et al.	Dear Editor, The article entitled “Angiotensin converting enzyme (ACE) dimerization is the initial step in the ACE inhibitor-induced ACE signaling cascade in endothelial cells” by Kohlstedt et al. (2006) provides evidence that ACE exists as both a monomer and a dimer, not only on the surface of transfected CHO cells, as demonstrated previously (Kost et al., 2003), but also on the surface of cultured human umbilical vein endothelial cells. Moreover, the amount of ACE dimers on the surface of human endothelial cells increased dramatically in the presence of the ACE inhibitor ramiprilat and this ligand binding initiates a signaling cascade involving phosphorylation of the ACE cytoplasmic region. This is the first evidence for cell signaling being mediated via ectodomain interaction(s) between the ACE monomeric forms and the study has important implications regarding our understanding of the unique roles of the ACE protein’s N- and C- domains. Kohlstedt et al. suggest that their experiments do not support our hypothesis of a link between ACE dimerization and shedding from the cell surface (Kost et al., 2003, Balyasnikova et al., 2005). We believe that there are three fundamental flaws in this reasoning, based on previous work in our laboratories. First, the authors were unable to prevent ramiprilat-induced ACE dimerization on the surface of human endothelial cells using different oligosaccharides (glucose, mannitol and galactose). We have shown (Kost et al., 1998, 2000) that ACE forms dimers in reverse micelles, whereas deglycosylated ACE failed to dimerize. Carbohydrates, especially Neu5Ac- and Gal-terminated saccharides, competitively inhibited ACE-ACE interaction. Moreover, the most effective glycan inhibitors of ACE dimerization were the total pool of ACE oligosaccharides and biantennary complex oligosaccharides from other glycoproteins. These findings allowed us to conclude that ACE possesses a specific carbohydrate-recognizing domain (CRD, lectin-like center). The putative region of this CRD was further localized on the N-domain by using truncated N- and C-domains of ACE and monoclonal antibodies to different epitopes of the N-domain (Kost et al. 2003). Furthermore, galactose not only prevented ACE dimerization in a biomembrane model system, but also affected antibody-induced ACE shedding from the surface of ACE-expressing CHO cells (Kost et al. 2003). Reduced glycosylation of somatic ACE also significantly increased the basal rate of ACE shedding. Two monoclonal antibodies (9B9 and 3G8) with overlapping epitopes that blocked ACE dimerization in the reverse micelles, also affected ACE shedding from the surface of CHO cells. Kohlstedt et al. did not observe any effect of galactose, glucose or mannitol on ramiprilat-induced ACE dimerization. These data are not surprising as glucose and mannitol have a four- and two-fold order of magnitude lower affinity for the CRD than galactose, respectively. The only monosaccharide used in Kohlstedt’s study that was likely to affect ACE dimerization was galactose. However, the galactose concentration (10 ìM) that was needed to block ACE dimerization in a biomembrane model system (Kost et al. 2000) is inappropriate for cell membrane experiments where galactose can be involved in numerous other glycan-glycan and glycan-protein interactions. Thus, only 100-fold higher concentrations of galactose affected basal and antibody-induced ACE shedding from ACE-expressing cells (Kost et al., 2003). Secondly, there is no reason to accept a priori that carbohydrate- controlled ACE dimers and ramiprilat-induced ACE dimers are the same. In fact, bovine ACE was able to form three types of carbohydrate-mediated dimers in reverse micelles and even formed a tetramer that was not controlled by carbohydrates (Kost et al. 2003, Grinshtein et al.1999). Human ACE was able to form only one compact carbohydrate-controlled dimer and larger dimer that was not formed via carbohydrate-recognizing domain (Kost et al. 2003). Finally, Kohlstedt et al exclude the possibility of ACE dimerization affecting ectodomain shedding based on their data showing that the antibody mAb 9B9 did not prevent ramiprilat–induced ACE dimerization on the cell surface. Once again, these data are not surprising and the conclusions not entirely appropriate since this antibody only inhibited dimerization in the reverse micelles. On the other hand, the mAb 3G8, which has an overlapping epitope with that of mAb 9B9 blocks both dimerization in reverse micelles and shedding from the surface of ACE- expressing cells (Kost et al. 2003). In contrast, mAb 9B9 dramatically increased ACE shedding from the surface of these cells (Balyasnikova et al. 2002, Balyasnikova et al. 2005, Kohlstedt et al. 2006). Important findings have emerged from our studies on the effects of mAbs on different functions of the ACE enzyme that are pertinent to this discussion: 1) the binding of mAbs to a region of the N domain, defined as the overlapping surface of epitopes 9B9 and 3A5, significantly induced ACE shedding; 2) the binding of mAbs to another region of the N domain, defined as the overlapping surface of epitopes 9B9 and 3G8, inhibited dimerization of the somatic ACE in the reverse micelles. Therefore, using a set of mAbs, we have identified a region on the N domain of ACE, which is very sensitive to the binding of monoclonal antibodies. mAb 3G8 results in inhibition of ACE shedding on the cell surface and prevention of dimerization in reverse micelles. Binding of mAb 3A5 to the N-domain resulted in conformational changes that prevented binding of other mAbs to the N domain, inhibition of ACE catalytic activity (Danilov et al., 1994), and induction of ACE shedding (Balyasnikova et al., 2002). However, binding of mAb 9B9 to the N domain prevented dimerization in reverse micelles (as with mAb 3G8) and increased ACE shedding (as with mAb 3A5) - Balyasnikova et al. (2005). Thus, the inability of mAb 9B9 to prevent ramiprilat-induced dimerization of ACE on the cell surface should not preclude the proposed influence of ACE dimerization on ACE ectodomain shedding. Undoubtedly, the crosstalk between proteins is a complex and exquisitely controlled mechanism for triggering and controlling cell function. Further work in this field is needed to reveal the full impact of ACE and various types of signaling on the renin angiotensin system and cardiovascular health. Sergei M. Danilov (University of Illinois at Chicago, USA), Olga A. Kost (Moscow State University, Russia ), Edward E. Sturrock (University of Cape Town, South Africa). REFERENCES 1. Kohlstedt K, Gershome C, Friedrich M, Muller-Esterl W, Alhenc-Gelas F, Busse R, Fleming I. (2006) Angiotensin converting enzyme (ACE) dimerization is the initial step in the ACE inhibitor-induced ACE signaling cascade in endothelial cells. Mol. Pharm 69, 1725-1732. 2. Kost OA, Balyasnikova IV, Chemodanova EE, Nikolskaya II, Albrecht RF II, Danilov SM (2003). Epitope-dependent blocking of the angiotensin- converting enzyme dimerization by monoclonal antibodies to N-terminal domain of ACE: Possible Link of ACE dimerization and shedding from the cell surface. Biochemistry 42, 6965-6976. 3. Balyasnikova IV, Woodman ZL, Albrecht RFII, Natesh R, Acharya KR, Sturrock ED, Danilov SM. (2005) Localization of an N domain region of angiotensin-converting enzyme involved in the regulation of ectodomain shedding using monoclonal antibodies. J Proteome Res. 4, 258-267. 4. Kost OA, Orth TA, Nikolskaya II, Nametkin SN, Levashov AV. (1998) Carbohydrates regulate the dimerization of angiotensin-converting enzyme, Biochem. Mol. Biol. Int. 44, 535-542. 5. Kost OA, Bovin NV, Chemodanova EE, Nasonov VV, Orth TA. (2000) New feature of angiotensin-converting enzyme: carbohydrate-recognizing domain, J. Mol. Recognit. 13, 360-369. 6. Grinshtein SV, Nikolskaya II, Klyachko NL, Levashov AV, Kost OA (1999) Structural organization of membrane and soluble forms of somatic angiotensin-converting enzyme. Biochemistry (Moscow), 64, 571-580. 7. Danilov S, Jaspard E, Churakova T, Towbin H, Savoie, F, Lei W, Alhenc Gelas F. (1994) Structure-function analysis of angiotensin I-converting enzyme using monoclonal antibodies. J Biol Chem. 269, 26806-26814. 8. Balyasnikova IV, Karran EH, Albrecht RFII, Danilov, SM. (2002) Epitope- specific antibody-induced cleavage of angiotensin-converting enzyme from the cell surface. Biochem. J. 362, 585-595.
Authors' Response 5 July 2006
Ingrid Fleming Johann Wolfgang Goethe-Universitat, Frankfurt, Germany., Karin Kohlstedt Send letter to journal: Re: Authors' Response fleming{at}em.uni-frankfurt.de Ingrid Fleming, et al.	Dear Editor, In our recent article (Kohlstedt et al., 2006. Angiotensin-converting enzyme (ACE) dimerization is the initial step in the ACE inhibitor-induced ACE signaling cascade in endothelial cells. Mol Pharmacol 69:1725-1732) we reported that human somatic ACE can be detected in the monomeric as well as the dimeric form in human endothelial cells that endogenously express ACE and in porcine aortic endothelial cells stably overexpressing the human enzyme. Moreover, our study demonstrated that ACE inhibitors enhance dimmer formation and that this step is essential for the subsequent activation of the c-Jun N terminal kinase (JNK), a step we have previously shown to be involved in the “ACE signaling cascade” (Kohlstedt et al., 2004; Kohlstedt et al., 2005). To address the mechanisms involved in the regulation of ACE dimerization and the link to ACE inhibitor-induced JNK activation we assessed the effects of different antibodies and carbohydrates on ACE dimerization as previous studies, performed mainly in CHO cells and in a biomembrane model system, had led to the identification of a carbohydrate- recognizing domain in the N terminus of ACE and had linked enzyme dimerization with its cleavage/secretion (Kost et al., 2000; Kost et al., 2003; Balyasnikova et al., 2005). We found that treating ACE expressing endothelial cells with antibodies to specific epitopes in the N domain (Danilov et al., 1994) exerted relatively discrete effects on enzyme dimerization that were not significantly different from that detected in solvent-treated cells. The ACE inhibitor, ramiprilat on the other hand, induced a pronounced ACE dimerization that was unaffected by any of the antibodies tested, including one previously reported to prevent ACE dimerization in reverse micelles (Kost et al., 2003). The ACE antibodies tested did however result in the cleavage/secretion of human somatic ACE from endothelial cells, a finding which is consistent with the findings of Balyasnikova et al., (2002). It seems that the link between carbohydrate- induced dimerization and ACE secretion is likely to be a complex one as an antibody that promoted ACE secretion (9B9) as well as an antibody that prevented this process (3G8) were both reported to prevent enzyme dimerization in reverse micelles (Kost et al., 2003). To address the role of the putative carbohydrate-recognizing domain on ACE dimerization in endothelial cells we performed experiments with sugars including galactose but were only able to use concentrations up to 10 µmol/L as higher concentrations significantly compromised endothelial cell viability. Thus, we may have missed an effect of this carbohydrate on ACE dimerization reported in reverse micelles (Kost et al., 2000) and which these authors now point out can only be assessed in intact ACE- expressing cells using 100-fold higher concentrations of galactose. However, we also addressed which domain of the ACE enzyme is implicated in ACE inhibitor-induced enzyme dimerization using a series of ACE mutants. We found that specific mutation of the C but not the N domain, attenuated both basal ACE dimerization and that induced by the application of an ACE inhibitor (Kohlstedt et al., 2006). These data tend to rule out a role for the N terminal domain in regulating ACE inhibitor-induced ACE dimerization but say nothing about enzyme dimerization elicited by high concentrations of carbohydrates, a response we did not assess. In summary, we respectfully disagree that there are “fundamental flaws” in our reasoning and while we acknowledge the significant input of Dr’s Danilov, Kost and Sturrock to this topic, we feel it is necessary to exert caution when extrapolating the results of studies obtained in experimental systems in which the human enzyme is overexpressed (e.g. CHO cells) or studies based on the use of a biomembrane model system. This is because the extent and sites of protein glycosylation can vary markedly from cell type to cell type as well as between native and in cultured cells (Bevilacqua et al., 1996). Indeed, somatic ACE contains 17 potential sites for N-glycosylation, mainly of the complex type (Das and Soffer, 1975) and deglycosylated ACE as well as sequentially desialylated and degalactosylated ACE fail to dimerize (Kost et al., 2000). We however wholeheartedly agree with the comment that there is no reason to assume that carbohydrate-controlled ACE dimers and ramiprilat-induced ACE dimers are the same. Indeed, it is clear that human ACE can dimerize via a mechanism that is independent of the carbohydrate-recognizing domain (Kost et al. 2003), indicating that distinct mechanisms may be involved in the regulation of ACE dimerization in response to specific stimuli. Finally, while the physiological/pathophysiological role of ACE dimerization via the carbohydrate-recognizing domain remains to be determined, our data (Kohlstedt et al., 2006) highlight the importance of the C terminal domain of ACE in enzyme dimerization in response to a specific clinically- relevant stimulus i.e., an ACE inhibitor, and link this phenomenon to functional initiation of ACE signaling. Karin Kohlstedt and Ingrid Fleming Johann Wolfgang Goethe-Universität, Frankfurt, Germany. References Balyasnikova IV, Karran E H, Albrecht R F and Danilov S M (2002) Epitope-specific antibody-induced cleavage of angiotensin-converting enzyme from the cell surface. Biochem J 362:585-595. Balyasnikova IV, Woodman Z L, Albrecht R F, Natesh R, Acharya K R, Sturrock E D and Danilov S M (2005) Localization of an N-domain region of angiotensin-converting enzyme involved in the regulation of ectodomain shedding using monoclonal antibodies. J Proteome Res 4:258-267. Bevilacqua M, Vago T, Rogolino A, Conci F, Santoli E and Norbiato G (1996) Affinity of angiotensin I-converting enzyme (ACE) inhibitors for N- and C-binding sites of human ACE is different in heart, lung, arteries, and veins. J Cardiovasc Pharmacol 28:494-499. Danilov S, Jaspard E, Churakova T, Towbin H, Savoie F, Wei L and Alhenc-Gelas F (1994) Structure-function analysis of angiotensin I- converting enzyme using monoclonal antibodies. Selective inhibition of the amino-terminal active site. J Biol Chem 269:26806-26814. Das M and Soffer R L (1975) Pulmonary angiotensin-converting enzyme. Structural and catalytic properties. J Biol Chem 250:6762-6768. Kohlstedt K, Brandes R P, Muller-Esterl W, Busse R and Fleming I (2004) Angiotensin-converting enzyme is involved in outside-in signaling in endothelial cells. Circ Res 94:60-67. Kohlstedt K, Busse R and Fleming I (2005) Signaling via the angiotensin-converting enzyme enhances the expression of cyclooxygenase-2 in endothelial cells. Hypertension 45:126-132. Kohlstedt K, Gershome C, Friedrich M, Muller-Esterl W, Alhenc-Gelas F, Busse R and Fleming I (2006) Angiotensin-converting enzyme (ACE) dimerization is the initial step in the ACE inhibitor-induced ACE signaling cascade in endothelial cells. Mol Pharmacol 69:1725-1732. Kost OA, Balyasnikova I V, Chemodanova E E, Nikolskaya I I, Albrecht R F and Danilov S M (2003) Epitope-dependent blocking of the angiotensin- converting enzyme dimerization by monoclonal antibodies to the N-terminal domain of ACE: possible link of ACE dimerization and shedding from the cell surface. Biochemistry 42:6965-6976. Kost OA, Bovin N V, Chemodanova E E, Nasonov V V and Orth T A (2000) New feature of angiotensin-converting enzyme: carbohydrate-recognizing domain. J Mol Recognit 13:360-369.