Identification of Membrane-bound Variant of Metalloendopeptidase Neurolysin (EC 3.4.24.16) as the Non-angiotensin Type 1 (Non-AT1), Non-AT2 Angiotensin Binding Site

Wangler, Naomi J.; Santos, Kira L.; Schadock, Ines; Hagen, Fred K.; Escher, Emanuel; Bäder, Michael; Speth, Robert C.; Karamyan, Vardan T.

doi:10.1074/jbc.m111.273052

Cited by 34 publications

(39 citation statements)

References 63 publications

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“…As shown in Figure 1, optimal unmasking was observed at PCMB concentrations of 100–300 μM. The optimal PCMB concentration are slightly lower than our initial report [26], but is consistent with subsequent more recent studies [33,56]. Two-way ANOVA of the K D values indicated that the K D for testis was lower at 100 μM than at 300 μM (p<0.01) and that there was a main effect of higher affinity at 100 μM versus 300 μM (p=0.0033).…”

Section: Resultssupporting

confidence: 91%

“…Of primary interest for assessment of the PCMB activation of 125 I-SI Ang II binding is whether PCMB mimics naturally occurring physiological or pathophysiological conditions. While these studies were in progress, we determined that this potential new member of the RAS was a membrane associated variant of neurolysin (E.C.3.4.24.16) based upon studies in neurolysin transfected cells and neurolysin knockout mouse brains [33]. Neurolysin has a broad tissue distribution [49] and is highly expressed in rat testis membranes as well as the brain [50].…”

Section: Discussionmentioning

confidence: 99%

“…Recently, we identified this binding site as neurolysin (E.C. 3.4.24.16) a member of the M3 zinc metallopeptidase family [33]…”

Section: Introductionmentioning

confidence: 99%

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The effects of para-chloromercuribenzoic acid and different oxidative and sulfhydryl agents on a novel, non-AT1, non-AT2 angiotensin binding site identified as neurolysin

Santos¹,

Vento²,

Wright³

et al. 2013

Regulatory Peptides

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A novel, non-AT1, non-AT2 brain binding site for angiotensin peptides that is unmasked by p-chloromercuribenzoate (PCMB) has been identified as a membrane associated variant of neurolysin. The ability of different organic and inorganic oxidative and sulfhydryl reactive agents to unmask or inhibit 125I-Sar1Ile8 angiotensin II (SI-Ang II) binding to this site was presently examined. In tissue membranes from homogenates of rat brain and testis incubated in assay buffer containing losartan (10 μM) and PD123319 (10 μM) plus 100 μM PCMB, 5 of the 39 compounds tested inhibited 125I-SI Ang II binding in brain and testis. Mersalyl acid, mercuric chloride (HgCl2) and silver nitrate (AgNO3) most potently inhibited 125I-SI Ang II binding with IC50’s ~1–20 μM This HgCl2 inhibition was independent of any interaction of HgCl2 with angiotensin II (Ang II) based on the lack of effect of HgCl2 on the dipsogenic effects of intracerebroventricularly administered Ang II and 125I-SI Ang II binding to AT1 receptors in the liver. Among sulfhydryl reagents, cysteamine and reduced glutathione (GSH), but not oxidized glutathione (GSSG) up to 1 mM, inhibited PCMB-unmasked 125I-SI Ang II binding in brain and testis. Thimerosal and 4-hydroxymercuribenzoate moderately inhibited PCMB-unmasked 125I-SI Ang II binding in brain and testis at 100 μM; however, they also unmasked non-AT1, non-AT2 binding independent of PCMB. 4-hydroxybenzoic acid did not promote 125 I-SI Ang II binding to this binding site indicating that only specific organomercurial compounds can unmask the binding site. The common denominator for all of these interacting substances is the ability to bind to protein cysteine sulfur. Comparison of cysteines between neurolysin and the closely related enzyme thimet oligopeptidase revealed an unconserved cysteine (cys650, based on the full length variant) in the proposed ligand binding channel (Brown et al., 2001) [1] near the active site of neurolysin. It is proposed that the mercuric ion in PCMB and closely related organomercurial compounds binds to cys650, while the acidic anion forms an ionic bond with a nearby arginine or lysine along the channel to effect a conformational change in neurolysin that promotes Ang II binding.

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Section: Resultssupporting

confidence: 91%

Section: Discussionmentioning

confidence: 99%

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The effects of para-chloromercuribenzoic acid and different oxidative and sulfhydryl agents on a novel, non-AT1, non-AT2 angiotensin binding site identified as neurolysin

Santos¹,

Vento²,

Wright³

et al. 2013

Regulatory Peptides

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“…This suggests the existence of a non-AT 1 , non-AT 2 , non-neurolysin Ang II binding site with a different pattern of expression in the mouse brain. A much smaller amount of 125 I-SI Ang II binding (∼17% that of the wild-type strain) was observed in our previous study using membrane homogenates obtained from the brains of neurolysin knockout mice [40]. A possible explanation for this disparity may be the differences in tissue preparation.…”

Section: Discussionmentioning

confidence: 58%

Distribution of Non-AT1, Non-AT2 Binding of 125I-Sarcosine1, Isoleucine8 Angiotensin II in Neurolysin Knockout Mouse Brains

et al. 2014

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The recent identification of a novel binding site for angiotensin (Ang) II as the peptidase neurolysin (E.C. 3.4.24.16) has implications for the renin-angiotensin system (RAS). This report describes the distribution of specific binding of 125I-Sarcosine1, Isoleucine8 Ang II (125I-SI Ang II) in neurolysin knockout mouse brains compared to wild-type mouse brains using quantitative receptor autoradiography. In the presence of p-chloromercuribenzoic acid (PCMB), which unmasks the novel binding site, widespread distribution of specific (3 µM Ang II displaceable) 125I-SI Ang II binding in 32 mouse brain regions was observed. Highest levels of binding >700 fmol/g initial wet weight were seen in hypothalamic, thalamic and septal regions, while the lowest level of binding <300 fmol/g initial wet weight was in the mediolateral medulla. 125I-SI Ang II binding was substantially higher by an average of 85% in wild-type mouse brains compared to neurolysin knockout brains, suggesting the presence of an additional non-AT1, non-AT2, non-neurolysin Ang II binding site in the mouse brain. Binding of 125I-SI Ang II to neurolysin in the presence of PCMB was highest in hypothalamic and ventral cortical brain regions, but broadly distributed across all regions surveyed. Non-AT1, non-AT2, non-neurolysin binding was also highest in the hypothalamus but had a different distribution than neurolysin. There was a significant reduction in AT2 receptor binding in the neurolysin knockout brain and a trend towards decreased AT1 receptor binding. In the neurolysin knockout brains, the size of the lateral ventricles was increased by 56% and the size of the mid forebrain (−2.72 to +1.48 relative to Bregma) was increased by 12%. These results confirm the identity of neurolysin as a novel Ang II binding site, suggesting that neurolysin may play a significant role in opposing the pathophysiological actions of the brain RAS and influencing brain morphology.

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“…In neurons, but not astrocytes, approximately 10 % of the enzymatic activity is membrane-bound [59]. Wangler et al [60] report that “membrane- anchoring mechanism (s)” are not understood. Clearly, analytical techniques with the objective of measuring membrane-bound (ecto) enzyme activity would be useful.…”

Section: Introductionmentioning

confidence: 99%

Electroosmotic perfusion of tissue: sampling the extracellular space and quantitative assessment of membrane-bound enzyme activity in organotypic hippocampal slice cultures

Sandberg

et al. 2014

Anal Bioanal Chem

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This review covers recent advances in sampling fluid from the extracellular space of brain tissue by electroosmosis (EO). Two techniques, EO sampling with a single fused-silica capillary and EO push–pull perfusion, have been developed. These tools were used to investigate the function of membrane-bound enzymes with outward-facing active sites, or ectoenzymes, in modulating the activity of the neuropeptides leu-enkephalin and galanin in organotypic-hippocampal-slice cultures (OHSCs). In addition, the approach was used to determine the endogenous concentration of a thiol, cysteamine, in OHSCs. We have also investigated the degradation of coenzyme A in the extracellular space. The approach provides information on ectoenzyme activity, including Michaelis constants, in tissue, which, as far as we are aware, has not been done before. On the basis of computational evidence, EO push–pull perfusion can distinguish ectoenzyme activity with a ~100 µm spatial resolution, which is important for studies of enzyme kinetics in adjacent regions of the rat hippocampus.

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Identification of Membrane-bound Variant of Metalloendopeptidase Neurolysin (EC 3.4.24.16) as the Non-angiotensin Type 1 (Non-AT1), Non-AT2 Angiotensin Binding Site

Cited by 34 publications

References 63 publications

The effects of para-chloromercuribenzoic acid and different oxidative and sulfhydryl agents on a novel, non-AT1, non-AT2 angiotensin binding site identified as neurolysin

The effects of para-chloromercuribenzoic acid and different oxidative and sulfhydryl agents on a novel, non-AT1, non-AT2 angiotensin binding site identified as neurolysin

Distribution of Non-AT1, Non-AT2 Binding of 125I-Sarcosine1, Isoleucine8 Angiotensin II in Neurolysin Knockout Mouse Brains

Electroosmotic perfusion of tissue: sampling the extracellular space and quantitative assessment of membrane-bound enzyme activity in organotypic hippocampal slice cultures

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