Fine Epitope Mapping of Monoclonal Antibody 5F1 Reveals Anticatalytic Activity toward the N Domain of Human Angiotensin-Converting Enzyme

Danilov, Sergei M.; Watermeyer, Jean M.; Balyasnikova, Irina V.; Gordon, Kerry; Kugaevskaya, E. V.; Elisseeva, Yulia E.; Albrecht, Ronald F.; Sturrock, Edward D.

doi:10.1021/bi700489v

Cited by 25 publications

(24 citation statements)

References 44 publications

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“…4B). Y465 might participate directly in mAb 5F1 binding to ACE, because it is localized close to/within the epitope for mAb 5F1 [53] (Fig. 5), or the mutation has resulted in gross conformational changes that have altered the mAb 5F1 epitope.…”

Section: Resultsmentioning

confidence: 99%

An Angiotensin I-Converting Enzyme Mutation (Y465D) Causes a Dramatic Increase in Blood ACE via Accelerated ACE Shedding

et al. 2011

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BackgroundAngiotensin I-converting enzyme (ACE) metabolizes a range of peptidic substrates and plays a key role in blood pressure regulation and vascular remodeling. Thus, elevated ACE levels may be associated with an increased risk for different cardiovascular or respiratory diseases. Previously, a striking familial elevation in blood ACE was explained by mutations in the ACE juxtamembrane region that enhanced the cleavage-secretion process. Recently, we found a family whose affected members had a 6-fold increase in blood ACE and a Tyr465Asp (Y465D) substitution, distal to the stalk region, in the N domain of ACE.Methodology/Principal FindingsHEK and CHO cells expressing mutant (Tyr465Asp) ACE demonstrate a 3- and 8-fold increase, respectively, in the rate of ACE shedding compared to wild-type ACE. Conformational fingerprinting of mutant ACE demonstrated dramatic changes in ACE conformation in several different epitopes of ACE. Cell ELISA carried out on CHO-ACE cells also demonstrated significant changes in local ACE conformation, particularly proximal to the stalk region. However, the cleavage site of the mutant ACE - between Arg1203 and Ser1204 - was the same as that of WT ACE. The Y465D substitution is localized in the interface of the N-domain dimer (from the crystal structure) and abolishes a hydrogen bond between Tyr465 in one monomer and Asp462 in another.Conclusions/SignificanceThe Y465D substitution results in dramatic increase in the rate of ACE shedding and is associated with significant local conformational changes in ACE. These changes could result in increased ACE dimerization and accessibility of the stalk region or the entire sACE, thus increasing the rate of cleavage by the putative ACE secretase (sheddase).

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Section: Resultsmentioning

confidence: 99%

An Angiotensin I-Converting Enzyme Mutation (Y465D) Causes a Dramatic Increase in Blood ACE via Accelerated ACE Shedding

et al. 2011

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“…Eight mAbs recognize epitopes on the catalytically active N-domain [15], [30], [32]–[34] and 8 mAbs recognize epitopes localized on the C-domain of ACE [16], [36]. We recently demonstrated that the pattern of precipitation of ACE activity (conformational fingerprinting of ACE) using this set of mAbs provides a sensitive means of identifying changes in local conformation of ACE due to inactivation, inhibition, mutations, etc.…”

Section: Resultsmentioning

confidence: 99%

“…After washing with PBS containing 0.05% Tween 20, the wells were incubated with different anti-ACE mAbs (2 µg/ml) directed to 16 different epitopes on the N and C domain of ACE [15]–[16], [30], [32]–[34] in PBS + 0.1 mg/ml BSA for 2 hrs at RT and washed. Wells were then incubated with 50 µl of soluble ACE secreted from CHO cells transfected with wild type or mutant ACE or with lysates from these cells, resembling a membrane-bound form of ACE (wt and mutant).…”

Section: Methodsmentioning

confidence: 99%

Angiotensin I-Converting Enzyme Gln1069Arg Mutation Impairs Trafficking to the Cell Surface Resulting in Selective Denaturation of the C-Domain

et al. 2010

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BackgroundAngiotensin-converting enzyme (ACE; Kininase II; CD143) hydrolyzes small peptides such as angiotensin I, bradykinin, substance P, LH-RH and several others and thus plays a key role in blood pressure regulation and vascular remodeling. Complete absence of ACE in humans leads to renal tubular dysgenesis (RTD), a severe disorder of renal tubule development characterized by persistent fetal anuria and perinatal death.Methodology/Principal FindingsPatient with RTD in Lisbon, Portugal, maintained by peritoneal dialysis since birth, was found to have a homozygous substitution of Arg for Glu at position 1069 in the C-terminal domain of ACE (Q1069R) resulting in absence of plasma ACE activity; both parents and a brother who are heterozygous carriers of this mutation had exactly half-normal plasma ACE activity compared to healthy individuals. We hypothesized that the Q1069R substitution impaired ACE trafficking to the cell surface and led to accumulation of catalytically inactive ACE in the cell cytoplasm. CHO cells expressing wild-type (WT) vs. Q1069R-ACE demonstrated the mutant accumulates intracellularly and also that it is significantly degraded by intracellular proteases. Q1069R-ACE retained catalytic and immunological characteristics of WT-ACE N domain whereas it had 10–20% of the nativity of the WT-ACE C domain. A combination of chemical (sodium butyrate) or pharmacological (ACE inhibitor) chaperones with proteasome inhibitors (MG 132 or bortezomib) significantly restored trafficking of Q1069R-ACE to the cell surface and increased ACE activity in the cell culture media 4-fold.Conclusions/SignificanceHomozygous Q1069R substitution results in an ACE trafficking and processing defect which can be rescued, at least in cell culture, by a combination of chaperones and proteasome inhibitors. Further studies are required to determine whether similar treatment of individuals with this ACE mutation would provide therapeutic benefits such as concentration of primary urine.

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“…We think that some of the changes in pattern of ACE immunoprecipitation by 16 mAbs seen in mutants lacking transmembrane anchor are likely due to the changes in glycosylation pattern of mutated ACE. The time of traffic of mutated ACEs through endoplasmic reticulum is much shorter than a wild-type ACE [41]–[42], [29] and as a result the glycosylation pattern of mutated ACEs is different from wild-type ACE; this could result in differential binding of some mAbs (i1A8, i2H5, and 5F1), because glycosylation sites on Asn residues are included in their epitopes [40], [43]–[45].…”

Section: Resultsmentioning

confidence: 99%

Angiotensin I-Converting Enzyme Mutation (Trp1197Stop) Causes a Dramatic Increase in Blood ACE

et al. 2009

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BackgroundAngiotensin-converting enzyme (ACE) metabolizes many peptides and plays a key role in blood pressure regulation and vascular remodeling. Elevated ACE levels may be associated with an increased risk for different cardiovascular or respiratory diseases, including asthma. Previously, a molecular mechanism underlying a 5-fold familial increase of blood ACE was discovered: Pro1199Leu substitution enhanced the cleavage-secretion process. Carriers of this mutation were Caucasians from Europe (mostly Dutch) or had European roots.Methodology/Principal FindingsWe have found a family of African-American descent whose affected members' blood ACE level was increased 13-fold over normal. In affected family members, codon TGG coding for Trp1197 was substituted in one allele by TGA (stop codon). As a result, half of ACE expressed in these individuals had a length of 1196 amino acids and lacked a transmembrane anchor. This ACE mutant is not trafficked to the cell membrane and is directly secreted out of cells; this mechanism apparently accounts for the high serum ACE level seen in affected individuals. A haplotype of the mutant ACE allele was determined based on 12 polymorphisms, which may help to identify other carriers of this mutation. Some but not all carriers of this mutation demonstrated airflow obstruction, and some but not all have hypertension.Conclusions/SignificanceWe have identified a novel Trp1197Stop mutation that results in dramatic elevation of serum ACE. Since blood ACE elevation is often taken as a marker of disease activity (sarcoidosis and Gaucher diseases), it is important for clinicians and medical scientists to be aware of alternative genetic causes of elevated blood ACE that are not apparently linked to disease.

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Fine Epitope Mapping of Monoclonal Antibody 5F1 Reveals Anticatalytic Activity toward the N Domain of Human Angiotensin-Converting Enzyme

Cited by 25 publications

References 44 publications

An Angiotensin I-Converting Enzyme Mutation (Y465D) Causes a Dramatic Increase in Blood ACE via Accelerated ACE Shedding

An Angiotensin I-Converting Enzyme Mutation (Y465D) Causes a Dramatic Increase in Blood ACE via Accelerated ACE Shedding

Angiotensin I-Converting Enzyme Gln1069Arg Mutation Impairs Trafficking to the Cell Surface Resulting in Selective Denaturation of the C-Domain

Angiotensin I-Converting Enzyme Mutation (Trp1197Stop) Causes a Dramatic Increase in Blood ACE

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