A model of the ACE2 structure and function as a SARS-CoV receptor

The angiotensin-converting enzyme 2 (ACE2) is an important regulator of the renin-angiotensin system and was very recently identified as a functional receptor for the SARS virus. The ACE2 sequence is similar (sequence identities 43% and 35%, and similarities 61% and 55%, respectively) to those of the testis-specific form of ACE (tACE) and the Drosophila homolog of ACE (AnCE). The high level of sequence similarity allowed us to build a robust homology model of the ACE2 structure with a root-mean-square deviation from the aligned crystal structures of tACE and AnCE less than 0.5A. A prominent feature of the model is a deep channel on the top of the molecule that contains the catalytic site. Negatively charged ridges surrounding the channel may provide a possible binding site for the positively charged receptor-binding domain (RBD) of the S-glycoprotein, which we recently identified [Biochem. Biophys. Res. Commun. 312 (2003) 1159]. Several distinct patches of hydrophobic residues at the ACE2 surface were noted at close proximity to the charged ridges that could contribute to binding. These results suggest a possible binding region for the SARS-CoV S-glycoprotein on ACE2 and could help in the design of experiments to further elucidate the structure and function of ACE2.     

Structural details on the binding of antihypertensive drugs captopril and enalaprilat to human testicular angiotensin I-converting enzyme

Angiotensin converting enzyme (ACE) plays a critical role in the circulating or endocrine renin-angiotensin system (RAS) as well as the local regulation that exists in tissues such as the myocardium and skeletal muscle. Here we report the high-resolution crystal structures of testis ACE (tACE) in complex with the first successfully designed ACE inhibitor captopril and enalaprilat, the Phe-Ala-Pro analogue. We have compared these structures with the recently reported structure of a tACE-lisinopril complex [Natesh et al. (2003) Nature 421, 551-554]. The analyses reveal that all three inhibitors make direct interactions with the catalytic Zn(2+) ion at the active site of the enzyme: the thiol group of captopril and the carboxylate group of enalaprilat and lisinopril. Subtle differences are also observed at other regions of the binding pocket. These are compared with N-domain models and discussed with reference to published biochemical data. The chloride coordination geometries of the three structures are discussed and compared with other ACE analogues. It is anticipated that the molecular details provided by these structures will be used to improve the binding and/or the design of new, more potent domain-specific inhibitors of ACE that could serve as new generation antihypertensive drugs.     

High-Resolution Crystal Structures of Drosophila melanogaster Angiotensin-Converting Enzyme in Complex with Novel Inhibitors and Antihypertensive Drugs

Angiotensin I-converting enzyme (ACE), one of the central components of the renin-angiotensin system, is a key therapeutic target for the treatment of hypertension and cardiovascular disorders. Human somatic ACE (sACE) has two homologous domains (N and C). The N- and C-domain catalytic sites have different activities toward various substrates. Moreover, some of the undesirable side effects of the currently available and widely used ACE inhibitors may arise from their targeting both domains leading to defects in other pathways. In addition, structural studies have shown that although both these domains have much in common at the inhibitor binding site, there are significant differences and these are greater at the peptide binding sites than regions distal to the active site. As a model system, we have used an ACE homologue from Drosophila melanogaster (AnCE, a single domain protein with ACE activity) to study ACE inhibitor binding. In an extensive study, we present high-resolution structures for native AnCE and in complex with six known antihypertensive drugs, a novel C-domain sACE specific inhibitor, lisW-S, and two sACE domain-specific phosphinic peptidyl inhibitors, RXPA380 and RXP407 (i.e., nine structures). These structures show detailed binding features of the inhibitors and highlight subtle changes in the orientation of side chains at different binding pockets in the active site in comparison with the active site of N- and C-domains of sACE. This study provides information about the structure-activity relationships that could be utilized for designing new inhibitors with improved domain selectivity for sACE.     

Crystal structure of a phosphonotripeptide K-26 in complex with angiotensin converting enzyme homologue (AnCE) from Drosophila melanogaster

Angiotensin-I converting enzyme (ACE, a zinc dependent dipeptidyl carboxypeptidase) is a major target of drugs due to its role in the modulation of blood pressure and cardiovascular disorders. Here we present a crystal structure of AnCE (an ACE homologue from Drosophila melanogaster with a single enzymatic domain) in complex with a natural product-phosphonotripeptide, K-26 at 1.96 Å resolution. The inhibitor binds exclusively in the S₁ and S₂ binding pockets of AnCE (coordinating the zinc ion) through ionic and hydrogen bond interactions. A detailed structural comparison of AnCE·K-26 complex with individual domains of human somatic ACE provides useful information for further exploration of ACE inhibitor pharmacophores involving phosphonic acids.     

Structure of testis ACE glycosylation mutants and evidence for conserved domain movement

Human angiotensin-converting enzyme is an important drug target for which little structural information has been available until recent years. The slow progress in obtaining a crystal structure was due to the problem of surface glycosylation, a difficulty that has thus far been overcome by the use of a glucosidase-1 inhibitor in the tissue culture medium. However, the prohibitive cost of these inhibitors and incomplete glucosidase inhibition makes alternative routes to minimizing the N-glycan heterogeneity desirable. Here, glycosylation in the testis isoform (tACE) has been reduced by Asn-Gln point mutations at N-glycosylation sites, and the crystal structures of mutants having two and four intact sites have been solved to 2.0 A and 2.8 A, respectively. Both mutants show close structural identity with the wild-type. A hinge mechanism is proposed for substrate entry into the active cleft, based on homology to human ACE2 at the levels of sequence and flexibility. This is supported by normal-mode analysis that reveals intrinsic flexibility about the active site of tACE. Subdomain II, containing bound chloride and zinc ions, is found to have greater stability than subdomain I in the structures of three ACE homologues. Crystallizable glycosylation mutants open up new possibilities for cocrystallization studies to aid the design of novel ACE inhibitors.     

Homologous substitution of ACE C-domain regions with N-domain sequences: effect on processing, shedding, and catalytic properties

Angiotensin-converting enzyme (ACE) exists as two isoforms: somatic ACE (sACE), comprised of two homologous N and C domains, and testis ACE (tACE), comprised of the C domain only. The N and C domains are both active, but show differences in substrate and inhibitor specificity. While both isoforms are shed from the cell surface via a sheddase-mediated cleavage, tACE is shed much more efficiently than sACE. To delineate the regions of tACE that are important in catalytic activity, intracellular processing, and regulated ectodomain shedding, regions of the tACE sequence were replaced with the corresponding N-domain sequence. The resultant chimeras C1-163Ndom-ACE, C417-579Ndom-ACE, and C583-623Ndom-ACE were processed to the cell surface of transfected Chinese hamster ovary (CHO) cells, and were cleaved at the identical site as that of tACE. They also showed acquisition of N-domain-like catalytic properties. Homology modelling of the chimeric proteins revealed structural changes in regions required for tACE-specific catalytic activity. In contrast, C164-416Ndom-ACE and C191-214Ndom-ACE demonstrated defective intracellular processing and were neither enzymatically active nor shed. Therefore, critical elements within region D164-V416 and more specifically I191-T214 are required for the processing, cell-surface targeting, and enzyme activity of tACE, and cannot be substituted for by the homologous N-domain sequence.     

Molecular Dynamics Simulation and Molecular Docking Studies of Angiotensin Converting Enzyme with Inhibitor Lisinopril and Amyloid Beta Peptide

Angiotensin converting enzyme (ACE) cleaves amyloid beta peptide. So far this cleavage mechanism has not been studied in detail at atomic level. Keeping this view in mind, we performed molecular dynamics simulation of crystal structure complex of testis truncated version of ACE (tACE) and its inhibitor lisinopril along with Zn²⁺ to understand the dynamic behavior of active site residues of tACE. Root mean square deviation results revealed the stability of tACE throughout simulation. The residues Ala 354, Glu 376, Asp 377, Glu 384, His 513, Tyr 520 and Tyr 523 of tACE stabilized lisinopril by hydrogen bonding interactions. Using this information in subsequent part of study, molecular docking of tACE crystal structure with Aβ-peptide has been made to investigate the interactions of Aβ-peptide with enzyme tACE. The residues Asp 7 and Ser 8 of Aβ-peptide were found in close contact with Glu 384 of tACE along with Zn²⁺. This study has demonstrated that the residue Glu 384 of tACE might play key role in the degradation of Aβ-peptide by cleaving peptide bond between Asp 7 and Ser 8 residues. Molecular basis generated by this attempt could provide valuable information towards designing of new therapies to control Aβ concentration in Alzheimer’s patient.     

Dipeptidase-inactivated tACE action in vivo: selective inhibition of sperm-zona pellucida binding in the mouse

The angiotensin-converting enzyme (ACE) plays a crucial role in male fertilization and is a key regulator of blood pressure. Testicular ACE (tACE), the germinal specific isozyme expressed on different promoters, exclusively carries out the role of ACE in fertility, although the site and mode of action are not well known. To investigate the contribution of tACE in fertilization, we produced transgenic mouse lines carrying a dipeptidase-inactivated mutant. Although the transgenic mice showed normal blood pressure, kidney morphology, and fertility, reduced fertilization was observed after in vitro fertilization (IVF). The sperm-zona pellucida (ZP) binding was exclusively impaired in these lines in a manner similar to that observed in an Ace knockout mouse. The dipeptidase activity was reduced in epididymal ingredients but not in the testis. Furthermore, direct application of mutant protein did not suppress sperm-ZP binding of intact sperm during IVF, implying that the dipeptidase-inactivated mutant affects sperm modification in the epididymis for ZP binding. Our results indicate that the dipeptidase-inactivated tACE acts in vivo, suggesting that tACE contributes to fertilization as a dipeptidase at least in the epididymis.     

In vitro degradation of the Neb-Trypsin modulating oostatic factor (Neb-TMOF) in gut luminal content and hemolymph of the grey fleshfly, Neobellieria bullata

The unblocked hexapeptidic Trypsin Modulating Oostatic Factor of the fleshfly, an inhibitor of both trypsin and ecdysone biosynthesis, resists very well proteolytic breakdown by enzymes present in the lumen of the gut of previtellogenic fleshflies. However, when incubated in hemolymph of adult flies, females and males, its half-life time is a mere 0.5 min. In hemolymph of last instar larvae, this value increases to about 1.5 min. Whereas PMSF, a potent inhibitor of serine proteases has no effect, captopril and lisinopril, both known to be specific inhibitors of mammalian angiotensin I converting enzyme (ACE), effectively inhibit TMOF breakdown in fly hemolymph. Digestion of Neb-TMOF by recombinant Drosophila AnCE on itself results in identical degradation products as with total hemolymph. In both cases ESI-Qq-oa-Tof mass spectrometry demonstrated the appearance of peptide fragments with the sequences NPTN, LH and NP. These observations not only confirm the reported presence of circulating ACE-like activity in flies but also strongly suggest that in flies this hemolymph ACE-like activity might be involved in the regulation of the oostatic activity as exerted by Neb-TMOF.     

The structure of testis angiotensin-converting enzyme in complex with the C domain-specific inhibitor RXPA380

Angiotensin I-converting enzyme (ACE) is central to the regulation of the renin-angiotensin system and is a key therapeutic target for combating hypertension and related cardiovascular diseases. Currently available drugs bind both active sites of its two homologous domains, although it is now understood that these domains function differently in vivo. The recently solved crystal structures of both domains (N and C) open the door to new domain-specific inhibitor design, taking advantage of the differences between these two large active sites. Here we present the first crystal structure at a resolution of 2.25 A of testis ACE (identical to the C domain of somatic ACE) with the highly C-domain-specific phosphinic inhibitor, RXPA380. Testis ACE retains the same conformation as seen in previously determined inhibitor complexes, but the RXPA380 central backbone conformation is more similar to that seen for the inhibitor captopril than enalaprilat. The RXPA380 molecule occupies more subsites of the testis ACE active site than the previously determined inhibitors and possesses bulky moieties that extend into the S2' and S2 subsites. Thus the high affinity of RXPA380 for the testis ACE/somatic ACE C domain is explained by the interaction of these bulky moieties with residues unique to these domains, specifically Phe 391, Val 379, and Val 380, that are not found in the N domain. The characterization of the extended active site and the binding of a potent C-domain-selective inhibitor provide the first structural data for the design of truly domain-specific pharmacophores.     

Characteristics of monoclonal antibody binding with the C domain of human angiotensin converting enzyme

Binding of a panel of eight monoclonal antibodies (mAbs) with the C domain of angiotensin converting enzyme (ACE) to human testicular ACE (tACE) (corresponding to the C domain of the somatic enzyme) was studied and the inhibition of the enzyme by the mAb 4E3 was found. The dissociation constants of complexes of two mAbs, IB8 and 2H9, with tACE were 2.3 +/- 0.4 and 2.5 +/- 0.4 nM, respectively, for recombinant tACE and 1.6 +/- 0.3 nM for spermatozoid tACE. Competition parameters of mAb binding with tACE were obtained and analyzed. As a result, the eight mAbs were divided into three groups, whose binding epitopes did not overlap: (1) 1E10, 2B11, 2H9, 3F11, and 4E3; (2) 1B8 and 3F10; and (3) IB3. A diagram demonstrating mAb competitive binding with tACE was proposed. Comparative analysis of mAb binding to human and chimpanzee ACE was carried out, which resulted in revealing of two amino acid residues, Lys677 and Pro730, responsible for binding of three antibodies, 1E10, 1B8, and 3F10. It was found by mutation of Asp616 located close to Lys677 that the mAb binding epitope 1E10 contains Asp616 and Lys677, whereas mAbs 1B8 and 3F10 contain Pro730.     

Possible mechanism of captopril induced endothelium-dependent relaxation in isolated rabbit aorta

The mechanism of captopril, an angiotensin converting enzyme (ACE) inhibitor with sulfhydryl group (SH) in its structure, to produce an endothelium-dependent vasorelaxation was studied. In rabbit aorta with intact endothelium and precontracted with phenylephrine, captopril and superoxide dismutase (SOD) produced dose-dependent relaxation. Lisinopril, an ACE inhibitor without a -SH group in its structure, did not produce endothelium-dependent relaxation. It was observed that captopril, like SOD, produced the relaxation by protecting the EDRF from getting inactivated by superoxide anions as pyrogallol and methylene blue inhibited both the captopril and SOD-mediated relaxation. The free radical scavenging action of captopril is further substantiated by the observation that captopril, but not lisinopril, inhibited FeCl₃/ascorbic acid-induced lipid peroxidation in whole tissue homogenates of rabbit aorta to a level comparable to that of SOD. These results suggest that endothelium-dependent vasodilation produced by captopril may be due to its ability to scavenge superoxide anion and this property may be ascribed to the -SH group present in its structure.     

Characteristics of monoclonal antibody binding with the <Emphasis Type="Italic">C</Emphasis> domain of human angiotensin converting enzyme

Binding of a panel of eight monoclonal antibodies (mAbs) with the C domain of angiotensin converting enzyme (ACE) to human testicular ACE (tACE) (corresponding to the C domain of the somatic enzyme) was studied, and the inhibition of the enzyme by the mAb 4A3 was found. The dissociation constants of complexes of two mAbs, 1B8 and 2H9, with tACE were 2.3 ± 0.4 and 2.5 ± 0.4 nM, respectively, for recombinant tACE and 4.7 ± 0.5 and 1.6 ± 0.3 nM for spermatozoid tACE. Competition parameters of mAb binding with tACE were obtained and analyzed. As a result, the eight mAbs were divided into three groups, whose binding epitopes did not overlap: (1) 1E10, 2B11, 2H9, 3F11, and 4E3; (2) 1B8 and 3F10; and (3) 1B3. A diagram demonstrating mAb competitive binding with tACE was proposed. Comparative analysis of mAb binding to human and chimpanzee ACE was carried out, which resulted in revealing of two amino acid residues, Lys677 and Pro730, responsible for binding of three antibodies, 1E10, 1B8, and 3F10. It was found by mutation of Asp616 located close to Lys677 for Leu that the mAb binding epitope 1E10 contains Asp616 and Lys677, whereas mAbs 1B8 and 3F10 contain Pro730.     

The role of glycosylation and domain interactions in the thermal stability of human angiotensin-converting enzyme

Abstract The N and C domains of somatic angiotensin-converting enzyme (sACE) differ in terms of their substrate specificity, inhibitor profiling, chloride dependency and thermal stability. The C domain is thermally less stable than sACE or the N domain. Since both domains are heavily glycosylated, the effect of glycosylation on their thermal stability was investigated by assessing their catalytic and physicochemical properties. Testis ACE (tACE) expressed in mammalian cells, mammalian cells in the presence of a glucosidase inhibitor and insect cells yielded proteins with altered catalytic and physicochemical properties, indicating that the more complex glycans confer greater thermal stabilization. Furthermore, a decrease in tACE and N-domain N-glycans using site-directed mutagenesis decreased their thermal stability, suggesting that certain N-glycans have an important effect on the protein's thermodynamic properties. Evaluation of the thermal stability of sACE domain swopover and domain duplication mutants, together with sACE expressed in insect cells, showed that the C domain contained in sACE is less dependent on glycosylation for thermal stabilization than a single C domain, indicating that stabilizing interactions between the two domains contribute to the thermal stability of sACE and are decreased in a C-domain-duplicating mutant.     

ACE-dependent and chymase-dependent angiotensin II generation in normal and glucose-stimulated human mesangial cells

High glucose (HG) increases angiotensin II (AngII) generation in mesangial cells (MC). Chymase, an alternative AngII-generating enzyme, is upregulated in the glomeruli of diabetic kidneys. In this study, we examined AngII synthesis by human MC via angiotensin-converting enzyme (ACE)-dependent and chymase-dependent pathways under normal glucose (NG, 5 mM) and HG (30 mM) conditions. NG cells expressed ACE and chymase mRNA. Under NG conditions the chymase inhibitor chymostatin reduced AngII levels in cell lysates and in the culture medium, and the ACE inhibitor captopril had no effect. HG induced a 3-fold increase in chymase mRNA and protein but not in ACE mRNA; however, HG induced a 10-fold increase in intracellular ACE activity. The increase in AngII generation induced by HG was found in the cell lysate but not in the culture medium. The rise in intracellular AngII was not prevented by captopril or by chymostatin. Moreover, captopril inhibited extracellular ACE activity but failed to block intracellular ACE activity; these results suggested that captopril was unable to reach intra-cellular ACE. Losartan did not change the intracellular AngII content in either NG or HG conditions, and this lack of change suggested that the increase in AngII was due to intracellular generation. Together these results suggest that chymase may be active in human MC and that both ACE and chymase are involved in increased AngII generation during the HG stimulus by different mechanisms, including an upregulation of chymase mRNA and a rise in intracellular ACE activity, favoring the generation and accumulation of intracellular AngII.     

Inhibitor analysis of angiotensin I-converting and kinin-degrading activities of bovine lung and testicular angiotensin-converting enzyme.

Inhibition of bovine lung and testicular angiotensin-converting enzyme (ACE) by some well-known ACE inhibitors (lisinopril, captopril, enalapril), new substances (Nalpha-carboxyalkyl dipeptides PP-09, PP-35, and PP-36), and phosphoramidon was investigated using Cbz-Phe-His-Leu and FA-Phe-Phe-Arg (C-terminal analogs of angiotensin I and bradykinin, respectively) as the substrates. The somatic (two domains) and testicular (single domain) isoenzymes demonstrated different kinetic parameters for hydrolysis of these substrates. All of the inhibitors were competitive inhibitors of both ACE isoforms, and the Ki values were substrate-independent. The relative potencies of the inhibitors for both enzymes were: lisinopril > captopril > PP-09 > enalapril > PP-36 > PP-35 > phosphoramidon. The inhibition efficiency of PP-09 was comparable with those of the well-known ACE inhibitors. Captopril was more effectively bound to the somatic ACE (Ki = 0.5 nM) than to the testicular isoform (Ki = 6.5 nM).     

Presence of angiotensin converting enzyme isoforms in larval lepidoptera (Spodoptera littoralis)

In this research the presence of angiotensin converting enzyme (ACE) in larvae of the lepidopteran Spodoptera littoralis was evaluated. Making use of the substrate Abz-FRK-(Dnp)P-OH and the specific inhibitor captopril at 10 microM, ACE activity was determined in a fluorescence assay for intact larvae, hemolymph, head, midgut and dorsal tissue. In dorsal tissue and hemolymph, ACE activity was highest. These data are consistent with a possible role for ACE in contractions of the dorsal vessel and metabolism of circulating peptide hormones in the hemolymph. After the presence of ACE was confirmed, a sequential procedure of anion exchange and size exclusion chromatography was applied to purify ACE from whole wandering larvae (last stage). With this procedure, three different ACE pools were collected that cleaved the fluorogenic substrate Abz-FRK-(Dnp)P-OH. Activity could be inhibited by a final concentration of 2.5 microM captopril. In addition, two out of three samples eluted at different salt concentration and thus ACE 1, 2 and 3 represent at least two different ACE isoforms. These data reveal that ACE is present in S. littoralis and that at least two out of three isolated ACE forms are truly isoforms.     

In Vitro fertilization failure of normozoospermic men: search for a lack of testicular isozyme of angiotensin-converting enzyme

Background

Angiotensin converting enzyme (ACE) is a metalloprotease with two isoforms. The somatic isoform is a key component of the renin-angiotensin system; its main function is to hydrolyse angiotensin I into angiotensin II. The germinal or testicular isoform (tACE) located at the plasma membrane of the spermatozoa, plays a crucial role in the spermatozoa-oocyte interaction during in vivo fertilization, in rodents. Disruption of the tACE in mice has revealed that homozygous male tACE?/? sire few pups despite mating normally. Few spermatozoa from these tACE?/? mice are bound to the zona pellucida (ZP) despite normal semen parameters. Based on these findings in mice models, we hypothesized that some infertile men that have the same phenotype as the tACE?/? mice, ie normal semen parameters and a lack of sperm bind to the ZP in vitro, may have a tACE defect.

Methods

Twenty four men participated to this study. The case subjects (n?=?10) had normal semen parameters according to the WHO guidelines (WHO 1999) but a total in vitro fertilization failure with absence of sperm fixation to the ZP. The control subjects (n?=?14) also had normal semen parameters and a normal fertilization rate ≥65%. We investigated the tACE expression in spermatozoa by Western-Blot and performed a DNA sequencing of the tACE gene.

Results

Three case-subjects and one control-subject had no tACE expression. There were no statistic differences between the two groups. No mutation was detected in the tACE DNA sequence.

Conclusions

Our results didn’t show any involvement of tACE in human fertilization especially in ZP binding.     

The angiotensin converting enzyme inhibitor captopril reduces oviposition and ecdysteroid levels in Lepidoptera

The role of angiotensin converting enzyme (ACE, peptidyl dipeptidase A) in metamorphic- and reproductive-related events in the Egyptian cotton leafworm, Spodoptera littoralis (Lepidoptera, Noctuidae) was studied by using the selective ACE inhibitor captopril. Although oral administration of captopril had no effect on larval growth, topical administration to new pupae resulted in a large decrease of successful adult formation. Oviposition and overall appearance of adults emerging from treated larvae did not differ significantly from those emerging from non-treated larvae. In contrast, topical or oral administration of captopril to newly emerged adults caused a reduction in oviposition. By evaluating the effect of captopril on ecdysteroid titers and trypsin activity, we revealed an additional physiological role for ACE. Captopril exerted an inhibitory effect on ecdysteroid levels in female but not in male adults. Larvae fed a diet containing captopril exhibited increased trypsin activity. A similar captopril-induced increase in trypsin activity was observed in female adults. In male adults, however, captopril elicited reduced levels of trypsin activity. Our results suggest that captopril downregulates oviposition by two independent pathways, one through ecdysteroid biosynthesis regulation, and the other through regulation of trypsin activity. Apparently, fecundity is influenced by a complex interaction of ACE, trypsin activity, and ecdysteroid levels.     

Testicular Angiotensin-converting enzyme with different glycan modification: characterization on glycosylphosphatidylinositol-anchored protein releasing and dipeptidase activities

We have previously found that the angiotensin-converting enzyme (ACE) carries GPI-anchored protein releasing activity (GPIase) as well as dipeptidase activity. Testicular ACE (tACE), the male germinal specific isozyme, plays a crucial role in male fertilization. The amino-terminal region of this isozyme is different from that of somatic isozyme (sACE) and contains potential O-linked glycosylation sites. By multiple mutagenesis after an in silico prediction, amino acid residues acquiring O-glycans were assigned. Both GPIase and dipeptidase activities were compared between O-glycan null mutant and wild-type molecules, but no differences were found. Furthermore, the wild-type tACE was produced in two different cells (COS7 and CHO) and its activities compared. The GPIase activity, but not dipeptidase, was apparently higher for CHO-derived molecule than COS7. Sensitivity to neuraminidase and O-glycosidase digestions and the profile of glycosylation were quite different between these two molecules. Moreover, serial digestions with neuraminidase and O-glycosidase have no influence on GPIase activity of both molecules, suggesting that the sialylation and the presence of O-glycan has no influence on tACE enzyme activities, while the set of glycans modulate GPIase activity.