Angiotensin-converting enzyme-like activity in crab gills and its putative role in degradation of crustacean hyperglycemic hormone

Archives of Insect Biochemistry and Physiology 68:171–180 (2008) Angiotensin-Converting Enzyme-Like Activity in CrabGills and Its Putative Role in Degradation ofCrustacean Hyperglycemic Hormone J. Sook Chung1* and S.G. Webster2
Angiotensin-converting enzyme-like enzyme activity (ACELA) was found in Carcinus maenas using reverse phase high perfor-mance liquid chromatography (RP-HPLC) analysis of degradation kinetics of a synthetic substrate (Hippuryl-histidyl-leucine)and a specific inhibitor (captopril). Gills contained the highest ACELA, then brain, muscle, and testis, respectively, while noactivity was detected in the following tissues: hepatopancreas, hindgut, hypodermis, heart, and hemolymph. ACELA present ingill membranes exhibited a Km of 0.23 mM and Vmax of 7.6 nmol with synthetic substrate. The enzyme activity was dependenton Cl– concentration and was markedly inhibited by captopril, lisinopril, and EDTA. Addition of Zn2+ to membranes previouslytreated with EDTA restored 89% activity, suggesting that C. maenas ACELA is a Zn2+ metalloenzyme. Gill membranes pre-pared from premolt crabs showed similar levels of ACELA to those of the intermolt animals. Administration of captopril in vivolengthened the half life of circulating CHH, while in vitro incubation of gill membranes with captopril reduced CHH. Theseresults suggest that C. maenas ACELA present in gills is likely to be involved in degradation of this neuropeptide. Arch. InsectBiochem. Physiol. 68:171–180, 2008.
KEYWORDS: angiotensin-converting enzyme-like activity; crab gills; crustacean hyperglycemichormone; inactivation of hormone INTRODUCTION
iting hormone (MIH), and crustacean cardioactivepeptide (CCAP) in hemolymph are low, ranging in concentration from <10–11 to 10–10 M (Webster, 1996; lymph or blood are transient and at low concentra- Chang et al., 1998; Chung et al., 1999; Webster and tion, yet they are crucial for initiating signalling Chung, 1999; Phlippen et al., 2000; Chung and pathway resulting in physiological action. In crus- Webster, 2005; Nakatsuji and Sonobe, 2003).
taceans, crustacean hyperglycemic hormone (CHH) Compared with crustacean neuropeptides (5– is usually released in immediate response to stresses 10 min), insect neuropeptides, despite relatively (e.g., hyperthermal, hypoxia, and emersion), but smaller size, appear to have much longer half lives stays only a short time in the hemolymph (Webster, in circulation. For example, adipokinetic hormone, 1996; Chang et al., 1998; Chung and Webster, hypertrehalosemic hormone, allatostatins, and 2005). The circulating levels of CHHs, molt inhib- bombyxin, when injected into various species of 1Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, Maryland2School of Biological Sciences, Bangor University, Bangor, Gwynedd, United Kingdom Grant sponsor: Biotechnology and Biological Sciences Research Council, UK.
Abbreviations used: ACE = angiotensin converting enzyme (dipeptidyl carboxypeptidase I, EC; AG-I = angiotensin-I; CHH = crustaceanhyperglycemic hormone.
*Correspondence to: J. Sook Chung, Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, MD 21202.
E-mail: Received 1 December 2007; Accepted 4 March 2008 2008 Wiley-Liss, Inc.
DOI: 10.1002/arch.20247Published online in Wiley InterScience ( insects, showed half lives of approximately 18– Here we report that ACELA is, indeed, present 53 min (Rayne and O’Shea, 1992; Strey et al., 1993; in gills of C. maenas. The results obtained from in Garside et al., 1997; Goldsworthy et al., 2002) and vitro incubation of CHH with gill membranes and up to 170 min (Suenobu et al., 2004) in hemo- an in vivo half life study suggest that ACELA may lymph. Such short half lives of crustacean neuro- be an enzyme involved in regulation of CHH titres peptides, compared to insects, imply that crustaceans in hemolymph, and CHH can be an endogenous may have mechanisms to rapidly remove circulat- ing neuropeptides. However, very little is knownabout enzymes and tissues in crustaceans that MATERIALS AND METHODS
may be involved in processes of metabolic inac- tivation, degradation, or in clearance of circu-lating neuropeptides.
C. maenas were collected with baited traps Much of the information available in inverte- from the Menai Strait (Wales, UK). They were kept brates regarding the degradation of neuropeptides in re-circulated seawater at 12–15°C under ambi- or neurohormones was initially gathered through ent light conditions. Adult male crabs at intermolt homologous studies of vertebrate systems. For ex- (carapace width: 45–55 mm) were used for all ample, angiotensin-converting enzyme (ACE) activity in invertebrates has been suggested to inac-tivate neuropeptides (Lamango and Issac, 1994; Chemicals
Laurent and Salzet, 1996a,b; Lamango et al., 1996).
Moreover, the wide distribution of ACE activity, in- Hippuryl-1-histidyl-1-leucine (Hip-His-Leu), cluding central nervous tissues, reproductive tissues, hippuric acid, captopril {1-[(2S)-3-mercapto-2 hemocytes, and hemolymph, may further implicate methylproprionyl]-L-proline}, and lisinopril {(S)- ACE in a putative role in other physiological actions 1{N2-(1-carboxy-3-phenylpropyl)-L-lysyl]-L-pro- such as metamorphosis, reproduction, and immu- line dihydrate} were purchased from Sigma (St.
nity in corresponding tissues (Vandenbroeke et al., Louis, MO). Angiotensin I was obtained from 1997; Hens et al., 2002; Vandingenen et al., 2002; Ekbote et al., 2003; Salzet and Verger-Bocquet, 2001;Macours et al., 2003; Laurent et al., 1997; Leung et Purification, Quantification,
al., 1992). In crustaceans, angiotensin-converting and Iodination of CHH
enzyme-like activity (ACELA) was initially reportedin the gills of the blue crab, Callinectes sapidus The methods for isolation, purification, and (Smiley and Doig, 1994) and in the terrestrial crab, quantification of C. maenas CHH and iodination Chasmagnathus granulatus (Delorenzi et al., 1996).
procedures were as described in Webster (1993).
Our initial clearance experiment to determine a half-life of CHH using [125I]CHH (previously in- Briefly, the extracts of sinus glands were first iso- jected into crabs) showed that it was rapidly re- lated on a Phenyl column (4.6 × 250 mm, Wa- moved from hemolymph, and accumulated in the ters) using a gradient 30–80% B over 60 min at a gills. This result indicated that gills may play a role flow rate of 1.0 ml/ min. (A = 0.11% trifluoroacetic in degradation of CHH. Our preliminary observa- acid [TFA] in H2O; B = 0.1% TFA in 60% acetoni- tion, together with previous reports that ACE ac- trile.) The fractions containing CHH were then tivity was found in gills of other species of crabs pooled and re-run on a C18 column (4.6 × 250 (Smiley and Doig, 1994; Delorenzi et al., 1996), mm, Phenomenex) using a gradient 45–60% B led us to investigate ACELA in C. maenas gills and over 30 min at same flow rate. Peaks were manu- its putative role in regulating neuropeptide levels ally collected. The quantification of neuropeptides was performed by amino acid analysis of hydroly- Archives of Insect Biochemistry and Physiology sates obtained by gas-phase hydrolysis in vacuo gently overlaid on 200 µl of the same buffer con- at 150°C for 1 h using azeotropic hydrochloric taining 0.06% (w/v) Triton X-114 and 6% (w/v) acid containing a trace of phenol. Vacuum-dried sucrose and incubated for 5 min at 37°C. Phase hyrolysates were then quantified for amino acid separation of Triton X-114 was obtained by imme- composition by o-phthalaldehyde pre-column diate centrifugation at 13,000 rpm for 5 min at derivertization. Then, 300 pmol of CHH was iodi- 20°C. The supernatant was collected and the de- nated with 300 µCi of [125I](Amersham) using the tergent layer was re-suspended in an equal volume of buffer (50 mM Tris/HCl, 150 mM NaCl, pH7.4). Both detergent-rich and detergent-poor frac- Preparation of Membranes
Anterior and posterior gills were dissected from ACE Assay
crabs (n = 6), after being pre-chilled on ice for 30min. After rinsing in ice-cold crustacean saline (Webster and Keller, 1986), gills were homogenized brane preparations (20 µg/20 µl final incubation with a Polytron® in ice-cold buffer containing 50 volume) in the presence of 3 nmol of Hip-His- mM HEPES, either in the presence or absence of Leu in the assay buffer described above, either in 300 mM NaCl (pH 8.3). Homogenates were cen- the presence or absence of captopril (final concen- trifuged at 1,000g for 10 min at 4°C and superna- tration at 18.75 µM) for 30 min at 37°C. The en- tants were further centrifuged at 30,000g for 40 min zyme activity was calculated by the estimation of at 4°C. The resulting pellets, once re-suspended in the difference between amounts of remaining Hip- 50 mM HEPES, were estimated for protein con- His-Leu at times 0 and 30 min after incubation.
centration using a modified Lowry method (Lowry At the beginning or end of incubation, samples et al., 1951). Aliquots of membrane protein (120– were acidified with glacial acetic acid (to give a 150 µg/µl) were immediately frozen in liquid ni- final concentration of 2 N in 100 µl final volume).
trogen and kept at –20°C until used.
The samples were mixed and centrifuged at 13,000rpm for 5 min at room temperature. The superna- Treatment of Gill Membranes With
tant (~100 µl) was applied onto a RP-HPLC con- Ethyldiaminetetraacetic Acid (EDTA)
nected to a C18 column (Bakerbond WP C18, 4.6× 250 mm, J.T. Baker Co.), with a gradient of 30– Membranes, pre-incubated with 5 mM EDTA in 80% B over 30 min (A = 0.11% TFA in H2O, B = the assay buffer (50 mM HEPES and 300 mM NaCl, 0.1% TFA in 60% acetonitrile). Absorbance was pH 8.3) for 30 min at 37°C, were centrifuged at monitored at 210 nm with a flow rate of 1 ml/ min.
30,000g for 40 min at 4°C. Pellets were washed The samples of AG-I and CHH were chromato- twice with the assay buffer and the final pellets were re-suspended in the same buffer.
Degradation of Angiotensin-I and CHH
Phase Separation of Membrane Proteins
by C. maenas ACELA in Gills and
With Triton X-114
Amino Acid Analysis of Peaks
Membranes were initially incubated on ice (30 Substrate specificity of C. maenas ACELA in gills min with occasional shaking) with 10 mM Tris was tested using synthetic ACE substrate, AG I, and buffer (pH 7.4) containing 150 mM NaCl and CHH. Each substrate (3 nmol) except CHH (300 1.5% (w/v) Triton X-114. Detergent-insoluble ma- pmol) was incubated with a membrane concen- terial was removed by centrifugation at 30,000g for tration of 40 µg/50 µl at 37°C for 30 min, either 40 min at 4°C. Portions of the supernatant were in the presence or in the absence of captopril (360 Archives of Insect Biochemistry and Physiology µM). The samples were treated as described above Gill membranes prepared from premolt crabs for RP-HPLC separation. The degradation rate of showed similar levels of enzyme activity (0.995 ± each substrate was calculated by comparing peak 0.025 nmol/min/mg protein) to that of intermolt areas of substrate in the presence or in the absence (1.044 ± 0.05 nmol/min/mg protein).
of captopril. Peaks were collected manually andanalyzed for amino acid composition as described Characteristics of ACELA in Crab Gills
The saturation curve analyses of Lineweaver- Statistics
Burk plot (Fig. 2) of the hydrolysis of Hip-His-Leuby ACELA in gill preparation gave values of Km, Statistical analysis was carried out using either 0.23 ± 0.012 mM and Vmax, 7.6 nmol/min/mg Student’s t-test or ANOVA (GraphPad InStat ver- protein, respectively. All substrate concentrations sion 3.0, GraphPad Software, San Diego, CA).
used were well below the Km value and in no casewas more than 10% of the substrate consumed dur- ing the experimental period. Captopril inhibited The Tissue Distribution of ACELA in Crabs
ACE activity in a dose-dependent manner with avalue of IC50 < 1 µM (Fig. 2B).
The following tissues from male intermolt C. To determine the effect of Cl– concentration on maenas were examined for ACELA: muscle (from the hydrolysis of ACE substrate by gill membrane walking legs), hypodermis, hindgut, heart, midget preparations, NaCl was added to the incubation gland, brain, hemolymph, and gills. Of these tis- medium to give final concentrations of Cl– rang- sues tested, gills displayed by far the highest level ing from 0 to 300 mM. ACELA was highly depen- of ACELA (Fig. 1). Brains, and to a lesser extent dent on Cl– concentrations. The maximal activity testis and muscle membranes, showed much less was observed at 25 mM of Cl–, double the activity enzyme activity than that of gill membranes (Fig.
compared to that at 0 mM (Fig. 3). Increasing Cl– 1). Gill membranes prepared from the edible crab, concentration to over 300 mM did not change the Cancer pagurus, also exhibited ACELA, with approxi- mately 30% of that in C. maenas gills.
A: Michaelis-Menten plot of the hydrolysis of
Tissue distribution of ACELA. Enzyme activity synthetic ACE substrate by Carcinus gill membranes. B:
(pmol/min/mg protein) was presented as mean ± 1 SE, n ACELA was inhibited by captopril in a dose-dependent = 5–8. ND = non-detected. Statistical significance was manner. Each point in A and B was shown as mean ± 1 tested using nonparametric ANOVA (Kruskal-Wallis Test).
Archives of Insect Biochemistry and Physiology In Vivo Clearance Rate of CHH
Initially, 120 fmol of [125I] CHH (~200,000 DPM) was injected into crabs. Two minutes afterinjection (to allow distribution of the injected bo-lus), the radioactivity in the hemolymph was mea-sured and was considered to be at a reference time0 (100%). At 5, 10, and 30 min, small hemolymphsamples (100 µl) were removed and radioactivitywas measured by a gamma counter (Wallac 1470).
The hormone was cleared very rapidly, with an es-timated half-life of 8 min (Fig. 4). When crabs werefirst injected with captopril, to give an estimated Effect of Cl– concentrations on ACELA in gills.
circulating concentration of 500 µM, followed by Each point was presented as mean ± 1 SE (n = 4–6).
hormone injection, the half-life increased to 13min. At sampling points of 15 and 30 min, crabs Effect of a Divalent Chelating Agent (EDTA) and
injected with captopril showed a significantly high Divalent Metal Ions on the ACELA
recovery of [125I]CHH in their hemolymphs com-pared to controls (P > 0.01 and P > 0.05, respec- EDTA (100 µM) -treated gill membranes con- tively). Subsequent dissection of major tissues from tained 21% of enzyme activity compared with un- these crabs (gills, hindgut, hepatopancreas, testis, treated controls. Inclusion of the following bivalent heart, muscle, and hypodermis) followed by the metal ions (Co2+, Cu2+, Ca2+, Mg2+, Mn2+, Fe2+, andZn2+) was carried out to determine whether enzymeactivity could subsequently be retrieved from thegill membranes pre-treated with 5 mM of EDTA. Theaddition of 100 µM of either Zn2+ or Co2+ restoredapproximately 89 and 56% of enzyme activity, whileMn2+, Mg2+, or Fe2+ application regained less activ-ity (37, 19, and 13%, respectively). In contrast, theremaining ACELA in the membrane pre-treatedwith EDTA was completely abolished by the addi-tion of 100 µM of Cu+.
Phase Separation of C. maenas ACELA
The phase separation property of Triton X-114 The clearance of [125I] CHH from hemolymph, at >30°C in partitioning hydrophobic proteins into in vivo. Animals were injected with [125I]CHH and the detergent-rich phase has been used for isolat- hemolymphs were removed at the sampling points. Thesamples were counted for the radioactivity remaining in ing integral membrane proteins. ACELA was esti- hemolymphs. Closed circles: In the absence of captopril; mated after gill membrane was treated with Triton Open circles: in the presence of captopril. Each point was X-114, to determine if this enzyme activity was shown as mean ± 1 SE (n = 8). Statistical significance was associated with an integral membrane protein.
obtained by the comparison between in the absence and Approximately 80% of the activity was in the de- the presence of captopril at sampling points of 15 min tergent-poor phase, while the remaining activity (**) and 30 min (*), respectively. **P < 0.005, *P < 0.05 (20%) was recovered from the detergent-rich phase.
Archives of Insect Biochemistry and Physiology measurement of residual radioactivity revealed thatalmost 98% of the radioactivity was found in gills.
The radioactivity was equally distributed over gillpairs, and there was no difference between ante-rior and posterior gills (Fig. 5).
Degradation of Angiotensin-I and CHH by
C. maenas ACELA in Gills

To determine substrate specificity of ACELA in C. maenas gills, Hip-His-Leu, AG-I, and CHH wereincubated with gill membranes in the presence ofcaptopril. The presence of captopril at a concen-tration of 360 µM reduced the degradations of Hip-His-Leu by 46%, of AG-I, by 13%, and of CHH by83%. Peaks identified by amino acid analyses showin Figure 6A that C. maenas ACELA processed Ag-I(peak 4) to Ag-II (peak 3) by cleaving off His-Leu-OH (peak 1) from the C-terminus. Peak 2 (AG-III), the product of peak 3, was further cleaved byaminopeptidase. Amino acid composition of peak1 of Figure 6B (Table 1) showed that it was a de-graded product of CHH, while peak 2 was identi-fied as intact CHH.
RP-HPLC chromatogram showed cleavage of AG- I (A) and CHH (B) after incubation with membranes pre-
pared from gills. The results of amino acid composition
of each peak are as follows. A: Peak 1: His-Leu-OH; Peak
2 (AG-III): Arg-Val-Tyr-Ile-His-Pro-Phe-OH; Peak 3 (AG-
II): Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-OH; and Peak 4: H-
Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-OH. B: Peak 1:
Digested CHH; Peak 2: CHH intact. *Captopril. Note that
the peaks obtained from each HPLC do not accurately
represent amount of product formed due to a difference
in extinction coefficients.
The distribution of injected [125I] CHH in gills.
Gills were collected 30 min after the injection of radioac-tive CHH. After rinsing and drying, the radioactivity was The present study using the synthetic substrate measured. Anterior gills (4) were presented as A1–A4; pos-terior gills, P1–P4. Bar was presented as mean of mammalian ACE (Hip-His-Leu-OH) and cap- 8). There was no statistical significance (at P < 0.05) topril, a selective specific inhibitor, demonstrated among the values of [125I] CHH obtained from gills (non- ACELA in gill, brains, and testis of the shore crab, parametric ANOVA, Kruskal-Wallis Test).
C. maenas. The highest ACELA was detected in the Archives of Insect Biochemistry and Physiology TABLE 1. The Amino Acid Composition of Each Peak (Fig. 6A and B)* 1997). However, ACELA was not detected in C. maenas hemolymph, while similar activity was de- scribed in hemolymph of Mytilus edulis (Leung et By injection of [125I] CHH into C. maenas, it was possible to monitor the clearance rate from hemolymph as well as tissue-specific accumulation of this radiolabelled peptide. The measured half- life of C. maenas [125I] CHH in hemolymph was 5–10 min, in agreement with our previous results obtained from non-radiolabelled CHH injection into crabs (Webster, 1996; Chung and Webster, 1996). However, in the presence of captopril, the half-life of this peptide was increased by more than *Amino acids (AA) are presented as three letter abbreviation. Numbers shown in 5 min. Over 99% of the radioactivity was recov- upper layer were obtained from the amino acid analysis of the peaks shown inFigure 6A and B, while those in parentheses are based on the amino acid se- ered from the gill tissues, in which it was distrib- quences of peptides and their fragments.
a uted equally between the left and right sides of the gills. Posterior gills showed slightly higher ra-dioactivity than those of anterior gills, but the dif- gill membranes. Enzyme activities in gills of pre- ference was not statistically different. This implies molt crabs were similar to activities of intermolt that the distribution of ACELA is ubiquitous, animals, suggesting that this enzyme is probably throughout posterior and anterior gills.
not involved in the molting process. The wide dis- C. maenas ACELA in gills produced AG-II by tribution of ACELA in various tissues of C. maenas cleaving the dipeptide from the C terminus of AG- is in agreement with previous results found in the I (Fig. 6A). Interestingly, AG-II was further pro- blue crab, C. sapidus (Smiley and Doig, 1994). In- cessed by an aminopeptidase that was co-present terestingly in C. granulatus, ACELA found in CNS in gill membranes, yielding a further product, and gills was co-exited with AGII (Delorenzi et al., peak 2 (AG-III). The combination of selective in- 1996). Additionally, the gill membranes of the ed- hibitors of neprilysin (phosphoramidon and 1,10 ible crab, C. pagurus, also exhibited ACELA, sug- o-phenanthroline) and aminopeptidase (ama- gesting that ACELA in gills may be a common statin) with these substrates showed that each of these peptides can be degraded by more than one Based on our results, the properties of C. maenas enzyme in the tissue membrane extract (Masler ACELA were also similar to those of mammalians et al., 1996; Leung et al., 1992). Surely, synthetic and invertebrates including houseflies and leeches ACE substrate containing a single ACE-specific (Salzet and Verger-Bocquet, 2001, Ekbote et al., cleavage site can be the only substrate for ACE, 2003; Macours et al., 2003). The identical proper- whereas AG-I might be a substrate for neprilysin ties are listed as follows: (1) Zn2+ metalloenzyme, in the presence of captopril. As a result, the in- (2) captopril and lisinopril sensitive, and (3) Cl–- hibitory effect of captopril on AG-I degradation dependent activity. Furthermore, C. maenas ACELA was much smaller than that of the synthetic sub- seemed to be a membrane-bound protein, but it strate. This result is congruent with a finding from could be easily dissociated from the membrane.
the leech (Ekbote et al., 2003), also suggesting After phase separation with Triton X-114, most of the presence of multiple enzymes in the gill mem- the enzyme activity was found in the detergent- poor phase, like the one in the leech and housefly The highest inhibition (83%) of ACE in the pres- (Salzet and Verger-Bocquet, 2001; Laurent et al., ence of captopril was observed with C. maenas CHH Archives of Insect Biochemistry and Physiology pathway of AG-I (A) and pu-
tative cleavage sites of ACELA
in CHH (B). Amino acids itali-
cized: cleavage sites; amino ac-
ids underlined: parts possibly
compared with AG-I and synthetic ACE substrate.
in gills agreed with a broad range of enzyme activ- Considering the nature of known ACE substrates, ity including endopeptidase function on a wide CHH would seem an unlikely candidate since it is substrate specificity (Isaac et al., 1998).
large (72 amino acid residues), blocked at both Crustacean gills, which are multifunctional tis- N- and C-termini, and has three intradisulphide sues composed of at least six different cell types bridges (Kegel et al., 1989). The solution structures (Mantel and Farmer, 1983; Lawson et al., 1994), of CHH neuropeptides (CHH and MIH) obtained seem to serve as a target tissue of the CHH recep- by circular dichroism consisted of a very high per- tor (Chung and Webster, 2006) as well as a site of centage of α-helices and β-strands (Chung and degradation. It will be of interest to localize the Webster, 1996; Katayama et al., 2003). A close ex- amination of the primary sequence of CHH reveals In conclusion, we propose that ACELA present that there are more than five putative ACE cleav- in the gill membranes of C. maenas participates in age sites (Fig. 7B), but these putative cleavage sites regulating the levels of circulating neuropeptides.
may not be readily available to ACE since they may Additionally, since ACELA is found in the gills of not be exposed to the surface of the molecule. Nev- following species, C. maenas, C. pagurus, C. sapidus, ertheless, our preliminary data obtained from the and C. granulatus, we believe that this enzyme may amino acid analysis of two peaks of CHH cleaved be ubiquitously present in all crustaceans with at by ACELA indicated that the cleavage occurred at least a function in the processing/inactivation/deg- a part of the C-terminus and potentially the area radation of circulating neuropeptides.
between disulphide bridges, resulting in frag-mented and inactivated CHH. However, we are un- ACKNOWLEDGMENTS
able to locate the exact cleavage site of CHH. Morework is required to obtain detailed information on This work was supported by a grant to S.G.W.
the degradation pathway of CHH. Our initial study from the Biotechnology and Biological Sciences into the substrate specificity of crab ACELA present Archives of Insect Biochemistry and Physiology LITERATURE CITED
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Archives of Insect Biochemistry and Physiology


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