Sequence Analysis, Kinetic Constants, and Anion Inhibition Profile of the Nacrein-Like Protein (CgiNAP2X1) from the Pacific Oyster Magallana gigas (Ex-Crassostrea gigas)
Abstract
:1. Introduction
2. Results and Discussion
2.1. Amino Acid Sequence Analysis
2.2. Phylogenetic Analysis
2.3. Production and Characterization of the Recombinant Enzyme (CgiNAP2X1)
2.4. Enzyme Kinetics
2.5. Anion Inhibition Profile
- The anions with the smallest propensity to coordinate metal ions, perchlorate and tetrafluoroborate, and inhibitors such as fluorosulfonate, iminodisulfonate, perrhuthenate, perrhenate, bisulfite, divanadate, tellurate, azide, and cyanide were not inhibitors of CgiNAP2X1 up to concentrations of 100 mM. With the exception of perchlorate and tetrafluoroborate, a different profile was shown for the human isoforms hCA I and II (Table 3).
- Several other anions; fluoride, chloride, bromide, iodide, cyanate, thiocyanate, bicarbonate, carbonate, nitrate, nitrite, hydrogen sulfite, stannate, selenate, peroxydisulfate, tetraborate, selenocyanate, trithiocarbonate, and trifluorometansolfonato were also weak inhibitors of CgiNAP2X1, with inhibition constants in the range of 3.1–20.7 mM (Table 3). Interesting to note is that most of these inhibitors were micro and/or submicromolar inhibitors for the human enzymes. It should be noted that the weak affinity of CgiNAP2X1 for bicarbonate and carbonate might be a positive feature for the physiological role of this enzyme in the deposition of the molluskan shell, since the enzyme is not appreciably inhibited in the presence of rather high concentrations of these two anions. Moreover, for the same reason, the enzyme could have also a potential biotechnological application in the CO2 capture processes.
- The best anion inhibitors of CgiNAP2X1 were diethyldithiocarbamate, sulfamide, sulfamate, phenylboronic acid, and phenylarsonic acid, which showed a micromolar affinity for this enzyme, with KIs in the range of 76–87 μM (Table 3). Diethyldithiocarbamate was recently shown to lead to highly potent inhibitors directed against many α- or β-CAs, and some X-ray crystal structures also revealed their binding mode, which is achieved by coordination to the metal ion via a negatively charged sulfur atom [47,48,49,50,51]. It is conceivable that a similar binding mode is achieved also within the CgiNAP2X1 active site. Sulfamide and sulfamate also coordinate (via an ionized nitrogen atom) to the Zn(II) ion within the hCA II active site, and probably the same inhibition mechanism is valid against the oyster enzyme [47,48,49,50,51].
- Most of these anions are weak, millimolar CgiNAP2X1 inhibitors probably because the enzyme is in contact with sea water, rich in many inorganic anions (chloride, sulfate, etc.) which may interfere with the enzyme’s activity. Similar behavior was observed earlier for other marine organism CAs [37].
3. Materials and Methods
3.1. Gene Identification, Synthesis, and Cloning
3.2. Protein Expression and Purification
3.3. SDS-PAGE and Protonography
3.4. Amino Acid Sequence and Phylogenetic Analysis
3.5. Enzyme Kinetics
4. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Acronym | Enzyme Type | Organism | Accession Number |
---|---|---|---|
PfuNA | Nacrein | Pinctada fucata | BAA11940.1 |
PmaNA | Nacrein-like | Pinctada maxima | BAF42330.1 |
PmarNA | Nacrein | Pinctada margaritifera | AEC03970.1 |
CgiNAPre | Nacrein precursor | Crassostrea gigas | NP_001292235.1 |
CgiNAF | Nacrein isoform F | Crassostrea gigas | EKC37961.1 |
CgiNAX4 | Nacrein isoform X4 | Crassostrea gigas | XP_011426380.1 |
CgiNAP2X1 | Nacrein-like isoform P2X1 | Crassostrea gigas | XP_011428406.1 |
CgiNA | Nacrein-like | Crassostrea gigas | XP_011428408.1 |
CgiNA_Mit | Mitochondrial nacrein | Crassostrea gigas | EKC36446.1 |
TmaNA | Nacrein | Turbo marmoratus | BAB91157.1 |
HtuCA | α-CA | Haliotis tuberculata | AEL22200.1 |
HgiCA | α-CA | Haliotis gigantea | BAH58350.1 |
SsalCA | α-CA | Streptococcus salivarius] | WP_002888224.1 |
NgonCA | α-CA | Neisseria gonorrhoeae | WP_003688976.1 |
SspCA | α-CA | Sulfurihydrogenibium yellowstonense | WP_012459296.1 |
HpylCA | α-CA | Helicobacter pylori | WP_010882609.1 |
CruCA4 | α-CA isoform 4 | Corallium rubrum | KU557746.1 |
SpiCA2 | α-CA isoform 2 | Stylophora pistillata | ACE95141.1 |
HumCAI | α-CA isoform 1 | Homo sapiens | NP_001729.1 |
HumCAII | α-CA isoform 2 | Homo sapiens | AAH11949.1 |
PokCA | α-CA | Phreagena okutanii | BAO01209.1 |
MgaCA | α-CA | Mytilus galloprovincialis | ALF62133.1 |
CgiCA2 | α-CA isoform 2 | Crassostrea gigas | EKC31880.1 |
CgiCA2X1 | α-CA isoform 2X1 | Crassostrea gigas | XP_011447892.1 |
CgiCA2X3 | α-CA isoform 2X3 | Crassostrea gigas | XP_011447895.1 |
SpCA1 | α-CA isoform 1 | Stylophora pistillata | ACA53457.1 |
TgiCA | α-CA | Tridacna gigas | AAX16122.1 |
CgiCA12 | α-CA isoform 12 | Crassostrea gigas | EKC34762.1 |
ObiCA9X | α-CA isoform 9X | Octopus bimaculoides | XP_014786584.1 |
HblCA | α-CA | Heterololigo bleekeri | BAL42244.1 |
Enzyme | Organism | Class | kcat 1 (s−1) | kcat/KM1 (M−1 × s−1) | KIs (acetazolamide) 1 (nM) |
---|---|---|---|---|---|
HumCAI | Homo sapiens | α | 2.0 × 105 | 5.0 × 107 | 250 |
HumCAII | Homo sapiens | α | 1.4 × 106 | 1.5 × 108 | 12 |
CgiNAP2X1 | Crassotrea gigas | Nacrein-like | 1.0 × 106 | 1.2 × 108 | 495 |
SpiCA1 | Stylophora pistillata | α | 3.1 × 105 | 4.6 × 107 | 16 |
SpiCA2 | Stylophora pistillata | α | 5.6 × 105 | 8.3 × 107 | 74 |
CruCA4 | Corallium rubrum | α | 2.4 × 105 | 5.2 × 107 | 450 |
MgaCA | Mytilus galloprovincialis | α | 4.1 × 105 | 3.6 × 107 | 380 |
rec-MgaCA | Mytilus galloprovincialis | α | 4.2 × 105 | 3.5 × 107 | 361 |
Inhibitor 1 | KIs [mM] 2 | ||
---|---|---|---|
hCA I | hCA II | CgiNAP2X1 | |
F− | >100 | >100 | 12.1 |
Cl− | 6 | >100 | 10.8 |
Br− | 4 | 63 | 16.6 |
I− | 0.3 | 26 | 8.86 |
CNO− | 0.0007 | 0.03 | 3.11 |
SCN− | 0.2 | 1.60 | 5.88 |
CN− | 0.0005 | 0.02 | >100 |
N3− | 0.0012 | 1.51 | >100 |
HCO3− | 12 | 85 | 1.98 |
CO32− | 15 | 73 | 6.62 |
NO3− | 7 | 35 | 10.3 |
NO2− | 8.4 | 63 | 7.14 |
HS− | 0.0006 | 0.04 | 8.19 |
HSO3− | 18 | 89 | >100 |
SnO32− | 0.57 | 0.83 | 3.47 |
SeO42− | >100 | >100 | 8.74 |
TeO42− | 0.66 | 0.92 | >100 |
OsO52− | 0.92 | 0.95 | >100 |
P2O74− | 25.8 | 48.5 | 9.47 |
V2O74− | 0.54 | 0.57 | >100 |
B4O72− | 0.64 | 0.95 | 8.85 |
ReO4− | 0.11 | 0.75 | >100 |
RuO4− | 0.101 | 0.69 | >100 |
S2O82− | 0.107 | 0.084 | 20.7 |
SeCN− | 0.085 | 0.086 | 8.21 |
CS32− | 0.0087 | 0.0088 | 31.7 |
Et2NCS2− | 0.00079 | 0.0031 | 0.76 |
CF3SO3− | >100 | >100 | 8.68 |
PF6− | >100 | >100 | >100 |
SO42− | 63 | >100 | 14.5 |
ClO4− | >100 | >100 | >100 |
BF4− | >100 | >100 | >100 |
FSO3− | 0.79 | 0.46 | >100 |
NH(SO3)22− | 0.31 | 0.76 | >100 |
H2NSO2NH2 | 0.31 | 1.13 | 0.076 |
H2NSO3H | 0.021 | 0.39 | 0.073 |
Ph-B(OH)2 | 58.6 | 23.1 | 0.072 |
Ph-AsO3H2 | 31.7 | 49.2 | 0.087 |
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Perfetto, R.; Del Prete, S.; Vullo, D.; Sansone, G.; Barone, C.M.A.; Rossi, M.; Supuran, C.T.; Capasso, C. Sequence Analysis, Kinetic Constants, and Anion Inhibition Profile of the Nacrein-Like Protein (CgiNAP2X1) from the Pacific Oyster Magallana gigas (Ex-Crassostrea gigas). Mar. Drugs 2017, 15, 270. https://doi.org/10.3390/md15090270
Perfetto R, Del Prete S, Vullo D, Sansone G, Barone CMA, Rossi M, Supuran CT, Capasso C. Sequence Analysis, Kinetic Constants, and Anion Inhibition Profile of the Nacrein-Like Protein (CgiNAP2X1) from the Pacific Oyster Magallana gigas (Ex-Crassostrea gigas). Marine Drugs. 2017; 15(9):270. https://doi.org/10.3390/md15090270
Chicago/Turabian StylePerfetto, Rosa, Sonia Del Prete, Daniela Vullo, Giovanni Sansone, Carmela M. A. Barone, Mosè Rossi, Claudiu T. Supuran, and Clemente Capasso. 2017. "Sequence Analysis, Kinetic Constants, and Anion Inhibition Profile of the Nacrein-Like Protein (CgiNAP2X1) from the Pacific Oyster Magallana gigas (Ex-Crassostrea gigas)" Marine Drugs 15, no. 9: 270. https://doi.org/10.3390/md15090270
APA StylePerfetto, R., Del Prete, S., Vullo, D., Sansone, G., Barone, C. M. A., Rossi, M., Supuran, C. T., & Capasso, C. (2017). Sequence Analysis, Kinetic Constants, and Anion Inhibition Profile of the Nacrein-Like Protein (CgiNAP2X1) from the Pacific Oyster Magallana gigas (Ex-Crassostrea gigas). Marine Drugs, 15(9), 270. https://doi.org/10.3390/md15090270