Biochemical and molecular characterization of N66 from the shell of Pinctada mazatlanica

Total RNA extraction and RNA reverse transcription

Total RNA was extracted with TRIzol^® (Invitrogen, Carlsbad, CA, U.S.A.) according to the manufacturer’s instructions. Samples were homogenized using a glass pestle; then, two consecutive extractions of each sample were made. RNA purity and concentration were determined by spectrophotometry using a NanoDrop ND-2000 (Thermo Scientific, Waltham, MA, U.S.A.) at 260/280 and 260/230 nm absorbance ratios (range, 1.9–2.0). The RNA integrity was assessed on a 1% (w/v) agarose-synergel gel. To ensure complete DNA absence, a direct PCR was performed using one μL (50 ng/μL) of each RNA preparation with 28S ribosomal specific primers as a non-amplified control. After that, one μg of total RNA was used from each verified RNA sample for cDNA synthesis using the cloned AMV First-Strand cDNA Synthesis Reaction (Invitrogen, Carlsbad, CA, U.S.A.) and oligo-dT primer, afterwards cDNAs were stored at −80 °C until use. Control reactions were performed without template or non-reverse transcribed RNA to determine the presence of DNA.

Amplification of the N66 coding sequence from mantle

A search for mollusk sequences encoding N66 available at GenBank yielded three homologous sequences, N66 Pinctada maxima (GenBank: AB032613), N44 Pinctada maxima (GenBank: FJ913472), and SP-S Mizuhopecten yessoensis (GenBank: AB185328). Based on these sequences, specific primers were designed to amplify the coding sequence of N66 (Table 1). PCR amplification of cDNA fragments encoding N66 was done in a final volume of 12.5 μL containing 10 pmol of each forward and reverse primer, 50 ng cDNA, 10 nmol dNTPs mixture, 1.25 μL 10X Taq Buffer, and 1 U Taq DNA polymerase (11615010A; Invitrogen, Carlsbad, CA, USA). PCR amplification were carried out for 5 min at 94 °C, followed by five cycles of 1 min at 94 °C, 1 min at 40 °C, and 2 min at 72 °C, followed of 35 cycles of 1 min at 94 °C, 1 min at 50 °C, and 2 min at 72 °C. In the last cycle, the extension step at 72 °C lasted 10 min. PCR products were analyzed on 1% (w/v) agarose-synergel gels and visualized under UV light after staining with UView loading dye (166-5112; Bio-Rad, Hercules, CA, USA). PCR products were cloned using the TOPO-TA cloning kit (K4500-01; Invitrogen, Carlsbad, CA, USA) and both strands were sequenced. The obtained sequence was submitted to the National Center for Biotechnology Information (NCBI) database for BLAST searching and other informatic analysis.

In silico analyses

Identification of the open reading frame was performed using the ORF finder software (https://www.ncbi.nlm.nih.gov/orffinder/) and sequence alignment with homologous sequences was performed using CLUSTAL Omega. Identification of putative protein motifs was performed using the MotifScan (Pfam HMMs global models database) available at the Swiss Institute of Bioinformatic (http://myhits.isb-sib.ch/cgi-bin/motif_scan). Identification of signal peptide was achieved by using SignalP 4.1 Server (Petersen et al., 2011). Theoretical molecular weight, isoelectric point (pI), and amino acid composition of the protein were calculated using the ProtParam software from Expert Protein Analysis System (ExPASy; https://www.expasy.org/). Putative glycosylation and phosphorylation sites of N66 were determined using NetOGly4.0 (Steentoft et al., 2013), NetNGlyc 1.0 (http://www.cbs.dtu.dk/services/NetNGlyc/), and NetPhos2.0 (Blom, Gammeltoft & Brunak, 1999).

Vector construction and recombinant protein expression

The coding region of the N66 cDNA, without the signal peptide, was synthesized by PCR with two primers (N66_HindF and N66_HindR) to introduce the restriction enzyme sites (HindIII, AAGCTT) at the 5′ and 3′ ends (Table 1). The PCR product was directly digested with HindIII (NEB, New England, Ipswich, MA, USA) and isolated from gel electrophoresis, purified and ligated into a polyhistidine fusion protein expression vector pTrcHis C (Invitrogen Inc., Carlsbad, CA, USA) that was linearized with the same restriction enzyme, yielding pTrcHis C/N66. Escherichia coli TOP10 cells were transformed with the construct and cultured in six mL of LB medium containing 100 μg/mL ampicillin at 37 °C with shaking at 150 rpm overnight. Five mL of the culture medium were transferred to a flask containing 245 mL of the same medium and further incubated. When OD₆₀₀ of the bacterial cells reached ∼0.7, isopropyl β-D-thiogalactosidase was added to a final concentration of one mM and incubated at 37 °C with constant shaking at 150 rpm for 4 h. The bacterial cells were harvested by centrifugation (7,500 rpm, 25 min at room temperature) and stored at −20 °C. The frozen bacterial pellet was thawed, dispersed in ice-cold Tris–HCl buffer (50 mM, pH 7.4) and disrupted with glass beads for 5 min of vigorous vortex. The bacterial lysate was centrifuged (12,000×g rpm, 15 min, 4 °C), and the supernatant was recovered. Recombinant His-tagged proteins were purified from the supernatant using a Sepharose-Ni column (GE Healthcare, Chicago, IL, USA).

Shell matrix extraction

The organic matrix of prismatic and nacreous layers was crushed into a fine powder. The powdered matrix (20 g) was suspended in 100 mL of cold acetic acid (4 °C, 10% v/v) and incubated for 24 h with continuous stirring. Soluble (ASM) and insoluble (AIM) matrix fractions were separated by centrifugation at 14,000×g rpm for 20 min. The AIM was rinsed with distilled water and lyophilized. After that, the ASM was dialyzed 24 h against cold acetic acid (4 °C, 1% v/v), afterward the ASM was dialyzed another 24 h against distilled water. The dialyzed ASM was concentrated by lyophilization. Protein concentration was determined with the Lowry method (Lowry et al., 1951).

N66 purification by affinity chromatography

Native and recombinant N66 were isolated by affinity chromatography using a Sepharose-Ni column (GE Healthcare, Chicago, IL, USA; 1.45 × 5.0 cm). The protein sample (one mL) was loaded onto the column, previously equilibrated with 50 mM Tris–HCl pH 7.4, 300 mM NaCl buffer, mixed and incubated for 1 h at 25 °C. The unbound enzyme was washed with five bed volumes of 50 mM Tris–HCl pH 7.5, 300 mM NaCl buffer. The bound enzyme was eluted by washing the column with elution buffer containing 50 mM Tris–HCl pH 7.5, 300 mM NaCl, 300 mM Imidazole. Fractions of one mL were collected from the elution and evaluated by electrophoresis. Fractions containing the N66 protein were pooled and concentrated through centrifugal filters (AMICON Ultra-10; Millipore, Burlington, MA, USA) at 4,000×g for 20 min at 25 °C to a final volume of 500 μL. The concentrated sample was desalted using a PD-10 desalting column (Amershan Pharmacia, Little Chalfont, UK). Glycerol was added to the enzyme solution (final concentration 50%), and the sample was stored at −20 °C until use. Protein content was quantified with the Lowry method (Lowry et al., 1951) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purified protein was performed.

SDS-PAGE and staining

Native and recombinant N66 proteins were analyzed under reducing conditions using 10% SDS-PAGE (Mini-PROTEAN; Bio-Rad, Hercules, CA, USA), as described by Laemmli (1970) and stained with silver nitrate (Merril & Washart, 1998) in 10% acrylamide gels. Samples were diluted (1:4) with sample buffer (0.125M Tris–HCl, 2% SDS, 20% v/v glycerol, 0.04% bromophenol blue, 5% β-mercaptoethanol at pH 6.8) and heated for 10 min at 100 °C. The electrophoresis was performed at 80V constant voltage. To detect protein bands, gels were stained with 0.1% Coomassie Brilliant Blue R-250 in 7.5% acetic acid and 5% methanol at room temperature and distained in the 10% acetic acid and 40% methanol.

Glycosylation was also analyzed by electrophoresis under reducing conditions (Thornton, Carlstedt & Sheehan, 1994). The gel was first incubated in a fixing solution (5% TCA) for 5 min, then incubated in an oxidant solution (0.8% periodic acid in 0.3% sodium acetate) and rinsed with water. Then, the gel was incubated in Schiff’s reagent for 10 min in the dark. Finally, the gel was washed three times with 3% nitric acid for 3 min. The gel was stored in 7.5% acetic acid, 5% v/v methanol. Ovoalbumin (Sigma-Aldrich, St. Louis, MO) was used as positive control.

Mass spectrometry analyses

To identify the mass spectrometric profile of the native N66, 20 μg of protein were separated by 10% SDS-PAGE. After electrophoresis, the gel was stained with Coomassie Blue R-250 and bands were manually cut from the gel. After tryptic digestion, the resulting peptides were subjected to Liquid Chromatography-Mass Spectrometry (LC-MS). The LC-MS was performed at the Proteomic Unit Facility at the Biotechnology Institute, in Cuernavaca, Mexico. The resulting data were compared with the proteomic database at GenBank and with the deduced amino acid sequence from cDNA of N66 from Pinctada mazatlanica.

Carbonic anhydrase assay

Carbonic anhydrase activity from native and recombinant N66 was assayed qualitatively and quantitatively. In the qualitative assay, 1% agarose plates were prepared with borate buffer (0.05M boric acid, 0.025M NaCl). Samples (10 μL with 0.2 mg protein/mL) were loaded on the agar plate. After sample diffusion 0.1% bromocresol purple (dissolved in 0.1M Tris–HCl pH 8.5) was added and incubated for 10 min at room temperature. Afterwards, the solution was discarded and the agar plate was washed with distilled water. Finally, saturated CO₂ water was added as substrate; the production of hydrogen ions during the CO₂ hydration reaction lowers the pH of the solution until the formation of yellow halo zones which indicate CA activity while dark zones indicate lack of activity (Manchenko, 2002). Bovine CA (Sigma-Aldrich, St. Louis, MO) was used as positive control and it was boiled to function as a negative control.

The quantitative assay for CA activity is described in detail by Wilbur & Anderson (1948). CA activity was measured by the decrease of pH resulting from the hydration of CO₂ to HCO₃⁻ and H⁺ after the addition of the substrate. All experiments were performed at 4 °C. To run the assay, five mL of 0.012M Tris–HCl buffer (pH 8.3) were transferred to a small beaker and one mL of the homogenate diluted in 0.012M Tris–HCl buffer was added to obtain 20 μg of protein. The mixture was constantly stirred with a magnetic stirring bar. Afterwards, four mL of cold saturated CO₂ solution were added, and the decrease in pH was recorded by a pH probe. CA activity was calculated as (t₀−t)/t, where t₀ is the time needed for the non-catalyzed reaction and t is the time for the catalyzed reaction to obtain a pH decrease from 8.0 to 6.0. Units of enzyme activity (U) were normalized to the total protein content.

Crystallization assay with native and recombinant N66 proteins

Three crystallization solutions were prepared, two induces calcite (calcitic crystallization solution) and one induces aragonite (aragonite crystallization solution). Calcitic crystallization solutions (40 mM CaCl₂, pH 8.2, 100 mM NaHCO₃, and 100 mM CaCO₃) and aragonite crystallization solution (40 mM MgCl₂, pH 8.2, 100 mM NaHCO₃) were prepared following the protocol of Weiss et al. (2000) and Hillner et al. (1992), respectively. Native and recombinant N66 (10 μL of 0.2 mg/mL) were added to 50 μL of the saturated solution. As a control, buffer solution without protein was used. Each experiment was made in triplicates and incubated at 4 °C for 21 days in a cover slide situated at the bottom of a six-hole microplate which was sealed with parafilm to avoid contamination. The morphology of the crystals produced were analyzed by scanning electron microscopy (SEM) at the Electronic Microscopy Laboratory at CIBNOR.

Native N66 from Pinctada mazatlanica shells

The organic matrix of the shell from Pinctada mazatlanica was separated by SDS-PAGE revealing a wide range of abundant molecular weight proteins in the ASM fraction (Fig. 3A). The ASM fraction was used to isolate the N66 by affinity chromatography using a Sepharose-Ni matrix (Fig. S2) since the active site of the CA domain contains three histidines involved in metal ion binding which has been shown to be conserved in N66 from Pinctada mazatlanica (this study) and Pinctada maxima (Kono, Hayashi & Samata, 2000). The fractions containing the eluted N66 (Fig. S2) were pooled and desalted. Two bands in SDS-PAGE and silver stained gels were observed (Fig. 3B) with molecular weights of ∼60 and 64 kDa. A flow sheet of the purification process for N66 is presented in Table 3. The enzyme was enriched 3.39-fold and the yield was 56.52%. Electrophoretic analysis of N66 with PAS stain, demonstrated that N66 is glycosylated (Fig. 3B); however, the type of carbohydrate associated was not analyzed. The results of LC-MS of the bands matched the deduced amino acid sequence of the N66 of the mantle of Pinctada mazatlanica (Table 4), as described before (Fig. 1), and allowed us to identify it as a N66, a SMPs from the shell of Pinctada mazatlanica.

Recombinant N66 from Pinctada mazatlanica

The recombinant N66 protein was expressed as a soluble form at 37 °C with one mM IPTG in E. coli after a 4 h induction, and the bands corresponding to the protein in SDS-PAGE analysis agreed with the native N66 of ASM from Pinctada mazatlanica (Fig. 3B). The recombinant N66 was successfully purified by affinity chromatography using Ni-Sepharose columns (Fig. S3). The electrophoretic analysis showed two protein bands of ∼60 and 64 kDa which agreed with the theoretical molecular weight of the native N66 of ASM from Pinctada mazatlanica (Fig. 3B). Recombinant N66 did not show glycosylation after staining with PAS (data not shown).

Carbonic anhydrase activity of native and recombinant N66 proteins

The CA activity of the native and recombinant N66 was confirmed qualitatively (Fig. 4) and quantitatively. Figure 4 showed comparable yellow halos obtained with the native and recombinant N66, indicating CA activity. Commercial bovine CA was used as positive control. The comparable CA activity of the native and recombinant N66 proteins was confirmed with the Wilbur and Anderson method (1948). The results showed significant differences between the native (161 ± 12.5 U/mg protein) and the recombinant protein (197 ± 7.5 U/mg protein), being the recombinant protein the one with higher activity.

In vitro crystallization by native and recombinant N66

To elucidate the effect of native and recombinant N66 on the growth of calcium carbonate crystals, three individual in vitro crystallization assays testing the formation of aragonite and calcite were established and analyzed by SEM (Fig. 5). When no protein was added (negative control) to the three salts tested (NaHCO₃ + MgCl₂, NaHCO₃ + CaCl₂, and CaCO₃), different crystal morphology was observed. The crystals formed were unorganized when sodium bicarbonate was in the presence of magnesium chloride, while in the presence of calcium chloride, crystals become compact and thicker, and with calcium carbonate, the formed crystals exhibited the typical rhombohedral form of calcite (Figs. 5A–5C). In the presence of 0.2 mg protein/mL of native N66 to the three salt preparations, polycrystalline aggregates of aragonite and calcite in presence of MgCl₂ and CaCO₃ respectively were shown, however in the presence of CaCl₂, crystals were small structures of calcite and in a lesser quantity per area (Figs. 5D–5F). In contrast to the native N66, the recombinant form of N66 produced bigger and organized crystal structures plates (Figs. 5G–5I).