MipLAAO, a new L-amino acid oxidase from the redtail coral snake Micrurus mipartitus

Isolation of MipLAAO

MipLAAO, the fraction identified as LAAO in proteomic analysis of M. mipartitus venom (Rey-Suárez et al., 2011), was isolated using size exclusion chromatography in a high performance liquid chromatographic system (SEC-HPLC). For this, two mg of lyophilized venom (obtained from specimens maintained in Serpentarium of Universidad de Antioquia from the western region of Antioquia, Colombia), were dissolved in PBS (phosphate-buffered saline; 0.12 M NaCl, 40 mM sodium phosphate, pH 7.2) and separated on a Biosec S-2000 column (Phenomenex, 5 µm particle diameter; 300 × 7.8 mm), using a Shimadzu Prominence-20A chromatograph, monitored at 215 nm. Elution was performed at 0.7 mL/min using the same buffer. Five chromatographic separations were repeated and the LAAO fraction was collected, immediately concentrated, and finally desalted with Amicon Ultra filters (MWCO 10,000 membrane).

To assess the purity of MipLAAO, the protein was submitted to analytical reverse-phase high performance liquid chromatographic (RP-HPLC) using a C₁₈ column (Pinnacle, 5 µm particle diameter; 250 × 4.6 mm), eluted at 1 mL/min with a linear gradient from 0 to 70% solution B (0.1% TFA, 99.9% acetonitrile) in 25 min. Electrophoretic homogeneity was evaluated by SDS-PAGE (Laemmli, 1970) after reduction with 5% 2-mercaptoethanol at 100 °C for 5 min.

Molecular mass determination

The molecular mass of MipLAAO was determined by MALDI-TOF mass spectrometry. The protein (1 µg) was diluted in water-TFA (0.1%), mixed at 1:1 with saturated sinapinic acid in 50% acetonitrile, 0.1% TFA, and spotted (1 µL) onto an OptiToF-384 plate for MALDI-TOF MS analysis. Spectra were acquired on an Applied Biosystems 4800-Plus instrument (Foster City, CA, USA), using 500 shots/spectrum and a laser intensity of 4,200, in linear positive mode, over the m/z range 10,000–80,000.

Protein identification by MALDI-TOF/TOF peptide sequencing

For MS peptide sequencing, MipLAAO (40 µg) was reduced with dithiothreitol (10 mM), and alkylated with iodoacetamide (50 mM), followed by digestion with sequencing grade trypsin for 24 h at 37 °C, as described (Rey-Suárez et al., 2012). The resulting peptides were separated by RP-HPLC on a C₁₈ column (2.1 × 150 mm; Phenomenex), eluted at 0.3 mL/min with a 0–70% acetonitrile gradient over 40 min, manually collected, and dried in a vacuum centrifuge (Vacufuge, Eppendorf). Peptides were redissolved in 50% acetonitrile, 0.1% TFA, and analyzed by MALDI-TOF/TOF (4,800 Plus Proteomics Analyzer; Applied Biosystems, Foster City, CA, USA) using α-cyano-hydroxycinnamic acid as matrix, at 2 kV in positive reflectron mode, 500 shots/spectrum, and a laser intensity of 3,000. Fragmentation spectra were initially searched using the Paragon^® algorithm of ProteinPilot 4.0 (AB; Sciex, Framingham, MA, USA) against the UniProt/SwissProt serpentes database, and further they were manually interpreted de novo.

Snake venom gland cDNA synthesis and sequencing

mRNA was extracted from M. mipartitus venom gland of a specimen which died in captivity in the Serpentarium of Universidad de Antioquia, using the trizol method (Life Technologies, Carlsbad, CA, USA). The cDNA was obtained using Maxima First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). The conditions used in thermocycler were 95 °C for 8 min, followed by 10 min at 95 °C, 10 min at 53 °C and 2 min at 72 °C and 7 min at 72 °C to complete the cycle.

The LAAO-cDNA was obtained using the forward primer 5′-GAT GAA TGT CTT CTT TAT GTT CTC-3′, and the reverse 5′-GCA AGA GAT GTG AAT CGT GCT-3′ and PCR Supermix (Invitrogen), and conditions described by Pierre et al. (2005). The purified product was ligated to the pGEM-T Easy cloning vector (Promega, Madison, WI, USA) and used to transform Escherichia coli competent cells DH5-α and TOP10. The cells were cultured on LB (Luria-Bertani) agar, and transformed colonies were used to the obtain the plasmid using the QIA prep Spin Miniprep kit (QIAGEN, Hilden, Germany). The extracted product was sent to Macrogen Korea, specifying that it corresponded to complete plasmids with gene inserted at the multiple cloning site, to be sequenced from the vector promoter T7 and SP6 in order to confirm the direction in which the construct was inserted into the vector.

Bioinformatics procedures

The edition of cDNA sequences was performed in BioEdit version 7.0 (Hall, 1999). Nucleotide sequences were translated into amino acids to evaluate the reading frame and ensure the absence of premature stop codons or other nonsense mutation using GeneDoc software (Nicholas, Nicholas & Deerfield, 1997). SignalP 4.01 server available at http://www.cbs.dtu.dk/services/SignalP/ was used for signal peptide prediction (Petersen et al., 2011) and the posterior analyses were performed only with the predicted mature protein. NetNglyc (http://www.cbs.dtu.dk/services/NetNGlyc/) was used to predict the glycosylation sites.

Phylogenetic relationships and genetic distance

Bayesian inference (BI) algorithms implemented in MrBayes v3.0B4 (Ronquist & Huelsenbeck, 2003) was used to infer phylogenetic trees. A total of 48 related amino acid sequences of venom LAAOs, including 17 from Elapidae, 30 from Viperidae and one from Polychrotidae were obtained in two protein databases, BLAST (BLASTp http://blast.ncbi.nlm.nih.gov/Blast.cgi) and Uniprot (http://www.uniprot.org/blast/). These sequences were selected with E-values close to zero and with percentage of identity >30% (Pearson, 2013) (Supplementary material S1).

The multiple alignment of amino acid sequences of mature chains was performed in PRALINE (Heringa, 1999) using default parameters. After including gaps to maximize alignments, the final number of amino acid positions was 500. For phylogenetic analysis we used Anolis carolinensis sequence (R4GD21) as outgroup. The best-fitting model of amino acid substitution was selected using Bayesian Information Criterion (BIC) (Neath & Cavanaugh, 2012) implemented in the program MEGA version 7 (Kumar, Stecher & Tamura, 2016). These results gave a best fit for the JTT + Γ (Jones, Taylor & Thornton, 1992) amino acid substitution model.

We executed three parallel MCMC runs simultaneously, each one was run for 30 × 10⁶ generations with four Markov chains (one cold and three heated chains). We used Tracer v1.6 (Rambaut & Drummond, 2013) for visualizing output parameters to ascertain stationarity and whether or not the duplicated runs had converged on the same mean likelihood. Nodes were considered supported if posterior probabilities (PP) > 0.95. Trees were visualized using FigTree v1.1 (Rambaut, 2009) available at http://tree.bio.ed.ac.uk/software/figtree/.

In order to establish the genetic distance among LAAOs obtained in this study with LAAOs corresponding to Micrurus species used in the phylogenetic analysis, a matrix distance from aligned Micrurus sequences was obtained in Mega Version 7 (Supplemental Information 2).

Enzymatic characterization

LAAO activity was determined using the method of Kishimoto & Takahashi (2001). In brief, increasing doses (0.9 µg–3.0 µg) of MipLAAO (in 10 µL of water) were added to 90 µL of a reaction mixture (250 µM L-Leucine, 2 mM o-phenylenediamine, and 0.8 U/ml horseradish peroxidase) in 50 mM Tris pH 8.0 buffer. After incubation at 37 °C for 60 min, the reaction was stopped with 50 µL of 2 M H₂SO₄, and absorbances were recorded at 492 nm.

To determine the substrate specificity of MipLAAO, L-Leu was replaced with other L-amino acids (L-His, L-Ser, L-Arg, L-Ala, L-Trp, L-Glu, L-Cys, L-Lys, L-Tyr, L-Val, L-Ile, L-Thr), under standard assay conditions. The amount of MipLAAO in the reaction mixture was 1.5 µg. LAAO activity on L-Leu was also evaluated at different pH values (5.0–11.0).

Antimicrobial assay

Antimicrobial assays were performed according to the National Committee for Clinical Laboratory Standards (Clinical and Laboratory Standards, 2009) using two methods: the first, agar diffusion assays, in which 5 µL (10 µg) of MipLAAO was added to media (Muller-Hinton medium) with a suspension of 1.5 ×10⁶ colony forming units (CFU)/mL of E. coli (ATCC 25922) or S. aureus (ATCC 25923) and incubated at 37 °C for 24 h. Sterile saline solution and chloramphenicol (10 µg; Phyto Technology Laboratories, Lenexa, KS, USA) were used as negative and positive controls, respectively. Diameters of the bacterial growth inhibition zones were measured (Vargas et al., 2013). Each assay was performed in triplicate.

The second method was the broth microdilution in 96-well plates (Clinical and Laboratory Standards, 2009). Inoculum suspensions of S. aureus cultures were prepared and adjusted to a density of 1.5 ×10⁵ CFU/mL. In each well, 50 µL of the bacterial suspension and 50 µL of MipLAAO (0.01–7.0 µg/mL) were mixed. Plates were incubated at 37 °C for 24 h, and the minimum inhibitory concentration (MIC) was defined as the lowest concentration of enzyme that prevented visible growth (absence of turbidity) in the broth. Assays were performed in triplicate. Sterile saline solution and chloramphenicol were used as controls.

Statistical analysis

Results were expressed as mean ± SD. Analysis of variance (ANOVA- Kruskal-Wallis test) followed by Bonferroni post-test was employed to evaluate the statistical significance of data on LAAO activity, substrate preference, and pH effects. Differences were considered statistically significant when p < 0.05.

Isolation, determination of molecular mass, cloning and amino acid sequencing of MipLAAO

The fraction identified as LAAO in M. mipartitus venom (Rey-Suárez et al., 2011), here named MipLAAO, was isolated using SEC-HPLC, with an retention time of 4.5 min, (Fig. 1A). This fraction corresponds to 6% of the chromatogram with a final yield of 100 µg per run. The protein eluted as a homogeneous peak by RP-HPLC analysis, at 19.6 min (Fig. 1B). SDS-PAGE of MipLAAO under reducing (Fig. 1B, insert), or non reducing conditions (not shown) both presented a single band, with a migration at ∼57 kDa, indicating that it is a monomeric protein.

The cDNA obtained from the venom gland mRNA of M. mipartitus evidenced a product of ∼1,503 bp, corresponding to the expected molecular mass for the LAAO coding sequence. The primers amplified a DNA fragment, which was purified, ligated and used to transform E. coli cells. Eight positive clones, randomly selected, were sequenced and six coding sequences, including signal peptide, were obtained and designated as MipLAAO-1 to MipLAAO-6 (Fig. 2). These sequences were deposited in Genbank, with access codes (MH010800, MH010801, MH010802, MH010803, MH010804, MH010805).

Protein sequences deduced from the six cDNA clones consist of 501 amino acids, with a segment of 18 amino acids corresponding to signal peptide, and 483 amino acids corresponding to mature chain (Fig. 2). Few changes among the sequences were observed. MALDI-TOF/TOF peptide sequences obtained de novo matched the amino acid sequences deduced from cDNA (Fig. 2). These sequences evidenced high identity values compared to other LAAOs from elapid and viperid snakes (Supplemental Information 1). Highest identities were obtained against elapid LAAOs, especially from other Micrurus, the LAAOs of which have been predicted by transcriptomic studies, such as M. spixi LAAO (89% identity; Supplemental Information 1).

The observed isotope-averaged molecular mass of the isolated MipLAAO was 59,100.6 Da, by MALDI-TOF MS (Fig. 1C). The theoretical molecular masses predicted (https://web.expasy.org/peptide_mass/) from the nucleotide-sequenced clones MipLAAO-1 to -6 vary from 55,121.02 Da to 55,010.94 Da. These variations correspond to few amino acid substitutions among them (Fig. 2). The difference between these theoretical mass values and that determined for the isolated protein are suggestive of post-translational modification of the enzyme, for example glycosylation. MALDI-TOF/TOF peptide sequencing matched the predicted MipLAAO cDNA-deduced sequence, covering 56% (Fig. 2). In addition, the deduced amino acid sequences of MipLAAO isoforms predict these to be basic proteins, according to the Compute Mw/pI tool at https://web.expasy.org/compute_pi/, with pI values within the range of 8.85–8.93.

Phylogenetic relationships

The deduced protein sequences of six MipLAAO isoforms obtained in this study and 48 other related LAAOs were aligned to infer phylogenetic relationships. Our analysis recovered a deep split of LAAOs into two lineages (Clades A and B, Fig. 3), separating all Elapidae family enzymes in Clade A from those of Viperidae family in Clade B, except for the King Cobra (Ophiophagus hannah) LAAO, which shares between 49.0% and 50.8% identity with other Elapidae LAAOs, and looped out with Anolis carolinesis LAAO (outgroup). This split, in clear agreement with snake taxonomy, is associated to differences in the amino acid residue at position 223, occupied by Ser in all LAAOs from clade A (with the exception of M. surinamensis and Bungarus sp., having Asp and Asn, respectively) or by His in all members of clade B.

Within Clade A (Elapidae family), all LAOOs from Micrurus conform a well-supported group (PP = 1.00) with genetic distance range between 12% with M. spixii and 18.9% with M. tener (Supplemental Information 2). MipLAAOs sequences here obtained present variations with other Micrurus LAAOs at positions 175 (His instead of Tyr), 301 (His instead of Arg) and 384 (His instead of Leu).

Functional characterization of MipLAAO

MipLAAO was obtained in active form, presenting a conspicuous concentration-dependent enzymatic activity upon L-Leu (Fig. 4A). This enzyme only oxidized hydrophobic amino acids like L-Trp, L-Tyr, and L-Leu (Fig. 4B). Furthermore, the enzyme maintained its activity within the pH range 7.0–10.0, being optimal at pH 8.0 (Fig. 4C).

Antibacterial activity of MipLAAO

MipLAAO exhibited antibacterial activity against S. aureus, but not against E. coli. Against the former, the enzyme produced a bacterial growth inhibition halo of 19.8 ± 0.6 mm, while chloramphenicol used as a control produced a halo of 12 ± 1 mm. The MIC against S. aureus evaluated by the broth microdilution method was 2 µg/mL.