Materials
Trunk wash was collected from one male (Tembo, born 1985) and five female (Tonga, 1984; Numbi, 1992; Mongu, 2003; Iqhwa, 2013; Kibali, 2019) African elephants at the Vienna Zoo during routine procedures. Briefly, 100 mL of a sterile 0.9% saline solution is injected in each nostril of the trunk, which is kept in a lifted position, so that the solution is running up to the base of the trunk. The mixture of the solution and trunk mucus is collected in sterile plastic bags by active blowing of the elephant. Chemicals were all from Merck, Austria, unless otherwise stated. Restriction enzymes and kits for DNA extraction and purification were from New England Biolabs, USA. Oligonucleotides and synthetic genes were custom synthesised at Eurofins Genomics, Germany.
Ethics declaration
We confirm that the trunk wash performed to provide a sample of the mucus was carried out as a routine procedure to monitor the health of elephants at the Vienna Zoo and in accordance with relevant guidelines and regulations.
Trunk wash fractionation
Trunk wash was centrifuged for 1 h at 10,000 g, the supernatant was dialyzed against 50 mM Tris–HCl buffer, pH 7.4 and concentrated by ultrafiltration in the Amicon stirred cell, then fractionated by anion-exchange chromatography on HiPrep-Q 16/10 column, 20 mL (Bio-Rad), along with standard protocols.
Protein alkylation and digestion, and mass spectrometry analysis
SDS-PAGE gel portions of proteins from whole elephant trunk wash (for component identification), chromatographic fractions of the elephant trunk wash (for PTMs analysis) or SDS-PAGE gel bands of LafrOBP1 expressed in P. pastoris were in parallel triturated, washed with water, in gel-reduced, S-alkylated, and digested with trypsin (Sigma, sequencing grade). Resulting peptide mixtures were desalted by μZip-TipC18 (Millipore) using 50% (v/v) acetonitrile, 5% (v/v) formic acid as eluent, vacuum-dried by SpeedVac (Thermo Fisher Scientific, USA), and then dissolved in 20 μL of aqueous 0.1% (v/v) formic acid for subsequent MS analyses by means of a nanoLC-ESI-Q-Orbitrap-MS/MS system, comprising an UltiMate 3000 HPLC RSLC nano-chromatographer (Thermo Fisher Scientific) interfaced with a Q-ExactivePlus mass spectrometer (Thermo Fisher Scientific) mounting a nano-Spray ion source (Thermo Fisher Scientific). Chromatographic separations were obtained on an Acclaim PepMap RSLC C18 column (150 mm × 75 μm ID; 2 μm particle size; 100 Å pore size, Thermo Fisher Scientific), eluting the peptide mixtures with a gradient of solvent B (19.92/80/0.08 v/v/v water/acetonitrile/formic acid) in solvent A (99.9/0.1 v/v water/formic acid), at a flow rate of 300 nL/min. In particular, solvent B started at 3%, increased linearly to 40% in 45 min, then achieved 80% in 5 min, remaining at this percentage for 4 min, and finally returned to 3% in 1 min. The mass spectrometer operated in data-dependent mode in positive polarity, carrying out a full MS1 scan in the range m/z 345–1350, at a nominal resolution of 70,000, followed by MS/MS scans of the 10 most abundant ions in high energy collisional dissociation (HCD) mode. Tandem mass spectra were acquired in a dynamic m/z range, with a nominal resolution of 17,500, a normalized collision energy of 28%, an automatic gain control target of 50,000, a maximum ion injection time of 110 ms, and an isolation window of 1.2 m/z. Dynamic exclusion was set to 20 s36.
Bioinformatics for peptide identification and post-translational modification assignment
Raw mass data files were searched by Proteome Discoverer v. 2.4 package (Thermo Fisher Scientific), running the search engine Mascot v. 2.6.1 (Matrix Science, UK), Byonic™ v. 2.6.46 (Protein Metrics, USA) and Peaks Studio 8.0 (BSI, Waterloo, Ontario, Canada) software, both for peptide assignment/protein identification and for post-translational modification analysis.
In the first case, analyses were carried out against a customized database containing protein sequences downloaded from NCBI (https://www.ncbi.nlm.nih.gov/) for superorder Afrotheria (consisting of 192,838 protein sequences, December 2021) plus the most common protein contaminants and trypsin. Parameters for database searching were fixed carbamidomethylation at Cys, and variable oxidation at Met, deamidation at Asn/Gln, and pyroglutamate formation at Gln. Mass tolerance was set to ± 10 ppm for precursors and to ± 0.05 Da for MS/MS fragments. Proteolytic enzyme and maximum number of missed cleavages were set to trypsin and 3, respectively. All other parameters were kept at default values. In the latter case, raw mass data were analyzed against a customized database containing LafrOBP1 (XP_023395442.1) protein sequence plus the most common protein contaminants and trypsin, allowing to search Lys-acetylation (Δm = + 42.01), Ser/Thr/Tyr-phosphorylation (Δm = + 79.97), and the most common mammals N-linked glycans at Asn and O-linked glycans at Ser/Thr/Tyr, using the same parameters previously set. The max PTM sites per peptide was set to 2.
Proteome Discoverer peptide candidates were considered confidently identified only when the following criteria were satisfied: (i) protein and peptide false discovery rate (FDR) confidence: high; (ii) peptide Mascot score: > 30; (iii) peptide spectrum matches (PSMs): unambiguous; (iv) peptide rank (rank of the peptide match): 1; (v) Delta CN (normalized score difference between the selected PSM and the highest-scoring PSM for that spectrum): 0. Byonic peptide candidates were considered confidently identified only when the following criteria were satisfied: (i) PEP 2D and PEP 1D: < 1.0 × 10−5; (ii) FDR: 0; (iii) q-value 2D and q-value 1D: < 1.0 × 10−5. An FDR value of 1% was specified as the cut-off of false discovery rate for peptides identification by Peaks Studio. Manual interpretation and verification of the candidate spectra were always performed.
Yeast expression and purification of proteins
The gene encoding OBP1 was cloned between XhoI and NotI restriction sites into the pPIC9 vector downstream of the α-factor secretion signal peptide. The following set of primers was used: 5’-AACTCGAG AAA AGA ATG CTG GAA GAA CC-3’; 5’-AAGCGGCCGC TTA GGT CAG TGT TTC CGG-3’ both for EmaxOBP1 and LafrOBP1. The construct was linearized with BglII and used to transform electrocompetent yeast cells (strain GS115) by electroporation (1.5 kV, 25 µF). Transformed cells were cultured on minimal dextrose (MD) agar plates (1.5% agar, 1.34% w/v yeast nitrogen base (YNB), 2% glucose, 4 × 10–5% biotin) for 48 h at 30 °C. Then, colonies were transferred onto minimal methanol (MM) agar plates (1.5% agar, 1.34% w/v yeast nitrogen base (YNB), 0.5% methanol, 4 × 10–5% biotin) and incubated at 30 ºC for further 48 h. Several colonies were selected from MM plates and screened for protein expression. First, colonies were grown in 10 mL of buffered minimal glycerol (BMGY) medium (1% w/v yeast extract, 2% w/v peptone, 1.34% w/v yeast nitrogen base with ammonium sulfate without amino acids (YNB), 4 × 10–5% biotin, 100 mM potassium phosphate, pH 6.0, 1% v/v glycerol) at 29 ºC, shaking at 240 rpm for 24 h. To induce protein expression, cells were harvested by centrifugation (15 min, 3,000 g, room temperature), re-suspended in 50 mL of buffered minimal methanol (BMM) medium (1.34% w/v YNB, 4 × 10–5% biotin, 100 mM potassium phosphate, pH 6.0, 1% v/v methanol) and incubated at 30 ºC for 72 h. To maintain protein expression, every 24 h methanol was added in amounts of 1.5% v/v. At the same intervals, samples of the supernatants were analyzed on SDS-PAGE for protein expression. On the basis of the results obtained, large-scale expression was performed under the above conditions for 72 h. The supernatant containing the recombinant OBP was centrifuged for 1 h at 10,000 g, dialyzed against 50 mM Tris–HCl buffer, pH 7.4, and concentrated by ultrafiltration in the Amicon stirred cell. Purification was performed by anion-exchange chromatography on HiPrep-Q 16/10, 20 mL (Bio-Rad) column, followed by elution from a Phenyl Sepharose column with the linear gradient of 0.6—0 M ammonium sulfate in 50 mM Tris–HCl, pH 7.4.
Disulfide assignment
A sample of recombinant LafrOBP1 (20 μg) was dissolved in 0.1 M tetraethylammonium bicarbonate (TEAB), pH 6.5, containing 4 M guanidinium chloride, and then treated with iodoacetamide (0.5 M final concentration) for 30 min, in the dark. The protein was precipitated by the addition of 6 vol of cold acetone at −20 °C, overnight. After centrifugation at 12,000 rpm at 4 °C, for 20 min, supernatant was removed, and the recovered pellet was dried. Recovered protein was dissolved in 0.05 M TEAB, pH 6.5 (2 µg/μL final concentration), treated with trypsin (1:30 w/w enzyme/substrate) for 16 h, at 37 °C, and then with chymotrypsin (1:30 w/w enzyme/substrate) for 16 h, at 37 °C. Protein digest was desalted with ZipTip C18 (Millipore, USA) and directly analyzed with a UltiMate 3000 HPLC RSLC nano-chromatographer (Thermo Fisher Scientific) linked to a Q-ExactivePlus mass spectrometer (Thermo Fisher Scientific), as reported above.
Dedicated BioPharma Finder v. 4.0 (Thermo Fisher Scientific) and pLink v. 2.3.9 software were used for database searching of mass spectrometric data, enabling the specific function of disulfide-linked peptides attribution, and applying the additional settings reported above for Proteome Discoverer, Byonic and Peaks Studio examinations. A confidence score > 95 for BioPharma Finder and/or an E-value < 1.0–10 for pLink results were considered for a reliable identification of disulfide-bridged peptides. Candidate spectra were always verified by manual interpretation.
Protein Model
A three-dimensional model of LafrOBP1 was obtained with the Swiss Model software30,31,32, using the allergen Bosd2 structure (PDB: 4WFU.1.A) as a template. The figure was generated with Chimera software37.
Ligand-binding assays
Affinities of the fluorescent probe N-phenyl-1-naphthylamine (1-NPN) to EmaxOBP1 and LafrOBP1 (both expressed in yeast) were evaluated by titrating a 2 μM solution of each protein in 50 mM Tris-HCl, pH 7.4 with 1 mM 1-NPN in methanol to final concentrations of 2–16 μM. Dissociation constants of other ligands were measured by displacement of the fluorescent reporter 1-NPN from the complex. Accordingly, solutions of protein and 1-NPN, both at the concentration of 2 μM in 50 mM Tris-HCl, pH 7.4, were titrated with 1 mM methanol solutions of each ligand to final concentrations of 2–16 μM. Emission spectra were recorded on a PerkinElmer FL 6500 spectrofluorimeter in a right-angle configuration, at room temperature, with slits of 5 nm for both excitation and emission, using 1 cm path quartz cuvettes. The excitation wavelength was set at 337 nm and intensities were recorded in correspondence with the peak maximum, around 390 nm. Data for 1-NPN were processed with Prism software.
Dissociation constants of other ligands were calculated from the corresponding [IC]50 values (the. concentration of each ligand halving the initial value of fluorescence), using the equation: Kd = [IC]50/1 + [1-NPN]/KNPN, where [1-NPN] is the concentration of free 1-NPN and KNPN the. dissociation constant of the complex OBP/1-NPN.
Digestion with phosphatase and glycosidase
Fractions 27 and 29 from trunk wash and a sample of LafrOBP1 expressed in P. pastoris were treated in parallel with Lambda phosphatase, O-glycosidase and PNGase along with the protocols provided by the manufacturers. The digestion products were analysed on SDS-PAGE. The product after digestion of recombinant LafrOBP1 with phosphatase was assayed for binding activity to (Z)-7-dodecenyl acetate in competitive fluorescent binding experiments, as reported above.
Source: Ecology - nature.com
