Alternative splicing events expand molecular diversity of camel CSN1S2 increasing its ability to generate potentially bioactive peptides

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Post-translational modifications – Phosphorylation

The term PTM (Post-Translational Modifications) denotes changes in the polypeptide chain due to either the addition or removal of distinct chemical moieties to amino acid residues, proteolytic processing of the protein termini, or the introduction of covalent cross-links between domains of the protein. PTMs are involved in most cellular processes including the maintenance of protein structure and integrity, regulation of metabolism and defense processes, and in cellular recognition events and morphology changes (Larsen et al., 2006). Phosphorylation of proteins is one of the most frequent PTM in eukaryotic cells. It has become a common knowledge that phosphorylation of CN occurs at S or T aa residues in tripeptide sequences S/T-X-A where X represents any aa residue and A is an acidic aa residue (Mercier, 1981). This consensus sequence is recognized by FAM20C, a Golgi CN-kinase, which phosphorylates secreted phosphoproteins, including both CN and members of the small integrin-binding ligand N-linked glycoproteins (SIBLING) protein family, which modulate biomineralization (Ishikawa et al., 2012). PTM, occurring in the endoplasmic reticulum and/or Golgi complex after synthesis of the polypeptide chain, play a critical role in micelle formation and stability (Holland, 2008).

Extracellular vesicles

Milk is usually consider as a complex biological liquid in which supramolecular structures (casein micelles and milk fat globules) are found beside minerals, vitamins and soluble proteins (whey proteins) as well as cells. It was recently shown that milk contains also extracellular vesicles that are released by cells as mediators of intercellular communication. Indeed, cell communicate with neighboring cells or with distant cells through the secretion of extracellular vesicles (Tkach & Théry, 2016). Phospholipid bilayer-enclosed extracellular vesicles (EVs) are naturally generated and released from several cell domains of life (Bacteria, Archaea, Eukarya) into the extracellular space under physiological and pathological conditions (Delcayre et al., 2005; G. Raposo, 1996). EVs are commonly classified according to their sub-cellular origin into three major subtypes, such as microvesicles, exosomes, and apoptotic bodies. Contents of vesicles vary with respect to mode of biogenesis, cell type, and physiologic conditions (Abels & Breakefield, 2016). Exosomes represent the smallest population among EVs, ranging in size from 30 to 150 nm in diameter (Hromada et al., 2017). They are generated inside multivesicular bodies in the endosomal compartment during the maturation of early late endosomes and are secreted when these compartments fuse with the plasma membrane (Figure 1.4) (van der Pol et al., 2012). Found in all biofluids exosomes harbor different cargos as a function of cell type and physiologic state (Abels & Breakefield, 2016). Milk is the sole source of nutrients for the newborn and very young offspring, as well as being an important means to transfer immune components from the mother to the newborn of which the immune system is immature (Abels & Breakefield, 2016; Hromada et al., 2017). Milk is therefore thought to play an important role in the development of the immune system of the offspring. Milk is also a source of delivers molecules, via exosomes and/or microvesicles, acting on immune modulation of neonates due to their specific proteins, mRNA, long non-coding RNA and miRNA contents. Exosomes and wider EVs have come in the limelight as biological entities containing unique proteins, lipids, and genetic material. It was shown that the RNA contained in these vesicles could be transferred from one cell to another, through an emerging mode of cell-to-cell (Colombo et al., 2014; Simons & Raposo, 2009). RNAs conveyed by EVs are translated into proteins within transformed cells (mRNA), and/or are involved in regulatory functions (miRNA). For this reason, EVs are recognized as potent vehicles for intercellular communication, capable for transferring messages of signaling molecules, nucleic acids, and pathogenic factors (Kabani & Melki, 2016). Over the last decade, EVs were widely explored as biological nanovesicles for the development of new diagnostic and therapeutic applications as a promising source for new biomarkers in various diseases (Kanada et al., 2015). For example, exosomes secreted by dendritic cells have been shown to carry MHC-peptide complexes allowing efficient activation of T lymphocytes, thus displaying immunotherapeutic potential as promoters of adaptive immune responses (Keller et al., 2006). Recently, cell culture studies showed that bovine milk-derived EVs act as a carrier for chemotherapeutic/chemopreventive agents against lung tumor xenografts in vivo (Munagala et al., 2016). Nevertheless, their physiological relevance has been difficult to evaluate because their origin, biogenesis and secretion mechanisms remained enigmatic.
Despite a significant number of publications describing the molecular characteristics and investigating the potential biological functions of milk-derived exosomes (Reinhardt et al., 2012; van Herwijnen et al., 2016), there are only one dealing with exosomes derived from camel milk (Yassin et al., 2016). These authors report for the first time isolation and characterization using proteomic (SDS-PAGE and western blot analysis) and transcriptomic analyses exosomes from dromedary milk at different lactation stages. However, there is no comprehensive investigation on exosomal protein variations and variability in composition between individual camels. Milk-derived EVs from Bactrian and hybrid milks have never been explored before.

Milk samples collection and preparation

In total 181 raw milk samples (Table 2.1.) were collected during morning milking on healthy dairy camels belonging to two camel species: C. bactrianus (n=72) and C. dromedarius (n=65), and their hybrids (n=42), at different lactation stages, ranging between 30 and 90 days postpartum. Bactrian camels were originating from Kazakh type whereas dromedary camels were from Turkmen Arvana breed. Unfortunately, the information about the nature and the level of hybridization of hybrids was not available. All species are well adapted to the local environment of Kazakhstan.
Camels grazed on four various natural pastures with the distance more than 3,500 kms between the regions at extreme points of Kazakhstan: Almaty (AL) at the foot of Tien Shan Mountain, Shymkent (SH) along deserts Kyzylkum and Betpak-Dala, Kyzylorda (KZ) on the edge of the steppe, and Atyrau (ZKO) at the mouth of the Caspian Sea (Figure 2.1). Whole- milk samples were centrifuged at 2,500 g for 20 min at 4oC (Allegra X-15R, Beckman Coulter, France) to separating fat from skimmed milk. Samples were quickly frozen and stored at -80°C (fat) and -20°C (skimmed milk) until analysis.

Selection of milk samples for analysis

Of the 181 milk samples collected, 63, including C. bactrianus (n=19), C. dromedarius (n=20), and hybrids (n=24) from four different regions of Kazakhstan were selected for SDS-PAGE analysis (Figure 2.2). Each Bactrian and dromedary camel group formed by 5 animals, except Bactrians of Atyrau regions (n=4). For hybrids, there were 4 groups comprising 10 animals (Kyzylorda and Shymkent regions), whereas there were only 1 and 3 animals for Almaty and Atyrau regions, respectively. This selection was based on lactation stages and number of parities (from 2 to 14) of each camel group composed by the species and grazing regions. It should be emphasized that data available on animals: breed, age, lactation stage and calving number, were estimated by a local veterinarian, since no registration of camels in farms is maintained. Due to the lack of sufficient information, dromedary milk samples (n=5) from Almaty region were excluded from subsequent analyses. Then, 8 of the 58 remaining milk samples from three different regions (C. bactrianus, n=3, C. dromedarius, n=3, and hybrids, n=2) exhibiting the most representative SDS-PAGE patterns were analyzed by LC-MS/MS after a tryptic digestion of excised gel bands. Additionally, 30 milk samples (C. bactrianus, n=10; C. dromedarius, n=10; hybrids, n=10), taken from the 63 milks analyzed by SDS-PAGE, were analyzed by LC-ESI-MS (Bruker Daltonics).

Coomassie blue (Bradford) protein assay

To estimate the concentration of total protein in a milk sample the Coomassie Blue Protein Assay was used (Bradford, 1976). Absorbance at 590 nm was measured using the UV-Vis spectrophotometer (UVmini-1240, Shimadzu). The reference standard curve was done with commercial bovine serum albumin (BSA) powder dissolved in MilliQ water and diluted to a concentration of 1 mg/mL. Series of dilutions (0.1, 0.2, 0.4, 0.6, and 0.8 µg/µL) were prepared from the stock solution, in duplicate to ensure the protein concentration is within the range of the assay.

1D sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

Both major and low-abundant proteins resolved by SDS-PAGE were identified after excision by mass analysis of the tryptic hydrolysate. The method used in the study was based on that from Laemmli (Laemmli, 1970). Twenty-five micrograms of each individual skimmed milk sample were loaded into 12.5% acrylamide resolving gel and subjected to electrophoresis. Samples were prepared with Laemmli Lysis-Buffer (Sigma-Aldrich). Separations were performed in a vertical electrophoresis apparatus (Bio-Rad, Marnes-la- Coquette, France). After GelCode Blue Safe Protein staining and gel scanning using Image Scanner iii (Epson ExpressionTM 10,000 XL, Sweden), resolved bands were excised from the gel and submitted to digestion by trypsin. Thereafter, tryptic peptides were analyzed by LC-MS/MS.

Identification of proteins by LC-MS/MS analysis

In order to identify the main protein contained in each electrophoretic band, mono dimensional electrophoresis (1D SDS-PAGE) followed by trypsin digestion and by LC-MS/MS analysis, was used essentially as described (Saadaoui et al., 2014). Briefly, after a 10 cm migration of samples in such an 1D SDS-PAGE, the 16 main electrophoretic bands (1.5 mm3) were cut on each gel lane, transferred into 96-well microtiter plates (FrameStar, 4titude, 0750/Las). Reduction of disulfide bridges of proteins was carried out by incubating at 37oC for one hour with dithiothreitol (DTT, 10 mM, Sigma), meanwhile the alkylation of free cysteinyl residues with iodoacetamide (IAM, 50 mM, Sigma) at RT for 45 min in total obscurity. After gel pieces were washed twice, first, with 100 µL 50% ACN/50 mM NH4HCO3 and then with 50 µL ACN, they were finally dried. The hydration was performed at 37oC overnight using digestion buffer 400 ng lys-C protease + trypsin. Hereby, peptides were extracted with 50% ACN/0.5% TFA and then with 100% ACN. Peptide solutions were dried in a concentrator and finally dissolved into 70 µL 2% ACN in 0.08% TFA. The identification of peptides was obtained using UltiMate™ 3000 RSLCnano System (Thermo Fisher Scientific) coupled either to LTQ Orbitrap XL™ Discovery mass spectrometer or QExactive (Thermo Fischer Scientific). Four µL of each sample was injected with flow of 20 µL/min on a precolumn cartridge (stationary phase: C18 PepMap 100, 5 µm; column: 300 µm x 5 mm) and desalted with a loading buffer 2% ACN and 0.08% TFA. After 4 min, the precolumn cartridge was connected to the separating RSLC PepMap C18 column (stationary phase: RSLC PepMap 100, 2 µm; column: 75 µm x 150 mm). Elution buffers were A: 2% ACN in 0.1% formic acid (HCOOH) and B: 80% ACN in 0.1% HCOOH. The peptide separation was achieved with a linear gradient from 0 to 35% B for 34 min at 300 nL/min. One run took 42 min, including the regeneration and the equilibration steps at 98% B.
Peptide ions were analyzed using Xcalibur 2.1 with the following machine set up in CID mode: 1) full MS scan in Orbitrap with a resolution of 15 000 (scan range [m/z] = 300-1600) and 2) top 8 in MS/MS using CID (35% collision energy) in Ion Trap. Analyzed charge states were set to 2-3, the dynamic exclusion to 30 s and the intensity threshold was fixed at 5.0 x 102.
Raw data were converted to mzXML by MS convert (ProteoWizard version 3.0.4601). UniProtKB Cetartiodactyla database was used (157,113 protein entries, version 2015), in conjunction with contaminant databases were searched by algorithm X!TandemPiledriver (version 2015.04.01.1) with the software X!TandemPipeline (version 3.4) developed by the PAPPSO platform (http://pappso.inra.fr/bioinfo/). The protein identification was run with a precursor mass tolerance of 10 ppm and a fragment mass tolerance of 0.5 Da. Enzymatic cleavage rules were set to trypsin digestion (“after R and K, unless P follows directly after”) and no semi-enzymatic cleavage rules were allowed. The fix modification was set to cysteine carbamido methylation and methionine oxidation was considered as a potential modification. Results were filtered using inbuilt X!TandemParser with peptide E-value of 0.05, a protein E-value of -2.6, and a minimum of two peptides.

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LC-ESI-MS

Fractionation of camel milk proteins and determination of their molecular masses, performed by coupling RP-HPLC to ESI-MS (micrOTOFTM II focus ESI-TOF mass spectrometer; Bruker Daltonics), were essentially as described (Saadaoui et al., 2014). In total 20 µL of skimmed milk samples were first clarified by the addition of 230 µL of clarification solution 0.1 M bis-Tris buffer pH 8.0, containing 8 M urea, 1.3% trisodium citrate, and 0.3% DTT. Clarified milk samples (25 µL) were directly injected onto a Biodiscovery C5 reverse phase column (300 Å pore size, 3 µm, 150 x 2.1 mm; Supelco, France). The mobile phase of the column corresponded to a gradient mixture of Solvent A (H2O/TFA 100:0.25, v/v) and Solvent B (ACN/TFA 100:0.20, v/v). Elution was achieved using a linear gradient from 5% to 27% B in 20 min, from 27% to 33% B in 0.1 min, from 33% to 34% B in 11.1 min, from 34% to 40% B in 0.1 min, from 40% to 41% B in 14.9 min, and from 41% to 90% B in 0.1 min. This gradient elution was followed by an isocratic elution at 90% B for 4.9 min, and a linear return to 5% B in 0.1 min. The temperature of the column was adjusted to 52oC and the flow rate to 0.2 mL/min. Eluted peaks were detected by UV-absorbance at 214 nm. The liquid effluent was introduced to the mass spectrometer. Positive ion mode was used, and mass scans were acquired over a mass-to-charge ratio (m/z) ranging between 600 and 3000 Da.

Milk fat globule collection and RNA extraction

Total RNA was extracted from MFG fraction stored at -80°C using Trizol (Invitrogen) following the protocol from the manufacturer as described by Brenaut et al., (2012).

First-strand cDNA synthesis and PCR amplification

First-strand cDNA was synthesized from 5 to 10 ng of total RNA primed with oligo(dT)20 and random primers (3:1, vol/vol) using Superscript III reverse transcriptase (Invitrogen Life Technologies Inc., Carlsbad, CA) according to the manufacturer’s instructions. One microliter of 2 U/µL RNase H (Invitrogen Life Technologies) was then added and the reaction mix was incubated for 20 min at 37°C to remove RNA from heteroduplexes. Single-strand cDNA thus obtained was stored at -20°C. cDNA samples covering the entire coding regions of caseins were amplified. PCR was performed in an automated thermocycler GeneAmp® PCR System 2,400 (Perkin-Elmer, Norwalk, USA) with GoTaq® G2 Flexi DNA Polymerase Kit (Promega Corporation, USA). Reactions were carried out with 0.2 mL thin-walled PCR tubes with flat cap strips (Thermo Scientific, UK), in 50 µL volumes containing 5X Green or Colorless GoTag® Flexi Buffer, MgCl2 Solution 25 mM, PCR Nucleotide Mix 10 mM each, GoTag® G2 Flexi DNA Polymerase (5 U/µL), 10 mM each oligonucleotide primer, template DNA and nuclease-free water, up to the final volume. Primer pairs, purchased from Eurofins (Eurofins genomics, Germany), were designed using published Camelus nucleic acid sequence (NCBI, NM_001303566.1). Sequencing of PCR fragments was performed with primer pairs used for PCR and sequenced from both strands, according to the Sanger method by Eurofins.

Identification of main milk proteins from 1D SDS-PAGE by LC-MS/MS

After first adjusting protein concentrations at the same value, 63 individual camel milk samples were separated onto SDS-PAGE. The comparative analysis of whole milk samples by SDS-PAGE displayed rather similar electrophoretic profiles with related migration characteristics and the same apparent molecular weights between individual milk samples of different species and regions. A typical gel pattern from which proteins were identified in individual C. bactrianus, C. dromedarius and hybrid milk samples of Kyzylorda region is shown in Figure 2.3.
Sixteen main bands relatively well-resolved were excised from the electrophoretic pattern. The most intense band observed around 26 kDa was identified as β-CN. Quantitative analyses on camel milk proteins carried out before have demonstrated significantly higher amounts of β-CN compared to the homologous bovine CN (Kappeler et al., 2003). The most representative other bands were characterized as being: WAP (12.5 kDa), α-LAC (14.3 kDa), GlyCAM 1 (15.4 kDa and 17.2 kDa), κ-CN (20.3 kDa), PGRP (21.3 kDa), αs2-CN (22.9 kDa), αs1-CN (25.7 kDa), neutrophil gelatinase (28.3 kDa), lipoprotein lipase (46.5 kDa), perilipin-2 (47.2 kDa), butyrophilin (51.0 kDa), amine oxidase (55.3 kDa), lactadherin (56.2 kDa), heat shock protein (70.0 kDa), LTF (77.1 kDa), lactoperoxidase (87.7 kDa), and xanthine oxidase (150 kDa). Masses mentioned above correspond to theoretical masses of proteins identified on the basis of tryptic profiles after LC-MS/MS analysis. Globally, the electrophoretic patterns of Kazakh camel milk samples agree with those reported recently for Israelian and Tunisian camel milk samples (Felfoul et al., 2017; Merin et al., 2001). However, surprisingly the prominent fact was the apparent absence in Kazakh milk samples of camel serum albumin (CSA), the major WP with a molecular mass equal to 66.0 kDa in camel colostrum (Merin et al., 2001). By contrast, this protein has been successfully identified, with the best E-value, in Tunisian fresh milk samples (Felfoul et al., 2017).

Table of contents :

Chapter 1 General Introduction
Camelids: the other non-cattle dairy species of arid and semiarid rangelands
Camel milk as a source of health promoting compounds
The protein fractions of camel milk
Caseins
Whey proteins
Milk fat globule membrane proteins
Factors responsible for the molecular complexity of milk proteins
Genetic variants
Alternative splicing
Post-translational modifications – Phosphorylation
Extracellular vesicles
Aim and outline of this study
Chapter 2 Combining different proteomic approaches to resolve complexity of the milk protein fraction of dromedary, Bactrian camels and hybrids, from different regions of Kazakhstan
Abstract
Introduction
Materials and Methods
Results
Total protein content
Identification of main milk proteins from 1D SDS-PAGE by LC-MS/MS
Qualitative proteome of camel skimmed milk by LC-MS/MS
Camel milk protein profiling by LC-ESI-MS
Multiple spliced variants of CSN1S1
Discussion
Interspecies in-depth proteomic analysis of camel milk proteins
Molecular diversity of camel caseins: genetic polymorphism and alternative splicing
Post-translational modifications of milk proteins: phosphorylation of caseins
Conclusions
Chapter 3 Alternative splicing events expand molecular diversity of camel CSN1S2 increasing its ability to generate potentially bioactive peptides
Abstract
Introduction
What gene(s) UP1 and UP2 arise from?
UP1 and UP2: new camel αs2-CN splicing variants
Cross-species comparison of the gene encoding αs2-CN and primary transcript maturation
Phosphorylation level enhances camel αs2-CN isoform complexity
Alternate splicing isoforms of camel αs2-CN increase its ability to generate potential bioactive
peptides
Chapter 4 The main WAP isoform usually found in camel milk arises from the usage of an improbable intron cryptic splice site in the precursor to mRNA in which a GC-AG intron occurs
Abstract
Background
Methods
Results
Nucleotide sequence of camel WAP cDNA
Identification and characterization of a new WAP genetic variant in Bactrian camel milk
Camel WAP may exist as two isoforms differing in size
Discussion
Camel WAP is phosphorylated
The usage of an unlikely intron cryptic splice site is responsible for the insertion of 4 amino acid
residues in the major camel WAP isoform
Intron 3 of camel WAP gene is a GC-AG intron type
Conclusions
Chapter 5 Comprehensive Proteomic Analysis of Camel Milk-derived Extracellular Vesicles
Abstract
Introduction
Materials and methods
Results and Discussion
Isolation of camel milk-derived EVs
Morphology of isolated camel milk-derived EVs
In-depth proteomic analysis of camel milk-derived EVs
Exosomes are a rich source of potential milk biomarkers
Conclusions
Chapter 6 General Discussion
Global analysis: complexity of the camel milk proteome
Complexity of the « casein » fraction: the case of αs2-CN and potential impact in terms of function
WAP: originality of the protein and of the gene
EVs: Beyond their role in the communication between cells, what possible effects on the
consumer (offspring or adult)
What should be implemented now?
Acknowledgements
Curriculum vitae
Résumés

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