Proteomic and RNA profiling of milk-derived exosomes from s1 …€¦ · Proteomic and RNA...
Transcript of Proteomic and RNA profiling of milk-derived exosomes from s1 …€¦ · Proteomic and RNA...
Proteomic and RNA profiling of milk-derived exosome s from αs1-casein deficient goats reveal that CSN1S1 genotype modulates the repertoire of molecules they conveyed
1GABI, INRA, AgroParisTech, Université Paris-Saclay, Jouy-en-Josas ; 2EXCILONE, Elancourt, 78990 France 3INRA, UMR MICALIS, Plateforme d’Analyse Protéomique Paris Sud Ouest (PAPPSO), Université Paris-Saclay, 78350 Jouy-en-Josas, France
BACKGROUND
Isolation and validation of milk-derived exosomes
RESULTS
Zuzana Krupova1-2, Christine Péchoux1, Céline Henry3, Marine Dumarest1, Pierre Defrenaix2 and Patrice Martin1
Extracellular vesicles (EVs), includingexosomes, have been identified in milkand shown to contain proteins, mRNAand microRNA (miRNA), which could betransferred to neighbouring cells. Theyare recognized as mediators ofintercellular communication and thoughtto be involved in the development of theyoung immune functions.
A “naturally occurring KO" of the genethat specifies αs1-casein (large deletion)has been reported in the goat species.Goats homozygous for such a null alleleat the CSN1S1 locus, display a chronicendoplasmic reticulum (ER) stress due tothe accumulation of the other (immature)caseins in this compartment, in themammary epithelial cell, impacting itssecretory phenotype.
Milk-derived extracellular vesicles
The objective of this study is to precisely assess the impact of αs1-casein deficit onexosomes content and composition, performing a comparative analysis of protein and RNAcontents of exosomes isolated from goat milk of extreme genotypes at the CSN1S1 locus.
The objective of this study is to precisely assess the impact of αs1-casein deficit onexosomes content and composition, performing a comparative analysis of protein and RNAcontents of exosomes isolated from goat milk of extreme genotypes at the CSN1S1 locus.
Workflow
Swelling of the Rough Endoplasmic Reticulum:Accumulation of immature caseins
CS
N1
S1
O/
O
OBJECTIVE
2. Immunofluorescence
1. Transmission Electron Microscopy A
nti-
CD
81
Ant
i-C
D63
CONCLUSIONS� The isolation method developed is effective and gives milk-derived exosome populations free of contamination by other EVs and milk components.� Nearly 280 proteins involved in biogenesis of exosomes and MVB formation, their adhesion and internalization as well as proteins associated with the membranetransport were identified of which 18 and 23 proteins differed between OO and AA genotypes, respectively.� Ongoing profiling of RNA from goat milk-derived exosomes has already identified more than 230 miRNA among which some (miR-148a-3p, miR30a-5p, miR-200aand miR-200b) are highly represented whatever the genotype, and confirms they originates from MEC (presence of mRNA encoding α-lactalbumin and αs2-casein).
This study was funded by the National Agency for Research (MilkChEST, ANR-12-BSV6-0013-04), by the GIS APIS-GENE.RNA quality control and qPCR were performed on the Microgenomics plateform, part of @BRIDGe core facility(Jouy-en-Josas, France).
Acknowledgements
RNA Quality control (mRNA : qPCR)
Protein concentration
Exosomal marker detection (WB, ELISA)
50 mL goat milkSkimming(low speed
centrifugation)
RNA-Seq analysis
RNA and proteinextraction
NGS (Exiqon, NextSeq500)
Ultra-centrifugation
(sucrose gradient)
LC-MS/MS Proteome (PAPPSO, LC-MS/MS)
Morphometry &size distribution(TEM, TRPS, DLS)
Microscopy Ant
i
Ant
i
Anti-HSP70 (70 kDa)
Anti-CD81(26 kDa)
Anti- β-CN(25 kDa)
5. Western Blot analysis
Detection by ELISA of exosomalmarkers (CD63, CD9) which were not detected by Western Blot analysis
4. Size distribution and concentration determination TRPS (IZON)
Particle count:
309545394003
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fr 10 fr 11 fr 12 moy
av OO
av AA
3. Protein concentration in exosomalfractions: CSN1S1 AA vs. OO milks
AAOO
F10 F11 F12 Average
Pro
tein
conc
entr
atio
n (µ
g/µl
)
Heat map and unsupervised hierarchical clustering by sample and microRNA
5 goats AA & 5 goats OO Reference genomeOrganism: Capra hircus. Secondary genome: Bos taurus. Annotation reference: miRBase 20 (http://www.mirbase.org/) Experimental design:Average number of reads: 20 million reads per sampl e Number of sequencing cycles (read length): 50 nt. Single-end read20 putative novel microRNA identified and 11 novel m icoRNApreviously reported in other species
6. LC-MS/MS
Enrichment in OO goat milk-derived exosomes
• Protein disulfide isomerase A3
• Gelsoline
• Endoplasmin
• Complement component C9, 3d
• Apolipoprotein A-IV
Enrichment in AA goat milk-derived exosomes
• Catenin alpha-1
• Integrin beta-2
• Flotilin-2
• Alkaline phosphatase
• Dipeptidyl peptidase 4
23
Goat exo
AA279
18
Goat exo
OO
Xanthine dehydrogenase/oxidase 43,50 enzymes, metabolismFatty acid synthase 13,62Sodium-dependent phosphate transport protein 2B 28,83
membrane traffickingMembrane cofactor protein 12,50Annexin A5 8,72Rab GDP dissociation inhibitor beta 5,54Actin, cytoplasmic 2 11,60 cytoskeletonCD81 antigen 11,00
targeting, adhesion
CD9 antigen 54,50CD59 molecule, complement regulatory protein 10,75CD36_BOVIN Platelet glycoprotein 4 58,11Glycosylation-dependent cell adhesion molecule 19,57Mucin-1 7,33Lactadherin 49,85Butyrophilin subfamily 1 member A1 93,95 MHC membre
Most abundant proteins observed in goat milk-derive d exosomes
8. miRNA: NGS
7. mRNA: qPCR
CSN1S1 CSN1S2 PPAI LALBA RPS24
AAOO
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