Proteomic and RNA profiling of milk-derived exosomes from s1 …€¦ · Proteomic and RNA...

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Proteomic and RNA profiling of milk-derived exosomes from αs1-casein deficient goats reveal that CSN1S1 genotype modulates the repertoire of molecules they conveyed 1 GABI, INRA, AgroParisTech, Université Paris-Saclay, Jouy-en-Josas ; 2 EXCILONE, Elancourt, 78990 France 3 INRA, 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 Krupova 1-2 , Christine Péchoux 1 , Céline Henry 3 , Marine Dumarest 1 , Pierre Defrenaix 2 and Patrice Martin 1 Extracellular vesicles (EVs), including exosomes, have been identified in milk and shown to contain proteins, mRNA and microRNA (miRNA), which could be transferred to neighbouring cells. They are recognized as mediators of intercellular communication and thought to be involved in the development of the young immune functions. A “naturally occurring KO" of the gene that specifies αs1-casein (large deletion) has been reported in the goat species. Goats homozygous for such a null allele at the CSN1S1 locus, display a chronic endoplasmic reticulum (ER) stress due to the accumulation of the other (immature) caseins in this compartment, in the mammary epithelial cell, impacting its secretory phenotype. Milk-derived extracellular vesicles The objective of this study is to precisely assess the impact of αs1-casein deficit on exosomes content and composition, performing a comparative analysis of protein and RNA contents 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 on exosomes content and composition, performing a comparative analysis of protein and RNA contents of exosomes isolated from goat milk of extreme genotypes at the CSN1S1 locus. Workflow Swelling of the Rough Endoplasmic Reticulum: Accumulation of immature caseins CSN1S1 O/O OBJECTIVE 2. Immunofluorescence 1. Transmission Electron Microscopy nti-CD81 nti-CD63 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 membrane transport 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-200a and 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 milk Skimming (low speed centrifugation) RNA-Seq analysis RNA and protein extraction NGS (Exiqon, NextSeq500) Ultra- centrifugation (sucrose gradient) LC-MS/MS Proteome (PAPPSO, LC-MS/MS) Morphometry & size distribution (TEM, TRPS, DLS) Microscopy An An Anti-HSP70 (70 kDa) Anti-CD81 (26 kDa) Anti-β-CN (25 kDa) 5. Western Blot analysis Detection by ELISA of exosomal markers (CD63, CD9) which were not detected by Western Blot analysis 4. Size distribution and concentration determination TRPS (IZON) Particle count: 3095 4539 4003 0 1 2 3 4 5 6 7 8 9 10 fr 10 fr 11 fr 12 moy av OO av AA 3. Protein concentration in exosomal fractions: CSN1S1 AA vs. OO milks AA OO F10 F11 F12 Average Protein concentration (μg/μl) Heat map and unsupervised hierarchical clustering by sample and microRNA 5 goats AA & 5 goats OO Reference genome Organism: Capra hircus. Secondary genome: Bos taurus. Annotation reference: miRBase 20 (http://www.mirbase.org/) Experimental design: Average number of reads: 20 million reads per sample Number of sequencing cycles (read length): 50 nt. Single-end read 20 putative novel microRNA identified and 11 novel micoRNA previously 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 AA 279 18 Goat exo OO Xanthine dehydrogenase/oxidase 43,50 enzymes, metabolism Fatty acid synthase 13,62 Sodium-dependent phosphate transport protein 2B 28,83 membrane trafficking Membrane cofactor protein 12,50 Annexin A5 8,72 Rab GDP dissociation inhibitor beta 5,54 Actin, cytoplasmic 2 11,60 cytoskeleton CD81 antigen 11,00 targeting, adhesion CD9 antigen 54,50 CD59 molecule, complement regulatory protein 10,75 CD36_BOVIN Platelet glycoprotein 4 58,11 Glycosylation-dependent cell adhesion molecule 19,57 Mucin-1 7,33 Lactadherin 49,85 Butyrophilin subfamily 1 member A1 93,95 MHC membre Most abundant proteins observed in goat milk-derived exosomes 8. miRNA: NGS 7. mRNA: qPCR CSN1S1 CSN1S2 PPAI LALBA RPS24 AA OO 0 1 2

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Page 1: Proteomic and RNA profiling of milk-derived exosomes from s1 …€¦ · Proteomic and RNA profiling of milk-derived exosomes from αs1-casein deficient goats reveal that CSN1S1 genotype

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

0

1

2

3

4

5

6

7

8

9

10

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

0

1

2