Domestic Fowl Breed Variation in Egg White Protein Expression

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Omics Technologies Applied to Agriculture and Food

Domestic fowl breed variation in egg white protein expression: application of proteomics and transcriptomics Barbora Bílková, Zuzana #widerská, Lukáš Zita, Denis Laloë, Mathieu Charles, Vladimir Benes, Pavel Stopka, and Michal Vinkler J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03099 • Publication Date (Web): 08 Oct 2018 Downloaded from http://pubs.acs.org on October 15, 2018

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Journal of Agricultural and Food Chemistry

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Domestic fowl breed variation in egg white protein

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expression: application of proteomics and transcriptomics

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Barbora Bílková (a), Zuzana Świderská (a,b), Lukáš Zita (c), Denis Laloë (d), Mathieu

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Charles (d), Vladimír Beneš (e), Pavel Stopka (a), Michal Vinkler (a)

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a) Charles University, Faculty of Science, Department of Zoology, Prague, Czech Republic,

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EU

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b) Charles University, Faculty of Science, Department of Cell biology, Prague, Czech

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Republic, EU

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c) Czech University of Life Sciences, Faculty of Agrobiology, Food and Natural Resources,

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Department of Animal Husbandry, Prague, Czech Republic, EU

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d) GABI, INRA, AgroParisTech, Université Paris-Saclay, Jouy-en-Josas, France, EU

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e) European Molecular Biology Laboratory, Heidelberg, Germany, EU

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List of abbreviations

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BGA - between group analysis

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CIA - co-inertia analysis

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PLGEM - power low global error model

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RPKM - Reads Per Kilobase Million

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Keywords 1 ACS Paragon Plus Environment


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antimicrobial peptides, avian oviduct transcriptome, bird albumen, chicken breed, egg white

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proteome

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Abstract

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Avian egg white is essential for protecting and nourishing bird embryos during their

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development. Being produced in the female magnum, variability in hen oviduct gene

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expression may affect egg white composition in domestic chickens. Since traditional poultry

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breeds may represent a source of variation, in the present study we describe the egg white

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proteome (mass spectrometry) and corresponding magnum transcriptome (high-throughput

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sequencing) for twenty hens from five domestic fowl breeds (large breeds: Araucana, Czech

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golden pencilled, Minorca and small breeds: Booted bantam, Rosecomb bantam). In total, we

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identified 189 egg white proteins and 16391 magnum-expressed genes. The majority of egg

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white protein content comprised proteins with an antimicrobial function. Despite general

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similarity, Between-class Principal Component Analysis revealed significant breed-specific

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variability in protein abundances, differentiating especially small and large breeds. Though

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we found strong association between magnum mRNA expression and egg white protein

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abundance across genes, co-inertia analysis revealed no transcriptome/proteome co-structure

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at the individual level. Our study is the first to show variation in protein abundances in egg

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white across chicken breeds with potential effects on egg quality, biosafety and chick

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development. The observed inter-individual variation probably results from post-

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transcriptional regulation creating a discrepancy between proteomic and transcriptomic data.

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Introduction

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The structure of the avian egg is a characteristic apomorphy of present-day birds. The egg is

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gradually developed by passing through specialised sections of the female oviduct, where

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distinct egg structures are progressively formed1. Given that the domestic chicken (Gallus

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gallus f. domestica) is both a model research-species in avian biology and an economically

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important agricultural species, most of our present knowledge about the egg formation

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process comes from this species. In chickens, it has previously been reported that distinct

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parts of the hen oviduct differ in the amount of proteins expressed, corresponding to their

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respective function in egg formation2. By volume, the egg white makes up the largest part of

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the chicken egg (ca. two thirds). Correspondingly, the magnum where the egg white is formed

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is the longest part of the hen oviduct. The magnum is covered with a mucosal tissue formed

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by tubular gland cells folded in a spiral pattern that enlarges the secreting surface1. During a

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three-hour period when egg yolk passages through the magnum, the complex egg white

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substance is secreted, which is involved in both protection and nourishment of the developing

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embryo.

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To date, only four studies have attempted to describe the complete composition of chicken

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egg white using modern proteomic methods4-6. Each of these studies was conducted using

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modern commercially produced eggs only, in which they found between 78 and 202 distinct

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egg white proteins. Earlier results gained by two-dimensional electrophoresis suggest that egg

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white protein abundances could differ importantly between both chicken breeds and

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populations7. Since the egg white contains large amounts of proteins involved in antimicrobial

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defence8, such variation could potentially affect egg quality, biosafety and chicken embryo

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development. 4 ACS Paragon Plus Environment

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Furthermore, the whole magnum transcriptome has not yet been described for chickens.

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While previous research has targeted experimentally induced changes in mRNA expression of

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genes within the oviduct9, associations between such changes and egg proteome composition

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in terms of both protein expression and their abundances remain largely unknown. As

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demonstrated by Kim and Choi10, experimental corticosterone treatment affects both magnum

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mRNA expression and egg white protein abundance of certain genes, though not necessarily

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in the same direction. In various systems, the integrative studies of whole proteome and

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transcriptome data have shown that despite their general consistency11-12 large discrepancies

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between mRNA expression and respective protein expression can be found13,14, suggesting an

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important role of post-transcriptional regulation of gene expression15.

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In this study, we attempt to improve our understanding of breed-specific variability in egg-

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white protein composition in terms of protein identification and quantification of relative

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protein abundances and fill gaps in our knowledge of the hen-magnum transcriptome-egg

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proteome relationship. We focus on traditional chicken breeds as these exhibit high

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phenotypic variability in morphological, physiological and immunological traits16,17 and egg

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production18. We selected five distinct traditional chicken breeds (Araucana, Booted bantam,

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Czech golden pencilled, Rosecomb bantam and Minorca) that highly differ in their phenotypic

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traits such as body size, weight and shape and also area of origin. We describe both the egg

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proteome and hen magnum transcriptome in order to highlight differences between breeds in

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both protein and transcript amounts and to test for associations between proteomic and

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transcriptomic data between individuals.



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Methods

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Animals

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A total of 535 fertilised eggs from hens of five different chicken breeds (Araucana, Booted

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bantam, Czech golden pencilled, Minorca and Rosecomb bantam) were kindly provided by

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small non-commercial breeders located in the Czech Republic, EU. All of the chosen breeds

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can be considered as layer breeds, with relatively high laying capacity from 90-190 eggs per

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year. Booted bantam and Rosecomb bantam also belong to dwarf fancy breeds, herein

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referred as the small breeds with adult body weight 0.4-0.8 kg; body weight of Araucana,

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Czech golden pencilled and Minorca breeds range from 1.2 to 1.7 kg, therefore these are

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referred to as the large breeds. The eggs were incubated to hatching in an OvaEasy 380

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Advance EX automatic egg incubator (Brinsea, Weston-super-Mare, UK) at a temperature of

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37.5°C and 50% humidity. The twenty hens used within this experiment were hatched

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between 13th May 2015 and 22nd June 2015 (Table S1) and immediately marked with two

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numbered wing-marks. All hens were housed under standardised conditions at the animal

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facility of the Czech University of Life Sciences in 0.50 × 0.88 × 0.45 m cages with access to

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food and water ad libitum (initially in breed-specific flocks of 5-10 animals, later

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individually). After reaching reproductive maturity (estimated by the onset of egg production;

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for all hens between 224 and 244 days of age), three eggs (excluding the first three) were

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collected from each hen. The hens were then euthanised by rapid cervical dislocation.

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Samples of the magnum tissue (the segment of the oviduct were egg white is formed) were

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taken within 15 min. after euthanasia, placed into RNAlater reagent (Quiagen, Hilden,

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Germany) and kept overnight at +4°C and afterwards stored at -80°C. This research was

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approved by the Ethical Committee of the Faculty of Science, Charles University (reference

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number 1373/2016-4). 6 ACS Paragon Plus Environment

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Egg white sample preparation

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All eggs were collected from hens within 12 hours after oviposition and immediately stored at

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5°C. To minimise changes in the abundance of some egg white proteins during storage19, we

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processed all eggs within two days of oviposition. First, the eggshells were opened with a

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sterilised knife and the egg whites separated from the yolks. The whites were then

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homogenised with a glass mechanical hand homogeniser for 60 seconds and divided into six

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250 µL aliquots that were kept at -20°C until analysis. Prior to analysis, all egg white samples

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from the three eggs of the same individual were pooled by mixing 4 µL of each sample (12µL

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of egg white in total). The pooled samples were then diluted in 12µL of PBS and precipitated

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with 112 µL of 98% ethanol. All precipitated samples were centrifuged at 2800 g and 4°C for

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15 min. After centrifugation, the supernatant was discarded and the samples dried for 30 min

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at 37°C. These were then re-suspended in 48 µL of digestion buffer (1% SDS, 100 mM

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triethylammonium bicarbonate (TEAB) – pH = 8.5) and were cleaved with trypsin (i.e., 1/50,

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trypsin/protein) at 37 °C overnight.

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nLC-MS2 analysis

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Nano Reversed phase columns were used to elute peptide cations using the same method as

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Cerna20. The eluting peptide cations were converted to gas-phase ions by electrospray

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ionisation and analysed on a Thermo Orbitrap Fusion mass spectrometer (Q-OT-qIT,

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Thermo). Survey scans of peptide precursors from 400 to 1600 m/z were performed at 120K

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resolution (at 200m/z) with a 5 × 105 ion count target. Tandem MS/MS was performed by



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isolation at 1.5 Th with the quadrupole, high-energy collision dissociation (HCD)

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fragmentation with normalized collision energy of 30, and rapid scan MS analysis in the ion

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trap. The MS/MS ion count target was set to 104 and the max injection time was 35ms. Only

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those precursors with a charge state of 2–6 were sampled for MS/MS. The dynamic exclusion

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duration was set to 45s with a 10 ppm tolerance around the selected precursor and its isotopes.

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Monoisotopic precursor selection was turned on and the instrument was run at top speed with

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2s cycles.

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Proteome data analysis

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All data were collected and quantified using MaxQuant software version 1.5.3.821. False

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discovery rate (FDR) was set to 1% for identification of all peptides and proteins. We set a

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minimum peptide length of seven amino acids. The Andromeda search engine was used for

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the MS/MS spectra search against the Uniprot database, with all duplicates removed. Enzyme

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specificity was set as C-terminal to Arg and Lys, also allowing cleavage at proline bonds and

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a maximum of two missed cleavages. Dithiomethylation of cysteine was selected as a fixed

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modification and N-terminal protein acetylation and methionine oxidation as variable

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modifications. Quantifications were performed with the label-free algorithms21.

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We were able to detect 266 proteins, of which 67 were assigned as contaminants. To

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standardise the total abundance of non-contaminating egg white proteins across samples, we

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normalised the amount of each protein according to the formula Cnet = Craw/(1-Pcont), where

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Cnet indicates the net amount of protein counts after normalisation, Craw the raw abundance of

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protein counts and Pcont the proportion of contamination in a given sample. In addition to

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chicken proteins, we also detected 10 proteins of avian specific viruses. We excluded all

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proteins occurring in three or less samples from further analysis, leaving a total dataset of 115 8 ACS Paragon Plus Environment

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egg

white

proteins.

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For protein classification, we used the online PANTHER library22. To find overrepresented

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Gene Ontologies (GOs) among the egg white proteins, we launched the PANTHER

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Overrepresentation Test with Bonferroni correction for multiple testing against the G. gallus

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(all genes in the database) reference list and annotation data sets, with the GO molecular

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function and GO biological process complete (GO Ontology database release date: 14. 08.

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2017).

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Transcriptomic data analysis

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Total RNA was isolated from magnum samples using the High Pure RNA Tissue Kit (Roche,

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Basel, Switzerland), according to the manufacturer’s instructions. The total amount of

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extracted RNA was quantified using an Agilent 2100 Bioanalyser with the Agilent RNA 6000

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Nano Kit (Agilent Technologies, California, USA). Library preparation and stranded paired-

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end mRNA sequencing was performed at The European Molecular Biology Laboratory

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(EMBL), Heidelberg. Barcoded stranded mRNA-seq libraries were prepared using the

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Illumina TruSeq RNA Sample Preparation v2 Kit (Illumina, San Diego, CA, USA),

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implemented on the liquid handling robot Beckman FXP2. The libraries obtained were pooled

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in equimolar amounts, with a 1.8 pM solution of this pool loaded on the NextSeq 500

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Illumina sequencer and sequenced bi-directionally, each read being 85 bases long, thereby

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generating ~25 million sequence pairs for each library. The sequencing results were submitted

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to the NCBI Sequence Read Archive (SRA Acc. No. SRP126816).

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Read sequences were trimmed of sequencing adaptors using Trim Galore! Software

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(Babraham Bioinformatics, Braham Institute, Cambridge, GB) and low-quality score bases

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(Phred quality score < 30) were removed from both (3’ and 5’) ends using SICKLE23 9 ACS Paragon Plus Environment


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software. Reads with a resulting trimmed sequence of less than 30 bp were discarded. The

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remaining trimmed reads of high quality sequences were then aligned to the G. gallus

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reference genome assembly Gallus_gallus-5.0 (GCA_000002315.3) using STAR software24

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for ultrafast transcript assembly. The percentage of uniquely mapped reads ranged from 91.82

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% to 94.34% (Table S2). Sequence reads were assigned and quantified at the gene level using

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the featureCounts program from the Subread package25. In order to compare mean expression

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between genes, the read counts obtained from the featureCounts program were converted to

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Reads Per Kilobase Million (RPKM).

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Statistical analysis

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Prior to analysis, we transformed both the proteomic and transcriptomic data using a natural

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logarithmic transformation (y ~log(x+1)). All data were then centred and scaled. First, we

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performed a PCA on the proteome and transcriptome datasets and analysed the effects of

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breed on proteome and transcriptome inter-individual variation. Second, we performed a

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Between Groups Component Analysis (BGA) on the proteomic dataset, with breed groups

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(breed; small breeds [Booted bantam and Rosecomb bantam]; large breeds [Araucana, Czech

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golden pencilled, Minorca]) used as grouping factors. BGA focuses on between-group

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variability by performing a PCA on group means. The importance of the difference between

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groups is assessed by the ratio of between-group inertia over total inertia26. The statistical

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significance of differences between groups was checked using the Monte-Carlo permutation

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test.

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We used the Power Low Global Error Model (PLGEM)27 to identify egg white proteins

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differing in abundance between small and large breeds. The signal-to-noise ratio was 10 ACS Paragon Plus Environment

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calculated as it explicitly takes unequal variances into account and penalises those proteins

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that have higher variance in each group more than those proteins that have a high variance in

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one group and a low variance in another27. As PLGEM can only be fitted on a set of replicates

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under the same experimental conditions, we first applied the test to the Czech golden

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pencilled data. Correlation between mean values and standard deviations was high (r2= 0.96,

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Pearson=0.883); hence, we continued with the resampled signal-to-noise ratio and calculated

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differences with the corresponding p-values between the small and large breed groups.

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For the purpose of consistency, we selected 97 genes-protein pairs that were common to both

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the datasets (Table S3) when comparing the proteomic and transcriptomic data. We calculated

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Spearman’s correlation between the mean mRNA expression values and mean protein

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abundance. To further describe relationships between the proteome and transcriptome

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datasets, we used Co-Inertia multivariate Analysis (CIA)28,29. CIA is useful for analysing the

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relationships between two tables (here representing proteomic and transcriptomic data) having

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the same samples in rows. This method finds the maximum shared structure between two

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datasets representing the same individual. CIA finds ordinations (dimension reduction

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diagrams) from the datasets that are most similar. This is done by finding successive axes

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from the two datasets with maximum covariance. Co-structure between proteome and

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transcriptome datasets is measured by the RV-coefficient, ranging from 0 to 1, where 1

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indicates highest and 0 the lowest degree of co-structure. The statistical significance of co-

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inertia was evaluated with a Monte Carlo permutation test. CIA can be applied to datasets

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where the number of variables far exceeds the number of samples (which is the case when

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applied to x-omics data; see29,30). All statistical analyses were undertaken in R software

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version 3.4.031with the ade4 package32.



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Results and discussion

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General description of the hen egg white proteome

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At the proteomic level, we detected 115 egg white proteins at 0.01 FDR, ranging from 109 to

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112 in each breed-specific proteome (Table S4). The successful identification of these

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proteins was due to the relatively high number of peptides per identification (9.45 ± 13.19,

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mean ± SD), high sequence coverage (27.02 ± 19.86 %) and high unique sequence coverage

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(26.08 ± 19.97%). Furthermore, we obtained a high and significant Spearman’s rank

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correlation (r = 0.720, P

Domestic Fowl Breed Variation in Egg White Protein Expression

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