Christelle Cebo

Major proteins of the goat milk fat globule membrane

Fat is present in milk as droplets of triglycerides surrounded by a complex membrane derived from the mammary epithelial cell called milk fat globule membrane (MFGM). Although numerous studies have been published on human or bovine MFGM proteins, to date few studies exist on MFGM proteins from goat milk. The objective of this study was thus to investigate the protein composition of the goat MFGM. Milk fat globule membrane proteins from goat milk were separated by 6% and 10% sodium dodecyl sulfate-PAGE and were Coomassie or periodic acid-Schiff stained. Most of MFGM proteins [mucin-1, fatty acid synthase, xanthine oxidase, butyrophilin, lactadherin (MFG EGF-8, MFG-E8), and adipophilin] already described in cow milk were identified in goat milk using peptide mass fingerprinting. In addition, lectin staining provided a preliminary characterization of carbohydrate structures occurring on MFGM proteins from goat milk depending on alpha(S1)-casein genotype and lactation stage. We provide here first evidence of the presence of O-glycans on fatty acid synthase and xanthine oxidase from goat milk. A prominent difference between the cow and the goat species was demonstrated for lactadherin. Indeed, whereas 2 polypeptide chains were easily identified by peptide mass fingerprinting matrix-assisted laser desorption/ionization-time of flight analysis within bovine MFGM proteins, lactadherin from goat milk consisted of a single polypeptide chain. Another striking observation was the presence of caseins associated with MFGM preparations from goat milk, whereas virtually no caseins were found in MFGM extracts from bovine milk. Taken together, these observations strongly support the existence of a singular secretion mode previously hypothesized in the goat.

Validation of RNA isolated from milk fat globules to profile mammary epithelial cell expression during lactation and transcriptional response to a bacterial infection

Mastitis, an inflammation of the mammary gland, is the most costly infectious disease of dairy ruminants worldwide. Although it receives considerable attention, the early steps of the host response remain poorly defined. Here, we report a noninvasive method using milk fat globules (MFG) as a source of mammary RNA to follow the dynamics of the global transcriptional response of mammary epithelial cells (MEC) during the course of a bacterial infection. We first assessed that RNA isolated from MFG were representative of MEC RNA; we then evaluated whether MFG RNA could be used to monitor the MEC response to infection. Sufficiently high yields of good-quality RNA (RNA integrity numbers ranging between 6.7 and 8.7) were obtained from goat MFG for subsequent analyses. Contamination of MFG by macrophages and neutrophils, which can be trapped during creaming, was assessed and when using quantitative real-time PCR for cell-type specific markers, was shown to be weak enough (<8%) to affect MFG gene expression profiling. Using microarrays, we showed that RNA extracted from MFG and from mammary alveolar parenchyma shared approximately 90% of the highlighted probes corresponding in particular to genes encoding milk proteins (CSN, BLG, LALBA) and enzymes involved in milk fat synthesis and secretion (FASN, XDH, ADRP, SCD, and DGAT1). In addition, a gene involved in the acute-phase reaction, coding for the serum amyloid A3 (SAA3) protein, was found within the first 50 most highly expressed genes in a noninfectious context in both mammary alveolar parenchyma and MFG, strongly suggesting that SAA3 is expressed in MEC. We took advantage of this noninvasive RNA sampling to follow the early proinflammatory response of MEC during the course of a bacterial infection and showed that the levels of mRNA encoding SAA3 sharply increased at 24h postinfection. Taken together, our results demonstrate that MFG represent a unique source of MEC RNA to noninvasively sample sufficient amounts of high-quality RNA to assess the dynamics of MEC gene expression in vivo, especially during the first steps of infection, thereby paving the way for the discovery of early biomarkers for the control of intramammary infections. Furthermore, this noninvasive technique could be used to provide mammary transcriptomic data on a large scale, thus filling the gap between genomic and phenotypic data.

Goat αs1-casein genotype affects milk fat globule physicochemical properties and the composition of the milk fat globule membrane

Milk fat secretion is a complex process that initiates in the endoplasmic reticulum of the mammary epithelial cell by the budding of lipid droplets. Lipid droplets are finally released as fat globules in milk enveloped by the apical plasma membrane of the mammary epithelial cell. The milk fat globule membrane (MFGM) thus comprises membrane-specific proteins and polar lipids (glycerophospholipids and sphingolipids) surrounding a core of neutral lipids (mainly triacylglycerols and cholesterol esters). We have recently described major proteins of the MFGM in the goat and we have highlighted prominent differences between goats and bovine species, especially regarding lactadherin, a major MFGM protein. Here, we show that, in the goat species, the well-documented genetic polymorphism at the α(s1)-casein (CSN1S1) locus affects both structure and composition of milk fat globules. We first evidenced that both milk fat globule size and ζ-potential are related to the α(s1)-casein genotype. At midlactation, goats displaying strong genotypes for α(s1)-casein (A/A goats) produce larger fat globules than goats with a null genotype at the CSN1S1 locus (O/O goats). A linear relationship (R(2)=0.75) between fat content (g/kg) in the milk and diameter of fat globules (μm) was established. Moreover, we found significant differences with regard to MFGM composition (including both polar lipids and MFGM proteins) from goats with extreme genotype at the CSN1S1 locus. At midlactation, the amount of polar lipids is significantly higher in the MFGM from goats with null genotypes for α(s1)-casein (O/O goats; 5.97±0.11mg/g of fat; mean ± standard deviation) than in the MFGM from goats with strong genotypes for α(s1)-casein (A/A goats; 3.96±0.12mg/g of fat; mean ± standard deviation). Two MFGM-associated proteins, namely lactadherin and stomatin, are also significantly upregulated in the MFGM from goats with null genotype for α(s1)-casein at early lactation. Our findings are discussed with regard to techno-functional properties and nutritional value of goat milk. In addition, the genetic polymorphism in the goat species appears to be a tool to provide clues to the lipid secretion pathways in the mammary epithelial cell.

Proteomics of the milk fat globule membrane from Camelus dromedarius

Camel milk has been widely characterized with regards to casein and whey proteins. However, in camelids, almost nothing is known about the milk fat globule membrane (MFGM), the membrane surrounding fat globules in milk. The purpose of this study was thus to identify MFGM proteins from Camelus dromedarius milk. Major MFGM proteins (namely, fatty acid synthase, xanthine oxidase, butyrophilin, lactadherin, and adipophilin) already evidenced in cow milk were identified in camel milk using MS. In addition, a 1D‐LC‐MS/MS approach led us to identify 322 functional groups of proteins associated with the camel MFGM. Dromedary MFGM proteins were then classified into functional categories using DAVID (the Database for Annotation, Visualization, and Integrated Discovery) bioinformatics resources. More than 50% of MFGM proteins from camel milk were found to be integral membrane proteins (mostly belonging to the plasma membrane), or proteins associated to the membrane. Enriched GO terms associated with MFGM proteins from camel milk were protein transport (p‐value = 1.73 × 10−14), translation (p‐value = 1.08 × 10−11), lipid biosynthetic process (p‐value = 6.72 × 10−10), hexose metabolic process (p‐value = 1.89 × 10−04), and actin cytoskeleton organization (p‐value = 2.72 × 10−04). These findings will help to contribute to a better characterization of camel milk. Identified MFGM proteins from camel milk may also provide new insight into lipid droplet formation in the mammary epithelial cell.

Identification of major milk fat globule membrane proteins from pony mare milk highlights the molecular diversity of lactadherin across species

Although several studies have been devoted to the colloidal and soluble protein fractions of mare milk (caseins and whey proteins), to date little is known about the milk fat globule membrane (MFGM) protein fraction from mare milk. The objective of this study was thus to describe MFGM proteins from Equidae milk and to compare those proteins to already described MFGM proteins from cow and goat milk. Major MFGM proteins (namely, xanthine oxidase, butyrophilin, lactadherin, and adipophilin) already described in cow or goat milk were identified in mare milk using mass spectrometry. However, species-specific peculiarities were observed for 2 MFGM proteins: butyrophilin and lactadherin. A highly glycosylated 70-kDa protein was characterized for equine butyrophilin, whereas proteins of 64 and 67 kDa were characterized for cow and goat butyrophilin, respectively. Prominent differences across species were highlighted for lactadherin. Indeed, whereas 1 or 2 polypeptide chains were identified, respectively, by peptide mass fingerprinting matrix-assisted laser desorption/ionization-time of flight analysis for caprine and bovine lactadherin, 4 isoforms (60, 57, 48, and 45 kDa) for lactadherin from mare milk were identified by 10% sodium dodecyl sulfate-PAGE. Polymerase chain reaction experiments on lactadherin transcripts isolated from milk fat globules revealed the existence of 2 distinct lactadherin transcripts in the horse mammary gland. Cloning and sequencing of both transcripts encoding lactadherin showed an alternative use of a cryptic splice site located at the end of intron 5 of the equine lactadherin-encoding gene. This event results in the occurrence of an additional alanine (A) residue in the protein that disrupts a putative atypical N-glycosylation site (VNGC/VNAGC) described in human lactadherin. Liquid chromatography coupled with tandem mass spectrometry analyses confirmed the existence of both lactadherin variants in mare MFGM. We show here that lactadherin from Equidae milk is much more complex than that from Bovidae milk (i.e., cow and goat milk), therefore raising questions regarding the precise function of these different isoforms, if any, in the equine mammary gland.

Combining proteomic tools to characterize the protein fraction of llama (Lama glama) milk

Llamas belong to the Camelidae family along with camels. While dromedary camel milk has been broadly characterized, data on llama milk proteins are scarce. The objective of this study was thus to investigate the protein composition of llama milk. Skimmed llama milk proteins were first characterized by a 2D separation technique coupling RP‐HPLC in the first dimension with SDS‐PAGE in the second dimension (RP‐HPLC/SDS‐PAGE). Llama milk proteins, namely caseins (αs1‐, αs2‐, β‐, and κ‐caseins), α‑lactalbumin, lactoferrin, and serum albumin, were identified using PMF. Llama milk proteins were also characterized by online LC‐ESI‐MS analysis. This approach allowed attributing precise molecular masses for most of the previously MS‐identified llama milk proteins. Interestingly, α‐lactalbumin exhibits distinct chromatographic behaviors between llama and dromedary camel milk. De novo sequencing of the llama α‐lactalbumin protein by LC coupled with MS/MS (LC‐MS/MS) showed the occurrence of two amino acid substitutions (R62L/I and K89L/I) that partly explained the higher hydrophobicity of llama α‐lactalbumin compared with its dromedary counterpart. Taken together, these results provide for the first time a thorough description of the protein fraction of Lama glama milk.

Milk Proteins | Inter-Species Comparison of Milk Proteins: Quantitative Variability and Molecular Diversity

In the last few years, developments in molecular biology, genomics, and proteomics have allowed remarkable progress in the understanding of milk protein structure and function, highlighting the extreme complexity and large variability (qualitative and quantitative) of the milk protein fraction, across, but also within, species. Meaningful examples taken in different species, including non-eutherian mammals, are discussed in this article to show how genomes and genetic polymorphisms may modulate the milk protein fraction by affecting different cellular processes, mainly at the posttranscriptional level (splicing of primary transcripts, posttranslational modifications), making milk a more complex biological fluid than previously assumed. However, the repertoire of minor milk proteins remains to be characterized in most species. Numerous substantiated or potential bioactive protein components have been found and many others still remain to be identified either as intact protein or as derived peptides, encrypted in the sequence of milk proteins. This is probably one of the greatest challenges facing milk science in the next years to provide the food industry and consumers with the basis for health-promoting properties of these proteins and peptides.

Phosphoproteomics of the goat milk fat globule membrane: New insights into lipid droplet secretion from the mammary epithelial cell

Mechanisms of milk lipid secretion are highly controversial. Analyzing the fine protein composition of the “milk fat globule membrane” (MFGM), the triple‐layered membrane surrounding milk lipid droplets (LDs) can provide mechanistic clues to better understand LD biosynthesis and secretion pathways in mammary epithelial cells (MECs). We therefore combined a high‐sensitive Q‐Exactive LC‐MS/MS analysis of MFGM‐derived peptides to the use of an in‐house database intended to improve protein identification in the goat species. Using this approach, we performed the identification of 442 functional groups of proteins in the MFGM from goat milk. To get a more dynamic view of intracellular mechanisms driving LD dynamics in the MECs, we decided to investigate for the first time whether MFGM proteins were phosphorylated. MFGM proteins were sequentially digested by lysine‐C and trypsin proteases and the resulting peptides were fractionated by a strong cation exchange chromatography. Titanium beads were used to enrich phosphopeptides from strong cation exchange chromatography eluted fractions. This approach lets us pinpoint 271 sites of phosphorylation on 124 unique goat MFGM proteins. Enriched GO terms associated with phosphorylated MFGM proteins were protein transport and actin cytoskeleton organization. Gained data are discussed with regard to lipid secretory mechanisms in the MECs. All MS data have been deposited in the ProteomeXchange with identifier PXD001039 (http://proteomecentral.proteomexchange.org/dataset/PXD001039).