Inter-Species Comparison of Milk Fat Globule Membrane Proteins Highlights the Molecular Diversity of Lactadherin

International Dairy Journal 24 (2012) 70e77

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International Dairy Journal

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Inter-species comparison of milk fat globule membrane proteins highlights the molecular diversity of lactadherin

Christelle Cebo*, Patrice Martin

INRA, UMR1313 Unité Génétique Animale et Biologie Intégrative, F-78350 Jouy-en-Josas, France article info abstract

Article history: Fat is present in milk as droplets of triacylglycerols surrounded by a complex membrane derived from the Received 30 June 2011 mammary epithelial cell called the milk fat globule membrane (MFGM). In-depth proteomic studies have Received in revised form been published for bovine MFGM proteins. However, to date, only sparse studies exist on MFGM proteins 23 September 2011 from non-cow milk. We have therefore characterized MFGM proteins in goat, sheep, horse and camel Accepted 25 September 2011 milk and we have highlighted prominent differences across species, especially for lactadherin, a major protein of the milk fat globule membrane. Indeed, lactadherin from goat and sheep milk appears as a single polypeptide chain in 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS- PAGE), whereas two and four polypeptide chains are respectively identiﬁed for lactadherin from camel and horse milk. Implications of the ﬁndings, especially with regard to bioactive properties of milk are discussed. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction studies on bovine MFGM proteins have been reported. Indeed, more than 120 proteins with diverse functions such as trafficking, Fat is present in milk as droplets of neutral lipids (mainly tri- signalling or immune response, have been identified in bovine acylglycerols) surrounded by a complex membrane, called the milk MFGM (Reinhardt & Lippolis, 2006, 2008). Although a report of fat globule membrane (MFGM). Formation of lipid droplets in the a study has been recently published for MFGM proteins from sheep mammary epithelial cell (MEC) is thought to occur by accumulation milk (Pisanu et al., 2011), studies on MFGM proteins from non-cow of lipids within the bilayer of the endoplasmic reticulum (ER). Lipid origin are sparse and are essentially devoted to the fat fraction of droplets dissociate from the ER by budding and are transported to milk. Consequently, based on our previous report on MFGM apical cell regions by as yet unknown mechanisms but probably proteins from goat milk (Cebo, Caillat, Bouvier, & Martin, 2010), we involving cytoskeletal components. These droplets are secreted in provide here an inter-species comparison of major MFGM proteins milk as fat globules by being progressively enveloped by the apical from cow, goat, sheep, horse and camel milk. Results show a high plasma membrane (Bauman, Mather, Wall, & Lock, 2006; Heid & species-to-species variation especially regarding lactadherin, Keenan, 2005). Hence, MFGM composition and structure reflect a major protein of the milk fat globule membrane. Implications of those of ER and plasma membranes from the MEC. the findings with regard to nutritional value of milk are discussed. Both technological and nutritional properties have been reported for most of MFGM components (Dewettinck et al., 2008; Spitsberg, 2005). MFGM acts as an emulsifying agent for lipids in the milk 2. Materials and methods aqueous phase. It has also been shown that several MFGM proteins can inhibit adhesion of pathogens to intestinal epithelia, by acting as 2.1. Animals and milk samples decoy receptors in the gastrointestinal tract (Schroten, 1998). Thus, increasing attention is being paid to MFGM components. Caprine milk samples were collected from A/A (homozygous MFGM proteins, mostly from bovine origin, have been exten- animals for as1-casein, allele A) primiparous Alpine dairy goats at sively reviewed (Mather, 2000). In addition, recent proteomic mid-lactation (149 17 days post-partum; INRA “Domaine de Galle” experimental farm, Avord, France). Preservatives (0.05 g L 1 potassium dichromate; 10 mM amino-caproic acid; 10 mM phenyl- fl * Corresponding author. Tel.: þ33 1 34 65 27 73; fax: þ33 1 34 65 29 26. methylsulfonyl uoride) were added to goat milk immediately after E-mail address: [email protected] (C. Cebo). milking to prevent proteolysis.

Ovine milk samples (Lacaune breed) were collected at the INRA University, Denmark). Blots were then washed (3 5 min followed experimental farm “La Fage” (UE0321, 12250 Roquefort-sur- by 3 15 min) in TBS 0.1% Tween-20 and incubated with goat anti- Soulzon, France). rabbit secondary antibodies coupled to horseradish peroxidase Equine milk samples were collected from five primiparous and (HRP) (1/5000, Interchim, Montluçon, France). Immunocomplexes one multiparous Welsh mares aged 3e11 years, with an average were revealed by the enhanced chemiluminescence (ECL) system body weight after foaling of 348.7 49.6 kg at 133 11 days post- (Dutscher, Brumath, France). partum from an INRA experimental farm (Unité Expérimentale de Physiologie animale de l’Orfrasière, Nouzilly, France). The mare and 2.3. Analysis and identification of proteins from 1D-PAGE by foal were separated for 1 h before milking to increase milk yield. MALDI-TOF mass spectrometry Then, the foal was brought back to the mare, but it was not allowed to suckle. Five hundred microlitres of an oxytocin solution Proteins were excised from 1D-gel and gel pieces were washed (OcytovemÔ,10UImL 1) were administered to the mare followed twice in ultrapure water followed by two washes in 50% acetonitrile by hand-milking in the presence of the foal. (ACN) and 50 mM ammonium bicarbonate (NH4HCO3) (v/v). After Camel (Camelus dromedarius) milk samples were collected from gel-drying for 10 min, the digestion was performed in 30 mLof two individuals at 150 days post-partum at the Experimental 50 mM NH4HCO3 (pH 8.0) with 0.2 mg of modified trypsin station of the Arid Land Institute of Medenine (Medenine, Tunisia). (sequencing grade; Promega, Charbonnières, France) for 16 h at Bovine milk samples were from two dairy cows at mid-lactation 37 C. The identity of peptides was obtained using a Voyager DE stage (Unité Mixte de Recherches Production du Lait, INRA Saint- super STR MALDI-TOF Mass Spectrometer (Applied Biosystems, Gilles, France). Milk samples were stored at 20 Cin50mL Foster City, CA, USA) equipped with a nitrogen laser emitting at aliquots. 337 nm. One microlitre of tryptic digest was mixed on the stainless For MFGM protein extraction, samples were incubated for steel MALDI plate with 1 mL of CHCA (alpha-cyano-4- 30 min at 37 C, then centrifuged at 1000 g for 15 min at 20 C hydroxycinnamic acid, SigmaeAldrich) at 5 mg mL 1 in ACN/0.3% followed by cooling for 30 min at 4 C to help skimming. Fat trifluoroacetic acid (TFA) (v/v) and dried at room temperature. globules were recovered in the supernatant layer and washed three Spectra were recorded in positive reflector mode with 20 kV as times with a buffer containing 0.9% (w/v) NaCl to remove residual accelerating voltage, a delayed extraction time of 120 ns, and a 63% caseins and whey proteins eventually adsorbed to fat globules. grid voltage. Internal mass calibration was performed using tryptic autolysis peptides ([M þ H]þ¼842.51 and [M þ H]þ¼2211.11). 2.2. Extraction and analysis of milk fat globular membrane proteins Mass spectra were treated by Data Explorer 4.2 (Applied Biosystems) with the following parameters: baseline correction, noise removal of MFGM proteins were extracted from milk fat with an SDS- 2, and peak resolution of 10,000. Spectral profiles were collected in containing solution as previously described (Cebo et al., 2010) the mass:charge ratio ¼ 800 to 3000 Da. All peptide masses were with some modifications. Briefly, lysis buffer comprising 63 mM assumed to be monoisotopic and protonated molecular ions TriseHCl, pH 9.0, and 2% sodium dodecyl sulphate (SDS) supple- [M þ H]þ. Proteins were identified among Swiss-Prot and TrEMBL mented with a protease inhibitor cocktail (Complete Mini, EDTA- databases using the Aldente Peptide Mass Fingerprinting tool from free, Roche Diagnostics, Meylan, France) was added to washed fat Expasy web site (http://www.expasy.org/tools/aldente/) with the globules then incubated for 1 h at room temperature (20 C) with following parameters: trypsin specificity, one missed cleavage, periodical vortexing (every 15 min) and centrifuged at 10,000 g 30 ppm mass accuracy, carbamidomethylation and methionine for 10 min. The floating cream layer was removed and lysates were oxidation as fixed and variable modifications, respectively. centrifuged again and then stored at 80 C for further analysis. Protein concentration was assessed with the Bio-Rad RC-DC Protein 2.4. RNA isolation and sequencing of lactadherin transcripts Assay according to the instructions of the manufacturer (Bio-Rad, Marnes-la-Coquette, France). Goats and sheep were slaughtered immediately after milking. MFGM proteins (20 mg) were resolved by 6% SDS-PAGE, followed Mammary tissues were collected in the secretory area containing by staining with Bio-Safe Coomassie (Bio-Rad). For total glycopro- lobulo-alveolar structures (acini). Samples were immediately tein analysis, 50 mg proteins were separated on 6% acrylamide gels frozen in liquid nitrogen and stored at 80 C. Total RNA was (4.5% acrylamide for the stacking gel) and revealed with the Schiff extracted using the TRIZOL reagent (Life Technologies, Villebon- reagent (SigmaeAldrich, Saint-Quentin-Fallavier, France) in accor- sur-Yvette, France). Alternatively, total RNA (camel and horse dance with the manufacturer’s protocol. Briefly, gels were fixed species) was extracted from milk fat globules material as previously overnight in 40% ethanol, 7% acetic acid. Gels were then washed described (Maningat et al., 2009). RNA concentration was deter- 30 min twice with fresh fixative solution. Carbohydrates were mined using a Nanodrop 1000 spectrophotometer (Thermo Fisher oxidized for 1 h by immersing gels in a solution of 1% periodic acid, Scientific, Rockford, Illinois, USA) and RNA quality was assessed 3% acetic acid, then washed ten times (10 min each) with water to with Agilent RNA 6000 LabChips (Agilent Technologies, Massy, remove traces of periodic acid. Gels were immersed for 1 h in the France). RNA integrity numbers (RIN) of all samples were at least Schiff’s reagent in the dark to reveal the glycoprotein bands, 7.0. RNA (500 ng) was reverse transcribed by the Superscript III washed 15 min with water. Images were immediately acquired reverse transcriptase and an equimolar mix of oligo-dT and random after washing. primers (Life Technologies). Prior to PCR amplification, cDNA For western blotting experiments, 20 mg of MFGM proteins were templates were denaturated at 95 C for 2 min. Each PCR reaction resolved by 10% SDS-PAGE and electro transferred onto nitrocel- was carried out with 30 cycles at 95 C for 1 min (denaturation), lulose. To avoid non-specific binding, blots were immersed for 1 h 58 C for 2 min (annealing), and 72 C for 3 min (extension). A final at room temperature (20 C) in a Tris Buffered Saline (TBS) solution extension step was added (72 C for 5 min). containing 1% (w/v) non fat dry milk and 1% (w/v) poly- Rapid amplification of cDNA ends (RACE) experiments was also vinylpyrrolidone (SigmaeAldrich). Blots were briefly rinsed in TBS performed to amplify the 30end of goat lactadherin transcript. First- and probed for 2 h in TBS 0.1% Tween-20 (SigmaeAldrich) with strand cDNA synthesis was initiated at the poly(A)-tail of mRNA antibodies against bovine lactadherin (1/10,000, kindly provided by using an oligo(dT)-anchor primer (sequence from 50 to 30: GAC CAC Dr J.T. Rasmussen, Department of Molecular Biology, Aarhus GCG TAT CGA TGT CGA CTT TTT TTT TTT TTT TTT V). Total RNA 72 C. Cebo, P. Martin / International Dairy Journal 24 (2012) 70e77

(1.5 mg) was then converted into cDNA by the Superscript III reverse chains are easily identified for bovine lactadherin by PMF MALDI- transcriptase (Life Technologies) using the oligo (dT)-anchor primer TOF analysis within bovine MFGM proteins as reported in litera- at a final concentration of 2.5 mM. Amplification of caprine lactad- ture (Cebo et al., 2010). Interestingly, ovine lactadherin was also herin was then directly conducted onto generated cDNA using the identified as a single protein of 56 kDa, i.e., slightly larger than the PCR anchor primer (sequence from 50 to 30: GAC CAC GCG TAT CGA goat lactadherin (54 kDa) (Figs. 1 and 2, this study). Lactadherin TGT CGA C) and a gene-specific primer (sequence from 50 to 30:GGT from camel milk was characterized as two equally expressed TTC CCT ACT ACG CAC GA). Sizing of amplified DNA fragments was polypeptide variants of 49 and 55 kDa. Up to four bands corre- performed with Agilent 2100 bioanalyzer (DNA 7500 kit, Agilent sponding to equine lactadherin can be identified within MFGM Technologies) on 1 mL sample. proteins in 10% SDS-PAGE (Cebo et al., unpublished data-b). According to manufacturer’s instructions, sizing resolution was Immunoblotting experiments using specific antibodies against 5% in the 100e1000 bp range and 15% in the 1000e7500 bp bovine lactadherin (a gift from Dr. Jan Trige Rasmussen, Depart- range. PCR products were sequenced in both directions by Eurofins ment of Molecular Biology, Aarhus University, Denmark) confirmed MWG GmbH (Ebersberg, Germany) and in silico translated by using our proteomic analyses (Fig. 2). Bovine lactadherin (lanes 1e2), and the Translate tool at the Swiss Institute of Bioinformatics (http:// to a lesser extent, caprine (lanes 3e4) and ovine (lanes 5e6) lac- www.expasy.org/tools/dna.html). Alignment of amino acid tadherin, were strongly stained by PAS 6/7 antibodies (Fig. 2A). sequences were performed by using the ClustalW2 tool at the EBI Lactadherin from camel and horse milk are hardly detected by site (http://www.ebi.ac.uk/Tools/msa/clustalw2/). N-glycosylation antibodies against bovine lactadherin. However, increased expo- sites were predicted using the NetNGlyc 1.0 Server (web site: http:// sure times to the ECL reagent allowed revealing two major bands www.cbs.dtu.dk/services/NetNGlyc/). for lactadherin from camel milk (lanes 7e8), together with the two upper bands of equine lactadherin (lane 9; Fig. 2B). 3. Results Butyrophilin from horse milk appears as a 70 kDa protein, whereas proteins of 64 kDa and 67 kDa were characterized for cow 3.1. Milk fat globular membrane proteins: a high species-to-species and goat butyrophilin, respectively. Molecular masses of ovine and variability bovine butyrophilin were comparable (around 64 kDa). A 63 kDa protein was identified for butyrophilin within MFGM proteins from Fig. 1 shows a representative 6% SDS-PAGE pattern of milk fat camel milk (Fig. 1). globule membrane (MFGM) samples extracted either from cow (lane 1), goat (lanes 2e3), sheep (lanes 4e5), camel (lanes 6e7) or 3.2. Amplification and sequencing of lactadherin transcripts from horse (lanes 8e9) milk. Major MFGM proteins were identified by goat, sheep, camel and horse mammary gland Peptide Mass Fingerprinting-Matrix-Assisted Laser Desorption/ Ionisation-time of flight (MALDI-TOF) mass spectrometry analysis Identification of several protein bands in SDS-PAGE for equine as xanthine oxidase (XDH/XO), butyrophilin (BTN), lactadherin lactadherin led us to search for splice variants for lactadherin in the (LDH), and adipophilin (ADRP). Although similarities across species horse species. We also attempted to amplify lactadherin transcripts can be highlighted for some MFGM proteins (mainly XO), the in the goat, sheep, and camel species. protein profile of MFGM proteins exhibits prominent differences The Equus caballus genome has been recently released (Wade from species to species, especially regarding two major proteins of et al., 2009). Eight exons covering a 1433 bp sequence were the MFGM, namely, butyrophilin and lactadherin. assigned for the lactadherin transcript from Eq. caballus (Ensembl Indeed, we have previously demonstrated that goat lactadherin gene ENSECAG00000018875). Goat genome sequencing is ongoing consists of a single polypeptide chain whereas two polypeptide and a sequence for caprine lactadherin (Guanzhong breed) has been recently released (GenBank Accession GU593981.1). Blast of bovine sequence for lactadherin (accession number NM_176610.1) led us to retrieve two non-overlapping EST sequences for sheep lactadherin (GenBank accession numbers DY499365.1 and GO744678.1) and one EST for dromedary lactadherin (accession

Fig. 1. Representative SDS-PAGE pattern of milk fat globule membrane (MFGM) Fig. 2. Identification of lactadherin protein through species by western blotting. proteins from several species. The MFGM proteins are from: lane 1, cow milk; lanes 2 MFGM proteins are from: lanes 1 and 2, bovine milk; lanes 3 and 4, caprine milk; lanes and 3, goat milk; lanes 4 and 5, sheep milk; lanes 6 and 7, camel milk; lanes 8 and 9, 5 and 6, ovine milk; lanes 7 and 8, dromedary milk; lane 9, equine milk. PAS 6/7 horse milk. MFGM proteins identified in samples were as follows: a, xanthine oxidase; antibodies react strongly with bovine, caprine and ovine lactadherin (panel A). b, butyrophilin; c, lactadherin; d, adipose differentiation related protein (adipophilin). Exposure times to enhanced chemiluminescence (ECL) reagent were greatly increased Positions of protein standards (kDa) are indicated to the left. Note that prominent to reveal the lower reactive lactadherin bands from camel or horse milk (panel B). differences were highlighted among species, especially for lactadherin (c) protein. Positions of molecular mass standards (kDa) are indicated to the left. C. Cebo, P. Martin / International Dairy Journal 24 (2012) 70e77 73 number CL5044contig1, Arabian Camel Genome project, http:// camel.kacst.edu.sa). Primers were thus designed to amplify specific regions of the lactadherin transcript in goat, sheep, camel and horse species (Table 1). A single-size transcript was amplified for most species, except in the horse species where two splice variants were amplified (Fig. 3). Cloning and sequencing of both transcripts encoding equine lactadherin led us to demonstrate the alternative use of a cryptic splice site located at the end of intron 5 of the equine lactadherin encoding gene. Interestingly, this event results in the occurrence of an additional alanyl (A) residue in the protein, disrupting a putative atypical N-glycosylation site (VNGC/VNAGC) (Cebo et al., unpublished data). Fig. 4 shows the alignment of amino acid sequences of lactadherin from cow (Q95114, UniprotKB/Swiss-Prot database), goat (ADD84517.1, Guanzhong dairy breed, Genbank database; JN016709, Alpine dairy breed, Genbank database), sheep (JN016710, Genbank database), horse (ENSECAP00000016644, Ensembl database; JF681042 (splice variant, our data), Genbank database; O77718 (testis), UniprotKB-Swiss-Prot database), and camel (GenBank JF423914). Among the differences between bovine with caprine or ovine lactadherin amino acid sequences, the insertion of an aspartic acid at position 63 of the mature bovine lactadherin (in the second EGF-like domain) can be noted. Surprisingly, in the lactadherin amino acid sequence for caprine lactadherin (Guanzhong dairy breed, Genbank # ADD84517), four amino acids are deleted within the first discoidin domain (corresponding sequence in the mature bovine lactadherin: Phe- eIleeGlyeAsn, starting at position 204 of the protein). In the horse species, at least three distinct proteins have been evidenced (Fig. 4). We demonstrated the occurrence of a cryptic splicing event for equine lactadherin that leads to the disruption of a putative AsneGlyeCys (NGC) N-glycosylation site, occupied with glycans in human lactadherin (Picariello, Ferranti, Mamone, Roepstorff, & Addeo, 2008) and conserved in the horse species (Fig. 4). In addition, the amino acid sequence RFELLGCEVNGCAEPLGLEDNSI located in equine lactadherin, and corresponding to the end of exon 5 and the beginning of exon 6, was deleted in the testis specific transcript (O77718).

3.3. Highly glycosylated proteins from cow, goat, and sheep milk

MFGM proteins (50 mg) from cow (lane 1), goat (lanes 2e3), sheep (lane 4e5), camel (lanes 6e7) and horse (lanes 8e9) milk were electrophoresed on 6% SDS-PAGE and stained with Periodic acid/Schiff reagent (PAS), which revealed only proteins with high carbohydrate contents (Fig. 5). Among PAS-stained proteins, MUC- 1, a high-molecular weight protein containing more than fifty percent carbohydrate, was identified as one or two protein bands Fig. 3. Electrophoregrams of PCR products for lactadherin from: panel A, goat; panel B, equally stained by the PAS reagent, depending on the fact that sheep; panel C, camel; panel D, horse. Primers used for PCR amplification are indicated animals were homozygous or heterozygous for MUC-1 (Campana, in Table 1. Lower marker (50 bp) and upper electrophoresis markers (10,380 bp) are Josephson, & Patton, 1992; Huott, Josephson, Hens, Rogers, & indicated. Note that two peaks are observed for equine lactadherin transcript whereas Patton, 1995). Indeed, since each co-dominant MUC-1 allele can a single peak is observed for caprine, ovine, and dromedary lactadherin transcripts. contain a variable number of tandem repeats (VNTR) encoding

a 20-amino acid motif, different sizes of MUC-1 can be observed by Table 1 Oligonucleotide primers used for the ampliﬁcation of lactadherin in goat, sheep, SDS-PAGE analysis. horse and camel species. MUC-1 from sheep milk (lanes 4e5) appears much larger than

0 0 MUC-1 from bovine milk (lane 1), but smaller in size than its Species Primers (sequence 5 > 3 ) Amplicon size (nt) caprine counterpart (lanes 2e3). Accordingly, analysis of the MUC- Goat, sheep Forward CCG GAC TCT TCG CCT TCT 1.154 Reverse CTG GAA CGG CAC CTC AAA TA 1 polymorphism in the ovine species showed that the sheep Camel Forward TTC TGT GAC TCC AGC CAG TG 1.099 repetitive region seemed to be smaller in size than the repetitive Reverse TAT CGA AGT TGC CAG GGA AG region of the goat (Rasero et al., 2007). MUC-1 proteins from camel Horse Forward GTG AAC CTC ATG CGG AAG AT 704 and horse milk were barely detectable. This may be due to a higher Reverse AGG AAG GGC GTC TCA AAC AT molecular weight of equine and dromedary mucins not allowing 74 C. Cebo, P. Martin / International Dairy Journal 24 (2012) 70e77

Fig. 4. Alignment of the amino acid sequences of lactadherin from cow, goat, sheep horse, and camel species. For clarity, signal peptides sequences (when available) are omitted. Functional domains according to bovine lactadherin, are indicated. N-glycosylation sites identiﬁed in bovine or human lactadherin are emboldened. Bta, Bos taurus; Chi, Capra hircus; Oar, Ovis aries; Hsa, Homo sapiens; Eca, Equus caballus; Cdr, Camelus dromedaries. The superscript 1 indicates partial coding sequence. C. Cebo, P. Martin / International Dairy Journal 24 (2012) 70e77 75

(Wheeler et al., 2009). A 50-amino acid amyloid-like peptide, called medin and corresponding to an integral proteolytic fragment of lactadherin has also been characterized in aortic tissues (Haggqvist et al., 1999). Two alternative splicing variants were shown to be expressed in mice mammary tissue. The variant predominantly expressed, during lactation, is longer with an additional 37-amino acid long domain with multiple O-glycosylation sites. By contrast, the short variant is ubiquitously present in several tissues and its expression decreased during lactation (Oshima et al., 1999). A cDNA splice variant was also identified in cattle, but bovine lactadherin is expressed as two glycosylation variants (Hvarregaard et al., 1996). We have recently shown that lactadherin from goat milk appears as a single polypeptide chain whereas two polypeptide fi fi Fig. 5. Highly glycosylated MFGM proteins from: lane 1, cow; lanes 2 and 3, goat; lanes chains are easily identi ed by peptide mass ngerprinting-matrix- 4 and 5, sheep; lanes 6 and 7, camel; lanes 8 and 9, horse. MFGM proteins (50 mg) were assisted laser desorption/ionisation-time of flight (PMF MALDI- separated on 6% SDS-PAGE and stained with periodic acid-Schiff reagent after oxida- TOF) analysis within bovine MFGM proteins (Cebo et al., 2010). tion of carbohydrate moieties by periodic acid. Positions of previously identified Similar findings are reported here for ovine lactadherin. In the proteins in bovine milk are indicated to the left: MUC, mucin; CD, cluster of differ- horse species, we have demonstrated both post-transcriptional (i.e., entiation; BTN, butyrophilin. Positions of protein standards (kDa) are indicated to the right. splicing variants) and post-translational isoforms for lactadherin (Cebo et al., unpublished data). Our recent work shows that two major protein variants exist for lactadherin in the camel species SDS-PAGE resolution of these glycoproteins. Accordingly, a previous (Saadaoui et al., unpublished data). Lactadherin structure is thus study reported extensive glycosylation of primates, horse, and species dependent, raising the question of the function of these camel MFGM proteins whereas true ruminants mucins (cow, goat, different isoforms in the biological function of lactadherin (Cebo & sheep) are characterized by a lower molecular weight (Welsch Martin, 2011). et al., 1990). Bands corresponding to MUC-15 (also known as PAS III), CD36, 4.2. Biological activities of lactadherin in the mammary gland and butyrophilin and lactadherin were visualized after PAS staining, other tissues thus accounting for the presence of carbohydrates. Interestingly, in PAS-stained gels, only one band corresponding to the heavily gly- A growing number of studies describe the biological roles cosylated lactadherin from goat and sheep milk is visualized mediated by lactadherin in the mammary gland but also in other whereas two bands corresponding to two distinct glycoproteins are tissues, including immune cells, intestinal cells, or the male genital visible for lactadherin from bovine (Hvarregaard, Andersen Lars tract (Raymond, Ensslin, & Shur, 2009). Lactadherin is able to Berglund, Rasmussen, & Petersen, 1996) or camel milk (Fig. 5). recognize cells undergoing apoptosis through its phospholipid- binding motifs. Since then, lactadherin is involved in numerous 4. Discussion apoptosis-related processes, including mammary gland remodelling (Hanayama & Nagata, 2005). Indeed, MFG-E8 is required in the 4.1. Lactadherin, a many-faceted protein clearance of apoptotic cells and milk fat globule during the mammary gland involution process. A deficiency of MFG-E8 leads Lactadherin (milk fat globule-epidermal growth factor; MFG- to the accumulation of milk fat globules in the mammary ducts E8) is a glycoprotein that was initially found in milk and during involution causing inflammation of mammary ducts and mammary epithelium cells (Stubbs et al., 1990). The main features mastitis. As a consequence, an aberrant redevelopment of the of lactadherin are the presence of two Epidermal Growth Factor mammary glands during subsequent lactation together with (EGF)-like domains in the N-terminal region of the protein with an a decrease of milk production is observed, thus demonstrating arginine-glycine-aspartic acid (RGD) sequence in the second EGF- a key role for lactadherin in mammary gland remodelling during like domain, and of two C-terminal regions of about 150 amino the involution process (Hanayama & Nagata, 2005). acids called F5/8 type C or C1/C2-like domains also present in A growing body of evidence also favours a role for lactadherin in coagulation factors V and VIII (Hvarregaard et al., 1996; Larocca milk fat secretion. A recent study (Qu et al., 2011) investigated et al., 1991; Stubbs et al., 1990). The C-terminal domain of the variations in the MFG-E8 gene and its associations with growth second F5/8 repeat has been shown to be responsible for traits and milk performance in the goat species (Capra hircus taxon, membrane binding through a phosphatidylserine-binding motif Guanzhong dairy breed). Interestingly, the authors identified four (Shi & Gilbert, 2003; Shi, Heegaard, Rasmussen, & Gilbert, 2004). single nucleotide polymorphisms (SNPs) in the goat MFG-E8 gene. 0 0 The RGD sequence is a cell-adhesion motif able to bind to avb3/5 They were in the 5 -Untranslated Region (5 -UTR), the fourth integrins therefore mediating cell adhesion as well as cell trans- intron, the seventh exon, the seventh intron. Among these SNP, the duction mechanisms (Taylor, Couto, Scallan, Ceriani, & Peterson, g.14892 T > C located in the seventh exon of the goat lactadherin 1997). gene was found to be significantly associated with the milk fat yield Multiple forms of lactadherin have already been characterized in the Guanzhong dairy breed (Qu et al., 2011). This is the in humans (Cavaletto et al., 1999; Giuffrida, Cavaletto, Giunta, Conti, demonstration of a relationship between lactadherin gene & Godovac-Zimmermann, 1998,). The 50 kDa protein is the full- expression and milk fat secretion process. Similarly, in as1-casein length protein also known as breast carcinoma protein BA46 that defective goats displaying secretory pathways dysfunction (Chanat, is highly expressed in human breast tumours (Larocca et al., 1991). Martin, & Ollivier-Bousquet, 1999; Martin, Szymanowska, Zwierz- The 30 kDa protein is a truncated form of BA46 consisting of the C- chowski, & Leroux, 2002), we have demonstrated that lactadherin terminal factor V/VIII-like domain that appears to anchor BA46 to is about two-fold overexpressed (Cebo et al., unpublished data). We the MFGM (Giuffrida et al., 1998). The existence of a new splice have also provided first evidence for a relationship between genetic variant lacking exon 7 has been recently reported in human kidney polymorphism at the CSN1S1 locus, milk fat globule biophysical 76 C. Cebo, P. Martin / International Dairy Journal 24 (2012) 70e77 properties, and expression level of lactadherin, thus suggesting human or bovine milk. More precisely, bovine O-linked oligosac- a role for lactadherin in the milk fat secretion process (Cebo et al., charides consists of mono- and disialylated core 1 oligosaccharides unpublished data). Accordingly, a possible role for lactadherin (Galb1-3GalNAc), while human O-glycans are monosialylated core in membrane secretion by the mammary epithelial cell has been type 2 oligosaccharides (Galb1-3(GlcNAcb1-6)GalNAc). Blood suggested by others (Oshima, Aoki, Kato, Kitajima, & Matsuda, group H structures (Fuca1-2Galb1-) and Lewis antigens were found 2002). to be expressed on human MFGM proteins, but not on MFGM Lactadherin has also been described as a potent anti-viral agent proteins isolated from bovine milk (Wilson et al., 2008). Because in rotavirus-induced gastroenteritis (Newburg et al., 1998). Indeed, inhibitory activities of lactadherin on rotaviruses are mainly due to breastfeeding is well-recognized as an important factor in protec- the oligosaccharides linked to the protein (Kvistgaard et al., 2004; tion against gastroenteric infections, which are mainly due to Yolken et al., 1992), observed discrepancies for carbohydrate rotaviruses. Rotaviruses enter the target cell through interactions composition of MFGM proteins isolated from human or bovine milk with a2b1, avb3,oraxb2 integrins (Isa, Realpe, Romero, López, & may explain the suppressive activity of human, but not bovine, Arias, 2004; Lopez & Arias, 2006; Zarate, Romero, Espinosa, Arias, lactadherin. From this point of view, additional experiments are & Lopez, 2004). Furthermore, it appears that rotavirus regulates mandatory to identify carbohydrate structures actually present on integrin expression in target cells thus resulting in an increased cell lactadherin from goat, sheep, horse and camel milk. adhesion and virus yield (Halasz, Holloway, Turner, & Coulson, From another point of view, it may be hypothesized that the 2008). Interestingly, human lactadherin binds to rotavirus and structure of lactadherin (especially with regard to glycosylation of inhibits its replication, both in vitro and in vivo (Newburg et al., the protein) affects its physico-chemical properties, particularly its 1998; Yolken et al., 1992). Because a common feature of lactad- resistance to digestion by gastric proteases. Indeed, the physical herin across species is the presence of an arginineeglycine- characteristics of the fat globules and the composition of the MFGM easpartic acid (RGD) sequence (a cell-adhesion motif able to bind proteins changed during incubation in simulated gastric fluid to integrins) in the second EGF-like domain of lactadherin, it may containing pepsin (Ye, Cui, & Singh, 2011). Butyrophilin was found be hypothesized that the RGD sequence of lactadherin interferes to be more resistant to gastric proteases than other MFGM glyco- with rotavirus attachment to intestinal cells. proteins like xanthine oxidase or bovine lactadherin (PAS 6/7). Outside of the mammary gland, lactadherin is also involved in Interestingly, PAS 6 (which contains an additional high-mannose- various intercellular interactions, including sperm-egg adhesion type N-glycan) was a little less sensitive than PAS 7 to during fertilization, maintenance of epithelia (including both 0.1 mg mL 1 pepsin treatment, thus demonstrating that the epididymal and intestinal epithelia), endothelial cells proliferation carbohydrate composition of the protein affects its resistance to and neovascularization (Raymond et al., 2009). Interestingly, lac- proteases from the gastrointestinal tract (Ye et al., 2011). Techno- tadherin is associated with exosomes, small membrane-bound logical processes like high-pressure treatment of raw milk may also vesicles released from many cell types, (mostly of immune origin) modulate MFGM protein composition (Ye, Anema, & Singh, 2004). upon fusion of vesicular bodies with the plasma membrane (Veron, Accordingly, PAS 6 and PAS 7 bands corresponding to bovine lac- Segura, Sugano, Amigorena, & Thery, 2005). Lactadherin is found to tadherin were seen at up to 700 MPa treatment of whole milk, but be highly present at the surface of immature dendritic cells (DC), PAS 6/7 glycoproteins were found to be less associated with the but its expression is considerably reduced in mature DC, thus MFGM after a 30 min treatment of milk at 800 MPa. Again, PAS 7 suggesting involvement of lactadherin in DC maturation processes was found to be slightly more sensitive to high-pressure treatment potentially amplifying the immune response (Veron et al., 2005). In than PAS 6. Taken together, these data favour a role for oligosac- addition, a recent study proposes a role for lactadherin in tumour charides linked to lactadherin in its bioactivity and bioavaibility, development by an immune-dependent mechanism (Sugano and highlight the need for a better characterization of carbohydrate et al., 2010). structures occurring on lactadherin protein from different species.

4.3. Lactadherin: a structureefunction relationship? 5. Conclusions

A structureefunction relationship has been reported for murine This study provides an inter-species comparison of major MFGM lactadherin (Oshima et al., 1999). Indeed, during lactation, a long proteins from cow, goat, sheep, camel and horse milk. Prominent lactadherin variant is produced from the in-frame inclusion of an differences were found across species, especially for lactadherin additional exon that encodes a Pro/Thr-rich domain of 37 amino protein. One (goat, sheep), two (cow, camel), and up to four (horse) acids long. The long variant is expressed predominantly in mouse distinct proteins can be characterized for lactadherin protein mammary gland and its expression is increased during late gesta- among the species studied to date. Although a splicing variant for tion and lactation, whereas the expression of the short lactadherin lactadherin has been characterized in the horse species, differences variant is ubiquitous. Interestingly, the additional Pro/Thr-rich reported here for lactadherin across species are mostly explained domain found in the long lactadherin variant possesses many by differential glycosylation of a single polypeptide backbone. putative O-glycosylation sites, as frequently found in mucin-type These observations raise questions, not only for the implication of glycoproteins, thus suggesting that O-glycosylation of murine lac- glycosylation of lactadherin in its biological activities on epithelia, tadherin might be involved in the function of MFG-E8 on milk fat but also for its impact on health especially with regard to anti- secretion (Oshima et al., 1999). infectious activities of lactadherin, as milk is the ﬁrst nourish- Involvement of oligosaccharides linked to lactadherin on its ment to the neonate. anti-rotaviral activities is also highly suspected. Indeed, chemical desialylation of the protein considerably reduces its anti-viral Acknowledgements activity, thus suggesting the involvement of sialic acids in the inhibition mechanism of lactadherin against pathogens (Newburg Dr. Jan Trige Rasmussen (Department of Molecular Biology, et al., 1998; Yolken et al., 1992). In addition, anti-rotaviral activi- Aarhus University, Denmark) is warmly acknowledged for the gift ties have not been established for bovine lactadherin (Kvistgaard of anti-lactadherin antibodies. Alicia Lopez (Master Student, Peru) et al., 2004). However, a recent study clearly shows differences in and Besma Saadaoui (PhD student, University of Gabès, Tunisia) are oligosaccharides found on MFGM proteins isolated either from acknowledged for their assistance to the work. C. Cebo, P. Martin / International Dairy Journal 24 (2012) 70e77 77

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