Glycoproteomics of Milk: Differences in Sugar Epitopes on Human

Pallesen , L. T.; Andersen , M. H.; Nielsen , R. L.; Berglund , L.; Petersen , T. E.; Rasmussen , L. K.; Rasmussen , J. T. Purification of MUC1 from b...
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Glycoproteomics of Milk: Differences in Sugar Epitopes on Human and Bovine Milk Fat Globule Membranes Nicole L. Wilson,†,§ Leanne J. Robinson,†,| Anne Donnet,‡ Lionel Bovetto,‡ Nicolle H. Packer,†,⊥ and Niclas G. Karlsson*,†,# Proteome Systems Limited, Locked Bag 2073, North Ryde, Sydney, NSW 1670, Australia, and Nestle´ Research Center, P.O. Box 44, CH-1000 Lausanne 26, Switzerland Received November 27, 2007

Oligosaccharides from human and bovine milk fat globule membranes were analyzed by LC-MS and LC-MS/MS. Global release of N-linked and O-linked oligosaccharides showed both to be highly sialylated, with bovine peak-lactating milk O-linked oligosaccharides presenting as mono- and disialylated core 1 oligosaccharides (Galβ1-3GalNAcol), while human milk had core type 2 oligosaccharides (Galβ13(GlcNAcβ1-6)GalNAcol) with sialylation on the C-3 branch. The C-6 branch of these structures was extended with branched and unbranched N-acetyllactosamine units terminating in blood group H and Lewis type epitopes. These epitopes were also presented on the reducing terminus of the human, but not the bovine, N-linked oligosaccharides. The O-linked structures were found to be attached to the high molecular mass mucins isolated by agarose-polyacrylamide composite gel electrophoresis, where MUC1 and MUC4 were present. Analysis of bovine colostrum showed that O-linked core 2 oligosaccharides are present at the early stage (3 days after birth) but are down-regulated as lactation develops. This data indicates that human milk may provide different innate immune protection against pathogens compared to bovine milk, as evidenced by the presence of Lewis b epitope, a target for the Helicobacter pylori bacteria, on human, but not bovine, milk fat globule membrane mucins. In addition, non-mucintype O-linked fucosylated oligosaccharides were found (NeuAc-Gal-GlcNAc1-3Fuc-ol in bovine milk and Gal-GlcNAc1-3Fuc-ol in human milk). The O-linked fucose structure in human milk is the first to our knowledge to be found on high molecular mass mucin-type molecules. Keywords: Milk • MUC1 • MUC4 • mucin • EGF • bacterial adhesion

Introduction The natural fat globules in milk are coated with a protective layer generally known as the Milk Fat Globule Membrane (MFGM). MFGM is composed of cholesterol, phospholipids, proteins and glycoproteins and represents 3-4% of the total volume of milk. The MFGM proteins make up 2-4% of the total protein content of human milk.1 The lipids in MFGM are secreted from the mammary gland cells and become enveloped in the apical plasma membrane of the alveolar cells, where they are released, together with the membrane bound proteins, from the cells by budding. Hence, MFGM proteins are a reflection of those in the membrane of the mammary gland alveolar cells. The glycoproteins in MFGM are thought to act as specific * To whom correspondence should be addressed. E-mail, Niclas.Karlsson@ nuigalway.ie; phone, +353(0)91 495606; fax, +353 (0)91 525700. † Proteome Systems Limited. § Current address: Allergan Australia Pty. Ltd., Gordon, Sydney NSW 2072, Australia. | Current address: The Walter & Eliza Hall Institute of Medical Research, Parkville,Victoria 3050 Australia. ‡ Nestle´ Research Center. ⊥ Current address: Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney NSW 2109, Australia. # Current address: Centre for BioAnalytical Sciences, Chemistry Department, NUI Galway, Ireland. 10.1021/pr700793k CCC: $40.75

 2008 American Chemical Society

bacterial and viral ligands which, when in the stomach of infants, contribute to the prevention of pathogenic organisms attaching to the intestinal mucosa.2,3 The extreme diversity of the glycans found in MFGM are thought to enable the glycoproteins to perform this function in the acidic environment of the stomach.4 The complex oligosaccharide moieties are synthesized by a variety of glycosyltransferases present in the mammary glands as well as in the stroma in which the mammary glands are imbedded. These oligosaccharide moieties present on the milk glycoproteins, with homology to epithelial mucus cell surface pathogen receptors in the stomach and intestine, may inhibit infection by competitively binding with the pathogens and clearing them from the infant gut.5 Proteomic and glycoproteomic techniques were used to study the glycoproteins and oligosaccharides present in the MFGM of both human and bovine milk. The oligosaccharides present on the high molecular weight mucin proteins as well as the global glycosylation profile of the MFGM glycoproteins were characterized. A highly glycosylated mucin protein, MUC1,6 has previously been purified and identified in milk, and monosaccharide analysis showed a difference in the composition of the O-linked oligosaccharides of MUC1 in bovine as compared to human milk, suggesting that significant structural differences may exist in the oligosaccharides.7 HuJournal of Proteome Research 2008, 7, 3687–3696 3687 Published on Web 07/15/2008

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Wilson et al. Table 1. Structural Prediction of N-Linked Oligosaccharide Alditols Globally Released by PNGase F from Human and Bovine Milk Fat Globule Membranes and Analyzed by LC-ESI-MS/MSb

Figure 1. Combined LC-ESI-MS spectra (RT 10-30 min) of globally released N-linked oligosaccharides from (A) bovine MFGM, and (B) human MFGM. Representative [M - 2H]2- ions correspond to identified structures, and the total proposed structures are presented in Table 1.

man MUC1 was shown to survive and maintain its integrity in the stomachs of humans and to inhibit the binding of Eschericha coli.2 We have analyzed in detail the different N- and O-linked oligosaccharides occurring in the MFGM of both human and bovine milk in order to compare the possible pathogenic protection that these structures may offer to newborns.

Experimental Procedures MFGM Preparation from Whole Milk. Human and bovine whole milk samples were ultracentrifuged at 13 000g for 30 min at 4 °C. This process separated the skim milk fraction from the cream. The cream was vortexed for 2 × 10 min intervals and allowed to rest between the vortexin intervals for approximately 2-3 min. The samples were incubated at 55 °C for 5 min, and then centrifuged at 2000g for 10 min at 25 °C in order to isolate the fat membranes from the butter oil. Approximately 1 mL of water was added to the membranes, the sample was vortexed and incubated at 55 °C for an additional 5 min, before being centrifuged at 2000g for 10 min at 25 °C. The water was removed and the insoluble fat membranes were lyophilized. Lyophilized MFGM proteins (5 mg) were dissolved in 1.0 mL of 1D gel electrophoresis sample8 buffer and were reduced for 15 min (95 °C) with 10 mM dithiotreitol, followed by alkylation (>2 h) in the dark with 25 mM iodoacetamide. The samples were either dot blotted onto PVDF membrane for global glycosylation analysis or were separated by composite polyacrylamide/agarose gel electrophoresis. Composite Polyacrylamide/Agarose Gel Electrophoresis (1D-AgPAGE). Reduced and alkylated human and bovine MFGM proteins were concentrated with 100 kDa molecular weight cutoff spin filters (Millipore, Bedford, MA) and resuspended with 1D sample buffer. Each sample (35 µL) was loaded onto high molecular weight 1D-AgPAGE gels8 and electrophoresed at 30 mA per gel for approximately 3 h. Gels were then either stained for glycoproteins with Periodic Acid Schiff’s (PAS) stain or electroblotted to PVDF PSQ and stained with Alcian Blue (Sigma-Aldrich Corp, ST Louis, MO). N-linked and O-linked oligosaccharides were released from the diffuse bands 3688

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a Lewis type structures. b × ) detected. Key: blue 9 ) GlcNAc, yellow 9 ) GalNAc, yellow b ) Gal, green b ) Man, red 2 ) Fuc, purple ( ) NeuAc.

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Figure 2. MS/MS spectra of a bovine N-linked oligosaccharide [M - 2H]2- ion of m/z 1059.6 containing a GalNAcβ1-4GlcNAcβ1- type terminal epitope (A) and human N-linked oligosaccharide [M - 2H]2- ion of m/z 1110.8.6 containing a Lewis a/x type terminal epitope (B). Major sequence ions are labeled.

Figure 3. Combined LC-ESI-MS spectra (RT 10-30 min) of globally released O-linked oligosaccharides from (A) bovine MFGM, and (B) human MFGM. Representative [M - H]- ions correspond to identified structures, and the total O-linked structures are presented in Table 2. The [M - H]- ion of m/z 472.1 is a degradation product (NeuAc-Galol).

by PNGase F digestion and reductive β-elimination, respectively, and analyzed by LC-ESI-MS. Enzymatic Release of N-Linked Oligosaccharides. Dot-blots were used to analyze the global glycosylation. The blotted proteins were cut from the PVDF membrane and placed in separate wells of a 96-well microtiter plate. The spots were then covered with 100 µL of 1% (w/v) poly(vinylpyrrolidone) 40 000 in 50% methanol, agitated for 10 min, and transferred to new wells after thoroughly washing with water. N-linked oligosaccharides were released from the proteins by incubation with 5 µL of PNGase F (0.5 units/µL) overnight at 37 °C. To recover the oligosaccharides, the samples were sonicated (in the 96 well plate) for approximately 5 min, and the released N-linked oligosaccharides transferred into clean wells and dried. The released N-glycans were treated with 10 µL of 50 mM KOH at room temperature for 15 min (to remove residual amines from PNGase F treatment on the reducing end) followed by reduction by adding 10 µL of 200 mM NaBH4 in 50 mM KOH and incubating for 2 h at 50 °C. The samples were then acidified by adding 1 µL of glacial acetic acid, and the reduced N-linked oligosaccharides were desalted by cation exchange chromatography.8 The N-linked oligosaccharides were resuspended in 10 µL of water for LC-ESI-MS analysis.

Chemical Release of O-Linked Oligosaccharides. After the removal of the N-linked oligosaccharides, the same cut out dot blots were rewet with approximately 2 µL of methanol. A solution of 50 mM KOH and 0.5 M NaBH4 (20 µL) was applied to each spot to release the O-linked oligosaccharides and the spots incubated at 50 °C for 16 h. The samples were then acidified and desalted by a cation exchange column using the method described above for reduced N-linked oligosaccharides. Alternatively, O-linked oligosaccharides were released in the same way from Alcian Blue-stained protein bands separated by gel electrophoresis and electroblotted to PVDF membrane. LC-MS of Oligosaccharide. Negative ion LC-ESI-MS and MS/ MS (ion trap LCQ DECA system (Thermo Electron Corp., Waltham, MA)) was used for the analysis of both the reduced N- and O-linked oligosaccharides. The samples were applied to a HyperCarb porous graphitized carbon column (5 µm HyperCarb, 0.32 × 150 mm) (Thermo Hypersil-Keystone, Bellefonte, PA) using a Surveyor autosampler (ThermoFinnigan, San Jose, CA). A linear water-acetonitrile gradient (0-25% acetonitrile in 30 min, followed by a 3 min wash with 90% acetonitrile containing 10 mM NH4HCO3 was used to separate the oligosaccharides. The flow rate was set at 200 µL/min, and a split ratio of 1/30 gave a final flow rate of approximately 6 µL/min through the column. Under these conditions, oligosaccharides were detected between 10-30 min in the chromatogram as individual components with an average peak width of about 30 s. LC-MS was performed in negative ion mode with three scan events: full scan with mass range m/z 320-2000, dependent zoom scan, and dependent MS/MS scan after collision induced fragmentation. Collision conditions used were normalized collision energy of 40% and an activation time of 30 ms. Dynamic exclusion of ions for zoom scan for 30 s was introduced after three selections within 30 s. Other mass spectrometric settings have been described previously.8 Data Analysis of LC-MS of Oligosaccharides. Pseudomolecular ions for potential oligosaccharides were retrieved from mass spectra averaged under the chromatographic peaks observed in the base peak trace. The composition of oligosaccharides was determined using the monoisotopic masses of detected components. The detected masses, together with their Journal of Proteome Research • Vol. 7, No. 9, 2008 3689

research articles Table 2. Structural Prediction of O-Linked Oligosaccharide Alditols Released by β-Elimination of Human and Bovine Milk Fat Globule Membranes (Global), and Their High Molecular Weight Protein Fraction (HMW), and Analyzed by LC-ESI-MS/ MSb

Wilson et al. eters of (1 amu and fragment masses with (0.5 amu to predict the possible oligosaccharides structures corresponding to these masses. Structures were assigned if they had a segmentation score of 1 and 2 based on the algorithms used in this software.9 Mass spectrometric relative intensities for comparison of individual oligosaccharides were obtained from summation of the intensities within a chromatographic peak of the isotopic cluster associated with each individual monoisotopic m/z corresponding to pseudomolecular ions of each detected oligosaccharide. Western Blotting. The medium molecular weight 1D-PAGE (3-8%) gels (Invitrogen Corp, Carlsbad, CA) and high molecular weight 1D-AgPAGE gels were electroblotted to PVDF PSQ and blocked overnight in a blocking solution of 1% (w/v) bovine serum albumin, and 0.1% (v/v) Tween 20 in phosphate buffered saline (PBS) (pH 7.5). Membranes were incubated for 1 h in the same blocking solution with monoclonal mouse antibodies against Lewis a and Lewis b (Sapphire Biosciences, Sydney, Australia) or mucins after deglycosylation:10 MUC1 (BC2),11 MUC4 (M4.275)12 ASGP-2 (35-4900, Zymed Laboratories, San Francisco, CA) and MUC313 (∼2 µg/mL working solutions). Membranes were washed 3 × 5 min in PBS (pH 7.5), 0.1% Tween 20 and then incubated for 1 h with anti-mouse Ig horse radish peroxidase conjugated secondary antibody (Silenus Labs, Melbourne, Australia) in blocking solution. After 5 × 5 min washes with PBS (pH 7.5) and 0.1% Tween 20, the membranes were incubated for ∼2 min in a solution containing equal amounts of Super Signal West Femto Chemiluminescent substrates (Pierce, Rockford, IL). ECL Hyperfilm (Amersham Pharmacia Biotech UK Ltd., Buckinghamshire, England) was exposed to membranes in cassette for ∼10 s to 5 min and the film was then developed in 16% Ilford Phenisol developer solution and fixed in 16% Ilford Hypam rapid fixer solution (Ilford Imaging UK Ltd., Cheshire, England). Film was washed in water and air-dried. For visual detection on the membrane, membranes were then washed in PBS (pH 7.5), 0.1% Tween 20 for 2 × 5 min and reincubated with primary antibody for 30 min. Membranes were washed 3 × 5 min in PBS (pH 7.5), 0.1% Tween 20 and then incubated for 1 h with anti-mouse Ig alkaline phosphatase conjugated secondary antibody (Sigma, St. Louis, MO) in blocking solution. After 5 × 5 min washes with PBS (pH 7.5) and 0.1% Tween 20, the membranes were incubated in NBT substrate (Sigma, St. Louis, MO) for 2-20 min (or until desirable color intensity achieved) and were then thoroughly washed in MilliQ water.

Results

a

Lewis type structures.

b

×) detected. Key: see Table 1.

associated MS/MS, were used in conjunction with GlycosidIQ9 software (Proteome Systems, Australia), which compares fragments produced by MS/MS with the theoretical fragmentation of those structures reported in GlycoSuiteDB, to determine the most probable oligosaccharide structure present. The combination of the elution time from the chromatography with the MS/MS data allowed the identification of isomers, which have the same composition but are structurally unique. Parent masses were searched by the GlycosidIQ software with param3690

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Global N-Linked Glycosylation Analysis of Human and Bovine Milk Fat Globule Membranes. N-linked oligosaccharides were released from the total MFGM fraction with PNGase F, reduced using sodium borohydride and analyzed by LC-ESIMS and LC-ESI-MS/MS. The combined mass spectra from LCMS separation are shown in Figure 1 and the structures interpreted by MS/MS are seen in Table 1. The N-linked oligosaccharides were found predominantly as doubly charged [M - 2H]2- ions. Both human and bovine MFGM samples contained bi-, tri- and tetra-antennary sialylated complex type N-linked oligosaccharide structures. Many of these structures showed increasing complexity by the addition of fucose either on the reducing end GlcNAc, or fucose and/or sialic acid on the exposed nonreducing end. As seen in the overall combined LC-MS spectra, there were significant differences between

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Figure 4. MS/MS spectrum of a human O-linked branched core 2 type oligosaccharides [M - 2H]2- ion of m/z 885.08 (the doubly charged ion of component m/z 1769.6-). Major sequence ions are labeled.

Figure 5. Comparison of the [M - H]- ion of m/z 1040.4 (core 2) peak intensity as a percentage of total O-linked oligosaccharides release, from different stages of bovine lactation (RT 10-30 min combined spectra).

human and bovine MFGM oligosaccharides. Fragmentation mass analysis showed that bovine N-linked oligosaccharides comprised structures displaying terminal diHexNAc epitopes ([M - 2H]2- of m/z 934.5, 986.4,1059.6 and 1080.2). In Figure 2A an example fragmentation of one of these isomers with a [M - 2H]2- of m/z 1059.6 is shown, where the presence of C2R′′ of m/z 422.8 identifies the diHexNAc epitope on one of the antennas. This is believed to correspond to the epitope GalNAcβ1-4GlcNAc which has previously been found on bovine N-linked oligosaccharides.14 This diHexNAc structure is masked by sialic acid where it occurs in the human milk structures ([M - 2H]2- of m/z 1132.0, 1205.0). Human oligosaccharide structures, on the other hand, express increased levels of what we believe are blood group H (FucR1-2 Galβ1-) and Lewis type antigens (Figure 2B). The fucose linkage of Lewis a (FucR14(Galβ1-3)GlcNAcβ1-) and x antigens (FucR1-3(Galβ1-4)GlcNAcβ1-) are indistinguishable by MS as are their fucosylated extended versions, Lewis b (FucR1-4(FucR1-2Galβ1-3)GlcNAcβ1-) and Lewis y (FucR1-3(FucR1-2Galβ1-4)GlcNAcβ1-) antigens. Both blood group H and Lewis type epitopes have been shown to be present in human N-linked milk oligosaccharides.15 Global O-Linked Mucin Type Glycosylation Analysis of Human and Bovine Milk Fat Globule Membranes. The combined mass spectra of O-linked oligosaccharides released

by β-elimination from bovine and human MFGM are shown in Figure 3 and the associated structures as interpreted from the MS/MS data are given as those obtained by global analysis in Table 2. Structural analysis shows that the bovine milk membrane protein oligosaccharides consist of singly charged, predominantly mucin-type, mono- and disialylated core 1 oligosaccharides (Galβ1-3GalNAc), while human oligosaccharides are monosialylated core 2 oligosaccharides (Galβ13(GlcNAcβ1-6)GalNAc), sialylated on the C-3 branch of GalNAc with complex extensions on the C-6 branch (an example of fragmentation analysis of these structures is shown in Figure 4). The difference in the core type structures between bovine and human O-linked oligosaccharides is highlighted by the relative mass spectrometric intensities of these structures found in each sample (Table 2). The MS data highlights that there is no terminal fucosylation detected on the bovine MGFM Olinked oligosaccharides. Analogous to the N-linked data, the fucose on the human MGFM O-linked oligosaccharides was found to be part of blood group H and Lewis type structures. Alteration of Global O-Linked Glycosylation Profile during Early Lactation. To determine whether the global profile of MFGM oligosaccharides changes during the onset of lactation, MFGM proteins from milk obtained from various stages of bovine lactation were analyzed for their oligosaccharide composition. Since the same components were present in each sample, we used the parent mass peak intensities for comparison of the samples. This method of relative quantitation is based on the similar structural and chemical properties of oligosaccharides which would be expected to display similar ionization and detection properties in ESI-MS.16 It was observed that the total O-linked oligosaccharide profile of the bovine MFGM fraction from the colostrum produced in the first period of lactation (3 and 7 days after birth) was different from the milk produced at peak lactation (6-8 weeks after giving birth). While at peak lactation bovine milk was shown to typically contain only small amounts of core 2 oligosaccharides (3% of total structures), the single sialylated core 2 oligosaccharide NeuAc-Gal-GlcNAc-6(Gal-3)GalNAcol with an [M - H]- ion of m/z 1040.4 appeared as a dominant peak in colostrum collected 3 days after birth (Figure 5). At 7 days after birthing, the relative intensity of this peak diminishes and is hardly apparent in milk collected at peak lactation. This indicates that a regulation of the core 2 GlcNAcβ1-6 glycosyltransferase enzyme occurs during early lactation, and may be an adaption to provide protection for the calf against specific pathogens. Journal of Proteome Research • Vol. 7, No. 9, 2008 3691

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Figure 6. Immunochemical identification and characterization of mucin type proteins from human and bovine MFGM using composite agarose-polyacrylamide gel electrophoresis. (A) Western blot analysis of MFGM with human MUC1, ASGP-2, MUC 4 and MUC 3 antibodies. (B) Oligosaccharide epitope identification with Lewis a and Lewis b antibodies.

Analysis of Human and Bovine MFGM Mucins. The major glycan-bearing components of the milk fat globule membranes are the high molecular weight mucin glycoproteins. The mucins of this fraction were separated by 1D composite AgPAGE gels and were stained by PAS and Alcian Blue to locate the heterogeneous mucin bands. Western blots of human MFGM high molecular mass glycoproteins were probed with antibodies against human mucins (Figure 6A), but did not show any crossreactivity with their bovine counterpart in bovine MFGM. The mucins, MUC1 and MUC4, (and ASGP-2 subunit of MUC4) were shown to be present in human MFGM, whereas there was no evidence of MUC 3. Both the MUC1 and MUC4 antibodies reacted with proteins present in the 0.8-1 MDa region. Since the predicted molecular weight of human MUC1 is approximately 100-200 kDa,6 this gel migration position indicates that there is a highly glycosylated form, with an apparent glycosylation level of 80-90% by weight, of MUC1 in human milk. The ASGP-2 MUC4 antibody detected bands at a lower molecular weight (∼180 kDa), showing that the MUC4 mucin domain (ASGP-1) is separated from its membrane bound domain (ASGP-2), at least in the reducing conditions used for SDS-AgPAGE. The human mucin antibodies did not react with any proteins in the bovine MFGM mucin separation, however mass spectrometric analysis indicated the presence of MUC1 (data not shown) in the Alcian-Blue stained protein band at around 200 kDa (Figure 7A). Bovine MUC1 is predicted to have a apomucin mass of around 50 kDa,7 whereas the human MUC1 apomucin has an increased number of repeat regions and is estimated to have a mass of approximately 200 kDa. The human milk MUC4 mucin could be estimated, based on its migration in the gel, to have a similar level of glycosylation to that of MUC1 as it has a similar apomucin size (230 kDa) (summarized in Figure 7B). O-linked oligosaccharides are the major glycan structures attached to mucins and these were released by β-elimination from the high molecular weight glycoproteins from human and bovine MFGM. The conditions of β-elimination can also 3692

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remove N-linked oligosaccharides,8 but none were detected in the MS profile. As in the global release of O-linked oligosaccharides, the mass spectrometric analysis of the oligosaccharides released from the high molecular weight bands of human MFGM between 0.8-1 MDa showed a large percentage of core 2 type structures, with smaller amounts of the core 1 type structures (Figure 7C). The fractionation of the MFGM into the enriched mucin subpopulation allowed more structures to be determined, particularly in the human MFGM (Table 2). The HMW protein region of the bovine MFGM showed the presence of three bands at approximately 170, 160, and 150 kDa (Figure 7A) which contained O-linked structures similar in number and type to those found in the global glycosylation analysis, with core 1 type mucin structures being the dominating structure (Figure 7C and Table 2). Western blots of these gel-separated MFGM mucins showed that both Lewis a (Galβ1-3(FucR1-4)GlcNAcβ1-) and Lewis b (FucR1-2Galβ1-3(FucR1-4)GlcNAcβ1-) are expressed on human milk mucins, but only weak staining could be detected on bovine mucins (Figure 6B), further confirming that cow’s milk does not express these fucosylated epitope structures. The mass spectrometric analysis also showed that Lewis type structures are only minor components of both N-linked and O-linked oligosaccharides on human MFGM proteins. Identification of O-Linked Fucose Type Oligosaccharide. In the global O-linked oligosaccharide structural analysis, we found evidence that some oligosaccharides are attached to the protein via a nonmucin-like O-linked fucose at the reducing terminus (Table 2 and Figure 8). The same core structure (Hex4HexNAc-Fuc) was seen in both human and bovine MGFM with the difference that the bovine structure was sialylated (NeuAc-Hex-4HexNAc-Fuc). The nonsialylated structure was seen in the global anaylsis of human MFGM and was located on the high molecular weight mucin proteins, while the bovine sialylated version was only found after global oligosaccharide release, indicating that it was probably present on lower molecular weight proteins of the bovine MFGM (Table 2). The

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Figure 7. Glycosylation of mucin type proteins from human and bovine MFGM after SDS-AgPAGE. (A) Proteins blotted from SDSAgPAGE and stained with Alcian Blue. (B) Cartoon depiction of the structure of the human and bovine milk fat globule membrane mucins. (C) MS spectra of oligosaccharides from the ∼1 MDa human, and ∼170 kDa bovine mucin bands marked with arrows in A.

amount detected in the mass spectrum ([M - H]- of m/z 530.2) of the released glycans from the human MFGM high molecular weight fraction (Figure 7C) indicates that it may be a significant component of the mucin oligosaccharides in the MFGM of human milk.

Discussion Glycosylation. A primary role of oligosaccharides, glycoproteins and mucins in milk appears to be in providing protection against pathogens by acting as competitive inhibitors for the binding sites on the epithelial surfaces of the intestine.17 Ongoing research is linking specific oligosaccharide structures with protection against specific pathogens and is also suggesting that some of these molecules (glycoproteins, glycoconjugates, etc.) may also act as growth promoters for the generation of a beneficial microflora in the colon. The data in this report show that the bovine milk glycosylation will provide a different protection against pathogens to that afforded by human milk. Structural analysis of milk fat globule membranes showed that O-linked oligosaccharides on bovine MFGM were primarily

based on core 1 type structures, whereas human MFGM O-linked oligosaccharides were almost exclusively core 2 structure based. The core 2 enzyme, GlcNAc β1-6 transferase, has been indicated as the key enzyme for generating more complex types of O-linked oligosaccharides.18 In Figure 9, we have shown a proposed biosynthetic route for the generation of Lewis a and Lewis b epitopes present in the human, but absent in bovine, MFGM. There was evidence that during the earlier stages of bovine lactation there was some synthesis of core 2 type mucin oligosaccharides (Figure 5), but that the content of this structure decreased rapidly and was low at peak lactation (Table 2). The samples analyzed in this study were from pooled milk. Further investigation of both bovine and human milk is needed to determine if this regulated glycosylation observed during lactation also holds true at an individual level. The core 2 structure provides a route for further extension of O-linked oligosaccharides, but significantly extended oligosaccharides were not detected in bovine MFGM by mass spectrometry and the glycosylation complexity found in human Journal of Proteome Research • Vol. 7, No. 9, 2008 3693

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Wilson et al. the ability to inhibit interactions between Lewis b and Helicobacter pylori,19 and sialylated oligosaccharides that are thought to inhibit the adhesion of the influenza A virus, enteropathogenic E. coli5 and H. pylori19 to their target cells. The detection of fucosylated oligosaccharides, in particular the Lewis type structures on mucins, provides an insight as to why breastfeeding may protect infants from H. pylori infections.22 Whether the presentation or clustering of oligosaccharides on a protein scaffold that occurs with glycoproteins confers increased interaction with potential pathogens compared to the interaction with free oligosaccharides in the milk requires further investigation.

Figure 8. MS/MS spectra and structural assignment of the O-linked fucose containing structures found in MFGM of (A) human: Hex-4HexNAc-Fuc-ol ([M _- H]- ion of m/z 530.2) and (B) bovine: NeuAc-Hex-4HexNAc1-Fuc-ol ([M - H]- ion of m/z 821.3).

Figure 9. Schematic of O-linked oligosaccharide biosynthesis. The O-linked biosynthesis in the Golgi compartment, including the biosynthesis of core 1, core 2 and extension into Lewis type epitopes by the action of fucosyltransferase 2 and 3 (FUT2 and 3). Asterik (*) indicates hexose polymer contamination.

milk was not seen. In this vein, it was found that the N-linked oligosaccharides of human MFGM were also decorated with these fucosylated terminal epitopes, while bovine was not. Other studies indicate a role for milk oligosaccharides and glycoconjugates that have structural homology to the glycan moieties of the intestinal mucosal cell surface and which may act as competitive inhibitors of pathogens binding to their glycoconjugate receptors.19–21 The protection breast milk has been known to offer in terms of preventing or reducing diarrhea may also be due to the presence of bioactive components of milk including sialylated and fucosylated oligosaccharides both as free oligosaccharides as well as on high molecular weight mucin-like proteins, thus, preventing or interfering with the binding and colonisation of microbial organisms. Examples of these include free oligosaccharides containing R1,2-linked fucose that inhibit the binding of E. coli in vitro and in vivo,4,18 fucosylated O-linked oligosaccharide glycoproteins that have 3694

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The finding of O-fucose oligosaccharide structures attached to human, but not bovine MFGM high molecular weight mucins, is an interesting difference between the two milks. O-fucosylation is an unusual form of O-linked glycosylation that was first found attached to the hydroxyl groups of serine or threonine residues on consensus sequences within epidermal growth factor (EGF)-like repeats.23 O-fucose modifications were reported to play a critical role in the modulating or receptor-ligand interactions,24 as well as being essential in the functioning of a family of Notch signaling proteins.25 O-fucose modifications have also been described in thrombospondin type 1 repeats (TSRs) and as with the EGF domains these small cysteine-knot motifs are thought to be involved in cellular signaling and adherence.26 In bovine milk, EGF domains have previously been found on lactadherin and contain O-linked fucose oligosaccharides.27 While the reasons for such oligosaccharides being present on human milk mucins is unclear, study of the high molecular mass fraction of the MFGM glycoproteins from other domestic animals (i.e., sheep, goats, pigs) has found the presence of this type of O-linked oligosaccharide only in human MFGM glycoproteins (data not shown). Bovine lactadherin has a consensus sequence for O-fucose that is not present in human lactadherin,21 and analysis of low molecular weight proteins showed that lactadherin was present in the MFGM fraction of both human and bovine milk (data not shown). This data could suggest that a high molecular weight glycoprotein may serve a similar function in human milk as lactadherin does in bovine. Mucin Expression. MUC1 has previously been reported to be a major constituent of human and bovine MFGM surrounding the lipid droplets secreted from the mammary gland epithelial cells.7,28 Previous studies suggest that these glycoproteins may prevent infections by microorganisms as MUC1 inhibits binding of S-fimbriated E. coli to buccal mucosa.2 Bovine MUC1 has been suggested to be present in the 160-170 kDa region,7 and MUC1 from human and bovine milk has been shown to differ not only in its oligosaccharide composition, but also in its mass. The amino acid sequence of human and bovine MUC1 has a 48% similarity where a large percentage of the difference is in the N-terminal and variable number tandem repeat (VNTR) regions. In humans, there is a large diversity in the VNTR repeat regions between individuals, and hence, the size difference between human and bovine MUC1 may not only be species dependent. MUC4 is a heterodimeric membrane mucin consisting of an O-glycosylated mucin subunit ASGP-1 (Ascites sialoglycoprotein-1), which is tightly, but not noncovalently, bound to an N-glycosylated transmembrane subunit ASGP-2.29 It has been identified as one of the milk membrane mucins.30 The transmembrane subunit ASGP-2 acts as an intermembrane ligand and activator for the receptor tyrosine kinase ErbB2, which has

research articles

Glycoproteomics of Milk been proposed to repress apopotosis in epithelial and cancer cells.29 In human milk, we found that we could detect O-linked fucosylation in the high molecular weight region, where MUC1 and MUC4 were detected. The MUC4 sequence has EGF repeat domains close to its membrane spanning domain, but the consensus sequence recorded for O-linked fucose is not present. Furthermore, this EGF domain is found in the ASGP-2 region, not in the mucin domain of MUC4 detected in the high molecular mass region (ASGP-1). Hence, this suggests that the O-linked fucose structure found in the high molecular weight fraction of human MFGM must be linked to a high molecular mass glycoprotein not yet identified in human milk. The data presented here indicates that both MUC1 and MUC4 in milk potentially have a role in pathogen adhesion. Since both membrane bound MUC1 and its ligand ICAM-1 and MUC4 with its interaction with ErbB2 also have been suggested to be involved in cellular signaling, the MUC1 and MUC4 found as “soluble” forms in the MFGM of milk may have the ability torefineligandinteractionsthatoccurwithmembrane-associated mucin forms in neonatal intestine and as such, modulate such biological processes. This function may also change over the lactation period in humans if there is a similar alteration of glycosylation on the milk mucins as is seen in bovine lactation. Recently, a highly glycosylated lower molecular weight mucin MUC15 (apomucin mass of 33 kDa) was identified in bovine MFGM and was shown to have glycans composed of fucose, galactose, mannose, N-acetylgalactosamine, N-acetylglycosamine, and sialic acid.31 In agreement with our data, the N-linked glycans were deduced by lectin and exoglycosidase analysis to contain sialylated N-glycans with terminal polylactosamine structures. The O-linked glycans were found to comprise only core 1 structures. In summary, the glycosylation and glycoconjugates of human and bovine MFGM differ significantly. The biochemical mantra is that structure and function is related, and part of the reason behind the difference in the bovine and human milk is probably related to the totally different microbial flora that needs to colonize the gut of healthy cows and humans, based on factors such as anatomy and diet. For example, the change in core 2 structures in bovine MFGM with time may reflect the change from a monogastric (preruminant) state to a ruminant state and the accompanying changes in digestive processes, bacterial flora and biological needs of the calf. This then determines the structural composition of the oligosaccharides presenting on the glycoproteins in their milk, which is designed as an innate protective means of clearing the unwanted pathogens from the gastrointestinal tract of the newborn offspring. This hypothesis is strengthened by the comparison of not only human and bovine milk glycosylation, but also of other species.32

Acknowledgment. Dr. Michael McGuckin is gratefully

(5) (6) (7)

(8) (9)

(10)

(11)

(12) (13) (14)

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(16)

(17) (18)

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acknowledged for providing mucin antibodies.

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