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Article

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 Nestl

Research Center, P.O. Box 44, CH-1000 Lausanne 26, Switzerland

J. Proteome Res., 2008, 7 (9), pp 3687–3696

DOI: 10.1021/pr700793k

Publication Date (Web): July 15, 2008

†

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.

‡

Nestl Research Center.

Current address: Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney NSW 2109, Australia.

* To whom correspondence should be addressed. E-mail, Niclas.Karlsson@nuigalway.ie; phone, +353(0)91 495606; fax, +353 (0)91 525700.

Current address: Centre for BioAnalytical Sciences, Chemistry Department, NUI Galway, Ireland.

Abstract

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 (Galβ1-3(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-mucin-type 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.

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