Glycoconjugate Journal

, Volume 22, Issue 3, pp 109–118 | Cite as

Carbohydrate phenotyping of human and animal milk glycoproteins

  • Anki Gustafsson
  • Imre Kacskovics
  • Michael E. Breimer
  • Lennart Hammarström
  • Jan Holgersson


Breast-milk has a well-known anti-microbial effect, which is in part due to the many different carbohydrate structures expressed. This renders it a position as a potential therapeutic for treatment of infection by different pathogens, thus avoiding the drawbacks of many antibiotics. The plethora of carbohydrate epitopes in breast-milk is known to differ between species, with human milk expressing the most complex one. We have investigated the expression of protein-bound carbohydrate epitopes in milk from man, cow, goat, sheep, pig, horse, dromedary and rabbit. Proteins were separated by SDS-PAGE and the presence of carbohydrate epitopes on milk proteins were analysed by Western blotting using different lectins and carbohydrate-specific antibodies. We show that ABH, Lewis (Le)x, sialyl-Lex, Lea, sialyl-Lea and Leb carbohydrate epitopes are expressed mainly on man, pig and horse milk proteins. The blood group precursor structure H type 1 is expressed in all species investigated, while only pig, dromedary and rabbit milk proteins carry H type 2 epitopes. These epitopes are receptors for Helicobacter pylori (Leb and sialyl-Lex), enteropathogenic (H type 1, Lea and Lex) and enterotoxic Escherichia coli (heat-stable toxin; H type 1 and 2), and Campylobacter jejuni (H type 2). Thus, milk from these animals or their genetically modified descendants could have a therapeutic effect by inhibiting pathogen colonization and infection. Published in 2005.

milk proteins carbohydrate epitopes pathogens 



endoplasmatic reticulum; MALT, mucosa-associated lymphoid-tissue; BSA, bovine serum albumin; Blood group antigens A: GalNAcα 1-3[Fucα 1-2]Galβ; B: Galα 1-3[Fucα 1-2]Galβ; H type 1: Fucα 1-2Galβ 1-3GlcNAc; H type 2: Fucα 1-2Galβ 1-4GlcNAc; Lewis a: Galβ 1-3[Fucα 1-4]GlcNAc; sialyl Lewis a: NeuAcα 2,3Galβ 1-3[Fucα 1-4]GlcNAc; Lewis b: Fucα 1-2Galβ 1-3[Fucα 1-4]GlcNAc; Lewis x: Galβ 1-4[Fucα 1-3]GlcNAc; sialyl Lewis x: NeuAcα 2,3Galβ 1-4[Fucα 1-3]GlcNAc; pk: Galα 1-4Galβ 1-4Glc; P1: Galα 1-4Galβ 1-4GlcNAc.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Ofek I, Sharon N, Adhesins as lectins: Specificity and role in infection, Curr Top Microbiol Immunol 151, 91–113 (1990).PubMedGoogle Scholar
  2. 2.
    Hamosh M, Protective function of proteins and lipids in human milk, Biol Neonate 74(2), 163–76 (1998).CrossRefPubMedGoogle Scholar
  3. 3.
    Newburg DS, Neubauer SH, Carbohydrates in Milks: Analysis, Quantities, and Significance (Academic Press, Inc, 1995), pp. 273–349.Google Scholar
  4. 4.
    Burgoyne RD, Duncan JS, Secretion of milk proteins, J Mammary Gland Biol Neoplasia 3(3), 275–86 (1998).CrossRefPubMedGoogle Scholar
  5. 5.
    Mather IH, Keenan TW, Origin and secretion of milk lipids, J Mammary Gland Biol Neoplasia 3(3), 259–73 (1998).CrossRefPubMedGoogle Scholar
  6. 6.
    Stromqvist M, Falk P, Bergstrom S, Hansson L, Lonnerdal B, Normark S, Hernell O, Human milk kappa-casein and inhibition of Helicobacter pylori adhesion to human gastric mucosa, J Pediatr Gastroenterol Nutr 21(3), 288–96 (1995).PubMedGoogle Scholar
  7. 7.
    Andersson B, Porras O, Hanson LA, Lagergard T, Svanborg-Eden C, Inhibition of attachment of Streptococcus pneumoniae and Haemophilus influenzae by human milk and receptor oligosaccharides, J Infect Dis 153(2), 232–7 (1986).PubMedGoogle Scholar
  8. 8.
    Peterson JA, Patton S, Hamosh M, Glycoproteins of the human milk fat globule in the protection of the breast-fed infant against infections, Biol Neonate 74(2), 143–62 (1998).CrossRefPubMedGoogle Scholar
  9. 9.
    Schwertmann A, Schroten H, Hacker J, Kunz C, S-fimbriae from Escherichia coli bind to soluble glycoproteins from human milk, J Pediatr Gastroenterol Nutr 28(3), 257–63 (1999).CrossRefPubMedGoogle Scholar
  10. 10.
    Holmgren J, Svennerholm AM, Lindblad M, Receptor-like glycocompounds in human milk that inhibit classical, El Tor Vibrio cholerae cell adherence (hemagglutination), Infect Immun 39(1), 147–54 (1983).PubMedGoogle Scholar
  11. 11.
    Newburg DS, Ashkenazi S, Cleary TG, Human milk contains the Shiga toxin and Shiga-like toxin receptor glycolipid Gb3, J Infect Dis 166(4), 832–6 (1992).PubMedGoogle Scholar
  12. 12.
    Herrera-Insua I, Gomez HF, Diaz-Gonzalez VA, Chaturvedi P, Newburg DS, Cleary TG, Human milk lipids bind Shiga toxin, Adv Exp Med Biol 501, 333–9 (2001).PubMedGoogle Scholar
  13. 13.
    Schnieke AE, Kind AJ, Ritchie WA, Mycock K, Scott AR, Ritchie M, Wilmut I, Colman A, Campbell KH, Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts, Science 278(5346), 2130–3 (1997).CrossRefPubMedGoogle Scholar
  14. 14.
    Brink MF, Bishop MD, Pieper FR, Developing efficient strategies for the generation of transgenic cattle which produce biopharmaceuticals in milk, Theriogenology 53(1), 139–48 (2000).CrossRefPubMedGoogle Scholar
  15. 15.
    Clark AJ, The mammary gland as a bioreactor: Expression, processing, and production of recombinant proteins, J Mammary Gland Biol Neoplasia 3(3), 337–50 (1998).CrossRefPubMedGoogle Scholar
  16. 16.
    Karatzas CN, Designer milk from transgenic clones, Nat Biotechnol 21(2), 138–9 (2003).CrossRefPubMedGoogle Scholar
  17. 17.
    Prieto PA, Mukerji P, Kelder B, Erney R, Gonzalez D, Yun JS, Smith DF, Moremen KW, Nardelli C, Pierce M, et al. Remodeling of mouse milk glycoconjugates by transgenic expression of a human glycosyltransferase, J Biol Chem 270(49), 29515–9 (1995).CrossRefPubMedGoogle Scholar
  18. 18.
    Ruiz-Palacios GM, Cervantes LE, Ramos P, Chavez-Munguia B, Newburg DS, Campylobacter jejuni binds intestinal H(O) antigen (Fuc alpha 1, 2Gal beta 1, 4GlcNAc), and fucosyloligosaccharides of human milk inhibit its binding and infection, J Biol Chem 278(16), 14112–20 (2003).CrossRefPubMedGoogle Scholar
  19. 19.
    Kunz C, Rudloff S, Schad W, Braun D, Lactose-derived oligosaccharides in the milk of elephants: Comparison with human milk, Br J Nutr 82(5), 391–9 (1999).PubMedGoogle Scholar
  20. 20.
    Mather IH, A review and proposed nomenclature for major proteins of the milk-fat globule membrane, J Dairy Sci 83(2), 203–47 (2000).PubMedGoogle Scholar
  21. 21.
    Mammen M, Choi S, Whitesides G, Polyvalent interactions in biological systems: Implications for design and use of multivalent ligands and inhibitors, Angew Chem Int Ed 37, 2754–2794 (1998).CrossRefGoogle Scholar
  22. 22.
    Lindhorst TK, Kieburg C, Krallmann-Wenzel U. Inhibition of the type 1 fimbriae-mediated adhesion of Escherichia coli to erythrocytes by multiantennary alpha-mannosyl clusters: The effect of multivalency, Glycoconj J 15(6), 605–13 (1998).CrossRefPubMedGoogle Scholar
  23. 23.
    Totani K, Kubota T, Kuroda T, Murata T, Hidari KI, Suzuki T, Suzuki Y, Kobayashi K, Ashida H, Yamamoto K, Usui T, Chemoenzymatic synthesis and application of glycopolymers containing multivalent sialyloligosaccharides with a poly(L-glutamic acid) backbone for inhibition of infection by influenza viruses, Glycobiology 13(5)315–26 (2003).CrossRefPubMedGoogle Scholar
  24. 24.
    Laemmli UK, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227(259), 680–5 (1970).PubMedGoogle Scholar
  25. 25.
    Kimura H, Shinya N, Nishihara S, Kaneko M, Irimura T, Narimatsu H, Distinct substrate specificities of five human alpha-1,3-fucosyltransferases for in vivo synthesis of the sialyl Lewis x and Lewis x epitopes, Biochem Biophys Res Commun 237(1), 131–7 (1997).CrossRefPubMedGoogle Scholar
  26. 26.
    Grabenhorst E, Nimtz M, Costa J, Conradt HS, In vivo specificity of human alpha1,3/4-fucosyltransferases III-VII in the biosynthesis of LewisX and Sialyl LewisX motifs on complex-type N-glycans. Coexpression studies from bhk-21 cells together with human beta-trace protein, J Biol Chem 273(47), 30985–94 (1998).CrossRefPubMedGoogle Scholar
  27. 27.
    Huang MC, Laskowska A, Vestweber D, Wild MK, The alpha (1,3)-fucosyltransferase Fuc-TIV, but not Fuc-TVII, generates sialyl Lewis X-like epitopes preferentially on glycolipids, J Biol Chem 277(49), 47786–95 (2002).CrossRefPubMedGoogle Scholar
  28. 28.
    Prieels JP, Monnom D, Dolmans M, Beyer TA, Hill RL, Co-purification of the Lewis blood group N-acetylglucosaminide alpha 1 goes to 4 fucosyltransferase, an N-acetylglucosaminide alpha 1 goes to 3 fucosyltransferase from human milk, J Biol Chem 256(20), 10456–63 (1981).PubMedGoogle Scholar
  29. 29.
    Blaser MJ, The bacteria behind ulcers, Sci Am 274(2), 104–7 (1996).PubMedGoogle Scholar
  30. 30.
    Crespo A, Suh B, Helicobacter pylori infection: Epidemiology, pathophysiology, and therapy, Arch Pharm Res 24(6), 485–98 (2001).PubMedGoogle Scholar
  31. 31.
    Clyne M, Thomas J, Weaver L, Drumm B, In vitro evaluation of the role of antibodies against Helicobacter pylori in inhibiting adherence of the organism to gastric cells, Gut 40(6), 731–8 (1997).PubMedGoogle Scholar
  32. 32.
    Hata Y, Kita T, Murakami M, Bovine milk inhibits both adhesion of Helicobacter pylori to sulfatide and Helicobacter pylori-induced vacuolation of vero cells, Dig Dis Sci 44(8), 1696–702 (1999).CrossRefPubMedGoogle Scholar
  33. 33.
    Wang X, Hirmo S, Willen R, Wadstrom T, Inhibition of Helicobacter pylori infection by bovine milk glycoconjugates in a BAlb/cA mouse model, J Med Microbiol 50(5), 430–5 (2001).PubMedGoogle Scholar
  34. 34.
    Borén T, Falk P, Roth KA, Larson G, Normark S, Attachment of Helicobacter pylori to human gastric epithelium mediated by blood group antigens, Science 262(5141), 1892–5 (1993).PubMedGoogle Scholar
  35. 35.
    Falk P, Roth KA, Boren T, Westblom TU, Gordon JI, Normark S, An in vitro adherence assay reveals that Helicobacter pylori exhibits cell lineage-specific tropism in the human gastric epithelium, Proc Natl Acad Sci USA 90(5), 2035–9 (1993).PubMedGoogle Scholar
  36. 36.
    Miller-Podraza H, Milh MA, Teneberg S, Karlsson KA, Binding of Helicobacter pylori to sialic acid-containing glycolipids of various origins separated on thin-layer chromatograms, Infect Immun 65(6), 2480–2 (1997).PubMedGoogle Scholar
  37. 37.
    Teneberg S, Miller-Podraza H, Lampert HC, Evans DJ, Jr., Evans DG, Danielsson D, Karlsson KA, Carbohydrate binding specificity of the neutrophil-activating protein of Helicobacter pylori, J Biol Chem 272(30), 19067–71 (1997).CrossRefPubMedGoogle Scholar
  38. 38.
    Angstrom J, Teneberg S, Milh MA, Larsson T, Leonardsson I, Olsson BM, Halvarsson MO, Danielsson D, Naslund I, Ljungh A, Wadstrom T, Karlsson KA, The lactosylceramide binding specificity of Helicobacter pylori, Glycobiology 8(4), 297–309 (1998).CrossRefPubMedGoogle Scholar
  39. 39.
    Roche N, Larsson T, Angstrom J, Teneberg S, Helicobacter pylori-binding gangliosides of human gastric adenocarcinoma, Glycobiology 11(11), 935–44 (2001).CrossRefPubMedGoogle Scholar
  40. 40.
    Ilver D, Arnqvist A, Ogren J, Frick IM, Kersulyte D, Incecik ET, Berg DE, Covacci A, Engstrand L, Boren T, Helicobacter pylori adhesin binding fucosylated histo-blood group antigens revealed by retagging, Science 279(5349), 373–7 (1998).CrossRefPubMedGoogle Scholar
  41. 41.
    Syder AJ, Guruge JL, Li Q, Hu Y, Oleksiewicz CM, Lorenz RG, Karam SM, Falk PG, Gordon JI, Helicobacter pylori attaches to NeuAc alpha 2,3Gal beta 1,4 glycoconjugates produced in the stomach of transgenic mice lacking parietal cells, Mol Cell 3(3), 263–74 (1999).CrossRefPubMedGoogle Scholar
  42. 42.
    Mahdavi J, Sonden B, Hurtig M, Olfat FO, Forsberg L, Roche N, Angstrom J, Larsson T, Teneberg S, Karlsson KA, Altraja S, Wadstrom T, Kersulyte D, Berg DE, Dubois A, Petersson C, Magnusson KE, Norberg T, Lindh F, Lundskog BB, Arnqvist A, Hammarstrom L, Boren T, Helicobacter pylori SabA adhesin in persistent infection and chronic inflammation, Science 297(5581), 573–8 (2002).CrossRefPubMedGoogle Scholar
  43. 43.
    Clarke SC, Haigh RD, Freestone PP, Williams PH, Virulence of enteropathogenic Escherichia coli, a global pathogen, Clin Microbiol Rev 16(3), 365–78 (2003).CrossRefPubMedGoogle Scholar
  44. 44.
    Cravioto A, Tello A, Villafan H, Ruiz J, del Vedovo S, Neeser JR, Inhibition of localized adhesion of enteropathogenic Escherichia coli to HEp-2 cells by immunoglobulin and oligosaccharide fractions of human colostrum and breast milk, J Infect Dis 163(6), 1247–55 (1991).PubMedGoogle Scholar
  45. 45.
    Gaastra W, Svennerholm AM, Colonization factors of human enterotoxigenic Escherichia coli (ETEC), Trends Microbiol 4(11), 444–52 (1996).CrossRefPubMedGoogle Scholar
  46. 46.
    Newburg DS, Pickering LK, McCluer RH, Cleary TG, Fucosylated oligosaccharides of human milk protect suckling mice from heat-stabile enterotoxin of Escherichia coli, J Infect Dis 162(5), 1075–80 (1990).PubMedGoogle Scholar
  47. 47.
    Newburg DS, Ruiz-Palacios GM, Altaye M, Chaturvedi P, Meinzen-Derr J, Guerrero Mde L, Morrow AL, Innate protection conferred by fucosylated oligosaccharides of human milk against diarrhea in breastfed infants, Glycobiology 14(3), 253–63 (2004).CrossRefPubMedGoogle Scholar
  48. 48.
    Korhonen TK, Vaisanen-Rhen V, Rhen M, Pere A, Parkkinen J, Finne J, Escherichia coli fimbriae recognizing sialyl galactosides, J Bacteriol 159(2), 762–6 (1984).PubMedGoogle Scholar
  49. 49.
    Prasadarao NV, Wass CA, Kim KS, Identification and characterization of S fimbria-binding sialoglycoproteins on brain microvascular endothelial cells, Infect Immun 65(7), 2852–60 (1997).PubMedGoogle Scholar

Copyright information

© Springer Science + Business Media, Inc. 2005

Authors and Affiliations

  • Anki Gustafsson
    • 1
    • 5
  • Imre Kacskovics
    • 3
  • Michael E. Breimer
    • 2
  • Lennart Hammarström
    • 4
  • Jan Holgersson
    • 4
  1. 1.Departments of Clinical ChemistrySahlgrenska University HospitalGöteborg
  2. 2.Departments of SurgerySahlgrenska University HospitalGöteborg
  3. 3.Department of Physiology and Biochemistry, Faculty of Veterinary ScienceSzent Istv’n UniversityBudapest
  4. 4.Division of Clinical Immunology, Karolinska InstitutetKarolinska University Hospital at HuddingeStockholmSweden
  5. 5.Division of Clinical ImmunologyKarolinska InstitutetStockholmSweden

Personalised recommendations