title: Human milk contains novel glycans that are potential decoy receptors for neonatal rotaviruses.
abstract: Human milk contains a rich set of soluble, reducing glycans whose functions and bioactivities are not well understood. Because human milk glycans (HMGs) have been implicated as receptors for various pathogens, we explored the functional glycome of human milk using shotgun glycomics. The free glycans from pooled milk samples of donors with mixed Lewis and Secretor phenotypes were labeled with a fluorescent tag and separated via multidimensional HPLC to generate a tagged glycan library containing 247 HMG targets that were printed to generate the HMG shotgun glycan microarray (SGM). To investigate the potential role of HMGs as decoy receptors for rotavirus (RV), a leading cause of severe gastroenteritis in children, we interrogated the HMG SGM with recombinant forms of VP8* domains of the RV outer capsid spike protein VP4 from human neonatal strains N155(G10P[11]) and RV3(G3P[6]) and a bovine strain, B223(G10P[11]). Glycans that were bound by RV attachment proteins were selected for detailed structural analyses using metadata-assisted glycan sequencing, which compiles data on each glycan based on its binding by antibodies and lectins before and after exo- and endo-glycosidase digestion of the SGM, coupled with independent MS(n) analyses. These complementary structural approaches resulted in the identification of 32 glycans based on RV VP8* binding, many of which are novel HMGs, whose detailed structural assignments by MS(n) are described in a companion report. Although sialic acid has been thought to be important as a surface receptor for RVs, our studies indicated that sialic acid is not required for binding of glycans to individual VP8* domains. Remarkably, each VP8* recognized specific glycan determinants within a unique subset of related glycan structures where specificity differences arise from subtle differences in glycan structures.
authors.affiliation: From the ‡Department of Biochemistry and the National Center for Functional Glycomics, Emory University School of Medicine, Atlanta, Georgia 30322;
authors.forename: Ying
authors.initials: Y
authors.lastname: Yu
authors.affiliation: From the ‡Department of Biochemistry and the National Center for Functional Glycomics, Emory University School of Medicine, Atlanta, Georgia 30322;
authors.forename: Yi
authors.initials: Y
authors.lastname: Lasanajak
authors.affiliation: From the ‡Department of Biochemistry and the National Center for Functional Glycomics, Emory University School of Medicine, Atlanta, Georgia 30322;
authors.forename: Xuezheng
authors.initials: X
authors.lastname: Song
authors.affiliation: §Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030;
authors.forename: Liya
authors.initials: L
authors.lastname: Hu
authors.affiliation: ¶Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas 77030;
authors.forename: Sasirekha
authors.initials: S
authors.lastname: Ramani
authors.affiliation: From the ‡Department of Biochemistry and the National Center for Functional Glycomics, Emory University School of Medicine, Atlanta, Georgia 30322;
authors.forename: Megan L
authors.initials: ML
authors.lastname: Mickum
authors.affiliation: ‖Glycomics Center, University of New Hampshire, Durham, New Hampshire 03824.
authors.forename: David J
authors.initials: DJ
authors.lastname: Ashline
authors.affiliation: §Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030; ¶Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas 77030;
authors.forename: B V Venkataram
authors.initials: BV
authors.lastname: Prasad
authors.affiliation: ¶Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas 77030;
authors.forename: Mary K
authors.initials: MK
authors.lastname: Estes
authors.affiliation: ‖Glycomics Center, University of New Hampshire, Durham, New Hampshire 03824.
authors.forename: Vernon N
authors.initials: VN
authors.lastname: Reinhold
authors.affiliation: From the ‡Department of Biochemistry and the National Center for Functional Glycomics, Emory University School of Medicine, Atlanta, Georgia 30322; dfsmith@emory.edu rdcummi@emory.edu.
authors.forename: Richard D
authors.initials: RD
authors.lastname: Cummings
authors.affiliation: From the ‡Department of Biochemistry and the National Center for Functional Glycomics, Emory University School of Medicine, Atlanta, Georgia 30322; dfsmith@emory.edu rdcummi@emory.edu.
authors.forename: David F
authors.initials: DF
authors.lastname: Smith
mesh_terms: D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism; D054794:Glycomics; D006801:Humans; D008895:Milk, Human / Q000737:chemistry*; D019158:N-Acetylneuraminic Acid / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011991:Receptors, Virus / Q000378:metabolism*; D012401:Rotavirus / Q000378:metabolism*; D013194:Staining and Labeling; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D036022:Capsid Proteins; D011134:Polysaccharides; D016601:RNA-Binding Proteins; D011991:Receptors, Virus; C060339:VP4 protein, Rotavirus; D017361:Viral Nonstructural Proteins; C079234:NS35 protein, rotavirus; D019158:N-Acetylneuraminic Acid