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.
Highlights
From the ‡Department of Biochemistry and the National Center for Functional Glycomics, Emory University School of Medicine, Atlanta, Georgia 30322; §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; ʈGlycomics Center, University of New Hampshire, Durham, New Hampshire 03824
The VP8*s of N155 and B223 have been analyzed on the Consortium for Functional Glycomics (CFG) defined glycan microarray [50, 60], which contains a wide assortment of glycans, including Nglycans, but is not useful for identifying human milk glycans (HMGs) that bind to viruses, because the CFG microarray contains only a few of the simple HMGs
The VP8* from bovine strain B233 bound only polylactosamines that terminated in a type 2 LacNAc, whereas the related human-bovine reassortant strain N155 bound the same type 2–terminating polylactosamines as strain B233 and bound polylactosamines terminating in GlcNAc1–3Gal, suggesting that this protein may bind
Summary
Materials—All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) and used without further purification unless otherwise indicated. Structural Analysis of Selected HMGs via MAGS—Based on the binding of the GST-tagged RV attachment proteins to HMGs on the HM-SGM-v2, 32 fractions were selected and printed in replicates of n ϭ 4 on a slide comprising 14 individual subarrays that were separated into individual wells for analysis using a silicon grid. This RVMAGS microarray comprised the 32 selected glycans, 12 defined milk glycans, 2 related structures obtained from the Consortium for Functional Glycomics (CFG), a biotin control, and a buffer control. 50000 40000 anti-Lea antibody (1 μg/ml) anti-H1 antibody (1:10) anti-Leb antibody (10 μg/ml)
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