Application of Microarrays for Deciphering the Structure and Function of the Human Glycome

Smith, David F.; Cummings, Richard D.

doi:10.1074/mcp.r112.027110

Cited by 61 publications

(55 citation statements)

References 93 publications

(75 reference statements)

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“…However, in order to identify a binding motif for these viral attachment proteins and understand the potential role of human milk glycans as decoy receptors for the corresponding RVs, definitive structural analysis was required. We retrieved 32 HMGs or glycan targets from the TGL in order to determine their structures via MAGS, as reported previously (25,75). The 32 HMGs included 7 glycans bound by VP8*N155 (HMG-13, -14, -18, -21, -27, -28, and -33), 4 glycans bound by VP8*N155 after they were treated by ␣1,2-fucosidase digestion (HMG-29, -34, -51, and -60), 11 glycans bound by VP8*RV3 (HMG-5, -8, -37, -41, -48, -49, -55, -56, -62, -69, and -76), and 10 glycans bound by VP8*B223 (HMG-16, -20, -23, -31, -45, -47, -54, -65, -66, and -67).…”

Section: Specific Lectins and Antibodies Identify Determinants Of Thementioning

confidence: 99%

“…Identification of these glycans is not trivial, and we approached this problem using a variety of classical methods to accumulate metadata on each individual glycan. We have termed this approach metadataassisted glycan sequencing (25,75) and have again demonstrated its utility in determining the structures of VP8*-binding glycans (Fig. 6).…”

Section: Specific Lectins and Antibodies Identify Determinants Of Thementioning

confidence: 99%

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Human Milk Contains Novel Glycans That Are Potential Decoy Receptors for Neonatal Rotaviruses

Lasanajak

Song

et al. 2014

Molecular & Cellular Proteomics

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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 metadataassisted 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. Molecular & Cellular Proteomics 13:

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Section: Specific Lectins and Antibodies Identify Determinants Of Thementioning

confidence: 99%

Section: Specific Lectins and Antibodies Identify Determinants Of Thementioning

confidence: 99%

Human Milk Contains Novel Glycans That Are Potential Decoy Receptors for Neonatal Rotaviruses

Lasanajak

Song

et al. 2014

Molecular & Cellular Proteomics

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117

126

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“…As glycan sequence and structure are not directly coded into the genome, our understanding of glycans and their functions in biological systems is much more primitive than that of DNA and proteins. Recently, printed glycan microarrays (glycoarrays) have emerged as powerful, high-throughput tools for screening glycan-protein interactions [1,2], and have been applied in disease detection [3], drug discovery [4], the study of immunity [5], and host-pathogen interactions [1,2], among others. Unfortunately, glycoarray applications are currently limited by the expensive and complex methods available to synthesize glycans or alternatively, by the challenges in identifying and tagging glycans from natural sources [6,7].…”

Section: This Work Was Supported In Part By National Institutes Of Hementioning

confidence: 99%

“…This exciting proof-of-concept work demonstrates that glycophages can be patterned on surfaces and may provide an attractive complement to well-established glycan microarrays such as the Consortium for Functional Glycomics (CFG) microarrays [2] and neoglycolipid (NGL)-based microarrays [1]. The novelty of this approach lies in the in vivo production in E. coli, and rapid precipitation-based purification of well-defined glycophages from culture supernatant.…”

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confidence: 97%

Introducing glycophage arrays: Facile production, purification and patterning of glycophages

Lajoie

Shusta

2014

Biotechnology Journal

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“…By interrogating these arrays with sera from different species infected with S. mansoni, we identified immunologically relevant fractions for further characterization. Using a combination of mass spectrometry and lectin and antibody binding studies (metadata-assisted glycan sequencing [MAGS]) (37,38), we have identified several glycan structures that represent the egg N-glycans most prominently recognized by these infected hosts. The shotgun glycan microarray led us to focus on one MAb, F2D2, which targets FLDNF and replicates the binding pattern of infected hosts.…”

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confidence: 99%

Identification of Antigenic Glycans from Schistosoma mansoni by Using a Shotgun Egg Glycan Microarray

et al. 2016

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f Infection of mammals by the parasitic helminth Schistosoma mansoni induces antibodies to glycan antigens in worms and eggs, but the differential nature of the immune response among infected mammals is poorly understood. To better define these responses, we used a shotgun glycomics approach in which N-glycans from schistosome egg glycoproteins were prepared, derivatized, separated, and used to generate an egg shotgun glycan microarray. This array was interrogated with sera from infected mice, rhesus monkeys, and humans and with glycan-binding proteins and antibodies to gather information about the structures of antigenic glycans, which also were analyzed by mass spectrometry. A major glycan antigen targeted by IgG from different infected species is the FLDNF epitope [Fuc␣3GalNAc␤4(Fuc␣3)GlcNAc-R], which is also recognized by the IgG monoclonal antibody F2D2. The FLDNF antigen is expressed by all life stages of the parasite in mammalian hosts, and F2D2 can kill schistosomula in vitro in a complement-dependent manner. Different antisera also recognized other glycan determinants, including core ␤-xylose and highly fucosylated glycans. Thus, the natural shotgun glycan microarray of schistosome eggs is useful in identifying antigenic glycans and in developing new anti-glycan reagents that may have diagnostic applications and contribute to developing new vaccines against schistosomiasis. Schistosomiasis is a major health problem in tropical and subtropical areas where it is endemic, with more than 200 million people actively infected and 800 million at risk of contracting the disease (1-3). Current treatment for disease is limited to the drug praziquantel (4), but cases of drug resistance have been reported (5). Decades of research on schistosomiasis vaccines have yielded only two candidates for clinical trials, and no encouraging results have been published yet (6-9). Thus, there is an urgent need to develop more sensitive diagnostic methods and to identify new vaccine candidates.Recent studies have shown that a major part of the host immune response to infection is directed against carbohydrate (glycan) antigens in glycoproteins and glycolipids (10-17). A wide variety of unusual antigenic determinants include glycans containing the LDN, fucosylated LDN sequences (LDNF, LDN-dF, FLDN, and FLDNF), Lewis X (Le x ), poly-Le x , core ␣3 fucose, and core ␤2 xylose structures (Fig. 1) (11, 14, 17), many of which are expressed by all developmental stages of schistosomes (18). Interestingly, monoclonal antibodies (MAbs) specific to these glycans recognize these antigens on the surface of 3-h-old schistosomula, and some anti-glycan antibodies can mediate killing in vitro in a complement-dependent fashion (18-21). Schistosoma mansoniinfected rhesus monkeys, which are known to self-cure after infection, have IgG to many glycan antigens, including Le x , LDN, LDNF, core fucose, and core xylose determinants, and their sera are effective in complement-mediated cytolysis of cells expressing Le x as well as schistosomulum larvae in ...

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Application of Microarrays for Deciphering the Structure and Function of the Human Glycome

Cited by 61 publications

References 93 publications

Human Milk Contains Novel Glycans That Are Potential Decoy Receptors for Neonatal Rotaviruses

Human Milk Contains Novel Glycans That Are Potential Decoy Receptors for Neonatal Rotaviruses

Introducing glycophage arrays: Facile production, purification and patterning of glycophages

Identification of Antigenic Glycans from Schistosoma mansoni by Using a Shotgun Egg Glycan Microarray

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