Yi Lasanajak

Novel Fluorescent Glycan Microarray Strategy Reveals Ligands for Galectins

Galectin-1 (Gal-1) and galectin-3 (Gal-3) are widely expressed galectins with immunoregulatory functions in animals. To explore their glycan specificity, we developed microarrays of naturally occurring glycans using a bifunctional fluorescent linker, 2-amino-N-(2-aminoethyl)-benzamide (AEAB), directly conjugated through its arylamine group by reductive amination to free glycans to form glycan-AEABs (GAEABs). Glycans from natural sources were used to prepare over 200 GAEABs, which were purified by multidimensional high-pressure liquid chromatography and covalently immobilized onto N-hydroxysuccinimide-activated glass slides via their free alkylamine. Fluorescence-based screening demonstrated that Gal-1 recognizes a wide variety of complex N-glycans, whereas Gal-3 primarily recognizes poly-N-acetyllactosamine-containing glycans independent of N-glycan presentation. GAEABs provide a general solution to glycan microarray preparation from natural sources for defining the specificity of glycan-binding proteins.

Shotgun glycomics: a microarray strategy for functional glycomics

Major challenges of glycomics are to characterize a glycome and identify functional glycans as ligands for glycan-binding proteins (GBPs). To address these issues we developed a general strategy termed shotgun glycomics. We focus on glycosphingolipids (GSLs), a class of glycoconjugates that is challenging to study, recognized by toxins, antibodies and GBPs. We derivatized GSLs extracted from cells with a heterobifunctional fluorescent tag suitable for covalent immobilization. We separated fluorescent GSLs by multidimensional chromatography, quantified them and coupled them to glass slides to create GSL shotgun microarrays. Then we interrogated the microarrays with cholera toxin, antibodies and sera from individuals with Lyme disease to identify biologically relevant GSLs that we subsequently characterized by mass spectrometry. Shotgun glycomics incorporating GSLs and potentially glycoprotein-derived glycans is an approach for accessing the complex glycomes of animal cells and is a strategy for focusing structural analyses on functionally important glycans.

A Sialylated Glycan Microarray Reveals Novel Interactions of Modified Sialic Acids with Proteins and Viruses

Many glycan-binding proteins in animals and pathogens recognize sialic acid or its modified forms, but their molecular recognition is poorly understood. Here we describe studies on sialic acid recognition using a novel sialylated glycan microarray containing modified sialic acids presented on different glycan backbones. Glycans terminating in β-linked galactose at the non-reducing end and with an alkylamine-containing fluorophore at the reducing end were sialylated by a one-pot three-enzyme system to generate α2–3- and α2–6-linked sialyl glycans with 16 modified sialic acids. The resulting 77 sialyl glycans were purified and quantified, characterized by mass spectrometry, covalently printed on activated slides, and interrogated with a number of key sialic acid-binding proteins and viruses. Sialic acid recognition by the sialic acid-binding lectins Sambucus nigra agglutinin and Maackia amurensis lectin-I, which are routinely used for detecting α2–6- and α2–3-linked sialic acids, are affected by sialic acid modifications, and both lectins bind glycans terminating with 2-keto-3-deoxy-d-glycero-d-galactonononic acid (Kdn) and Kdn derivatives stronger than the derivatives of more common N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Three human parainfluenza viruses bind to glycans terminating with Neu5Ac or Neu5Gc and some of their derivatives but not to Kdn and its derivatives. Influenza A virus also does not bind glycans terminating in Kdn or Kdn derivatives. An especially novel aspect of human influenza A virus binding is its ability to equivalently recognize glycans terminated with either α2–6-linked Neu5Ac9Lt or α2–6-linked Neu5Ac. Our results demonstrate the utility of this sialylated glycan microarray to investigate the biological importance of modified sialic acids in protein-glycan interactions.

Cross-comparison of protein recognition of sialic acid diversity on two novel sialoglycan microarrays.

Background: Various glycan microarrays are currently widely used, but systematic cross-comparisons are lacking. Results: We compare and contrast two sialoglycan microarrays using a variety of sialic acid-binding proteins. Conclusion: Diverse array formats can strengthen the quality of information, but differences between arrays may be observed. Significance: Glycan arrays with similar glycan structures cannot be simply assumed to give similar results. DNA and protein arrays are commonly accepted as powerful exploratory tools in research. This has mainly been achieved by the establishment of proper guidelines for quality control, allowing cross-comparison between different array platforms. As a natural extension, glycan microarrays were subsequently developed, and recent advances using such arrays have greatly enhanced our understanding of protein-glycan recognition in nature. However, although it is assumed that biologically significant protein-glycan binding is robustly detected by glycan microarrays, there are wide variations in the methods used to produce, present, couple, and detect glycans, and systematic cross-comparisons are lacking. We address these issues by comparing two arrays that together represent the marked diversity of sialic acid modifications, linkages, and underlying glycans in nature, including some identical motifs. We compare and contrast binding interactions with various known and novel plant, vertebrate, and viral sialic acid-recognizing proteins and present a technical advance for assessing specificity using mild periodate oxidation of the sialic acid chain. These data demonstrate both the diversity of sialic acids and the analytical power of glycan arrays, showing that different presentations in different formats provide useful and complementary interpretations of glycan-binding protein specificity. They also highlight important challenges and questions for the future of glycan array technology and suggest that glycan arrays with similar glycan structures cannot be simply assumed to give similar results.

Functional Glycomic Analysis of Human Milk Glycans Reveals the Presence of Virus Receptors and Embryonic Stem Cell Biomarkers

Background: Recognition of human milk glycans (HMGs) by lectins, antibodies, and pathogens is poorly understood. Results: Microarrays of isolated HMGs exhibited specific binding to proteins and pathogens. Conclusion: HMG microarray interrogation and novel metadata-assisted glycan sequencing provide a functional glycomics approach to discovering HMG function. Significance: HMGs represent a potential “liquid innate immune system” that is specifically recognized by antibodies and pathogens. Human milk contains a large diversity of free glycans beyond lactose, but their functions are not well understood. To explore their functional recognition, here we describe a shotgun glycan microarray prepared from isolated human milk glycans (HMGs), and our studies on their recognition by viruses, antibodies, and glycan-binding proteins (GBPs), including lectins. The total neutral and sialylated HMGs were derivatized with a bifunctional fluorescent tag, separated by multidimensional HPLC, and archived in a tagged glycan library, which was then used to print a shotgun glycan microarray (SGM). This SGM was first interrogated with well defined GBPs and antibodies. These data demonstrated both the utility of the array and provided preliminary structural information (metadata) about this complex glycome. Anti-TRA-1 antibodies that recognize human pluripotent stem cells specifically recognized several HMGs that were then further structurally defined as novel epitopes for these antibodies. Human influenza viruses and Parvovirus Minute Viruses of Mice also specifically recognized several HMGs. For glycan sequencing, we used a novel approach termed metadata-assisted glycan sequencing (MAGS), in which we combine information from analyses of glycans by mass spectrometry with glycan interactions with defined GBPs and antibodies before and after exoglycosidase treatments on the microarray. Together, these results provide novel insights into diverse recognition functions of HMGs and show the utility of the SGM approach and MAGS as resources for defining novel glycan recognition by GBPs, antibodies, and pathogens.

Human milk contains novel glycans that are potential decoy receptors for neonatal rotaviruses

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 MSn 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 MSn 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.

Fluorescent Glycosylamides Produced by Microscale Derivatization of Free Glycans for Natural Glycan Microarrays

A novel strategy for creating naturally derived glycan microarrays has been developed. Glycosylamines are prepared from free reducing glycans and stabilized by reaction with acryloyl chloride to generate a glycosylamide in which the reducing monosaccharide has a closed-ring structure. Ozonolysis of the protected glycan yields an active aldehyde, to which a bifunctional fluorescent linker is coupled by reductive amination. The fluorescent derivatives are easily coupled through a residual primary alkylamine to generate glycan microarrays. This strategy preserves structural features of glycans required for antibody recognition and allows development of natural arrays of fluorescent glycans in which the cyclic pyranose structure of the reducing-end sugar residue is retained.

Oxidative release of natural glycans for functional glycomics

Glycans have essential roles in biology and the etiology of many diseases. A major hurdle in studying glycans through functional glycomics is the lack of methods to release glycans from diverse types of biological samples. Here we describe an oxidative strategy using household bleach to release all types of free reducing N-glycans and O-glycan-acids from glycoproteins, and glycan nitriles from glycosphingolipids. Released glycans are directly useful in glycomic analyses and can be derivatized fluorescently for functional glycomics. This chemical method overcomes the limitations in glycan generation and promotes archiving and characterization of human and animal glycomes and their functions.

Glycan Microarray Analysis of P-type Lectins Reveals Distinct Phosphomannose Glycan Recognition

The specificity of the cation-independent and -dependent mannose 6-phosphate receptors (CI-MPR and CD-MPR) for high mannose-type N-glycans of defined structure containing zero, one, or two Man-P-GlcNAc phosphodiester or Man-6-P phosphomonoester residues was determined by analysis on a phosphorylated glycan microarray. Amine-activated glycans were covalently printed on N-hydroxysuccinimide-activated glass slides and interrogated with different concentrations of recombinant CD-MPR or soluble CI-MPR. Neither receptor bound to non-phosphorylated glycans. The CD-MPR bound weakly or undetectably to the phosphodiester derivatives, but strongly to the phosphomonoester-containing glycans with the exception of a single Man7GlcNAc2-R isomer that contained a single Man-6-P residue. By contrast, the CI-MPR bound with high affinity to glycans containing either phospho-mono- or -diesters although, like the CD-MPR, it differentially recognized isomers of phosphorylated Man7GlcNAc2-R. This differential recognition of phosphorylated glycans by the CI- and CD-MPRs has implications for understanding the biosynthesis and targeting of lysosomal hydrolases.

Shotgun glycomics of pig lung identifies natural endogenous receptors for influenza viruses.

Significance Studies using novel “shotgun glycan microarray” technology identify, for the first time to our knowledge, the endogenous receptors for influenza viruses from a natural host, the pig. Libraries of total N-glycans from pig lung were probed for binding properties using a panel of influenza viruses isolated from humans, birds, and swine. Natural glycan receptors were identified for all viruses examined, and although some displayed the rather broad α2,3 or α2,6 sialic acid linkage specificity conventionally associated with avian or human viruses, other strains were highly specific, revealing a complexity that has not been demonstrated previously. Because pigs are often implicated as intermediate hosts for pandemic viruses, these results and the approaches described will transform our understanding of influenza host range, transmission, and pathogenicity. Influenza viruses bind to host cell surface glycans containing terminal sialic acids, but as studies on influenza binding become more sophisticated, it is becoming evident that although sialic acid may be necessary, it is not sufficient for productive binding. To better define endogenous glycans that serve as viral receptors, we have explored glycan recognition in the pig lung, because influenza is broadly disseminated in swine, and swine have been postulated as an intermediary host for the emergence of pandemic strains. For these studies, we used the technology of “shotgun glycomics” to identify natural receptor glycans. The total released N- and O-glycans from pig lung glycoproteins and glycolipid-derived glycans were fluorescently tagged and separated by multidimensional HPLC, and individual glycans were covalently printed to generate pig lung shotgun glycan microarrays. All viruses tested interacted with one or more sialylated N-glycans but not O-glycans or glycolipid-derived glycans, and each virus demonstrated novel and unexpected differences in endogenous N-glycan recognition. The results illustrate the repertoire of specific, endogenous N-glycans of pig lung glycoproteins for virus recognition and offer a new direction for studying endogenous glycan functions in viral pathogenesis.