Publications

The protein glycome of individual cell types in the brain is unexplored, despite the critical function of these modifications in development and disease. In aggregate, the most abundant asparagine (N-) linked glycans in the adult brain are high mannose structures, and specifically Man5GlcNAc2 (Man-5), which normally exits the ER for further processing in the Golgi. Mannose structures are uncommon in other organs and often overlooked or excluded in most studies. To understand cell-specific contributions to the unique brain N-glycome and its abundance of Man-5, we performed RNAseq and MALDI-MS TOF protein N-glycomics at several timepoints during differentiation of multiple cell types. To this end, homogeneous cultures of glutamatergic neurons, GABAergic neurons, and brain-specific endothelial cells were generated from monoclonal human inducible pluripotent stem cells (hiPSCs) through cellular reprogramming. Small molecule induction of stably integrated synthetic transcription units driving morphogen expression generated differentiated cells with distinct patterns mirroring intact tissue. Comparing uninduced hiPSCs for each cell type revealed identical transcriptomic and glycomic profiles before differentiation, with low quantities of Man-5. In differentiated glutamatergic and GABAergic neurons, the most abundant N-glycans became Man-5 and its immediate precursor Man-6, despite the presence of transcripts encoding enzymes for their subsequent modification. Differentiation to brain-specific endothelial cells showed an opposite effect, with the N-glycome displaying an abundance of complex N-glycans and terminal modifications of the late secretory pathway. These results confirm that the restricted N-glycome profile of brain is programmed into neuronal differentiation, with regulation independent of the transcriptome and under tight evolutionary constraint.

Peptide-based therapeutics are recognized as potent and selective molecules but are often limited by short circulating half-lives, instability towards enzymatic degradation, and immunogenicity. To address these limitations and improve their pharmacological properties, peptides are commonly modified by the covalent attachment of polyethylene glycol (PEG). However, the large molecular weight and polydispersity of PEG chains complicate the interpretation of the full structure of PEGylated peptide therapeutics using standard analytical techniques. Here, we developed a mass spectrometric-based workflow in negative ion mode to identify and quantify GSnP-6, a P-selectin antagonist, with a linear 10 kDa PEG (PEG10) attached at the N-terminus of the glycopeptide. Intact mass analysis with multiple microscans allowed accurate measurements of precursor ions in complex biological mixtures with baseline resolution. Utilizing stepped collision energies improved sequence coverage and enabled identification of key amino acid modifications. We show the utility of this approach in evaluating the properties of PEG10-GSnP-6 in vitro and in vivo. Inhibitory capacity was preserved while extending the half-life of this glycopeptide, as shown by the reduction of P-selectin/PSGL-1 binding. By sustaining effective circulating concentrations, PEG conjugation of a P-selectin glycopeptide antagonist represents a promising therapeutic strategy to target diseases linked to inflammatory processes.

Hepatocytes synthesize a vast number of glycoproteins found in their membranes and secretions, many of which contain O-glycans linked to Ser/Thr residues. As the functions and distribution of O-glycans on hepatocyte-derived membrane glycoproteins and blood glycoproteins are not well understood, we generated mice with a targeted deletion of Cosmc (C1Galt1c1) in hepatocytes. Liver glycoproteins in WT mice express typical sialylated core 1 O-glycans (T antigen/CD176) (Galβ1-3GalNAcα1-O-Ser/Thr), whereas the Cosmc knockout hepatocytes (HEP-Cosmc-KO) lack extended O-glycans and express the Tn antigen (CD175) (GalNAcα1-O-Ser/Thr). Tn-containing glycoproteins occur in the sera of HEP-Cosmc-KO mice but not in WT mice. The LDL-receptor (LDLR), a well-studied O-glycosylated glycoprotein in hepatocytes, behaves as a ∼145kD glycoprotein in WT liver lysates, whereas it is reduced to ∼120 kDa in lysates from HEP-Cosmc-KO mice. Interestingly, the expression of the LDLR, as well as HMG-CoA reductase, which is typically altered in response to dysregulated cholesterol metabolism, are similar between WT and HEP-Cosmc-KO mice, indicating no significant effect by Cosmc deletion on either LDLR stability or cholesterol metabolism. Consistent with this, we observed no detectable phenotype in the HEP-Cosmc-KO mice regarding development, appearance or aging compared to WT. These results provide surprising, novel information about the pathway of O-glycosylation in the liver.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is a leading cause of death by an infectious disease globally, with no efficacious vaccine. Antibodies are implicated in Mtb control, but the mechanisms of antibody action remain poorly understood. We assembled a library of TB monoclonal antibodies (mAb) and screened for the ability to restrict Mtb in mice, identifying protective antibodies targeting known and novel antigens. To dissect the mechanism of mAb-mediated Mtb restriction, we optimized a protective lipoarabinomannan-specific mAb through Fc-swapping. In vivo analysis of these Fc-variants revealed a critical role for Fc-effector function in Mtb restriction. Restrictive Fc-variants altered distribution of Mtb across innate immune cells. Single-cell transcriptomics highlighted distinctly activated molecular circuitry within innate immune cell subpopulations, highlighting early activation of neutrophils as a key signature of mAb-mediated Mtb restriction. Therefore, improved antibody-mediated restriction of Mtb is associated with reorganization of the tissue-level immune response to infection and depends on the collaboration of antibody Fab and Fc.

ABO blood group antigens, which are complex carbohydrate moieties, and the first human polymorphisms identified, are critical in transfusion medicine and transplantation. Despite their discovery over a century ago, significant questions remain about the development of anti-ABO antibodies and the structural features of ABO antigens that cause hemolytic transfusion reactions. Anti-ABO antibodies develop naturally during the first few months of life, in contrast to other red blood cell (RBC) alloantibodies which form after allogeneic RBC exposure. Anti-ABO antibodies are the most common immune barrier to transfusion and transplantation, but the factors driving their formation are incompletely understood. Some studies suggest that microbes that express glycans similar in structure to the blood group antigens could play a role in anti-blood group antibody formation. While the role of these microbes in clinically relevant anti-blood group antibody formation remains to be defined, the presence of these microbes raises questions about how blood group-positive individuals protect themselves against blood group molecular mimicry. Recent studies suggest that galectins can bind and kill microbes that mimic blood group antigens, suggesting a unique host defense mechanism against microbial molecular mimicry. However, new models are needed to fully define the impact of microbes, galectins, or other factors on the development of clinically relevant naturally occurring anti-blood group antibodies.

Humans and other animals produce a diverse collection of antibodies, many of which bind to carbohydrate chains, referred to as glycans. These anti-glycan antibodies are a critical part of our immune systems' defenses. Whether induced by vaccination or natural exposure to a pathogen, anti-glycan antibodies can provide protection against infections and cancers. Alternatively, when an immune response goes awry, antibodies that recognize self-glycans can mediate autoimmune diseases. In any case, serum anti-glycan antibodies provide a rich source of information about a patient's overall health, vaccination history, and disease status. Glycan microarrays provide a high-throughput platform to rapidly interrogate serum anti-glycan antibodies and identify new biomarkers for a variety of conditions. In addition, glycan microarrays enable detailed analysis of the immune system's response to vaccines and other treatments. Herein we review applications of glycan microarray technology for serum anti-glycan antibody profiling.

It is important to recognize the great diversity of monosaccharides commonly encountered in animals, plants, and microbes, as well as to organize them in a visually interesting style that also emphasizes their similarities and relatedness. This article discusses the nature of building blocks, monosaccharides, and monosaccharide derivatives-terms commonly used in discussing "glycomolecules" found in nature. To aid in awareness of monosaccharide diversity, here is presented a Periodic Table of Monosaccharides. The rationale is given for construction of the Table and the selection of 103 monosaccharides, which is largely based on those presented in the KEGG and SNFG websites of monosaccharides, and includes room to enlarge as new discoveries are made. The Table should have educational value and is intended to capture the attention and foster imagination of those not very familiar with glycosciences, and encourage researchers to delve deeper into this fascinating area.

Modern studies on binding of proteins to glycans commonly involve the use of synthetic glycans and their derivatives in which a small amount of the material is covalently printed onto a functionalized slide in a glycan microarray format. While incredibly useful to explore binding interactions with many types of samples, the common techniques involve drying the slides, which leads to irreversible association of the protein to the spots on slides to which they bound, thus limiting a microarray to a single use. We have developed a new technique which we term Microwave Assisted Wet-Erase (MAWE) glycan microarrays. In this approach we image the slides under wet conditions to acquire the data, after which the slides are cleaned of binding proteins by treatment with a denaturing SDS solution along with microwave treatment. Slides cleaned in this way can be reused multiple times, and an example here shows the reuse of a single array 15 times. We also demonstrate that this method can be used for a single-array per slide or multi-array per slide platforms. Importantly, the results obtained using this technique for a variety of lectins sequentially applied to a single array, are concordant to those obtained via the classical dry approaches on multiple slides. We also demonstrate that MAWE can be used for different types of samples, such as serum for antibody binding, and whole cells, such as yeast. This technique will greatly conserve precious glycans and prolong the use of existing and new glycan microarrays.

Glycans are key to host-pathogen interactions, whereby recognition by the host and immunomodulation by the pathogen can be mediated by carbohydrate binding proteins, such as lectins of the innate immune system, and their glycoconjugate ligands. Previous studies have shown that excretory-secretory products of the porcine nematode parasite Trichuris suis exert immunomodulatory effects in a glycan-dependent manner. To better understand the mechanisms of these interactions, we prepared N-glycans from T. suis and both analyzed their structures and used them to generate a natural glycan microarray. With this array, we explored the interactions of glycans with C-type lectins, C-reactive protein, and sera from T. suis-infected pigs. Glycans containing LacdiNAc and phosphorylcholine-modified glycans were associated with the highest binding by most of these proteins. In-depth analysis revealed not only fucosylated LacdiNAc motifs with and without phosphorylcholine moieties but phosphorylcholine-modified mannose and N-acetylhexosamine-substituted fucose residues, in the context of maximally tetraantennary N-glycan scaffolds. Furthermore, O-glycans also contained fucosylated motifs. In summary, the glycans of T. suis are recognized by both the innate and adaptive immune systems and also exhibit species-specific features distinguishing its glycome from those of other nematodes.