Publications

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.

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.

BACKGROUND: Posttranslational glycosylation of IgG can modulate its inflammatory capacity through structural variations. We examined the association of baseline IgG N-glycans and an IgG glycan score with incident cardiovascular disease (CVD).

METHODS: IgG N-glycans were measured in 2 nested CVD case-control studies: JUPITER (Justification for the Use of Statins in Prevention: an Intervention Trial Evaluating Rosuvastatin; NCT00239681; primary prevention; discovery; Npairs=162); and TNT trial (Treating to New Targets; NCT00327691; secondary prevention; validation; Npairs=397). Using conditional logistic regression, we investigated the association of future CVD with baseline IgG N-glycans and a glycan score adjusting for clinical risk factors (statin treatment, age, sex, race, lipids, hypertension, and smoking) in JUPITER. Significant associations were validated in TNT, using a similar model further adjusted for diabetes. Using least absolute shrinkage and selection operator regression, an IgG glycan score was derived in JUPITER as a linear combination of selected IgG N-glycans.

RESULTS: Six IgG N-glycans were associated with CVD in both studies: an agalactosylated glycan (IgG-GP4) was positively associated, while 3 digalactosylated glycans (IgG glycan peaks 12, 13, 14) and 2 monosialylated glycans (IgG glycan peaks 18, 20) were negatively associated with CVD after multiple testing correction (overall false discovery rate <0.05). Four selected IgG N-glycans comprised the IgG glycan score, which was associated with CVD in JUPITER (adjusted hazard ratio per glycan score SD, 2.08 [95% CI, 1.52-2.84]) and validated in TNT (adjusted hazard ratio per SD, 1.20 [95% CI, 1.03-1.39]). The area under the curve changed from 0.693 for the model without the score to 0.728 with the score in JUPITER (PLRT=1.1×10-6) and from 0.635 to 0.637 in TNT (PLRT=0.017).

CONCLUSIONS: An IgG N-glycan profile was associated with incident CVD in 2 populations (primary and secondary prevention), involving an agalactosylated glycan associated with increased risk of CVD, while several digalactosylated and sialylated IgG glycans associated with decreased risk. An IgG glycan score was positively associated with future CVD.

Sickle Cell Disease (SCD) is a severe genetic disorder causing vascular occlusion and pain by upregulating the adhesion molecule P-selectin on endothelial cells and platelets. It primarily affects infants and children, causing chronic pain, circulatory problems, organ damage, and complications. Thus, effective treatment and management are crucial to reduce SCD-related risks. Anti-P-selectin antibody Crizanlizumab (Crimab) has been used to treat SCD. In this study, the heavy and light chain (HC and LC) genes of anti-P-Selectin antibody Crimab were cloned into a plant expression binary vector. The HC gene was under control of the duplicated 35S promoter and nopaline synthase (NOS) terminator, whereas the LC gene was under control of the potato proteinase inhibitor II (PIN2) promoter and PIN2 terminator. Agrobacterium tumefaciens LBA4404 was used to transfer the genes into the tobacco (Nicotiana tabacum cv. Xanthi) plant. In plants the genomic PCR and western blot confirmed gene presence and expression of HC and LC Crimab proteins in the plant, respectively. Crimab was successfully purified from transgenic plant leaf using protein A affinity chromatography. In ELISA, plant-derived Crimab (CrimabP) had similar binding activity to P-selectin compared to mammalian-derived Crimab (CrimabM). In surface plasmon resonance, the KD (dissociation binding constant) and response unit values were lower and higher than CrimabP, respectively. Taken together, these results demonstrate that the transgenic plant can be applied to produce biofunctional therapeutic monoclonal antibody.

Although immune tolerance evolved to reduce reactivity with self, it creates a gap in the adaptive immune response against microbes that decorate themselves in self-like antigens. This is particularly apparent with carbohydrate-based blood group antigens, wherein microbes can envelope themselves in blood group structures similar to human cells. In this study, we demonstrate that the innate immune lectin, galectin-4 (Gal-4), exhibits strain-specific binding and killing behavior towards microbes that display blood group-like antigens. Examination of binding preferences using a combination of microarrays populated with ABO(H) glycans and a variety of microbial strains, including those that express blood group-like antigens, demonstrated that Gal-4 binds mammalian and microbial antigens that have features of blood group and mammalian-like structures. Although Gal-4 was thought to exist as a monomer that achieves functional bivalency through its two linked carbohydrate recognition domains (CRDs), our data demonstrate that Gal-4 forms dimers and that differences in the intrinsic ability of each domain to dimerize likely influences binding affinity. While each Gal-4 domain exhibited blood group binding activity, the C-terminal domain (Gal-4C) exhibited dimeric properties, while the N-terminal domain (Gal-4N) failed to similarly display dimeric activity. Gal-4C not only exhibited the ability to dimerize, but also possessed higher affinity toward ABO(H) blood group antigens and microbes expressing glycans with blood group-like features. Furthermore, when compared to Gal-4N, Gal-4C exhibited more potent antimicrobial activity. Even in the context of the full-length protein, where Gal-4N is functionally bivalent by virtue of Gal-4C dimerization, Gal-4C continued to display higher antimicrobial activity. These results demonstrate that Gal-4 exists as a dimer and exhibits its antimicrobial activity primarily through its C-terminal domain. In doing so, these data provide important insight into key features of Gal-4 responsible for its innate immune activity against molecular mimicry.

The development and function of the brain requires N-linked glycosylation of proteins, which is a ubiquitous modification in the secretory pathway. N-glycans have a distinct composition and undergo tight regulation in the brain, but the spatial distribution of these structures remains relatively unexplored. Here, we systematically employed carbohydrate binding lectins with differing specificities to various classes of N-glycans and appropriate controls to identify glycan expression in multiple regions of the mouse brain. Lectins binding high-mannose-type N-glycans, the most abundant class of brain N-glycans, showed diffuse staining with some punctate structures observed on high magnification. Lectins binding specific motifs of complex N-glycans, including fucose and bisecting GlcNAc, showed more partitioned labeling, including to the synapse-rich molecular layer of the cerebellum. Understanding the spatial distribution of N-glycans across the brain will aid future studies of these critical protein modifications in development and disease of the brain.

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.