Alterations of protein glycosylation during disease progression have been described in many previous studies, particularly in cancer and autoimmune diseases. Consequently, glycans represent novel pathological biomarkers for the early diagnosis of diseases (Dube and Bertozzi 2005). Seo et al. investigated N-glycan profiles of sera samples taken from healthy donors and patients diagnosed with Behçet’s disease (BD), a rare immune disorder that can cause inflammation in multiple parts of the body. Native N-glycans of serum glycoproteins were released and analyzed by MALDI-TOF MS for glycoform profiling and PGC-LC-MS for isomer-specific monitoring of sialylated glycans. Based on their sensitivity to α2,3-sialidase and elution patterns on PGC-LC using reference glycans carrying α2,6-sialic acid, the authors demonstrated the differences of isomeric sialyl-N-glycans between healthy and diseased groups. In comparison to healthy donors, BD patients possessed more α2,3-linked sialic acid on their N-glycans, a novel, potential molecular signature for the diagnosis of BD.

Mass spectrometry has become the most useful tool for studying glycomes. Different analytical approaches have been established to describe, in addition to glycan profiles based on monosaccharide composition, also linkage and anomeric information. Previously, Dr. Kay-Hooi Khoo and collaborators have demonstrated that precise glycomic profiles of different zebrafish organs can be mapped using mass spectrometry (Yamakawa et al., 2018). As a complementary study, Tseng et al. employed a nano-LC-MS/MS-based analytical workflow to re-examine zebrafish N- and O-glycans that have undergone permethylation. This approach is highly sensitive due to an increased ionization efficiency of glycans after derivatization and is advantageous for identifying isomeric structures using diagnostic fragmentation ions. Interestingly, a repertoire of glycans with sulfate substitution was revealed in the negative ion mode, including sulfated Lac(di-)NAc and sialyl-LacNAc epitopes on N-glycans and sulfated core 1 O-glycan structures. Using a very similar analytical approach, Aoki et al. compared the serum N-glycomes of five fish species (teleost and chondrostrean). Despite the genomes remaining unsequenced, the analysis showed these fishes share basic N-glycan biosynthetic pathways with other vertebrates, as indicated by the presence of high-mannose type and complex-type N-glycans. The authors discovered novel motifs on the N-glycan antennae, featuring different sialylation patterns, degrees of O-acetylation of sialic acid and galactose modifications. To provide linkage details of the terminal residues, the authors also employed exo-glycosidases to digest glycopeptides prior to glycan analysis. This led to the discovery of a species-specific α-galactose modification, which was only found in the two sturgeon spices.

In contrast to sialic acid-bearing glycans that are most frequently observed in vertebrates and some pathogens, the deoxyhexose sugar, fucose, has been found in a wider variety of organisms. Thomès and Bojar looked into the distribution of fucose-containing glycans in over 2,000 organisms covering all taxonomic kingdoms. Using a machine learning approach, the authors thoroughly analyzed glycan structures stored in public databases and described in peer-reviewed literature. Interestingly, bacterial and fungal species were able to make glycans with the highest number of fucose residues, whereas fucose was often attached to N-acetyl-glucosamine in all kingdoms except fungi. The authors also explored the correlation of fucose usage in different E. coli strains and their growing environment as well as infection potential.

The galactose-α-1,3-galactose (α-Gal) is a carbohydrate antigen abundantly expressed on glycoproteins and glycolipids in cells of non-primate mammals, prosimians, and New World monkeys. However, humans and crown catarrhines evolved with the inability to synthesize the glycan molecule due to the functional inactivation of the α1,3-galactosyltransferase (α1,3GalT). In turn, they can naturally produce antibodies specific to α-Gal that may be harnessed for the development of novel immunotherapies. In his comprehensive review article, Galili described the immunological processes associated with anti-Gal/α-Gal epitope interactions and suggested α-Gal therapies based on these immune reactions, which include i) amplification of whole virus vaccine immunogenicity by α-Gal epitopes; ii) conversion of autologous tumors into antitumor vaccines by expression of α-gal epitopes on tumor cell membranes; iii) accelerating healing of external and internal injuries by α-Gal nanoparticles which decrease the healing time and diminish scar formation; and iv) increasing anti-Gal–mediated protection against pathogens carrying α-Gal or α-Gal-like epitopes.

Despite the potential beneficial effects of the α-Gal immunity, exposure to the α-Gal epitope from the saliva of certain tick species induces a complex allergic disease (i.e. α-Gal syndrome or red meat allergy) characterized by the development of specific anti-α-Gal IgE antibodies after mammalian meat consumption. However, the origin of the glyco-epitope in ticks is still a matter of scientific debate. The work by Sharma et al. investigated the functional role of the α-D-galactosidase (ADGal) and β-1,4 galactosyltransferase (β-1,4GalT) in endogenous α-Gal production, carbohydrate metabolism, and N-glycans profile in the Amblyomma americanum tick. Their study provides an insight into tick intrinsic factors involved in synthesizing or recycling of α-Gal and its possible involvement in the onset of the α-Gal syndrome.

We hope that the articles in this series will provide the glyco-crowd with examples of the power of high-resolution mass spectrometry in characterizing novel glyco-epitopes and will improve the understanding of the structural diversity of glycans in different organisms, while bringing more insights into the biosynthesis and biological roles of the fascinating sugar molecules.