Glycans (monosaccharides and oligosaccharides) and their conjugates (glycoproteins, glycolipids, and proteoglycans) are structurally diverse biomolecules that are involved in many biological processes of health and disease. The structural diversity of glycans and glycoconjugates is owed to their monosaccharide composition, anomeric state, glycosidic linkage, modification (phosphorylation, sulfation, acetylation, etc.) and aglycone (protein, lipid, etc.). These diverse structures are controlled by complex glycosylation processes in cells, which are mediated by various glycosyltransferases and glycosidases. Glycosylation processes can be chemically regulated by inhibition of glycosyltransferases or glycosidases with natural and synthetic molecules. Treatment of cells with inhibitors of these enzymes results in the production of glycans or glycoconjugates containing missing or altered glycan chains. This approach is highly useful for examining the potential functional role(s) of glycans and glycoconjugates in cells or tissues, and in biological processes of health and disease. Eventually, it will provide novel mechanisms for disease treatment. This review highlights recent developments in chemical regulation of glycosylation processes with specific targets including: inhibition of (1) N-glycosylation, (2) O-glycosylation, (3) O-linked GlcNAc glycosylation, (4) proteoglycan biosynthesis, (5) glycolipid biosynthesis, and (6) terminal glycosylation. The goal of this review is to provide researchers with more competent choices in their research and lay a foundation on which continued advancements can be made to promote further explorations in glycoscience and biomedical research and applications.

A novel lectin, termed “MytiLec,” was isolated and characterized from the mussel Mytilus galloprovincialis, an important food and environmental indicator species found in marine coastal areas worldwide. MytiLec binds to the sugar moiety of globotriose (Gb3), an α-galactoside, leading to apoptosis of Gb3-expressing Burkitt’s lymphoma cells. The amino acid sequence of MytiLec is unusual, but 3-dimensional structural analysis reveals the presence of β-trefoil fold, a well-known feature in “R-type” lectins, a family of galactose-binding proteins found in many types of organisms. To date, MytiLec has been found only in a few species of the mollusk family Mytilidae and the phylum Brachiopoda, which also express typical R-type lectins. In this minireview, we discuss: (i) possible reasons for the unusual coexistence of two distinct lectin families in the same animal family; (ii) structural models of MytiLec that are useful for design of lectins with improved anti-cancer properties; (iii) construction of “Mitsuba,” an artificial lectin based on MytiLec that has similar carbohydrate-binding activity but a more stable monomeric form; (iv) regulation of cell growth by MytiLec and related lectins through binding to glycans.

Recently, homogenous glycoprotein has been prepared by using a chemoenzymatic approach to investigate the function of glycoproteins. Glycoproteins produced by cultured cells generally have heterogeneous glycans, so it is unclear which glycan is associated with the function of the glycoprotein. Moreover, because glycoproteins are heterogeneous, it is difficult to analyze their structure by X-ray crystal diffraction or NMR measurement. To solve this problem, transgenic cells have been developed to produce homogeneous glycoproteins through control of the biosynthetic pathway, but it is difficult to prepare glycoproteins with various kinds of homogeneous glycan by using this approach. Therefore, a chemoenzymatic approach using ENGase has been developed, and it enables the replacement of various glycans. Because antibody-dependent cell-mediated cytotoxicity (ADCC) exists as an example of glycan function in glycoproteins, there have been many studies of glycan remodeling of antibodies. Here, I introduce recent research and technologies, and their progress, in the field of glycan remodeling.

Avian IgGs, also called IgYs, are a major immunoglobulin in serum and egg yolk. Unlike mammalian IgGs, avian IgGs lack a highly flexible hinge between the Fab and Fc regions, but possess one additional immunoglobulin domain (CH2 domain) on the constant region of a heavy chain. By contrast, the CH3 domain of avian IgGs resembles the CH2 domain of mammalian IgGs, and one N-glycosylation site is located at a corresponding position in these two domains. N-Glycans at this position are located inside a cavity of two heavy chains, and are considered important for stabilization of the Fc region. However, only high mannose-type N-glycans including monoglucosylated forms (Glc₁Man_8–9GlcNAc₂), are present at the conserved N-glycosylation site in the CH3 domain of avian IgGs, whereas biantennary complex-type N-glycans are generally present on mammalian IgGs. On the other hand, the structures of complex-type N-glycans of avian IgGs, which are most likely located on the CH2 domain and variable regions, are highly heterogeneous due to alteration of glycan sequences at non-reducing termini. N-Glycan structures from chicken, quail, pigeon, gull, turkey, guineafowl, and peafowl IgGs, revealed that some contain Galα1-4Gal and/or Galβ1-4Gal sequences in a species-specific manner. The data suggested that the glycan structures of avian IgGs are very useful indicators to explore species-specific glycan differentiations among avian species.

C-linked glycosylation, one of the protein glycosylations, is a unique type of glycosylation in which an α-mannose is attached to the indole C2 carbon of a tryptophan residue via a C–C linkage and is so named C-mannosylation. C-mannosylation is enzymatically catalyzed in the endoplasmic reticulum (ER) lumen, and the N-terminal side Trp residue of the consensus amino acid sequence Trp-Xaa-Xaa-Trp/Cys (Xaa represents any amino acid) is often C-mannosylated. It has been reported that about 30 proteins are C-mannosylated, and the functions of C-mannosylation are becoming clear. In 2013, C. elegans dumpy-19 (dpy-19) was identified as a C-mannosyltransferase, and we revealed that DPY19L3, one of the human homologs of dpy-19, has similar activity in 2016. In this review, we describe previous studies about C-mannosylation, including our results and future research perspectives.

A novel lectin, termed “MytiLec,” was isolated and characterized from the mussel Mytilus galloprovincialis, an important food and environmental indicator species found in marine coastal areas worldwide. MytiLec binds to the sugar moiety of globotriose (Gb3), an α-galactoside, leading to apoptosis of Gb3-expressing Burkitt’s lymphoma cells. The amino acid sequence of MytiLec is unusual, but 3-dimensional structural analysis reveals the presence of β-trefoil fold, a well-known feature in “R-type” lectins, a family of galactose-binding proteins found in many types of organisms. To date, MytiLec has been found only in a few species of the mollusk family Mytilidae and the phylum Brachiopoda, which also express typical R-type lectins. In this minireview, we discuss: (i) possible reasons for the unusual coexistence of two distinct lectin families in the same animal family; (ii) structural models of MytiLec that are useful for design of lectins with improved anti-cancer properties; (iii) construction of “Mitsuba,” an artificial lectin based on MytiLec that has similar carbohydrate-binding activity but a more stable monomeric form; (iv) regulation of cell growth by MytiLec and related lectins through binding to glycans.

Recently, homogenous glycoprotein has been prepared by using a chemoenzymatic approach to investigate the function of glycoproteins. Glycoproteins produced by cultured cells generally have heterogeneous glycans, so it is unclear which glycan is associated with the function of the glycoprotein. Moreover, because glycoproteins are heterogeneous, it is difficult to analyze their structure by X-ray crystal diffraction or NMR measurement. To solve this problem, transgenic cells have been developed to produce homogeneous glycoproteins through control of the biosynthetic pathway, but it is difficult to prepare glycoproteins with various kinds of homogeneous glycan by using this approach. Therefore, a chemoenzymatic approach using ENGase has been developed, and it enables the replacement of various glycans. Because antibody-dependent cell-mediated cytotoxicity (ADCC) exists as an example of glycan function in glycoproteins, there have been many studies of glycan remodeling of antibodies. Here, I introduce recent research and technologies, and their progress, in the field of glycan remodeling.

Avian IgGs, also called IgYs, are a major immunoglobulin in serum and egg yolk. Unlike mammalian IgGs, avian IgGs lack a highly flexible hinge between the Fab and Fc regions, but possess one additional immunoglobulin domain (CH2 domain) on the constant region of a heavy chain. By contrast, the CH3 domain of avian IgGs resembles the CH2 domain of mammalian IgGs, and one N-glycosylation site is located at a corresponding position in these two domains. N-Glycans at this position are located inside a cavity of two heavy chains, and are considered important for stabilization of the Fc region. However, only high mannose-type N-glycans including monoglucosylated forms (Glc₁Man_8–9GlcNAc₂), are present at the conserved N-glycosylation site in the CH3 domain of avian IgGs, whereas biantennary complex-type N-glycans are generally present on mammalian IgGs. On the other hand, the structures of complex-type N-glycans of avian IgGs, which are most likely located on the CH2 domain and variable regions, are highly heterogeneous due to alteration of glycan sequences at non-reducing termini. N-Glycan structures from chicken, quail, pigeon, gull, turkey, guineafowl, and peafowl IgGs, revealed that some contain Galα1-4Gal and/or Galβ1-4Gal sequences in a species-specific manner. The data suggested that the glycan structures of avian IgGs are very useful indicators to explore species-specific glycan differentiations among avian species.

C-linked glycosylation, one of the protein glycosylations, is a unique type of glycosylation in which an α-mannose is attached to the indole C2 carbon of a tryptophan residue via a C–C linkage and is so named C-mannosylation. C-mannosylation is enzymatically catalyzed in the endoplasmic reticulum (ER) lumen, and the N-terminal side Trp residue of the consensus amino acid sequence Trp-Xaa-Xaa-Trp/Cys (Xaa represents any amino acid) is often C-mannosylated. It has been reported that about 30 proteins are C-mannosylated, and the functions of C-mannosylation are becoming clear. In 2013, C. elegans dumpy-19 (dpy-19) was identified as a C-mannosyltransferase, and we revealed that DPY19L3, one of the human homologs of dpy-19, has similar activity in 2016. In this review, we describe previous studies about C-mannosylation, including our results and future research perspectives.