Biosynthesis of Mucin-Type O-glycans

There are many types of protein glycosylation. The type of O-glycosylation in which N-acetylgalactosaminogalactan (GalNAc) binds directly to the Ser/Thr residues of a polypeptide chain is called mucin-type O-glycosylation. This type of O-glycosylation is most common in humans and is evolutionarily conserved. Mucin-type O-glycosylation is present in certain fungi to mammals. The synthesis of mucin-type O-glycans involves the participation of several enzymes, including the N-acetylgalactosaminyltransferase (ppGalNAc-T), N-acetyl-β-galactosyltransferase 1 (C1Gal-T1), etc. Depending on the type of the second monosaccharide attached to the amino acid residue and the type of bonding connection, eight O-glycan chain core structures have been identified in mammals. After the formation of the O-glycan chain core structure, it is further modified by a variety of glycosyltransferases (GTs) to produce a variety of complex O-glycans. Abnormal synthesis of mucin-type O-glycans is associated with certain diseases.

Fig.1 Synthesis of mucin-type O-glycans. (Bergstrom & Xia, 2013)

Mucin Type O-glycan Glycoengineering Service at CD BioGlyco 

CD BioGlyco aims to facilitate clients in O-glycan glycoengineering. The O-glycans play a variety of structural and functional roles in organisms. We have developed a range of glycoengineering services for mucin-based O-glycans, including GT gene modification, O-glycan analysis, glycosylation site analysis, etc.

Fig.2 Mucin type O-glycan glycoengineering service. (CD BioGlyco)

• Targeted modification of mucin type O-glycan

The main cause of abnormal glycosylation is disordered GT expression. We influence the synthesis of various mucin type O-glycan by various gene editing techniques and ultimately achieve targeted modification of mucin type O-glycan. We detect the effect of GT on glycosylation by Knocking Out or Overexpressing synthetic-related GT genes. GT gene expression is different in different cancer cells. GT gene modification can also be used to detect the specific biological mechanism by which it affects the tumor phenotype.

• Mucin-type O-glycan and glycosylation site analysis service

Mucin-based O-glycan analysis includes O-glycopeptide/glycoprotein enrichment, O-glycan dissociation, O-glycan structure identification, and quantitative analysis. The steps of glycosylation site analysis include proteolytic digestion of glycoproteins, enrichment and isolation of glycopeptides, mass spectrometric analysis of the enriched O-glycopeptides, etc. We use chemical and enzymatic methods to release mucin-type O-glycans from glycoproteins. This is followed by derivatization using fluorescent labeling. Characterization of the glycans is easier after fluorescent labeling. We have abundant experimental instruments including hydrophilic interaction chromatography (HILIC), reversed-phase liquid chromatography-ion trap mass spectrometry coupled with mass spectrometry (RPLC-ITMS), electrospray ionization mass spectrometry (ESI-MS), etc.

Fig.3 Mucin-type O-glycan analysis process. (CD BioGlyco)

In addition to these, we also provide various mucin-type O-glycans synthesis services through in vitro synthesis methods.

Publication

Paper Title: The biological role of core 1β1-3galactosyltransferase (T-synthase) in mucin-type O-glycosylation in Silkworm, Bombyx mori

Technology: Liquid Chromatography-mass Spectrometry (LC-MS)

Journal: Insect Biochemistry and Molecular Biology

IF: 4.421

Published: 2023

Results: In this study, the overall structural characteristics of serum mucin-type O-glycans of Bombyx mori were analyzed by LC-MS and compared with recombinant Hs proteoglycan 4 (rHsPRG4). The results showed that (HexNAc)1 and (Hex)1(HexNAc)1 were the major components. (Hex)1(HexA)1(HexNAc)1 and (HexNAc)1(HexA)1 were minor components. Mucin-type O-glycans in silkworm serum are structurally identical to rHsPRG4. rPRG4 represents mucin-type O-glycosylation of secreted proteins in the silkworm.

Fig.4 Identification of mucin-type O-linked glycans in reporter protein expressed by BEVS and secreted proteins in silkworm serum by LC-MS. (Morio, et al., 2023)

Applications

• There are many types of mucin-type tumor-associated glycoantigens, including Tn antigen and sialyl Tn antigen. Glycoengineering techniques are used to study the role of glycosylation abnormalities in tumor migration, proliferation, etc.
• Glycosylation abnormalities can be caused by a variety of factors, including disordered expression of GTs, masking of glycan epitopes by substituent groups, competition between GTs for glycan binding sites, etc. Glycoengineering techniques are used to study the mechanisms by which glycosylation abnormalities occur.
• Glycoengineering techniques are used to analyze the molecular biological mechanisms by which GT expression affects disease development.

Advantages

• Glycoengineering techniques enable the synthesis of a variety of mucin-type O-glycans, contributing to the understanding of the biological functions of these complex compounds in living organisms.
• Our identification analysis is less time-consuming. The detection equipment is highly sensitive and reproducible.
• Multifaceted glycoengineering biotechnology helps researchers study the role played by O-glycans in various life activities from multiple aspects.

CD BioGlyco has been optimizing glycoengineering platforms and glycosylation analysis services. Please feel free to contact us for the details of our services. We are happy to help you with your challenges in glycomics.