Biosynthesis of Core 4

The core 4 structures are synthesized through the enzymatic action of C2GnT2, which facilitates the conversion of core 3 precursors. This conversion involves the addition of β1,6-GlcNAc from UDP-α-D-GlcNAc to the core 3 base structure. Core 4 O-glycans have been exclusively identified in secreted mucins present in specific mucin-secreting tissues like the bronchi, colon, and salivary glands. The gene encoding C2GnT2 is expressed in various organs, such as the stomach, colon, kidney, trachea, and testes. Notably, the expression of C2GnT2 in colon cancer cells, such as HCT116, has been found to suppress cell growth, adhesion, motility, and invasive properties. These observations suggest that the core 4 structure plays a critical protective role in colon and lung tissues.

Fig.1 Structure of O-glycan core 4. (CD BioGlyco)

O-glycan Core 4 Glycoengineering Service at CD BioGlyco

CD BioGlyco possesses a Glycoengineering Platform that supports us in installing core 4 O-glycan on the cell surface.

• We have cloned the cDNA that encodes for C4GnT, exhibiting a unique combination of activities, including a predominant C2GnT activity, a minor I-branching activity, and the conventional C4GnT activity.
• We also utilize genetic engineering techniques to modify the cDNA sequences, such as site-directed mutagenesis or gene synthesis, to optimize the activity or expression levels of the enzymes.
• Subsequently, cDNA sequences are transferred on the cell surface by transfection or viral transduction.
• The expression of the C2GnT2 is confirmed by using PCR.

Furthermore, we have a suitable mouse strain as the research model. By manipulating the expression or function of these glycosyltransferases in the mouse genome, we can modify and regulate the O-glycan core 4 chain on the cell face.

We also provide an approach to explore the distribution of O-glycan core 4 in the mouse cells. Capillary (graphitized carbon) liquid chromatography-mass spectrometry (LC-MS) analysis is utilized for the structural characterization of O-glycan core 4. The sensitivity and specificity of this approach have been demonstrated for O-glycans. The overall goal of this methodology is to characterize and investigate the core 4 O-glycan composition within the mucin fractions obtained from different regions.

• We extract mucins by using guanidinium hydrochloride (GuHCl), which yields the GuHCl-insoluble mucin fractions. These fractions are then subjected to reduction and alkylation before being separated using composite gel electrophoresis. Subsequently, Alcian Blue staining is applied to the gel to visualize the negatively charged mucins, specifically the muc2 mono- and oligomers.
• The GuHCl-insoluble and soluble fractions are separated using composite gels and then subjected to PAS staining. The GuHCl-soluble fraction reveals a prominent glycosylated protein band. After excision of the stained protein bands, they are subjected to trypsinization, and the resulting peptides are analyzed using mass spectrometry.
• Furthermore, both the GuHCl-insoluble mucin fractions and the GuHCl-soluble fraction are separated on composite gels and transferred onto a polyvinylidene fluoride (PVDF) membrane. The protein bands corresponding to those observed on the stained membranes are excised. The O-glycans attached to these proteins are subsequently released through reductive β-elimination. The released glycans are desalted and then analyzed using LC-MS.

Fig.2 The workflow diagram shows the five stages of the characterization. (CD BioGlyco)

Applications

• Our glycoengineering service has the potential to be applied in the research of cancer pathology.
• Our method can be used to study O-glycans from various sources and regions for comparative studies and identify potential variations in O-glycan composition.
• Through the analysis of mucin structure and glycosylation patterns, this method can offer valuable information about their functions, interactions, and contributions to various biological processes.
• This method can be employed to analyze changes and irregularities in mucins during disease states, facilitating the identification of potential biomarkers and therapeutic targets.

Advantages

• This method allows for the comprehensive analysis and characterization of O-glycan, including their size, glycosylation patterns, and regional differences in composition.
• The Alcian Blue staining specifically targets and visualizes negatively charged mucins, providing a clear and targeted visualization of these components.
• The combination of composite gel electrophoresis and mass spectrometry allows for the analysis of released O-glycans, providing insights into the glycosylation patterns present in the mucin fractions.

CD BioGlyco is widely recognized for its contributions to the field of glycobiology. Our research team is skilled at glycoengineering. CD BioGlyco strives to offer a comprehensive Cell Surface Glycoengineering Service by using state-of-the-art technologies and pioneering approaches. If you have any inquiries or need further details regarding our offerings, please don't hesitate to contact us.