Get to Know N-Glycosylation in Three Minutes

Structural Features and Modification Essence of N-Glycosylation

As one of the most universal post-translational modifications in eukaryotes, N-glycosylation links oligosaccharide chains to the side-chain amino group of asparagine (Asn) via β-glycosidic bonds. The modification site must conform to the conserved sequence N-X-S/T (where X represents any amino acid except proline). The core structure of this modification originates from the Glc₃Man₉GlcNAc₂ precursor assembled in the endoplasmic reticulum (ER), which undergoes cleavage and elongation to form highly diverse glycan structures. According to the composition of sugar chains surrounding the pentasaccharide core (Man₃GlcNAc₂), N-glycosylation is classified into three types: high-mannose (retaining ≥5 mannose residues), complex (containing extended structures like N-acetylglucosamine, galactose, and sialic acid), and hybrid (combining features of both). This structural diversity enables N-glycosylation to regulate protein folding, stability, and cellular localization. Its macroheterogeneity (multiple modification sites on the same protein) and microheterogeneity (different glycan chains at a single site) together constitute the complex regulatory network of the glycoproteome.

Biosynthetic and Dynamic Processing Pathways of N-Glycans

N-glycosylation initiation occurs in the ER, using dolichol as a lipid carrier to assemble the Glc₃Man₉GlcNAc₂ precursor through cascade catalysis by glycosyltransferases. The oligosaccharyltransferase (OST) complex recognizes the N-X-S/T motif in nascent polypeptide chains, transferring the complete precursor to Asn residues. Glucosidases then sequentially remove three glucose residues, serving as signals for the molecular chaperones Calnexin/Calreticulin to mediate proper protein folding. Misfolded proteins are cleared via the ER-associated degradation (ERAD) pathway, while correctly folded proteins enter the Golgi apparatus for subsequent processing. In the Golgi, α-mannosidases first trim excess mannose residues, followed by sequential actions of N-acetylglucosaminyltransferases, galactosyltransferases, sialyltransferases, etc., to add different sugar molecules based on cell types and physiological states. Notably, this processing exhibits species specificity: plant glycans often contain β1,2-xylose and α1,3-fucose; mammalian glycans are dominated by α2,6-sialic acid; and the α1,3-fucose modification common in insect cells is absent in humans due to the lack of corresponding enzymes, a difference of great significance in selecting recombinant protein expression systems.

Mass Spectrometry-Based Analytical System for N-Glycosylation

Modern N-glycosylation research relies on integrated technical platforms for separation-enrichment, glycan release, and mass spectrometry (MS) identification. During glycopeptide enrichment, lectin affinity chromatography uses concanavalin A (ConA) for specific recognition of high-mannose glycans or wheat germ agglutinin (WGA) for binding to N-acetylglucosamine to achieve targeted capture; boronic acid affinity chromatography relies on the reversible covalent interaction between cis-diol structures and boronic acid groups, suitable for separating glycans with vicinal diol structures. Glycan release is primarily achieved via enzymatic hydrolysis and chemical methods: PNGase F specifically cleaves the Asn-GlcNAc bond, converting Asn to Asp and introducing a mass shift of 0.9847 Da, and can perform O-18 labeling under deuterium oxide conditions for quantitative analysis; while hydrazinolysis efficiently releases glycans, it destroys Asn residues and makes modification site localization difficult, limiting its application. MS analysis typically employs a strategy combining HILIC (hydrophilic interaction chromatography) with LC-MS/MS, coupled with HCD (high-energy collision dissociation) or ECD (electron transfer dissociation) fragmentation modes, enabling not only the analysis of monosaccharide composition and linkage patterns of glycans but also the localization of modification sites through mass differences between peptide precursor ions and fragment ions. The recently developed hydrogen-deuterium exchange MS (HDX-MS) further reveals the impact of glycosylation on protein conformational dynamics.

Regulatory Network of N-Glycosylation in Immune Responses

N-glycosylation regulates the immune system in a multi-level and multi-target manner. Take IgG antibodies as an example: N-glycosylation of the Fc region directly affects effector functions. The degree of galactosylation determines the binding efficiency of antibodies to C1q, thereby regulating complement-dependent cytotoxicity (CDC); sialylation modifies can reduce ADCC effects by inhibiting FcγRⅢa binding. Notably, serum IgG from rheumatoid arthritis patients shows significantly reduced galactose and sialic acid content, leading to enhanced pro-inflammatory activity. During antigen presentation, N-glycosylation of the α1-α2 domain in MHC class I molecules maintains the conformational stability of the peptide-binding groove, influencing the loading efficiency and affinity of antigen peptides. Glycan modifications of CD molecules (e.g., CD44) participate in lymphocyte homing and intercellular adhesion, where sialylation and fucosylation alter cell surface charge and hydrophobicity to regulate immune cell migration trajectories. It is worth noting that pathogens can evade immune recognition by mimicking host glycosylation patterns—for instance, glycosylation site variations in influenza virus hemagglutinin can shield neutralizing epitopes, while high-mannose glycan clusters on HIV envelope proteins promote viral infection through abnormal binding to DC-SIGN receptors, providing new targets for vaccine design.

Molecular Mechanisms of N-Glycosylation-Driven Tumor Progression

Altered N-glycosylation profiles in tumor cells are closely associated with their malignant phenotypes, characterized by significantly increased glycan branching, core fucosylation, and sialylation levels. At the signaling level, β1,6-branching of N-glycans prolongs the activation time of epidermal growth factor receptor (EGFR), enhancing PI3K-AKT-mTOR pathway activity. This modification is mediated by N-acetylglucosaminyltransferase Ⅴ encoded by the Mgat5 gene, which is highly expressed in breast cancer, lung cancer, and other tumors. Core fucosylation (α1,6-fucose modification) promotes cell proliferation by regulating the Ras-MAPK pathway, and the expression level of its key enzyme FUT8 is negatively correlated with the prognosis of liver cancer patients. During invasion and metastasis, sialylated glycans weaken E-cadherin-mediated intercellular adhesion by increasing cell surface negative charge and promote the secretion of matrix metalloproteinases (MMPs). Abnormal N-glycosylation of E-cadherin can lead to its mislocalization from the cell membrane to the cytoplasm, triggering the epithelial-mesenchymal transition (EMT) program. More notably, the low-glucose and hypoxic conditions in the tumor microenvironment can upregulate the transcription of glycosyltransferase genes by activating HIF-1α, forming a positive feedback loop of metabolism-glycosylation-tumor progression. Targeting key glycosylation enzymes (e.g., inhibiting FUT8 or ST6Gal1) has shown potential to suppress tumor growth and metastasis in animal models.

Clinical Translation and Future Prospects of Glycosylation Research

The value of N-glycosylation as a disease diagnostic marker has been validated in multiple fields: altered glycosylation patterns of serum NSE in small cell lung cancer, increased fucosylation levels of alpha-fetoprotein (AFP) in liver cancer serum, and abnormal sialylation modifications of CA125 in ovarian cancer patients have all been applied to clinical auxiliary diagnosis. In therapy, glycosylation engineering is driving the optimization of therapeutic antibodies—for example, knocking out the fucosyltransferase gene in CHO cells can significantly enhance antibody ADCC effects. Vaccines targeting tumor-specific glycoepitopes (e.g., Tn antigen, sLeX) have also entered clinical trial stages. However, many mysteries remain in the dynamic regulation of the glycome: the spatiotemporal specific regulatory mechanisms of glycan modifications, the cross-talk between glycosylation and other post-translational modifications (e.g., phosphorylation), and the functional differences of organelle-specific glycosylation all require in-depth exploration by integrating single-cell glycoproteomics and gene editing technologies. With advancements in CRISPR-Cas9-mediated glycosyltransferase gene editing and high-resolution MS, N-glycosylation research will transition from descriptive analysis to mechanistic interpretation, providing new diagnostic markers and therapeutic targets for precision medicine.

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