Exploring N-Glycosidases from Multiple Dimensions
As a key tool enzyme in glycobiology research, N-glycosidase plays an irreplaceable role in protein glycosylation analysis, disease diagnosis, biopharmaceuticals, and other fields. This review comprehensively discusses the molecular characteristics and catalytic mechanisms of N-glycosidases, their core applications in glycoprotein research, the discovery and engineering of novel enzymes, as well as technological innovations and clinical translation prospects based on N-glycosidases. From the classic PNGase F to the novel PNGase F-II, from plant-derived bifunctional enzymes to engineered immobilized enzymes, the N-glycosidase family is driving the leapfrog development of glycobiology from basic research to practical applications with its unique catalytic properties. With the deepening understanding of the role of N-glycosylation in disease occurrence and development, N-glycosidases are not only continuously optimized as research tools but also become important breakthroughs for developing new diagnostic methods and targeted therapeutic strategies.
Molecular Characteristics and Catalytic Mechanisms of N-Glycosidases
Structural features and classification form the basis for understanding the functional diversity of N-glycosidases. N-glycosidases are a class of enzymes that specifically hydrolyze the β-glycosidic bond between asparagine (Asn) and N-acetylglucosamine (GlcNAc) in N-glycoproteins, and can be divided into multiple subclasses according to their sources and substrate specificities. The most classic PNGase F (peptide-N-glycosidase F), derived from Elizabethkingia miricola, has a molecular weight of approximately 36 kDa and can efficiently hydrolyze the innermost GlcNAc-Asn bond of almost all types of N-glycans (high-mannose, hybrid, and complex types), releasing complete N-glycan chains. In contrast, plant-derived PNGase A specifically acts on N-glycans containing core α1-3 fucose, a modification commonly found in plant and insect glycoproteins. Notably, the team led by Liu Xianwei from Shandong University discovered a unique bifunctional protein in the Arabidopsis thaliana genome, which exhibits both N-glycosidase activity (catalyzing amide bond cleavage) and transglutaminase activity (catalyzing amide bond formation). This dual function implies a potential complex regulatory relationship between N-glycosylation and protein cross-linking networks.
Catalytic mechanisms and substrate specificities determine the application scope of N-glycosidases. Studies have shown that the active center of PNGase F consists of three key amino acid residues: D60N, E206, and E118. Mutations in these residues can lead to the loss of hydrolytic activity or changes in substrate affinity. During catalysis, PNGase F first recognizes specific glycan structures on glycoproteins, then hydrolyzes the bond between GlcNAc and Asn, while converting Asn to Asp. It is particularly noteworthy that traditional PNGase F is inactive against N-glycoproteins containing core α1-3 fucose. This limitation has prompted scientists to isolate a novel N-glycosidase, PNGase F-II, from Elizabethkingia meningoseptica. This enzyme can remove glycan chains from α1-3 core-fucosylated N-glycoproteins, expanding the range of analyzable glycoproteins. Substrate specificity studies have shown that PNGase F-II can act on various substrates such as HRP glycoproteins and hybrid glycan N-glycoproteins, with broad substrate specificity and efficient enzymatic cleavage ability, providing new tools for glycan structure analysis and functional research.
Expression, purification, and engineering modification are key approaches to optimizing the performance of N-glycosidases. Traditional PNGase F often forms inactive inclusion bodies when expressed in E. coli, leading to difficult purification and high costs. Dr. Zhang Liang from Huazhong University of Science and Technology obtained recombinant His-PNGase F inclusion bodies accounting for more than 40% of the total bacterial protein by optimizing induction conditions, and successfully obtained high-purity active enzyme through urea denaturation and renaturation methods. Another innovative strategy is to introduce a co-fusion glutamine tag (Q tag) at the terminal of PNGase F, significantly improving soluble expression and facilitating direct purification. In enzyme engineering, immobilization technology has greatly enhanced the usage efficiency of PNGase F—non-covalent oriented immobilization increases the loading capacity of His-PNGase F by approximately 120 times, while covalent oriented immobilized enzymes based on Q tags still retain 78.6% activity after five repeated uses. More notably, combining immobilized PNGase F with microwave-assisted enzymatic digestion can achieve complete release of N-glycans within 5 minutes, drastically shortening the reaction time from the traditional 10-18 hours.
Studies on structure-function relationships provide a theoretical basis for rational design. Through crystal structure analysis and site-directed mutagenesis, scientists have gradually revealed the structural characteristics of the PNGase F active center and its interaction patterns with substrates. The crystal structure of PNGase F-II shows that it shares conserved catalytic residues with classic PNGase F but differs in the substrate recognition region, which may be the structural basis for its ability to act on α1-3 core-fucosylated substrates. Homology modeling and point mutation studies of bifunctional N-glycosidase/transglutaminase have found that specific amino acids and non-active center extended domains at both ends play an important role in balancing the two enzymatic activities. These structural biology insights not only deepen the understanding of N-glycosidase catalytic mechanisms but also provide a molecular blueprint for designing engineered enzymes with specific properties, such as improved thermal stability, altered substrate preferences, or the development of new catalytic functions.
Core Applications of N-Glycosidases in Glycoprotein Research
Glycan structure analysis represents the most classic application field of N-glycosidases. Through PNGase F treatment, researchers can completely release N-glycans from glycoproteins for compositional and structural analysis using mass spectrometry, liquid chromatography, and other techniques. The introduction of novel PNGase F-II has filled the gap in traditional enzyme analysis of α1-3 core-fucosylated glycan chains, making glycoform analysis of complex biological samples more comprehensive. Experimental procedures show that for common monomeric N-glycoproteins, after denaturation by heat treatment at 100°C for 10 minutes, PNGase F can complete deglycosylation within 1 hour at 37°C; for special proteins such as human immunoglobulin (IgG), the reaction needs to be extended to more than 6 hours under non-denaturing conditions to ensure complete enzymatic cleavage. Periodic acid-Schiff (PAS) staining and mass spectrometry are common methods to verify deglycosylation effects. The former intuitively reflects glycan removal by detecting changes in red signals of glycoprotein bands, while the latter accurately determines the molecular weight and structural characteristics of released glycan chains. These technical combinations provide reliable tools for studying the microheterogeneity of glycoproteins, helping to reveal the correlation between glycoforms and protein functions.
Glycosylation site identification is an important part of proteomics research. Mass spectrometry comparison of samples before and after N-glycosidase treatment can accurately determine the location and modification degree of glycosylation sites. In a glycoproteomics study of colorectal cancer, scientists established a high-quality database containing 7,125 complete glycopeptides corresponding to 704 glycoproteins through systematic analysis of cancerous and adjacent tissues, revealing the characteristics of N-glycosylation-mediated metabolic reprogramming. The construction of this multi-dimensional association network of "glycoform-site-protein" relies on the efficient release of glycopeptides by PNGase F and subsequent mass spectrometry analysis. Of particular note, glycosylation site-specific information provides a new dimension for discovering disease-related biomarkers—the identified glycoforms of CLCA1 and OLFM4 can serve as diagnostic markers for colorectal cancer, while specific glycoforms at the APMAP N196 site are closely related to disease progression. These findings highlight the key value of N-glycosidases in translational medicine research.
Functional glycomics analysis is moving from population-level to single-cell resolution. Facing the challenge that a single cell contains only picogram-level proteins, the team from the Academy of Military Medical Sciences innovatively adopted a "signal carrier" strategy, using N-glycopeptides enriched from constant samples by hydrophilic interaction chromatography (HILIC) as carrier channels, mixing them with trace/single-cell samples (TMT-labeled) for mass spectrometry detection, and achieving reliable analysis of ultra-trace samples through signal correction. This method does not require separate glycopeptide enrichment for single cells but can achieve considerable analysis depth, successfully revealing glycosylation heterogeneity among immune cell subsets and within tumor cell populations. Combined with FAIMS Pro interfaces and MSFragger-glyco software, multi-level quality control (FDR<1%) from peptide spectrum matching to glycan composition level has been realized, establishing a standardized process for studying glycosylation regulatory networks at single-cell resolution. This high-sensitivity analysis technology will promote in-depth understanding of tumor microenvironment heterogeneity and immune cell functional plasticity.
In glycoprotein function research, N-glycosidases provide precise tools for manipulating glycosylation status. By comparing the physicochemical properties and biological activities of proteins before and after deglycosylation, scientists can evaluate the functional contributions of specific glycoforms. In the study of Thermoascus aurantiacus β-glucosidase (Bgl3A), site-directed mutagenesis-generated deglycosylation mutants T44A, S228A, and S299A showed significantly different secretion levels and enzymatic properties— the S228A mutation resulted in minimal protein secretion and almost complete loss of activity, indicating that glycosylation at this site is crucial for enzyme expression and function; the S299A mutation improved the enzyme's thermal stability and catalytic efficiency towards cellobiose, demonstrating the fine regulation of glycosylation modifications on enzyme performance. Similar strategies have also been applied to antibody drug development. PNGase F treatment can distinguish glycosylation differences between Fc and Fab segments, guiding engineering modifications to optimize effector functions. These studies not only deepen the understanding of the biological significance of glycosylation but also provide a theoretical basis for rationally designing glycoprotein therapeutics with excellent performance.
Discovery and Engineering of Novel N-Glycosidases
The discovery of PNGase F-II has filled the technical gap in glycan structure analysis. Traditional PNGase F cannot act on N-glycoproteins containing core α1-3 fucose, limiting in-depth research on plant and insect glycoproteins. The novel N-glycosidase PNGase F-II isolated from the prokaryote Elizabethkingia meningoseptica breaks through this limitation, efficiently removing glycan chains from high-mannose, complex, hybrid, and α1-3 core-fucosylated N-glycoproteins. Enzymatic characterization shows that PNGase F-II exhibits optimal activity at 37°C and pH 7.0-8.0, requiring only 1 hour for enzymatic cleavage of heat-treated substrates, while natural IgG requires more than 6 hours of reaction time. Mass spectrometry analysis confirms that substrates such as ribonuclease B (RNase B), ovalbumin, human IgG, and horseradish peroxidase (HRP) can all release complete glycan chains after PNGase F-II treatment, with efficiency equivalent to or even better than that of PNGase F. This broad substrate specificity makes PNGase F-II a new sharp tool for glycobiology research, particularly suitable for the analysis of plant-derived glycoproteins and surface glycoproteins of certain pathogens.
The discovery of bifunctional N-glycosidase/transglutaminase has revealed potential links between glycosylation and protein cross-linking. The team from Shandong University identified a special enzyme in the Arabidopsis thaliana genome with both N-glycosidase (catalyzing amide bond cleavage) and transglutaminase (catalyzing amide bond formation) activities. Through prokaryotic and eukaryotic recombinant expression, glycosylation analysis, and point mutation studies, scientists have gradually clarified the determining conditions and catalytic mechanisms of this dual activity. Interestingly, based on the catalytic properties of the protein superfamily to which this enzyme belongs, researchers speculate that it may catalyze the cross-linking reaction between oligosaccharides and peptide chains, providing new ideas for glycopeptide and glycoprotein synthesis. By regulating reaction conditions (such as activating substrates, changing pH, or ionic strength), transglycosidation reactions are expected to be achieved, creating brand-new approaches for the synthesis of homogeneous glycoproteins and solving key bottlenecks in glycoprotein functional research and drug development.
Engineering modification of sugar-binding proteins has expanded the toolbox for N-glycan analysis. In addition to hydrolases, proteins that specifically recognize glycan chains also hold important value in glycomics research. The team from Huazhong University of Science and Technology systematically modified an F-box mutant protein (Fg) that recognizes glycan chains, verifying the necessity of the core pentasaccharide structure of N-glycans for its recognition. Fusing Fg with glutathione S-transferase (GST) allows convenient immobilization on glutathione-modified agarose microspheres, forming an efficient glycopeptide enrichment material—17 and 11 glycopeptides were identified from the enzymatic digestion products of IgG and HRP, respectively, demonstrating excellent selectivity and anti-interference ability in complex samples. More ingeniously, a fluorescent probe (GFP-Fg) constructed by fusing Fg with green fluorescent protein (GFP) can specifically label N-glycans on the cell membrane with a labeling efficiency of up to 99.9%, providing a powerful tool for dynamic studies of glycosylation in living cells. These innovations demonstrate the broad prospects of multifunctional protein engineering in glycobiology research.
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