The efficient expression of soluble glycosyltransferases from mammalian sources in Escherichia coli (E. coli) remains a significant challenge, often resulting in misfolding and the formation of inclusion bodies. In this study, we investigated strategies to enhance the solubility and catalytic activity of human GalNAc-T2 and GalNAc-T11, two O-glycosyltransferases involved in O-glycosylation of glycoproteins. We found that fusion with maltose-binding protein (MBP) and cellulase catalytic domain (Cel-CD), which led to majority of the fusion proteins being soluble, could increase the solubility of the recombinant proteins. Enzyme activity assays revealed that the fusion glycosyltransferase exhibited significantly higher catalytic efficiency than non-fused enzymes. In addition, the influence of GalNAc-T11 lectin domain on substrate specificity was also determined. The presence of lectin domain had no influence on the recognition of specific substrate and the specific activity of GalNAc-T11. This work offers an efficient approach for the large-scale production of human glycosyltransferases with enhanced bioactivity, highlighting its potential for glycosylation engineering of glycoprotein drugs.

Perfluorooctanoic acid (PFOA) is a well-known thyroid disruptor that has been found to induce hypothyroidism. However, the exact molecular mechanism by which PFOA reduces thyroid hormone levels remains unclear. In this study, we have discovered that PFOA disrupts the glycosylation process of the sodium/iodide symporter (NIS), which inhibits the translocation of NIS onto the plasma membrane of thyroid follicular cells. Our results also demonstrate that PFOA disrupts thyroid stimulating hormone (TSH)-dependent signaling pathways involved in cellular glycosylation, impairing NIS glycosylation and reducing the ability of iodine uptake. This leads to an insufficiency of iodine for thyroid hormone production inside the follicular cells of the thyroid, resulting in lower-than-normal thyroxine levels detected in zebrafish larvae. These findings are consistent with our previously published data, which showed that PFOA induces neural behavior changes during the early stages of neuronal development in zebrafish. This new discovery provides valuable insights into the molecular characteristics of endocrine-disrupting chemicals (EDCs) that are known to affect the thyroid. It may also contribute to a better understanding of how altered glycosylation could be a potential risk factor for the association between exposure to specific per- and polyfluoroalkyl substances (PFAS) and various health effects in humans.

The vast majority of proteins that traverse the mammalian secretory pathway become N-glycosylated in the endoplasmic reticulum (ER). The bulky glycan protein modifications, which are conserved in fungi and humans, act as maturation and quality-control tags. In this Review, we discuss findings published in the past decade that have rapidly expanded our understanding of the transfer and processing of N-glycans, as well as their role in protein maturation, quality control and trafficking in the ER, facilitated by structural insights into the addition of N-glycans by the oligosaccharyltransferases A and B (OST-A and OST-B). These findings suggest that N-glycans serve as reporters of the folding status of secretory proteins as they traverse the ER, enabling the lectin chaperones to guide their maturation. We also explore how the emergence of co-translational glycosylation and the expansion of the glycoproteostasis network in metazoans has expanded the role of N-glycans in early protein-maturation events and quality control.

Serum glycoprotein glycosylation changes can indicate disease onset and progression. However, the site-specific N-glycosylation of monoclonal immunoglobulins (M-proteins) in multiple myeloma (MM) and its clinical implications are unclear. In this study, we isolated pathogenic micromonoclonal IgA or IgG (approximately 2 μg) from IgA-MM patients (n = 22) and IgG-MM patients (n = 30), and normal polyclonal IgA and IgG from healthy controls (HCs) (n = 16). Using EThcD-sceHCD-MS/MS, the N-glycosylation dynamics of serum M-proteins in MM were determined. Compared with polyclonal IgA1 from HCs, monoclonal IgA1 from IgA-MM patients had higher fucosylation (58.1% vs 32.1%, p < 0.001), sialylation (68.0% vs 50.8%, p = 0.011), and mannosylation (1.5% vs 0.3%, p < 0.001). While, monoclonal IgG1 from IgG-MM patients had higher fucosylation (97.8% vs 95.3%, p < 0.001). In addition, specific N-glycan abundances correlated with MM clinical features: for IgA1, HexNAc5Hex5Fuc1NeuAc1 was associated with hypocomplementemia; for IgG1, HexNAc4Hex3Fuc1 was associated with the serum albumin level (r = -0.363, p = 0.049) and estimated glomerular filtration rate (r = -0.433, p = 0.017); and HexNAc4Hex5 was associated with therapeutic prognosis. In conclusion, monoclonal IgA1 and IgG1 in MM patients and their polyclonal isotypes in HCs have distinct N-glycosylation profiles, and specific N-glycans of M-proteins are associated with MM characteristics and therapeutic prognosis.

Glycosylation plays a critical role in regulating activity, stability, and correct folding of enzymes. In this study, recombinant Aspergillus niger α-L-rhamnosidase (r-Rha1) was employed to explore the impact of glycosylation in Pichia pastoris on the enzymatic properties and protein folding. β-elimination reaction and deglycosylase treatment assays demonstrated that r-Rha1 undergoes primarily N-glycosylation. The deglycosylated r-Rha1 was prepared in two ways: treating with Endoglycosidase F1 after expression (referred to as r-Rha1-vitro), or inhibiting intracellular glycosylation using tunicamycin (referred to as r-Rha1-vivo). Deglycosylation resulted in a 0.22-fold decrease in activity for r-Rha1-vitro and due to its slower turnover rate, r-Rha1-vivo showed a 0.73-fold decrease in activity. r-Rha1-vitro maintained the similar optimal temperature as r-Rha1, r-Rha1-vivo displayed a 10 °C lower optimal temperature. Compared to the decreased extent of r-Rha1-vitro in t1/2 at 55 °C, 60 °C, and 65 °C and Tm, chemical interferent deglycosylation in vivo showed a more profound impact on r-Rha1. Analyses based on circular dichroism, fluorescence spectroscopy, and differential scanning calorimetry revealed significant changes in the structure and thermodynamic stability of r-Rha1-vivo, accounting for its marked decline in activity and stability. The significant and unpredictable structure changes of r-Rha1-vivo proved the essential role of glycosylation for appropriate folding in P. pastoris.

Understanding the dynamic behavior of glycoproteins is crucial for deciphering their biological roles. This review explores the synergistic use of experimental and computational methods to address this complex challenge. Glycans, with their inherent flexibility and structural diversity, pose significant obstacles to traditional structural analysis. Innovative experimental techniques offer valuable snapshots of glycan conformations, but often lack the context of a physiological environment. Computational simulations provide atomic-level detail and explore the full range of dynamic motions, but require extensive resources and validation. Integrating these approaches, by using experimental data to refine and validate computational models, is essential for accurately capturing the complex interplay between glycans and proteins. This combined strategy promises to unlock a deeper understanding of glycoprotein function and inform the design of novel therapeutics.

Glycosylation is one of the most ubiquitous post-translational modifications observed in peptides and proteins. It affects the structural and functional characteristics of these macromolecules, thereby exerting a profound influence on a multitude of biological processes. N-Glycans are expected to be a beneficial modifier for increasing the solubility and in vivo half-life, and reducing the aggregation and immunogenicity of native bioactive peptides and proteins, which have seen limited clinical utility due to their short blood half-life and unsuitable physicochemical properties. Chemoselective glycosylation reactions that can be conducted post-synthesis and in aqueous conditions are a promising strategy for the high-throughput development of peptide/protein drugs. This "glycoconjugation" approach is particularly advantageous in that manipulation of glycan protecting groups is not necessary, thereby allowing conjugation reactions to be carried out between target molecules and unprotected glycans. By providing a single glycosylation profile, i. e., glycan structure, number, and position, glycoconjugation not only allows the beneficial properties of N-glycans to be exploited, but also facilitates the investigation of N-glycan function.

The PI3K/AKT signaling pathway is frequently dysregulated in cancer and controls key cellular processes such as survival, proliferation, metabolism and growth. Protein glycosylation is essential for proper protein folding and is also often deregulated in cancer. Cancer cells depend on increased protein folding to sustain oncogene-driven proliferation rates. The N-glycosyltransferase asparagine-linked glycosylation 3 homolog (ALG3), a rate-limiting enzyme during glycan biosynthesis, catalyzes the addition of the first mannose to glycans in an alpha-1,3 linkage. Here we show that ALG3 is phosphorylated downstream of the PI3K/AKT pathway in both growth factor-stimulated cells and PI3K/AKT hyperactive cancer cells. AKT directly phosphorylates ALG3 in the amino terminal region at Ser11/Ser13. CRISPR/Cas9-mediated depletion of ALG3 leads to improper glycan formation and induction of endoplasmic reticulum stress, the unfolded protein response, and impaired cell proliferation. Phosphorylation of ALG3 at Ser11/Ser13 is required for glycosylation of cell surface receptors EGFR, HER3 and E-cadherin. These findings provide a direct link between PI3K/AKT signaling and protein glycosylation in cancer cells.

Glycoproteomics is a rapidly developing field, and data analysis has been stimulated by several technological innovations. As a result, there are many software tools from which to choose; and each comes with unique features that can be difficult to compare. This work presents a head-to-head comparison of five modern analytical software: Byonic, Protein Prospector, MSFraggerGlyco, pGlyco3, and GlycoDecipher. To enable a meaningful comparison, parameter variables were minimized. One potential confounding variable is the glycan database that informs glycoproteomic searches. We performed glycomic profiling of the samples and used the output to construct matched glycan databases for each software. Up to 17,000 glycopeptide spectra were identified across three replicates of wild-type SH-SY5Y cells. There was overlap among all software for glycoproteins identified, locations of glycosites, and glycans; but there was no clear winner. Incorporation of several comparative criteria was critically important for learning the most information in this study and should be used more broadly when assessing software. A single criterion, such as number of glycopeptide spectra found, is not sufficient. We present evidence that suggests Byonic reports many spurious results at the glycoprotein and glycosite level. Overall, our results indicate that glycoproteomic searches should involve more than one software, excluding the current version of Byonic, to generate confidence by consensus. It may be useful to consider software with peptide-first approaches and with glycan-first approaches.

About one-third of the proteins synthesized in eukaryotic cells are directed to the secretory pathway, where close to 70% are being N-glycosylated. N-glycosylation is a crucial modification for various cellular processes, including endoplasmic reticulum (ER) glycoprotein folding quality control, lysosome delivery, and cell signaling. The defects in N-glycosylation can lead to severe developmental diseases. For the proteins to be glycosylated, they must be translocated to the ER through the Sec61 translocon channel, either via co-translationally or post-translationally. N-glycosylation not only could accelerate post-translational translocation but may also enhance protein stability, while protein folding can assist in their movement into the ER. However, the precise mechanisms by which N-glycosylation and folding influence translocation remain poorly understood. The chaperone BiP is essential for post-translational translocation, using a "ratchet" mechanism to facilitate protein entry into the ER. Although research has explored how BiP interacts with protein substrates, there has been less focus on its binding to glycosylated substrates. Here, we review the effect of N-glycosylation on protein translocation, employing single-molecule studies and ensembles approaches to clarify the roles of BiP and N-glycosylation in these processes. Our review explores the possibility of a direct relationship between translocation and a ratchet effect of glycosylation and the importance of BiP in binding glycosylated proteins for the ER quality control system.

The online version contains supplementary material available at 10.1007/s12551-025-01313-x.

X-linked myopathy with excessive autophagy (XMEA) is a rare neuromuscular disorder caused by mutations in the VMA21 gene, encoding a chaperone protein present in the endoplasmic reticulum (ER). In yeast and human, VMA21 has been shown to chaperone the assembly of the vacuolar (v)-ATPase proton pump required for the acidification of lysosomes and other organelles. In line with this, VMA21 deficiency in XMEA impairs autophagic degradation steps, which would be key in XMEA pathogenesis. Recent years have witnessed a surge of interest in VMA21, with the identification of novel mutations causing a congenital disorder of glycosylation (CDG) with liver affection, and its potent implication in cancer predisposition. With this, VMA21 deficiency has been further linked to defective glycosylation, lipid metabolism dysregulation and ER stress. Moreover, the identification of two VMA21 isoforms, namely VMA21-101 and VMA21-120, has opened novel avenues regarding the pathomechanisms leading to XMEA and VMA21-CDG. In this review, we discuss recent advances on the clinical spectrum associated with VMA21 deficiency and on the pathophysiological roles of VMA21.

N-glycosylation is a prevalent post-translational modification in eukaryotes, essential for regulating protein secretion. In Saccharomyces cerevisiae, glycosylation mutants have been shown to enhance the secretion of heterologous glycosylated proteins. However, whether these mutants can also increase the secretion of non-glycosylated proteins and whether the growth defects associated with glycosylation mutations can be mitigated remains unclear. This study aimed to characterize and optimize enhanced secretory expression in the promising yeast host Kluyveromyces marxianus by deleting MNN11, which encodes a subunit of the mannose polymerase II complex responsible for elongating α-1,6-linked mannose chains.

Compared to wild-type cells, the mnn11Δ cells significantly increased the secretion activities of four glycosylated enzymes and three non-glycosylated enzymes in flasks, with increases ranging from 29 to 668%. Transcriptomic analysis of mnn11Δ mutant revealed upregulation of genes related to essential protein secretion processes, including vesicle coating and tethering, protein folding, translocation, and glycosylation. Additionally, genes involved in vacuolar amino acid transport and amino acid biosynthesis were upregulated, suggesting an amino acid shortage, which might contribute to the observed severe growth defect of the mnn11Δ mutant in a synthetic medium with inorganic nitrogen. Supplementation of the synthetic medium with amino acids or low concentrations of yeast extract alleviated this growth defect, reducing the specific growth rate difference between wild-type strain and mnn11Δ cells from 65% to as little as 2%. During high-density fermentation, the addition of 0.5% yeast extract substantially reduced the lag phase of mnn11Δ mutants and increased the secretory activities of α-galactosidase, endoxylanase, and β-glucanase, by 11%, 18%, and 36%, respectively, compared to mnn11Δ mutant grown without yeast extract.

In K. marxianus, deletion of MNN11 enhances the secretion of both glycosylated and non-glycosylated proteins by improving key protein secretion processes. The growth defect in the mnn11Δ mutant is closely tied to insufficient amino acid supply. Supplementing the synthetic medium with low concentrations of organic nitrogen sources effectively alleviates this growth defect and enhances secretory expression. This strategy could be applied to optimize the expression of other glycosylation mutants.

It is widely acknowledged that dietary habits have profound impacts on human health and diseases. As the most important sweeteners and energy sources in human diets, hexoses take part in a broad range of physiopathological processes. In recent years, emerging evidence has uncovered the crucial roles of hexoses, such as glucose, fructose, mannose, and galactose, in controlling the differentiation or function of immune cells.

Herein, we reviewed the latest research progresses in the hexose-mediated modulation of immune responses, provided in-depth analyses of the underlying mechanisms, and discussed the unresolved issues in this field.

Owing to their immunoregulatory effects, hexoses affect the onset and progression of various types of immune disorders, including inflammatory diseases, autoimmune diseases, and tumor immune evasion. Thus, targeting hexose metabolism is becoming a promising strategy for reversing immune abnormalities in diseases.

The relationship between cancer development and alterations in IgG N-glycosylation has been well-established. However, comprehensive profiling of the N-glycome and N-glycoproteome in gastric cancer (GC) remains limited. Furthermore, the prognostic potential of IgG N-glycan patterns in identifying precursors to GC has yet to be fully elucidated.

The IgG N-glycome in GC was characterized using a custom high-throughput orthogonal mass spectrometry approach. Multivariate analysis was employed to identify and assess glycomic alterations. A comprehensive bioinformatics analysis was also conducted to investigate the differential expression of N-glycosylation-related genes and their potential roles in GC pathogenesis. Additionally, interleukin-11 (IL-11) levels were quantified using a standardized enzyme-linked immunosorbent assay (ELISA).

Galactosylation and sialylation of IgG decreased mainly in the IgG1 and IgG2 subclasses in GC, with subclass-specific changes in IgG3 and IgG4 galactosylation. These glycan modifications were represented by unique glycopeptides (IgG1_H5N5, IgG2_H4N3F1, IgG2_H4N4, IgG2_H4N4F1S1, IgG3/4_H4N4F1, IgG3/4_H4N4F1S1), which outperformed CA72-4 for GC diagnosis. Analysis of key glycogenes revealed differential expression patterns, implicating a functional role for IgG N-glycosylation in GC. Notably, the abundance of specific IgG glycosylation exhibited a significant correlation with serum level of IL-11.

Alterations in subclass-specific IgG N-glycosylation represent promising biomarkers for the detection and monitoring of GC progression, potentially influenced by cytokine-driven inflammation. Understanding these changes could improve our knowledge of molecular mechanisms, aiding in diagnostic improvements and therapeutic development.

Schistosomiasis is a neglected tropical disease and the second most common parasitic disease after malaria. While praziquantel remains the primary treatment, concerns about drug resistance highlight the urgent need for new drugs and effective vaccines to achieve sustainable control. Previous proteomic studies from our group revealed that the expression of Schistosoma japonicum glycosyltransferase and nicastrin as proteins was higher in single-sex males than mated males, suggesting their critical roles in parasite reproduction and their potential as vaccine candidates. In this study, bioinformatic tools were employed to analyze the structural and functional properties of these proteins, including their signal peptide regions, transmembrane domains, tertiary structures, and protein interaction networks. Recombinant forms of glycosyltransferase and nicastrin were expressed and purified, followed by immunization experiments in BALB/c mice. Immunized mice exhibited significantly elevated specific IgG antibody levels after three immunizations compared to adjuvant and PBS controls. Furthermore, immunization with recombinant glycosyltransferase and nicastrin significantly reduced the reproductive capacity of female worms and liver egg burden, though egg hatchability and adult worm survival were unaffected. These findings demonstrate that recombinant glycosyltransferase and nicastrin are immunogenic and reduce female worm fecundity, supporting their potential as vaccine candidates against schistosomiasis.

Keratoconus (KC) is an ectatic cornea disease with high prevalence and asymptomatic at early stage, leading to decreased visual acuity and even cornea transplantation. However, the etiology mechanism of keratoconus is still poorly understood. Oxidative stress (OS) and extracellular matrix (ECM) remodeling play critical roles in keratoconus development. Here, based on keratoconus datasets from GEO database, we obtained 454 differentially expressed genes (DEGs), which were further intersected with oxidative stress (OS) and extracellular matrix (ECM) genesets from MSigDB database. A total of 17 OS- and ECM-related DEGs (OEDEGs) were identified. Feature genes were screened by least absolute shrinkage and selection operator (LASSO) and support vector machine recursive feature elimination (SVM-RFE) algorithms, and a six-gene (COL1A1, CYP1B1, MMP3, HMOX1, FOS and GDF15) classification model was developed utilizing Logistic regression (LR), Support Vector Machine (SVM) and Naïve Bayes (NB) algorithms respectively, which was further verified in internal and external cohort. Subsequently, a predictive nomogram was constructed for KC patients. Six signature genes showed a strong correlation with the infiltration level of macrophages M1, neutrophils and eosinophils. Additionally, in vitro qRT-PCR validated the decreased expression of signature genes in either keratoconus clinical samples or human cornea epithelial (HCE) cells grown on soft hydrogel substrate. Finally, we revealed that CYP1B1 and GDF15 regulate cellular proliferation and response to oxidative stress. In conclusion, the developed combined OS and ECM gene signature showed excellent performance for keratoconus prediction, providing beneficial perspectives for keratoconus pathogenesis.

The relationship between membrane proteins and the lipid constituents of the membrane bilayer depends on finely-tuned atomic interactions, which itself depends on the precise distribution of amino acids within the 3D structure of the protein. In this regard, tryptophan (Trp), one of the least abundant amino acids, is found at higher levels in transmembrane proteins where it likely plays a role in helping anchor them to the membrane. We now re-evaluate Trp distribution in membrane proteins using all known proteins in the Swiss-Prot database and confirm that it is somewhat higher (∼1.7%) than in soluble proteins (∼1.0%), but not as high as in a well-quoted study (∼3.1%). However, the resident endoplasmic reticulum membrane protein, ceramide synthase (CerS), contains a higher abundance of Trp (3.4%). In the case of CerSs which contain a Hox-like domain, the Trp residues are asymmetrically distributed throughout the protein with a bias towards the lumenal side of the endoplasmic reticulum membrane. Mutation of these residues, even to other hydrophobic amino acids, leads to loss of activity, expression, and/or N-glycosylation. Moreover, five of the ten most conserved amino acids in the CerSs are Trp, and site-directed mutagenesis of numerous conserved Trp residues to alanine had distinct effects. Our data is consistent with other studies suggesting that Trp plays critical roles not only in membrane anchoring of transmembrane proteins but also in their activity and function.

Intestinal bacteria play a crucial role in human health, for example, by maintaining immune and metabolic homeostasis and protecting against pathogens. Survival in the human intestine depends on the bacterium's ability to utilize complex carbohydrates. Some species are known to use host-derived glycans; for example, Bifidobacteria can utilize O-glycan of mucin. However, there are few studies on intestinal bacteria utilizing host-derived N-glycan. Here, we identified the mechanism underlying the breakdown and utilization of complex-type N-glycan by the human intestinal bacterium Barnesiella intestinihominis. A growth assay showed that B. intestinihominis can utilize complex-type N-glycan as a carbon source, while RNA-seq analysis identified enzymes and transporters involved in the mechanism of N-glycan breakdown. In particular, the expression of three genes encoding glycoside hydrolase 85 endo-β-N-acetylglucosaminidase (endo-BIN1, endo-BIN2, and endo-BIN3) rose markedly in bacterial cells cultured in complex-type N-glycoprotein medium. We also found that the susC and susD genes, encoding the SusC/SusD membrane complex, form a gene cluster with endo-BIN genes, suggesting that SusC/SusD is involved in transportation of the glycan into the cell. Other genes encoding exo-type glycoside hydrolase enzymes showed elevated expression in cells grown in complex-type N-glycoprotein medium, suggesting that these enzymes function in further degradation of glycan for metabolism by the bacterium. Collectively, these findings suggest the survival strategy of an intestinal bacterium that has a unique metabolic pathway to use host-derived complex-type N-glycan as a nutrient.

Recent advances in mass spectrometry (MS)-based methods have significantly expanded the capabilities for quantitative glycoproteomics, enabling highly sensitive and accurate quantitation of glycosylation at intact glycopeptide level. These developments have provided valuable insights into the roles of glycoproteins in various biological processes and diseases. In this short review, we summarize pertinent studies on quantitative techniques and tools for site-specific glycoproteomic analysis published over the past decade. We also highlight state-of-the-art MS-based software that facilitate multi-dimension quantification of the glycoproteome, targeted quantification of specific glycopeptides, and the analysis of glycopeptide isomers. Additionally, we discuss the potential applications of these technologies in clinical biomarker discovery and the functional characterization of glycoproteins in health and disease. The review concludes with a discussion of current challenges and future perspectives in the field, emphasizing the need for more precise, high-throughput and efficient methods to further advance quantitative glycoproteomics and its applications.

Corn leaf blight and stem rot caused by Colletotrichum graminicola are significant diseases that severely affect corn crops. Glycosyltransferases (GTs) catalyze the transfer of sugar residues to diverse receptor molecules, participating in numerous biological processes and facilitating functions ranging from structural support to signal transduction. This study identified 101 GT genes through functional annotation of the C. graminicola TZ-3 genome. Subsequent analyses revealed differences among the C. graminicola GT (CgGT) genes. Investigation into subcellular localization indicated diverse locations of CgGTs within subcellular structures, while the presence of multiple domains in CgGTs suggests their involvement in diverse fungal biological processes through versatile functions. The promoter regions of CgGT genes are enriched with diverse cis-acting regulatory elements linked to responses to biotic and abiotic stresses, suggesting a key involvement of CgGT genes in the organism's multi-faceted stress responses. Expression pattern analysis reveals that most CgGT genes were differentially expressed during the interaction between C. graminicola and corn. Integrating gene ontology functional analysis revealed that CgGTs play important roles in the interaction between C. graminicola and corn. Our research contributes to understanding the functions of CgGT genes and investigating their involvement in fungal pathogenesis. At the same time, our research has laid a solid foundation for the development of sustainable agriculture and the utilization of GT genes to develop stress-resistant and high-yield crop varieties.

The unfolded protein response (UPR) is a sensing and signaling pathway that surveys the endoplasmic reticulum (ER) for protein folding challenges and responds whenever issues are detected. UPR activation leads to upregulation of secretory pathway chaperones and quality control factors, as well as reduces the nascent protein load on the ER, thereby restoring and maintaining proteostasis. This paradigm-defining view of the role of the UPR is accurate, but it elides additional key functions of the UPR in cell biology. In particular, recent work has revealed that the UPR can shape the structure and function of N- and O-glycans installed on ER client proteins. This crosstalk between the UPR's response to protein misfolding and the regulation of glycosylation remains insufficiently understood. Still, emerging evidence makes it clear that the UPR, and particularly the IRE1-XBP1s arm of the UPR, may be a central regulator of protein glycosylation with important biological consequences. In this review, we discuss the crosstalk between proteostasis, the UPR, and glycosylation, present progress towards understanding biological functions of this crosstalk, and examine potential roles in diseases such as cancer.

Transcription is a fundamental process involving the interaction of RNA polymerase II and related transcription factors. TFIIB is a transcription factor that plays a significant role in the formation and stability of the preinitiation complex in a precise orientation, as well as in the control of initiation and pre-elongation steps. At the initiation step, TFIIB interacts with three structures: the end of the TATA-binding protein, a GC-rich DNA sequence followed by the TATA box, and the C-terminal domain of RNA polymerase II. It is known that RNA polymerase II is a glycoprotein and contains O-GlcNAc sugar at the C-terminal domain during the initiation stage of transcription. However, it is unclear whether the transcription factors interacting with RNA polymerase II are glycoproteins or not. The study aims to determine the glycosylation (N- and/or O-linked glycosylations) of TFIIB by using bioinformatics in one invertebrate and seven vertebrate species and experimental methods in the sea urchin Paracentrotus lividus oocyte. Both bioinformatics and experimental analysis have shown that TFIIB is a glycoprotein. In addition, PNGase-F enzyme treatment, lectin blotting, and colloidal-gold conjugated lectin labeling results revealed that TFIIB contains O-linked GalNAc, mannose, GlcNAc, and α-2,3-linked sialic acid. Based on our results, we suggest that glycosylation modification may be involved in the transcription mechanism of the TFIIB protein.

Background/Objectives: Antibody light chains form amyloid fibrils that lead to progressive tissue damage in amyloid light chain (AL) amyloidosis. The properties of each patient's unique light chain appear to determine its propensity to form amyloid. One factor is N-glycosylation, which is more frequent in amyloid-associated light chains than in light chains from the normal immune repertoire. However, the mechanisms underlying this association are unknown. Here, we investigate the frequency and position within the light chain sequence of the N-glycosylation sequence motif, or sequon. Methods: Monoclonal light chains from AL amyloidosis and multiple myeloma were identified from the AL-Base repository. Polyclonal light chains were obtained from the Observed Antibody Space resource. We compared the fraction of light chains from each group harboring an N-glycosylation sequon, and the positions of these sequons within the sequences. Results: Sequons are enriched among AL-associated light chains derived from a subset of precursor germline genes. Sequons are observed at multiple positions, which differ between the two types of light chains, κ and λ, but are similar between light chains from AL amyloidosis and multiple myeloma. Positions of sequons map to residues with surface-exposed sidechains that are compatible with the folded structures of light chains. Within the known structures of λ AL amyloid fibrils, many residues where sequons are observed are buried, inconsistent with N-glycosylation. Conclusions: There is no clear structural rationale for why N-glycosylation of κ light chains is associated with AL amyloidosis. A better understanding of the roles of N-glycosylation in AL amyloidosis is required before it can be used as a marker for disease risk.

N-Acetylglucosaminyltransferases-IVa (GnT-IVa or MGAT4A) and -IVb (MGAT4B) are glycosyltransferase isozymes synthesizing the β1,4-GlcNAc branch in N-glycans, a glycan structure involved in diabetes. These enzymes uniquely have a non-catalytic lectin domain, which selectively recognizes the GnT-IV product N-glycan branch, but the role of this lectin domain has remained unclear. Here, using UDP-Glo enzyme assays, we discovered that this domain is required for activity toward glycoprotein substrates but not toward free glycans. Furthermore, we found that the lectin domain itself is decorated with an N-glycan, which can serve as a self-ligand and interact with the ligand binding site of the lectin domain in a glycan structure-dependent manner. Enzyme assays using glycan-remodeled GnT-IVa demonstrated that the interaction of the self-ligand with the lectin domain suppresses GnT-IVa activity toward glycoprotein substrates. These findings unveiled a lectin-assisted self-regulatory mechanism of glycosyltransferases, which deepens our understanding of the complex pathway of N-glycan biosynthesis.

Human immunoglobulin G (IgG) antibodies are one of the most important classes of biotherapeutic agents and undergo glycosylation at the conserved N297 site in the CH2 domain, which is critical for IgG Fc effector functions and anti-inflammatory activity. Hence, technologies for producing authentically glycosylated IgGs are in high demand. While attempts to engineer Escherichia coli for this purpose have been described, they have met limited success due in part to the lack of available oligosaccharyltransferase (OST) enzymes that can install N-linked glycans within the QYNST sequon of the IgG CH2 domain. Here, we identified a previously uncharacterized single-subunit OST (ssOST) from the bacterium Desulfovibrio marinus that exhibited greatly relaxed substrate specificity and, as a result, was able to catalyze glycosylation of native CH2 domains in the context of both a hinge-Fc fragment and a full-length IgG. Although the attached glycans were bacterial in origin, conversion to a homogeneous, asialo complex-type G2 N-glycan at the QYNST sequon of the E. coli-derived hinge-Fc was achieved via chemoenzymatic glycan remodeling. Importantly, the resulting G2-hinge-Fc exhibited strong binding to human FcγRIIIa (CD16a), one of the most potent receptors for eliciting antibody-dependent cellular cytotoxicity (ADCC). Taken together, the discovery of a unique ssOST from D. marinus provides previously unavailable biocatalytic capabilities to the bacterial glycoprotein engineering toolbox and opens the door to using E. coli for the production and glycoengineering of human IgGs and fragments derived thereof.

N-glycosylation is a highly heterogeneous post-translational modification that modulates protein function. Defects in N-glycosylation are directly linked to various human diseases. Despite the importance of quantifying N-glycans with high precision, existing glycoinformatics tools are limited. Here, we developed nQuant, a glycoinformatics tool that enables label-free and isotopic labeling quantification of N-glycomics data obtained via LC-MS/MS, ensuring a low false quantitation rate. Using the label-free quantification module, we profiled the N-glycans released from purified glycoproteins and HEK293 cells as well as the dynamic changes of N-glycosylation during mouse corpus callosum development. Through the isotopic labeling quantification module, we revealed the dynamic changes of N-glycans in acute promyelocytic leukemia cells after all-trans retinoic acid treatment. Taken together, we demonstrate that nQuant enables fast and precise quantitative N-glycomics.

Prostate cancer remains a major health concern, with prostate-specific antigen (PSA) being a key biomarker for its detection and monitoring. However, PSA levels often fall into a "gray zone", where PSA levels are not clearly indicative of cancer, thus complicating early diagnosis and treatment decisions. Glycosylation profiles, which often differ between healthy and diseased cells, have emerged as potential biomarkers to enhance the specificity and sensitivity of cancer diagnosis in these ambiguous cases. We propose the integration of two complementary techniques, namely quartz-crystal microbalance with dissipation (QCM-D) and surface-enhanced Raman scattering (SERS) to study PSA glycan profiles. QCM-D offers real-time operation, PSA mass quantification, and label-free detection with high sensitivity, as well as enhanced specificity and reduced cross-reactivity when using nucleic acid aptamers as capture ligands. Complementary SERS sensing enables the determination of the glycosylation pattern on PSA, at low concentrations and without the drawbacks of photobleaching, thereby facilitating multiplexed glycosylation pattern analysis. This integrated setup could retrieve a data set comprising analyte concentrations and associated glycan profiles in relevant biological samples, which may eventually improve early disease detection and monitoring. Prostate-specific antigen (PSA), a glycoprotein secreted by prostate epithelial cells, serves as our proof-of-concept analyte. Our platform allows multiplex targeting of PSA multiplex glycosylation profiles of PSA at "gray zone" concentrations for prostate cancer diagnosis. We additionally show the use of SERS for glycan analysis in PSA secreted from prostate cancer cell lines after androgen-based treatment. Differences in PSA glycan profiles from resistant cell lines after androgen-based treatment may eventually improve cancer treatment.

One of the main causes of poor prognoses in patient with glioblastoma (GBM) is drug resistance to current standard treatment, which includes chemoradiation and adjuvant temozolomide (TMZ). In addition, the concept of cancer stem cells provides new insights into therapy resistance and management also in GBM and glioblastoma stem cell-like cells (GSCs), which might contribute to therapy resistance. Bone morphogenetic protein-4 (BMP4) stimulates astroglial differentiation of GSCs and thereby reduces their self-renewal capacity. Exposure of GSCs to BMP4 may also sensitize these cells to TMZ. A recent phase I trial has shown that local delivery of BMP4 is safe, but a large variation in survival is seen in these treated patients and in features of their cultured tumors. We wanted to combine TMZ and BMP4 (TMZ + BMP4) therapy and assess the inter-tumoral variability in response to TMZ + BMP4 in patient-derived GBM cultures. A phase II trial could then benefit a larger group of patients than those treated with BMP4 only. We first show that simultaneous treatment with TMZ + BMP4 is more effective than sequential treatment. Second, when applying our optimized treatment protocol, 70% of a total of 20 GBM cultures displayed TMZ + BMP4 synergy. This combination induces cellular apoptosis and does not inhibit cell proliferation. Comparative bulk RNA-sequencing indicates that treatment with TMZ + BMP4 eventually results in decreased MAPK signaling, in line with previous evidence that increased MAPK signaling is associated with resistance to TMZ. Based on these results, we advocate further clinical trial research to test patient benefit and validate pathophysiological hypothesis.

Canine reproduction differs from that of many other domestic animals, and increased knowledge on biochemical changes during canine pregnancy is important for investigations of infertility or subfertility. The total glycosylation pattern, i.e., the glycome, of body fluids reflects cellular status in health and disease. The aim of the present pilot study was to investigate pregnancy-related changes of the serum N-glycome in bitches. A method based on Rapifluor HILIC-UPLC-FLR-MS was optimized and applied for analysis and quantification of N-glycans in canine serum. Serum samples from six pregnant and five non-pregnant bitches, collected at four well-defined time points, were included. The levels of sialylated and galactosylated complex glycans were significantly elevated in serum from pregnant bitches, consistent with previous reports on human pregnancy. The levels of fucosylated and agalactosylated glycans decreased significantly in pregnant dogs. In non-pregnant dogs, the glycosylation pattern did not change during the cycle. Pregnancy is an inflammatory state, but our findings during canine pregnancy are quite the opposite to changes that have previously been described for dogs with a known parasitic infection. Evaluation of the canine glycome may thus be valuable in studies of canine pregnancy, possibly differing inflammatory changes related to pregnancy to those caused by an infection.

The dolichyl-diphosphooligosaccharide-protein glycosyltransferase non-catalytic subunit (DDOST) is a key component of the oligosaccharyltransferase complex catalyzing N-linked glycosylation in the endoplasmic reticulum lumen. DDOST is associated with several cancers and congenital disorders of glycosylation. However, its role in pancreatic cancer remains elusive, despite its enriched pancreatic expression. Using quantitative mass spectrometry, we identify 30 differentially expressed proteins and phosphopeptides (DEPs) after DDOST knockdown in the pancreatic ductal adenocarcinoma (PDAC) cell line PA-TU-8988T. We evaluated DDOST / DEP protein-protein interaction networks using STRING database, correlation of mRNA levels in pancreatic cancer TCGA data, and biological processes annotated to DEPs in Gene Ontology database. The inferred DDOST regulated phenotypes were experimentally verified in two PDAC cell lines, PA-TU-8988T and BXPC-3. We found decreased proliferation and cell viability after DDOST knockdown, whereas ER-stress, ROS-formation and apoptosis were increased. In conclusion, our results support an oncogenic role of DDOST in PDAC by intercepting cell stress events and thereby reducing apoptosis. As such, DDOST might be a potential biomarker and therapeutic target for PDAC.

Gastric cancer is a remarkably heterogeneous tumor. Despite some advances in the diagnosis and treatment of gastric cancer in recent years, the precise treatment and curative outcomes remain unsatisfactory. Poor prognosis continues to pose a major challenge in gastric cancer. Therefore, it is imperative to identify effective targets to improve the treatment and prognosis of gastric cancer patients. It should be noted that glycosylation, a novel form of posttranslational modification, is a process capable of regulating protein function and influencing cellular activities. Currently, numerous studies have shown that glycosylation plays vital roles in the occurrence and progression of gastric cancer. As crucial enzymes that regulate glycan synthesis in glycosylation processes, glycosyltransferases are potential targets for treating GC. Hence, investigating the regulation of glycosyltransferases and the expression of associated proteins in gastric cancer cells is highly important. In this review, the related glycosyltransferases and their related signaling pathways in gastric cancer, as well as the existing inhibitors of glycosyltransferases, provide more possibilities for targeted therapies for gastric cancer.

Mass spectrometry (MS) has revolutionized analytical chemistry, enabling precise identification and quantification of chemical species, which is pivotal for biomarker discovery and understanding complex biological systems. Despite its versatility, the presence of background ions in MS analysis hinders the sensitive detection of low-abundance analytes. Therefore, studies aimed at lowering background ion levels have become increasingly important. Here, we utilized the commercially available Active Background Ion Reduction Device (ABIRD) to suppress background ions and assess its effect on the liquid chromatography-electrospray ionization (LC-ESI)-MS analyses of N-glycans on the Q Exactive HF mass spectrometer. We also investigated the effect of different solvent vapors in the ESI source on N-glycan analysis by MS. ABIRD generally had no effect on high-mannose and neutral structures but reduced the intensity of some structures that contained sialic acid, fucose, or both when methanol vapor filled the ESI source. Based on our findings on the highest number of identified N-glycans from human serum, methanol vapor in the ion source compartment may enhance N-glycan LC-ESI-MS analyses by improving the desolvation of droplets formed during the ESI process due to its high volatility. This protocol may be further validated and extended to advanced bottom-up proteomic/glycoproteomic studies for the analysis of peptide/glycopeptide ions by MS.

Membrane trafficking within the Golgi apparatus plays a pivotal role in the intracellular transportation of lipids and proteins. Dysregulation of this process can give rise to various pathological manifestations, including cancer. Exploiting Golgi defects, cancer cells capitalise on aberrant membrane trafficking to facilitate signal transduction, proliferation, invasion, immune modulation, angiogenesis, and metastasis. Despite the identification of several molecular signalling pathways associated with Golgi abnormalities, there remains a lack of approved drugs specifically targeting cancer cells through the manipulation of the Golgi apparatus. In the initial section of this comprehensive review, the focus is directed towards delineating the abnormal Golgi genes and proteins implicated in carcinogenesis. Subsequently, a thorough examination is conducted on the impact of these variations on Golgi function, encompassing aspects such as vesicular trafficking, glycosylation, autophagy, oxidative mechanisms, and pH alterations. Lastly, the review provides a current update on promising Golgi apparatus-targeted inhibitors undergoing preclinical and/or clinical trials, offering insights into their potential as therapeutic interventions. Significantly more effort is required to advance these potential inhibitors to benefit patients in clinical settings.

The enzyme UDP-glucose: glycoprotein glucosyltransferase (UGGT) is the gatekeeper of protein folding within the endoplasmic reticulum (ER). One-third of the human proteome traverses the ER where folding and maturation are facilitated by a complex protein homeostasis network. Both glycan modifications and disulfide bonds are of key importance in the maturation of these ER proteins. The actions of UGGT are intimately linked to the glycan code for folding and maturation of secretory proteins in the ER. UGGT selectively glucosylates the N-linked glycan of misfolded proteins so that they can reenter the lectin-folding chaperone cycle and be retained within the ER for further attempts at folding. An intriguing aspect of UGGT function is its interaction with its poorly understood cochaperone, the 15 kDa selenoprotein known as SELENOF or SEP15. This small protein contains a rare selenocysteine residue proposed to act as an oxidoreductase toward UGGT substrates. AlphaFold2 predictions of the UGGT1/SEP15 complex provide insight into this complex at a structural level. The predicted UGGT1/SEP15 interaction interface was validated by mutagenesis and coimmunoprecipitation experiments. These results serve as a springboard for models of the integrated action of UGGT1 and SEP15.

Glycosylation plays a pivotal role in tuning the folding and function of proteins. Because most human therapeutic proteins are glycosylated, understanding and controlling glycosylation is important for the design, optimization, and manufacture of biopharmaceuticals. Unfortunately, natural eukaryotic glycosylation pathways are complex and often produce heterogeneous glycan patterns, making the production of glycoproteins with chemically precise and homogeneous glycan structures difficult. To overcome these limitations, bacterial glycoengineering has emerged as a simple, cost-effective, and scalable approach to produce designer glycoprotein therapeutics and vaccines in which the glycan structures are engineered to reduce heterogeneity and improve biological and biophysical attributes of the protein. Here, we discuss recent advances in bacterial cell-based and cell-free glycoengineering that have enabled the production of biopharmaceutical glycoproteins with customized glycan structures.

Protein glycosylation, which involves the attachment of carbohydrates to proteins, is one of the most abundant protein co-translational and post-translational modifications. Advances in technology have substantially increased our knowledge of the biosynthetic pathways involved in protein glycosylation, as well as how changes in glycosylation can affect cell function. In addition, our understanding of the role of protein glycosylation in disease processes is growing, particularly in the context of immune system function, infectious diseases, neurodegeneration and cancer. Several decades ago, cell surface glycoproteins were found to have an important role in regulating ion transport across the cardiac sarcolemma. However, with very few exceptions, our understanding of how changes in protein glycosylation influence cardiovascular (patho)physiology remains remarkably limited. Therefore, in this Review, we aim to provide an overview of N-linked and O-linked protein glycosylation, including intracellular O-linked N-acetylglucosamine protein modification. We discuss our current understanding of how all forms of protein glycosylation contribute to normal cardiovascular function and their roles in cardiovascular disease. Finally, we highlight potential gaps in our knowledge about the effects of protein glycosylation on the heart and vascular system, highlighting areas for future research.

Congenital disorders of glycosylation (CDG) refer to monogenetic diseases characterized by defective glycosylation of proteins or lipids causing multi-organ disorders. Here, we investigate the clinical features and genetic variants of SSR4-CDG and conduct a preliminary investigation of its pathogenesis.

We retrospectively report the clinical data of a male infant with early life respiratory distress, congenital diaphragmatic eventration, cosmetic deformities, and moderate growth retardation. Peripheral blood was collected from the case and parents, genomic DNA was extracted and whole-exome sequencing was performed. The mRNA expression of SSR4 gene was quantified by Real-time Quantitative PCR. RNA sequencing analysis was subsequently performed on the case and a healthy child.

Whole-exome sequencing of the case and his parents' genomic DNA identified a hemizygous c.80_96del in SSR4, combined with the case's clinical features, the diagnosis of CDG was finally considered. In this case, the expression of SSR4 was downregulated. The case were present with 1,078 genes downregulated and 536 genes upregulated. SSR4 gene expression was significantly downregulated in the case. Meanwhile, gene set enrichment analysis (GSEA) revealed that SSR4-CDG may affect hemostasis, coagulation, catabolism, erythrocyte development and homeostatic regulation, and muscle contraction and regulation, etc. Improvement of growth retardation in case after high calorie formula feeding and rehabilitation training.

Our study expanded the SSR4-CDG variant spectrum and clinical phenotype and analyzed pathways potentially affected by SSR4-CDG, which may provide further insights into the function of SSR4 and help clinicians better understand this disorder.

Tandem mass spectrometry coupled with liquid chromatography (LC-MS/MS) has proven a versatile tool for the identification and quantification of proteins and their post-translational modifications (PTMs). Protein glycosylation is a critical PTM for the stability and biological function of many proteins, but full characterization of site-specific glycosylation of proteins remains analytically challenging. Collision-induced dissociation (CID) is the most common fragmentation method used in LC-MS/MS workflows, but the loss of labile modifications renders CID inappropriate for detailed characterization of site-specific glycosylation. Electron-based dissociation methods provide alternatives that retain intact glycopeptide fragments for unambiguous site localization, but these methods often underperform CID due to increased reaction times and reduced efficiency. Electron-activated dissociation (EAD) is another strategy for glycopeptide fragmentation. Here, we use a ZenoTOF 7600 SCIEX instrument to compare the performance of various fragmentation techniques for the analysis of a complex mixture of mammalian O- and N-glycopeptides. We found CID fragmentation identified the most glycopeptides and generally produced higher quality spectra, but EAD provided improved confidence in glycosylation site localization. Supplementing EAD with CID fragmentation (EAciD) further increased the number and quality of glycopeptide identifications, while retaining localization confidence. These methods will be useful for glycoproteomics workflows for either optimal glycopeptide identification or characterization.

TREK2, a two-pore domain potassium channel, is recognized for its regulation by various stimuli, including lipids. While previous members of the TREK subfamily, TREK1 and TRAAK, have been investigated to elucidate their lipid affinity and selectivity, TREK2 has not been similarly studied in this regard. Our findings indicate that while TRAAK and TREK2 exhibit similarities in terms of electrostatics and share an overall structural resemblance, there are notable distinctions in their interaction with lipids. Specifically, SAPI(4,5)P2,1-stearoyl-2-arachidonoyl-sn-glycero-3-phospho-(1'-myo-inositol-4',5'-bisphosphate) exhibits a strong affinity for TREK2, surpassing that of dOPI(4,5)P2,1,2-dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol-4',5'-bisphosphate), which differs in its acyl chains. TREK2 displays lipid binding preferences not only for the headgroup of lipids but also toward the acyl chains. Functional studies draw a correlation for lipid binding affinity and activity of the channel. These findings provide important insight into elucidating the molecular prerequisites for specific lipid binding to TREK2 important for function.

Glycosylation is one of the most ubiquitous post-translational modifications. It affects the structure and function of peptides/proteins and consequently has a significant impact on various biological events. However, the structural complexity and heterogeneity of glycopeptides/proteins caused by the diversity of glycan structures and glycosylation sites complicates the detailed elucidation of glycan function and hampers their clinical applications. To address these challenges, chemical and/or enzyme-assisted synthesis methods have been developed to realize glycopeptides/proteins with well-defined glycan morphologies. In particular, N-glycans are expected to be useful for improving the solubility, in vivo half-life and aggregation of bioactive peptides/proteins that have had limited clinical applications so far due to their short duration of action in the blood and unsuitable physicochemical properties. Chemical glycosylation performed in a post-synthetic procedure can be used to facilitate the development of glycopeptide/protein analogues or mimetics that are superior to the original molecules in terms of physicochemical and pharmacokinetic properties. N-glycans are used to modify targets because they are highly biodegradable and biocompatible and have structures that already exist in the human body. On the practical side, from a quality control perspective, close attention should be paid to their structural homogeneity when they are to be applied to pharmaceuticals.

With implications in several medical conditions, N-linked glycosylation is one of the most important posttranslation modifications present in all living organisms. Due to their nontemplate synthesis, glycan structures are extraordinarily complex and require multiple analytical techniques for complete structural elucidation. Mass spectrometry is the most common way to investigate N-linked glycans; however, with techniques such as liquid-chromatography mass spectrometry, there is complete loss of spatial information. Mass spectrometry imaging is a transformative analytical technique that can visualize the spatial distribution of ions within a biological sample and has been shown to be a powerful tool to investigate N-linked glycosylation. This review covers the fundamentals of mass spectrometry imaging and N-linked glycosylation and highlights important findings of recent key studies aimed at expanding and improving the glycomics imaging field.

Wnt/FZD-mediated signaling pathways are activated in more than 90% of hepatocellular carcinoma (HCC) cell lines. As a well-known secretory glycoprotein, Wnt3 can interact with FZD receptors on the cell surface, thereby activating the Wnt/β-catenin signaling pathway. However, the N-glycosylation modification site of Wnt3 and the effect of this modification on the biological function of the protein are still unclear.

To investigate the effect of Wnt3 N-glycosylation on the biological function of HCC cells.

Site-directed mutagenesis was used to verify the Wnt3 N-glycosylation sites, actinomycin D treatment was used to detect the stability of Wnt3 after site-directed mutation, the binding of the N-glycosylation site-directed mutant Wnt3 to FZD7 was observed by laser confocal microscopy, and the effects of the N-glycosylation site-directed mutation of Wnt3 on the Wnt/β-catenin signaling pathway and the progression of HCC cells were detected by western blot and cell function experiments.

Wnt3 has two N-glycosylation-modified sites (Asn90 and Asn301); when a single site at amino acid 301 is mutated, the stability of Wnt3 is weakened; the binding ability of Wnt3 to FZD7 decreases when both sites are mutated simultaneously; and the level of proteins related to the Wnt/β-catenin signaling pathway is downregulated. Cell proliferation, migration and invasion are also weakened in the case of single 301 site and double-site mutations.

These results indicate that by inhibiting the N-glycosylation of Wnt3, the proliferation, migration, invasion and colony formation abilities of liver cancer cells can be weakened, which might provide new therapeutic strategies for clinical liver cancer in the future.

N-glycosylation is one of the most universal and complex protein post-translational modifications (PTMs), and it is involved in many physiological and pathological activities. Owing to the low abundance of N-glycoproteins, enrichment of N-glycopeptides for mass spectrometry analysis usually requires a large amount of peptides. Additionally, oocyte protein N-glycosylation has not been systemically characterized due to the limited sample amount. Here, we developed a glycosylation enrichment method based on lectin and a single-pot, solid-phase-enhanced sample preparation (SP3) technology, termed lectin-based SP3 technology (LectinSP3). LectinSP3 immobilized lectin on the SP3 beads for N-glycopeptide enrichment. It could identify over 1100 N-glycosylation sites and 600 N-glycoproteins from 10 μg of mouse testis peptides. Furthermore, using the LectinSP3 method, we characterized the N-glycoproteome of 1000 mouse oocytes in three replicates and identified a total of 363 N-glycosylation sites from 215 N-glycoproteins. Bioinformatics analysis revealed that these oocyte N-glycoproteins were mainly enriched in cell adhesion, fertilization, and sperm-egg recognition. Overall, the LectinSP3 method has all procedures performed in one tube, using magnetic beads. It is suitable for analysis of a low amount of samples and is expected to be easily adaptable for automation. In addition, our mouse oocyte protein N-glycosylation profiling could help further characterize the regulation of oocyte functions.