Protein N-glycosylation Research Articles

Mutant Calreticulin in MPN: Mechanistic Insights and Therapeutic Implications.

More than a decade following the discovery of Calreticulin (CALR) mutations as drivers of myeloproliferative neoplasms (MPN), advances in the understanding of CALR-mutant MPN continue to emerge. Here, we summarize recent advances in mehanistic understanding and in targeted therapies for CALR-mutant MPN. Structural insights revealed that the mutant CALR-MPL complex is a tetramer and the mutant CALR C-terminus is exposed on the cell surface. Targeting mutant CALR utilizing antibodies is the leading therapeutic approach, while mutant CALR-directed vaccines are also in early clinical trials. Additionally, chimeric antigen receptor (CAR) T-cells directed against mutant CALR are under evaluation in preclinical models. Approaches addressing the cellular effects of mutant CALR beyond MPL-JAK-STAT activation, such as targeting the unfolded protein response, proteasome, and N-glycosylation pathways, have been tested in preclinical models. In CALR-mutant MPN, the path from discovery to mechanistic understanding to direct therapeutic targeting has advanced rapidly. The longer-term goal remains clonally-selective therapies that modify the disease course in patients.

Suppression of Dad1 induces cardiomyocyte death by weakening cell adhesion.

As cardiomyocyte loss causes heart failure, inhibition of cardiomyocyte death may be a therapeutic strategy against heart failure. In this study, we have identified defender against cell death 1 (Dad1) as a candidate regulator of cardiomyocyte death, using complementary DNA microarray and siRNA knockdown screening. Dad1 is a subunit of oligosaccharyltransferase (OST) complex that is responsible for protein N-glycosylation; however, its function in cardiomyocytes remains unknown. Importantly, the knockdown of Dad1 using siRNA reduced the viability of neonatal rat cardiomyocytes (NRCMs), accompanied by cleaved caspase3 expression, independent of endoplasmic reticulum stress. Dad1 knockdown impaired cell spreading and reduced myofibrillogenesis in NRCMs, suggesting that Dad1 knockdown induced anoikis, apoptosis by disrupting cell-matrix interactions. Consistently, knockdown of Dad1 impaired N-glycosylation of integrins α5 and β1, accompanied by inactivation of focal adhesion kinase. When cell adhesion was enhanced using adhesamine, fibronectin, or collagen type IV, cardiomyocyte death induced by Dad1 knockdown was reduced. Dad1 knockdown decreased the expression of staurosporine and temperature-sensitive 3 A (Stt3A), a catalytic subunit of OST complex. Interestingly, Stt3A knockdown using Stt3A siRNA reduced the expression of Dad1, indicating that both Dad1 and Stt3A were required for OST stabilization. In conclusion, Dad1 plays an important role in maintaining the expression of mature N-glycosylated integrins and their downstream signaling molecules to suppress cardiomyocyte anoikis.NEW & NOTEWORTHY This study found for the first time that the knockdown of Dad1 induced cardiomyocyte death, accompanied by impairment of myofibrillogenesis and cell spreading. Dad1 regulates the N-glycosylation of integrins in cooperation with Stt3A and preserves cell adhesion activity, promoting cardiomyocyte survival. This is the first demonstration that Dad1 contributes to the maintenance of cardiac homeostasis through the posttranslational modification of integrins, providing a novel insight into the biological significance of OST complex in cardiomyocytes.

ALG13-Related Epilepsy: Current Insights and Future Research Directions.

The ALG13 gene encodes a subunit of the uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) transferase enzyme, which plays a key role in the N-linked glycosylation pathway. This pathway involves the attachment of carbohydrate structures to asparagine (Asn) residues in proteins within the endoplasmic reticulum, by which N-glycosylated proteins produced participate a wide range of processes such as electrical gradients formation and neurotransmission. Mutations in the ALG13 gene have been identified as a causative factor for congenital disorders of glycosylation (CDG) and have been frequently associated with epilepsy in affected individuals. Several studies have demonstrated a strong correlation between abnormal N-glycosylation due to ALG13 deficiency and the onset of epilepsy. Despite these findings, the precise role of ALG13 in the pathogenesis of epilepsy remains unclear. This review provides a comprehensive overview of the current literature on ALG13-related disorders, with a focus on recent evidence regarding its role in epilepsy development and progression. Future research directions are also proposed to further elucidate the molecular mechanisms underlying this association.

Spatial Organization of the Sperm Cell Glycoproteome.

Sperm cells are terminally differentiated cells that are essential for reproduction in sexually reproducing species. Consistent with their highly specialized function, sperm cells harbor a unique proteome containing many proteins not expressed in somatic cells. In contrast, the post-translational landscape of the sperm proteome remains largely unexplored, limiting our understanding of how modifications such as glycosylation impact sperm function and sperm-egg interactions. Here, we used glycopeptide-centric glycoproteomics to comprehensively characterize protein N-glycosylation in sperm from three mammalian species, revealing clear conservation of glycosylation profiles. We find that glycosylation patterns in sperm proteins are distinct from those in plasma, with as clear distinctive features less sialyation and more paucimannosylation in sperm. Moreover, based on their subcellular location, sperm protein glycosylation varies, with paucimannose species enriched in the acrosomal vesicle, oligomannose species in the sperm head membrane, and complex glycan species in the acrosomal membrane.

Identification of serum N-glycans signatures in three major gastrointestinal cancers by high-throughput N-glycome profiling

BackgroundAlternative N-glycosylation of serum proteins has been observed in colorectal cancer (CRC), esophageal squamous cell carcinoma (ESCC) and gastric cancer (GC), while comparative study among those three cancers has not been reported before. We aimed to identify serum N-glycans signatures and introduce a discriminative model across the gastrointestinal cancers.MethodsThe study population was initially screened according to the exclusion criteria process. Serum N-glycans profiling was characterized by a high-throughput assay based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Diagnostic model was built by random forest, and unsupervised machine learning was performed to illustrate the differentiation between the three major gastrointestinal (GI) cancers.ResultsWe have found that three major gastrointestinal cancers strongly associated with significantly decreased mannosylation and mono-galactosylation, as well as increased sialylation of serum glycoproteins. A highly accurate discriminative power (> 0.90) for those gastrointestinal cancers was obtained with serum N-glycome based predictive model. Additionally, serum N-glycome profile exhibited distinct distributions across GI cancers, and several altered N-glycans were hyper-regulated in each specific disease.ConclusionsSerum N-glycome profile was differentially expressed in three major gastrointestinal cancers, providing a new clinical tool for cancer diagnosis and throwing a light upon the disease-specific molecular signatures.

GlycoPCT: Pressure Cycling Technology-Based Quantitative Glycoproteomics Reveals Distinctive N-Glycosylation in Human Liver Biopsy Samples of Nonalcoholic Fatty Liver Disease.

Protein N-glycosylation is vital in the human liver and influences functions such as lipid metabolism, apoptosis, and inflammation. However, site-specific N-glycosylation patterns and variations in liver biopsy samples between healthy individuals and those with nonalcoholic fatty liver disease (NAFLD) remain incompletely characterized, primarily due to the limitations of current clinical glycoproteomic methods, including a large demand for clinical samples, low efficiency of tissue protein extraction, and a low recovery rate of intact N-glycopeptides (IGPs). To address this issue, we developed GlycoPCT, a quantitative glycoproteomic method based on pressure cycling technology. It enables efficient recovery of IGPs and accurate analysis of trace liver biopsy samples. Our research revealed a total of 4,459 unique IGPs and 361 glycans from 758 glycoproteins. High-mannose type, complex type, fucosylation type, and sialylation type N-glycans were significantly upregulated in the NAFLD group (p < 0.001, t test). Notably, we also identified 182 upregulated IGPs from 67 proteins (p < 0.05, FC > 1.50) and 108 downregulated IGPs from 44 proteins (p < 0.05, FC < 0.67) in the NAFLD group. Furthermore, we highlighted an essential acute phase glycoprotein, alpha-1-acid glycoprotein 1 (A1TA), which is synthesized in the liver and plays a significant role in NAFLD progression. These novel glyco-signatures provide crucial clues for the diagnosis and pathogenesis of NAFLD.

Processing of N-glycans in the ER and Golgi influences the production of surface sialylated glycoRNA.

Glycoconjugates, including glycans on proteins and lipids, have obtained significant attention due to their critical roles in both intracellular and intercellular biological functions and processes. Notably, recent discoveries have revealed the presence of glycosylated RNAs (glycoRNAs) on cell surfaces. Despite the well-characterized roles of RNA modifications, RNA glycosylation remains relatively unexplored. In this study, we investigate the relationship between N-glycosylation and RNA glycosylation. Using a recombinant Siglec11-Fc as a probe, we detected surface sialylated glycoRNAs in human cell lines and identified their dependency on the catalytic isoforms of the oligosaccharyltransferase (OST) complex, implicating STT3A-dependent protein glycosylation as a predominant contributor for affecting indirect generation of glycoRNAs. Additionally, perturbations in N-glycan biosynthesis pathways or changes in N-glycan structure impact surface sialylated glycoRNA levels, indicating a regulatory role of glycan metabolic pathways in RNA glycosylation. Together, our results underscore the intricate relationship between protein N-glycosylation and processing and RNA biology.

The Possible Roles of Glucosamine-6-Phosphate Deaminases in Ammonium Metabolism in Cancer.

Nearly 5% of the glucose-6-phosphate (Glc6P) in cells is diverted into the hexosamine biosynthetic pathway (HBP) to synthesize glucosamine-6-phosphate (GlcN6P) and uridine diphosphate N-acetyl-glucosamine-6-phosphate (UDP-GlcN6P). Fructose-6-phosphate (Fru6P) is a common intermediary between glycolysis and the HBP. Changes in HBP regulation cause abnormal protein N-glycosylation and O-linked-N-acetylglucosamine modification (O-GlcNAcylation), affecting protein function and modifying cellular responses to signals. The HBP enzymes glucosamine-6-phosphate deaminases 1 and 2 (GNPDA1 and 2) turn GlcN6P back into Fru6P and ammonium, and have been implicated in cancer and metabolic diseases. Despite the plentiful literature on this topic, the mechanisms involved are just beginning to be studied. In this review, we summarize, for the first time, the current knowledge regarding the possible roles of the isoenzymes of both GNPDAs in the pathogenesis and development of metabolic diseases and cancer from a molecular point of view, highlighting their importance not only in supplying carbon from glycolysis, but also in ammonia metabolism.

Role of N-Glycosylation in Gastrointestinal Cancers.

Gastrointestinal cancers pose a significant global health challenge. N-glycosylation modulates various cellular processes, including key cancer-related mechanisms. Elucidating its involvement in the onset and advancement of these cancers can offer critical insights for enhancing diagnostic and therapeutic approaches. This review outlines the core process of protein N-glycosylation and highlights its contribution to the progression of gastrointestinal cancers, encompassing cell proliferation, survival, invasion, metastasis, and immune evasion, mainly through its impact on critical signaling pathways. Notably, aberrant N-glycosylation patterns have emerged as crucial biomarkers for the diagnosis and prognosis of various gastrointestinal cancers, providing the foundation for more personalized therapeutic approaches. Therapeutic strategies targeting N-glycosylation, such as glycosyltransferase inhibitors and glycoengineering, show significant promise in mitigating tumor aggressiveness and enhancing immune recognition. However, the clinical implementation of N-glycosylation biomarkers requires the standardization of glycosylation analysis techniques and solutions to challenges in sample processing and data interpretation. Future research efforts should concentrate on overcoming these obstacles to unlock the full potential of N-glycosylation in enhancing cancer management and advancing patient outcomes.

Proteomic and N-glycosylation analysis of fertile egg white during storage and incubation in chickens

Proteins in egg whites play vital roles in embryonic development. Simultaneously, protein modification is affected by the surrounding environment, which ultimately affects the structure and function of proteins. Here, we measured the phenotypes of eggs at different time points during storage and incubation and used 4D label-free quantitative proteomics technology and liquid chromatography/tandem mass spectrometry (LC-MS/MS)-technique to identify the differential proteins and N-glycosylation sites in egg whites during storage and incubation. We found that the differential N-glycoproteins in the early stage of storage were mainly related to protein structure changes, antibacterial activity, and cell proliferation, and that there were more protease inhibitors in egg whites, which decreased in the later stage of storage. Finally, eleven possible protein markers and N-glycosylation sites were identified to significantly change during storage and may exert an effect on hatchability, including the proteins involved in antibacterial activity (OVOA-N855, CLU-N154, ogchi-N82, PIGR-N290, WFDC2-N120), protein structure (LOC776816), and cell proliferation (ASAH1-N173). This study provides substantial insights into the physical and molecular compositional changes in egg whites under different storage times and revealed their potential effect on chick embryo development.

Chemical glycoproteomic profiling in rice seedlings reveals N-glycosylation in the ERAD-L machinery

As a ubiquitous and essential posttranslational modification occurring in both plants and animals, protein N-linked glycosylation regulates various important biological processes. Unlike the well-studied animal N-glycoproteomes, the landscape of rice N-glycoproteome remains largely unexplored. Here, by developing a chemical glycoproteomic strategy based on metabolic glycan labeling (MGL), we report a comprehensive profiling of the N-glycoproteome in rice seedlings. The rice seedlings are incubated with N-azidoacetylgalactosamine (GalNAz) - a monosaccharide analog containing a bioorthogonal functional group - to metabolically label N-glycans, followed by conjugation with an affinity probe via click chemistry for enrichment of the N-glycoproteins. Subsequent mass spectrometry analyses identify a total of 403 N-glycosylation sites and 673 N-glycosylated proteins, which are involved in various important biological processes. In particular, the core components of the endoplasmic reticulum (ER)-associated protein degradation (ERAD) machinery are N-glycosylated, and the N-glycosylation is important for the ERAD-L function. This work not only provides an invaluable resource for studying rice N-glycosylation, but also demonstrates the applicability of MGL in glycoproteomic profiling for crop species.

Comparison of Whey N-Glycoproteins in Yak Colostrum and Mature Milk Based on 4D Label-Free Technology.

Whey proteins are N-glycosylated proteins that play important roles in a variety of biological processes including immune defense. However, the N-glycosylation of yak colostrum (YC) and mature milk (YM) whey proteins is unknown. Therefore, this study systematically compared and analyzed YC and YM whey N-glycoproteomes using the 4D label-free technique. We identified 162 glycoproteins, 222 glycosylated peptides, and 234 glycosylation sites in YC and YM, of which 59 glycoproteins were differentially expressed in YC and YM. According to gene ontology annotation and KEGG pathway metabolism analysis, the differentially expressed N-glycoproteins were highly enriched in "cell adhesion", "extracellular region", and "calcium binding", and were mainly involved in the extracellular matrix (ECM)-receptor interaction pathway. The immunity-related N-glycoproteins, such as platelet glycoprotein 4 (CD36) and polymeric immunoglobulin receptor (PIGR), were observed to be different between YC and YM. The results revealed the glycosylation sites, composition, and biological functions of YC and YM whey N-glycoprotein, which supplemented our understanding of the N-glycosylation of yak whey proteins.

Oriental covalent immobilization of N-glycan binding protein via N-terminal selective modification

Lectin affinity chromatography is one of powerful tools for the study of protein glycosylation. Different lectin proteins can recognize different structures of monosaccharides or oligosaccharide units, allowing for the selective separation of glycopeptides or glycoproteins containing different polysaccharide structures. However, the N-glycans were only partially captured by most of common lectins, reducing the coverage rate of identifying N-glycoconjugates. Recently, it has been reported that the engineering variant of glycan binding protein Fbs1 has a high affinity for innermost Man3GlcNAc2 structure and is able to bind diverse types of N-glycans, which can be suitable for the analysis of protein N-glycosylation. However, efficient immobilization of protein to separation matrix is particularly challenging as it requires the functionality and integrity of the protein to be preserved. Herein, we describe a simple and robust strategy for oriental covalent immobilization of proteins on magnetic nanoparticles by N-terminal selective labeling techniques. We inserted the enterokinase cleavage site to produce the specific N- terminal glycine of protein. Under physiological conditions, the protein was immobilized on the surface of the MNPs by this glycine tag, and the enrichment process could be completed within 30 min. A whole enrichment and purification of glycan and glycopeptides were completed and analyzed by MALDI TOF-MS. The functional materials achieved stable enrichment of glycan structure in different enzyme digestion systems or complex samples, showing excellent anti-interference and applicability.

Systemic gene therapy corrects the neurological phenotype in a mouse model of NGLY1 deficiency.

The cytoplasmic peptide:N-glycanase (NGLY1) is ubiquitously expressed and functions as a de-N-glycosylating enzyme that degrades misfolded N-glycosylated proteins. NGLY1 deficiency due to biallelic loss-of-function NGLY1 variants is an ultrarare autosomal recessive deglycosylation disorder with multisystemic involvement; the neurological manifestations represent the main disease burden. Currently, there is no treatment for this disease. To develop a gene therapy, we first characterized a tamoxifen-inducible Ngly1-knockout (iNgly1) C57BL/6J mouse model, which exhibited symptoms recapitulating human disease, including elevation of the biomarker GlcNAc-Asn, motor deficits, kyphosis, Purkinje cell loss, and gait abnormalities. We packaged a codon-optimized human NGLY1 transgene cassette into 2 adeno-associated virus (AAV) capsids, AAV9 and AAV.PHPeB. Systemic administration of the AAV.PHPeB vector to symptomatic iNgly1 mice corrected multiple disease features at 8 weeks after treatment. Furthermore, another cohort of AAV.PHPeB-treated iNgly1 mice were monitored over a year and showed near-complete normalization of the neurological aspects of the disease phenotype, demonstrating the durability of gene therapy. Our data suggested that brain-directed NGLY1 gene replacement via systemic delivery is a promising therapeutic strategy for NGLY1 deficiency. Although the superior CNS tropism of AAV.PHPeB vector does not translate to primates, emerging AAV capsids with enhanced primate CNS tropism will enable future translational studies.

Molecular mechanism of infectious hematopoietic necrosis G protein N-glycosylation escaping from rainbow trout immunity

Infectious Hematopoietic Necrosis (IHN) is a viral disease that causes great economic losses in salmonid fish farming industry. It is known that the N-glycosylation site on the G protein affects the immune evasion and pathogenicity of the Infectious Hematopoietic Necrosis Virus (IHNV). In this study, a mutation in the N-glycosylation site of the G protein (438 amino acids) of IHNV resulted in an increased the survival rate of rainbow trout from 17.1 % to 75.3 %, and a decreased serum antibody IgM level after virus infection. In addition, this mutation decreased the expression of CD83 and CD209 on dendritic cells and enhanced the binding of G protein to TCR. This suggests that the activation of CD4+ T-cells was enhanced. We hypothesize that IHNV evades the host's immune system by impairing antigen presentation and T-cell responses through the N-glycosylation of G protein. This finding could provide new insights for developing live attenuated vaccines.

Physiologically Achievable Concentration of 2-Deoxy-D-Glucose Stimulates IFN-γ Secretion in Activated T Cells In Vitro.

2-deoxy-D-glucose (2DG) is a glycolysis and protein N-glycosylation inhibitor with promising anti-tumor and immunomodulatory effects. However, 2DG can also suppress T cell function, including IFN-γ secretion. Few human T cell studies have studied low-dose 2DG, which can increase IFN-γ in a Jurkat clone. We therefore investigated 2DG's effect on IFN-γ in activated human T cells from PBMCs, with 2DG treatment commenced either concurrently with activation or 48 h after activation. Concurrent 2DG treatment decreased IFN-γ secretion in a dose-dependent manner. However, 2DG treatment of pre-activated T cells had a hormetic effect on IFN-γ, with 0.15-0.6 mM 2DG (achievable in vivo) increasing and >2.4 mM 2DG reducing its secretion. In contrast, IL-2 levels declined monotonously with increasing 2DG concentration. Lower 2DG concentrations reduced PD-1 and increased CD69 expression regardless of treatment timing. The absence of increased T-bet or Eomes expression or IFNG transcription suggests another downstream mechanism. 2DG dose-dependently induced the unfolded protein response, suggesting a possible role in increased IFN-γ secretion, possibly by increasing the ER folding capacity for IFN-γ via increased chaperone expression. Overall, low-dose, short-term 2DG exposure could potentially improve the T cell anti-tumor response.

Exploring protein N-glycosylation in the green microalga Dunaliella salina

N-glycosylation is a major post-translational modification of proteins that has a crucial influence on cell targeting, activity, and half-life. This process starts in the endoplasmic reticulum where an oligosaccharide precursor is added to the newly synthesized protein and continues in the Golgi apparatus where the N-linked carbohydrate sequences are processed. Importantly, the most approved recombinant pharmaceutical proteins (so-called biologics) are glycoproteins mainly currently produced in mammalian cells which is a lengthy, costly, and complex process. Today, several microalgae such as the diatom Phaeodactylum tricornutum, and the green microalgae Chlamydomonas reinhardtii, Chlorella vulgaris, and Dunaliella salina are considered as efficient and eco-friendly alternative platforms for the production of biologics. However, unlike for C. reinhardtii, C. vulgaris, and P. tricornutum, there is to date no data reported regarding the protein N-glycosylation pathway in D. salina. Here, we first investigated the protein N-glycosylation in this green microalga by MALDI-TOF mass spectrometry. These analyses showed that proteins from D. salina are N-glycosylated with Man5GlcNAc2 oligomannoside. Using genome mining approaches, we then identified genes encoding proteins involved in the N-glycosylation pathways in D. salina. Genetic similarities and phylogenetic relationships of the putative sequences with homologues from C. reinhardtii, P. tricornutum, and humans were investigated. These data revealed that in D. salina the biosynthesis of nucleotide sugars and N-glycan biosynthesis share mainly similarities with the GnT I-independent pathway of C. reinhardtii that gives rise to the synthesis of a non-canonical oligomannoside Man5GlcNAc2. Although an α(1,3)-fucosyltransferase is identified in the D. salina genome, impairment of the cytosolic GDP-Fuc biosynthesis prevents the Golgi fucosylation of N-glycans. Taken together, these data demonstrated that proteins from D. salina are homogeneously N-glycosylated with a non-canonical Man5GlcNAc2.

N-Glycosylation-Induced Pathologic Protein Conformations as a Tool to Guide the Selection of Biologically Active Small Molecules.

Post-translational modifications such as protein N-glycosylation, significantly influence cellular processes. Dysregulated N-glycosylation, exemplified in Grp94, a member of the Hsp90 family, leads to structural changes and the formation of epichaperomes, contributing to pathologies. Targeting N-glycosylation-induced conformations offers opportunities for developing selective chemical tools and drugs for these pathologic forms of chaperones. We here demonstrate how a specific Grp94 conformation induced by N-glycosylation, identified previously via molecular dynamics simulations, rationalizes the distinct behavior of similar ligands. Integrating dynamic ligand unbinding information with SAR development, we differentiate ligands productively engaging the pathologic Grp94 conformers from those that are not. Additionally, analyzing binding site stereoelectronic properties and QSAR models using cytotoxicity data unveils relationships between chemical, conformational properties, and biological activities. These findings facilitate the design of ligands targeting specific Grp94 conformations induced by abnormal glycosylation, selectively disrupting pathogenic protein networks while sparing normal mechanisms.

Research Note: Comprehensive proteomic, phosphoproteomic, and N-glycoproteomic analysis of chicken egg yolk plasma

Chicken egg yolk plasma (EYP), the supernatant fraction of egg yolk obtained by water dilution and centrifugation, is a rich source of various bioactive substances and a significant bearer of yolk-emulsifying properties. This study utilized proteomics to conduct a comprehensive and in-depth analysis of both common and modified EYP proteins (phosphorylated proteins and N-glycosylated proteins). Total of 208 proteins were identified in EYP, including 42 phosphorylated proteins with 137 phosphorylation sites and 150 N-glycoproteins with 332 N-glycosylation sites. Among the phosphorylation sites, tyrosine accounted for 80.6%, while the N-glycosylation sites predominantly featured "N-X-T" motifs, accounting for 58.7%. Functional enrichment analysis revealed that most proteins were involved in regulating enzyme activity and inhibition with a particular focus on modulating peptidase activity. Notably, vitellogenins-2 (30 phosphorylation sites, 9 N-glycosylation sites) and apolipoprotein B (10 phosphorylation sites, 56 N-glycosylation sites) were the 2 proteins with the most modification sites. Additionally, EYP was found to contain the highly N-glycosylated complement proteins C3 and C4. These findings provide new insights into the protein composition of EYP and its roles in chicken embryo development and immune defense, offering a theoretical foundation for the application of EYP in various fields.

Consistent signatures in the human gut microbiome of longevous populations

ABSTRACT Gut microbiota of centenarians has garnered significant attention in recent years, with most studies concentrating on the analysis of microbial composition. However, there is still limited knowledge regarding the consistent signatures of specific species and their biological functions, as well as the potential causal relationship between gut microbiota and longevity. To address this, we performed the fecal metagenomic analysis of eight longevous populations at the species and functional level, and employed the Mendelian randomization (MR) analysis to infer the causal associations between microbial taxa and longevity-related traits. We observed that several species including Eisenbergiella tayi, Methanobrevibacter smithii, Hungatella hathewayi, and Desulfovibrio fairfieldensis were consistently enriched in the gut microbiota of long-lived individuals compared to younger elderly and young adults across multiple cohorts. Analysis of microbial pathways and enzymes indicated that E. tayi plays a role in the protein N-glycosylation, while M. smithii is involved in the 3-dehydroquinate and chorismate biosynthesis. Furthermore, H. hathewayi makes a distinct contribution to the purine nucleobase degradation I pathway, potentially assisting the elderly in maintaining purine homeostasis. D. fairfieldensis contributes to the menaquinone (vitamin K2) biosynthesis, which may help prevent age-related diseases such as osteoporosis-induced fractures. According to MR results, Hungatella was significantly positively correlated with parental longevity, and Desulfovibrio also exhibited positive associations with lifespan and multiple traits related to parental longevity. Additionally, Alistipes and Akkermansia muciniphila were consistently enriched in the gut microbiota of the three largest cohorts of long-lived individuals, and MR analysis also suggests their potential causal relationships with longevity. Our findings reveal longevity-associated gut microbial signatures, which are informative for understanding the role of microbiota in regulating longevity and aging.