Role Of Sialylation Research Articles

Neisseria gonorrhoeae scavenges host sialic acid for Siglec-mediated, complement-independent suppression of neutrophil activation.

Neisseria gonorrhoeae, the bacterium that causes gonorrhea, is an urgent global health concern due to increasing infection rates, widespread antibiotic resistance, and its ability to thwart protective immune responses. The mechanisms by which Gc subverts protective immune responses remain poorly characterized. One way N. gonorrhoeae evades human immunity is by adding sialic acid that is scavenged from the host onto its lipooligosaccharide, using the sialyltransferase Lst. Here, we found that sialylation enhances N. gonorrhoeae survival from neutrophil assault and inhibits neutrophil activation, independently of the complement system. Our results implicate bacterial binding of sialic acid-binding lectins (Siglecs) on the neutrophil surface, which dampens neutrophil antimicrobial responses. This work identifies a new role for sialylation in protecting N. gonorrhoeae from cellular innate immunity, which can be targeted to enhance the human immune response in gonorrhea.

The role of sialylation in gynecologic cancers.

Sialic acids (SA) are a kind of nine-carbon backbone sugars, serving as important molecules in cell-to-cell or cell-to-extra-cellular matrix interaction mediated by either O-linked glycosylation or N-linked glycosylation to attach the terminal end of glycans, glycoproteins, and glycolipids. All processes need a balance between sialylation by sialyltransferase (STs) and desialylation by sialidases (also known as neuraminidases, NEU). Although there is much in uncertainty whether the sialyation plays in cancer development and progression, at least four mechanisms are proposed, including surveillance of immune system, modification of cellular apoptosis and cell death, alteration of cellular surface of cancer cells and tumor associated microenvironment responsible carcinogenesis, growth and metastases. The current review focuses on the role of glycosylation in gynecologic organ-related cancers, such as ovarian cancer, cervical and endometrial cancer. Evidence shows that sialylation involving in the alternation of surface components of cells (tumor and cells in the microenvironment of host) plays an important role for carcinogenesis (escape from immunosurveillance) and dissemination (metastasis) (sloughing from the original site of cancer, migration into the circulation system, extravasation from the circulatory system to the distant site and finally deposition and establishment on the new growth lesion to complete the metastatic process). Additionally, modification of glycosylation can enhance or alleviate the aggressive characteristics of the cancer behaviors. All suggest that more understandings of glycosylation on cancers may provide a new therapeutic field to assist the cancer treatment in the near future.

Sialylation: A Cloak for Tumors to Trick the Immune System in the Microenvironment.

The tumor microenvironment (TME), where the tumor cells incite the surrounding normal cells to create an immune suppressive environment, reduces the effectiveness of immune responses during cancer development. Sialylation, a type of glycosylation that occurs on cell surface proteins, lipids, and glycoRNAs, is known to accumulate in tumors and acts as a "cloak" to help tumor cells evade immunological surveillance. In the last few years, the role of sialylation in tumor proliferation and metastasis has become increasingly evident. With the advent of single-cell and spatial sequencing technologies, more research is being conducted to understand the effects of sialylation on immunity regulation. This review provides updated insights into recent research on the function of sialylation in tumor biology and summarizes the latest developments in sialylation-targeted tumor therapeutics, including antibody-mediated and metabolic-based sialylation inhibition, as well as interference with sialic acid-Siglec interaction.

Targeting stromal cell sialylation reverses T cell-mediated immunosuppression in the tumor microenvironment

Immunosuppressive tumor microenvironments (TMEs) reduce the effectiveness of immune responses in cancer. Mesenchymal stromal cells (MSCs), precursors to cancer-associated fibroblasts (CAFs), promote tumor progression by enhancing immune cell suppression in colorectal cancer (CRC). Hyper-sialylation of glycans promotes immune evasion in cancer through binding of sialic acids to their receptors, Siglecs, expressed on immune cells, which results in inhibition of effector functions. The role of sialylation in shaping MSC/CAF immunosuppression in the TME is not well characterized. In this study, we show that tumor-conditioned stromal cells have increased sialyltransferase expression, α2,3/6-linked sialic acid, and Siglec ligands. Tumor-conditioned stromal cells and CAFs induce exhausted immunomodulatory CD8+ PD1+ and CD8+ Siglec-7+/Siglec-9+ Tcell phenotypes. Invivo, targeting stromal cell sialylation reverses stromal cell-mediated immunosuppression, as shown by infiltration of CD25 and granzyme B-expressing CD8+ Tcells in the tumor and draining lymph node. Targeting stromal cell sialylation may overcome immunosuppression in the CRC TME.

Insights into the Role of Sialylation in Cancer Metastasis, Immunity, and Therapeutic Opportunity.

Sialylation is an enzymatic process that covalently attaches sialic acids to glycoproteins and glycolipids and terminates them by creating sialic acid-containing glycans (sialoglycans). Sialoglycans, usually located in the outmost layers of cells, play crucial biological roles, notably in tumor transformation, growth, metastasis, and immune evasion. Thus, a deeper comprehension of sialylation in cancer will help to facilitate the development of innovative cancer therapies. Cancer sialylation-related articles have consistently increased over the last four years. The primary subjects of these studies are sialylation, cancer, immunotherapy, and metastasis. Tumor cells activate endothelial cells and metastasize to distant organs in part by the interactions of abnormally sialylated integrins with selectins. Furthermore, cancer sialylation masks tumor antigenic epitopes and induces an immunosuppressive environment, allowing cancer cells to escape immune monitoring. Cytotoxic T lymphocytes develop different recognition epitopes for glycosylated and nonglycosylated peptides. Therefore, targeting tumor-derived sialoglycans is a promising approach to cancer treatments for limiting the dissemination of tumor cells, revealing immunogenic tumor antigens, and boosting anti-cancer immunity. Exploring the exact tumor sialoglycans may facilitate the identification of new glycan targets, paving the way for the development of customized cancer treatments.

A Multiscale Molecular Dynamic Analysis Reveals the Effect of Sialylation on EGFR Clustering in a CRISPR/Cas9-Derived Model.

Bacterial and viral pathogens can modulate the glycosylation of key host proteins to facilitate pathogenesis by using various glycosidases, particularly sialidases. Epidermal growth factor receptor (EGFR) signaling is activated by ligand-induced receptor dimerization and oligomerization. Ligand binding induces conformational changes in EGFR, leading to clusters and aggregation. However, information on the relevance of EGFR clustering in the pattern of glycosylation during bacterial and viral invasion remains unclear. In this study, (1) we established CRISPR/Cas9-mediated GFP knock-in (EGFP-KI) HeLa cells expressing fluorescently tagged EGFR at close to endogenous levels to study EGF-induced EGFR clustering and molecular dynamics; (2) We studied the effect of sialylation on EGF-induced EGFR clustering and localization in live cells using a high content analysis platform and raster image correlation spectroscopy (RICS) coupled with a number and brightness (N&B) analysis; (3) Our data reveal that the removal of cell surface sialic acids by sialidase treatment significantly decreases EGF receptor clustering with reduced fluorescence intensity, number, and area of EGFR-GFP clusters per cell upon EGF stimulation. Sialylation appears to mediate EGF-induced EGFR clustering as demonstrated by the change of EGFR-GFP clusters in the diffusion coefficient and molecular brightness, providing new insights into the role of sialylation in EGF-induced EGFR activation; and (4) We envision that the combination of CRISPR/Cas9-mediated fluorescent tagging of endogenous proteins and fluorescence imaging techniques can be the method of choice for studying the molecular dynamics and interactions of proteins in live cells.

Sialylation of CD11b/CD18 regulates Neutrophil Transepithelial Migration and Neutrophil Inflammatory function in the Intestine

Neutrophils (PMNs) are the first immune responders to infection/inflammation playing critical roles in clearing invading microbes and promoting tissue repair. However, dysregulated trafficking of PMNs across mucosal surfaces is a pathological hallmark of diseases characterized by persistent or intermittent bursts of inflammation including inflammatory bowel disease (IBD). The critical final step in PMN trafficking into mucosal lined organs (including the gut) involves transepithelial migration (TEpM). The glycoprotein CD11b/CD18 is the predominant β2 integrin mediating PMN TEpM into the intestinal lumen. Furthermore, CD11b/CD18 also regulates key PMN inflammatory effector functions that are implicated in IBD pathogenesis including superoxide release. We have previously demonstrated that specific GlcNAc and Lewis X glycan structures on CD11b/CD18 can be targeted to reduce PMN intestinal trafficking. Recent mass spectrometry analyses revealed the presence of specific sialylated glycan structures on PMN CD11b. However the role of sialylation in regulating PMN CD11b/CD18 function is not well understood. Using lectins recognizing the most common linkages of sialic acid we show that sialidase mediated removal of α2‐3 sialic acid from CD11b/CD18 occurs during PMN migration across intestinal epithelia. Furthermore, specific inhibition of sialidase activity was found to reduce PMN intestinal migration in vitro and in vivo. In addition to limiting PMN intestinal trafficking, sialidase inhibition also reduced key PMN effector functions including degranulation and superoxide release. Finally, we demonstrate that sialidase inhibition impedes PMN CD11b activation and blocks downstream β2 integrin signaling mediated by Spleen tyrosine kinase (Syk). Therefore α2‐3 sialylated glycans on CD11b/CD18 represent novel targets for ameliorating PMN mediated intestinal tissue damage and reducing mucosal inflammation in IBD.

Involvement of N-acetylneuraminate cytidylyltransferase in Edwardsiella piscicida pathogenicity

Edwardsiella piscicida is a gram-negative bacterium that causes Edwardsiellosis in cultured fish. Edwardsiellosis is accompanied by symptoms such as skin lesions, hemorrhage, and necrosis in fish organs, which leads to significant economic losses in the aquaculture industry. Recently, we found that bacterial sialoglycoconjugates may be involved in the infectivity of E. piscicida. The more infectious strains of E. piscicida contain more sialic acid in the bacterial body, and the mRNA level of putative CMP-Neu5Ac synthase (css) is upregulated compared to that in the non-pathogenic strain. However, this putative css gene is yet to be cloned, and the involvement of CSS in E. piscicida pathogenicity remains unclear. Here, we cloned and transferred the css gene from E. piscicida into the FPC498 strain. CSS promoted infection in cultured cells originating from different fish species, and enhanced the mortality of E. piscicida-infected zebrafish larvae. CSS enhanced cell attachment and motility in E. piscicida, which differs from the decreased bacterial growth observed with the sialic acid-supplemented M9 medium. Both fractions (chloroform-methanol)-soluble and -insoluble fraction) prepared from E. piscicida pellet exhibited the increment of sialo-conjugates induced by CSS. Further, lectin blotting revealed the increment of Sia α2-3- and α2-6-, but not α2-8-, -linked glycoprotein in CSS-overexpressing E. piscicida. Overall, these findings indicate the physiological significance of CSS and the role of sialylation in E. piscicida pathogenicity.

Distinct N-Linked Immunoglobulin G Glycosylation Patterns Are Associated With Chronic Pathology and Asymptomatic Infections in Human Lymphatic Filariasis.

Lymphatic filariasis presents a complex spectrum of clinical manifestations ranging from asymptomatic microfilariaemic (MF+) to chronic pathology (CP), including lymphedema and elephantiasis. Emerging evidence suggests a link between the physiopathology of filarial infections and antibody properties. Post-translational glycosylation has been shown to play a key role in the modulation of antibodies’ effector functions. Here, we investigated the link between total IgG-N-glycosylation patterns and the physiopathology of human lymphatic filariasis using UPLC-FLD/ESI-MS comparison of N-glycan profiles of total IgG purified from endemic normals (EN), MF+, and CP patients. We detected a total of 19 glycans released from all IgG samples. Strikingly, agalactosylated glycan residues were more prominent in EN, whereas sialylation and bisecting GlcNac correlated with asymptomatic infections. While IgG from all three clinical groups expressed high levels of fucosylated residues, significantly lower expressions of afucosylated IgG were seen in MF+ individuals compared to EN and CP. Our data reveal distinct N-linked IgG glycan profiles in EN, MF+, and CP and suggest that IgG galactosylation and sialylation are associated with chronic pathology, whereas agalactosylation correlates with putative immunity. The results also indicate a role for sialylation, fucosylation, and bisecting GlcNac in immune tolerance to the parasite. These findings highlight the link between N-glycosylation and the physiopathology of lymphatic filariasis and open new research avenues for next-generation therapeutic formulations against infectious diseases.

Genome wide CRISPR screening reveals a role for sialylation in the tumorigenesis and chemoresistance of acute myeloid leukemia cells.

Aberrant activation of cytokine and growth factor signal transduction pathways confers enhanced survival and proliferation properties to acute myeloid leukemia (AML) cells. However, the mechanisms underlying the deregulation of signaling pathways in leukemia cells are unclear. To identify genes capable of independently supporting cytokine-independent growth, we employed a genome-wide CRISPR/Cas9-mediated loss-of-function screen in GM-CSF-dependent human AML TF-1cells. More than 182 genes (p<0.01) were found to suppress the cytokine-independent growth of TF-1cells. Among the top hits, genes encoding key factors involved in sialylation biosynthesis were identified; these included CMAS, SLC35A1, NANS, and GNE. Knockout of either CMAS or SLC35A1 enabled cytokine-independent proliferation and survival of AML cells. Furthermore, NSG (NOD/SCID/IL2Rγ-/-) mice injected with CMAS or SLC35A1-knockout TF-1cells exhibited a shorter survival than mice injected with wild-type cells. Mechanistically, abrogation of sialylation biosynthesis in TF-1cells induced a strong activation of ERK signaling, which sensitized cells to MEK inhibitors but conferred resistance to JAK inhibitors. Further, the surface level of α2,3-linked sialic acids was negatively correlated with the sensitivity of AML cell lines to MEK/ERK inhibitors. We also found that sialylation modulated the expression and stability of the CSF2 receptor. Together, these results demonstrate a novel role of sialylation in regulating oncogenic transformation and drug resistance development in leukemia. We propose that altered sialylation could serve as a biomarker for targeted anti-leukemic therapy.

The Paired Siglecs in Brain Tumours Therapy: The Immunomodulatory Effect of Dexamethasone and Temozolomide in Human Glioma In Vitro Model.

The paired sialic acid-binding immunoglobulin like lectins (Siglecs) are characterized by similar cellular distribution and ligand recognition but opposing signalling functions attributed to different intracellular sequences. Since sialic acid—Siglec axis are known to control immune homeostasis, the imbalance between activatory and inhibitory mechanisms of glycan-dependent immune control is considered to promote pathology. The role of sialylation in cancer is described, however, its importance in immune regulation in gliomas is not fully understood. The experimental and clinical observation suggest that dexamethasone (Dex) and temozolomide (TMZ), used in the glioma management, alter the immunity within the tumour microenvironment. Using glioma-microglia/monocytes transwell co-cultures, we investigated modulatory action of Dex/TMZ on paired Siglecs. Based on real-time PCR and flow cytometry, we found changes in SIGLEC genes and their products. These effects were accompanied by altered cytokine profile and immune cells phenotype switching measured by arginases expression. Additionally, the exposure to Dex or TMZ increased the binding of inhibitory Siglec-5 and Siglec-11 fusion proteins to glioma cells. Our study suggests that the therapy-induced modulation of the interplay between sialoglycans and paired Siglecs, dependently on patient’s phenotype, is of particular signification in the immune surveillance in the glioma management and may be useful in glioma patient’s therapy plan verification.

The Role of Sialylation in Respiratory Viral Infection and Treatment.

Respiratory infections caused by viruses such as influenza and coronavirus are a serious global problem due to their high infection rates and potential to spark pandemics, such as the current COVID-19 pandemic. Although preventing these infections by using vaccines has been the most successful strategy to date, effective vaccines are not always available. Therefore, developing broad-spectrum anti-viral drugs to treat such infections is essential, especially in the case of immunocompromised patients or for outbreaks of novel virus strains. Sialic acids have been highlighted as a key molecule in the viral infection cycle, with terminally sialylated glycans acting as a target for several viral proteins involved in infection, particularly respiratory infection. Inhibitors of one such protein, neuraminidase, are the only anti-influenza drugs currently on the market. Problems with neuraminidase inhibitors, including the development of resistance and a relatively narrow spectrum of activity, drive the need for an improved understanding of the viral infection cycle and the development of more resilient, broader-spectrum anti-viral treatments. Hence, this review outlines the various roles played by sialic acids in respiratory viral infection and provides examples of drugs that exploit sialic acids to inhibit viral infections. It has been concluded that drugs targeting host cell expression of sialic acid could be especially well suited to inhibiting a broad spectrum of respiratory infections. This warrants the continued design and improvement of such drugs in an attempt to lessen the burden of respiratory infections.

Slc35a1 deficiency causes thrombocytopenia due to impaired megakaryocytopoiesis and excessive platelet clearance in the liver.

Sialic acid is a common terminal residue of glycans on proteins and acidic sphingolipids such as gangliosides and has important biological functions. The sialylation process is controlled by more than 20 different sialyltransferases, many of which exhibit overlapping functions. Thus, it is difficult to determine the overall biological function of sialylation by targeted deletion of individual sialyltransferases. To address this issue, we established a mouse line with the Slc35a1 gene flanked by loxP sites. Slc35a1 encodes the cytidine-5’-monophosphate (CMP)-sialic acid transporter that transports CMP-sialic acid from the cytoplasm into the Golgi apparatus for sialylation. Here we report our study regarding the role of sialylation on megakaryocytes and platelets using a mouse line with significantly reduced sialylation in megakaryocytes and platelets (Plt Slc35a1– /–). The major phenotype of Plt Slc35a1–/– mice was thrombocytopenia. The number of bone marrow megakaryocytes in Plt Slc35a1–/– mice was reduced, and megakaryocyte maturation was also impaired. In addition, an increased number of desialylated platelets was cleared by Küpffer cells in the liver of Plt Slc35a1–/– mice. This study provides new insights into the role of sialylation in platelet homeostasis and the mechanisms of thrombocytopenia in diseases associated with platelet desialylation, such as immune thrombocytopenia and a rare congenital disorder of glycosylation (CDG), SLC35A1-CDG, which is caused by SLC35A1 mutations.

Role of sialylation in prion disease pathogenesis and prion structure.

Mammalian prion or PrPSc is a proteinaceous infectious agent that consists of a misfolded, self-replicating state of a sialoglycoprotein called the prion protein or PrPC. Sialylation of the prion protein, a terminal modification of N-linked glycans, was discovered more than 30 years ago, yet the role of sialylation in prion pathogenesis is not well understood. This chapter summarizes current knowledge on the role of sialylation of the prion protein in prion diseases. First, we discuss recent data suggesting that sialylation of PrPSc N-linked glycans determines the fate of prion infection in an organism and control prion lymphotropism. Second, emerging evidence pointing out at the role N-glycans in neuroinflammation are discussed. Thirds, this chapter reviews a mechanism postulating that sialylated N-linked glycans are important players in defining strain-specific structures. A new hypothesis according to which individual strain-specific PrPSc structures govern selection of PrPC sialoglycoforms is discussed. Finally, this chapter explain how N-glycan sialylation control the prion replication and strain interference. In summary, comprehensive review of our knowledge on N-linked glycans and their sialylation provided in this chapter helps to answer important questions of prion biology that have been puzzling for years.

Exploiting the molecular mimicry of Non-typeable Haemophilus influenzae with ketodeoxynonulosonic acid: Confusing the bacterium in its own game

Abstract The distinctly human mucosal commensal and opportunistic pathogen non-typeable Haemophilus influenzae (NTHi) is frequently responsible for acute exacerbations of chronic obstructive pulmonary disease (COPD) in adults, and causes otitis media in infants. A key tool in this organism’s pathogenic arsenal is its ability to mimic glycans on human cells, which like all vertebrate cells, display diverse glycans with terminal 9-carbon-backbone monosaccharides called sialic acids. NTHi pathogenesis is related to its remarkably efficient uptake of trace amounts of free host N-acetylneuraminic acid (Neu5Ac, the primary sialic acid in humans) allowing “cloaking” of surface glycans of lipooligosaccharides (LOS) with this monosaccharide. This helps evasion from host immune clearance by “self-mimicry”, while facilitating colonization and biofilm formation. Since the mechanism of NTHi sialic acid uptake evolved by convergent evolution and is not selective for Neu5Ac, we propose to exploit this mimicry using another sialic acid called 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (Kdn). While NTHi growth was unaffected by Kdn in media, we found glycosidically-bound Kdn in LOS, which made NTHi sensitive to human sera and whole blood killing. The role of sialylation in vivo was further explored using a novel mouse model, with a human-like sialic acid profile. This mouse showed better acute NTHi colonization of the respiratory tract following intranasal challenge compared to wild type mice. Since Kdn is a metabolic byproduct in humans and should be non-toxic, the possibility of using Kdn as a safe intervention against NTHi colonization and subsequent infection is currently being explored using this mouse model.

Hydrogen deuterium exchange reveals changes to protein dynamics of recombinant human erythropoietin upon N- and O- desialylation

Recombinant human erythropoietin (EPO) is a therapeutic glycoprotein widely used for treating anemia. EPO glycans carry extensive sialylation and the level of the modification is known to affect receptor binding, protein stability and pharmacokinetics. Nonetheless, a detailed understanding of the effects of sialylation on EPO conformation and dynamics is still lacking. Here we investigate the changes to EPO dynamics following enzymatic trimming of terminal sialic acid by amide hydrogen deuterium exchange mass spectrometry (HDX-MS). The results revealed that desialylation enhances structural flexibility near the glycosylation sites, with greater effects observed around the O-glycosylation site relative to the N-glycosylation sites. The affected regions are surface-exposed loops connecting the helix bundle, which do not appear to reduce the thermostability of the molecule as revealed from melting measurement. Our findings demonstrate the feasibility of HDX-MS technique in deciphering the function of specific type of glycosylation that can provide novel insights into the role of sialylation on protein therapeutics.

Multifaceted Role of Sialylation in Prion Diseases.

Mammalian prion or PrPSc is a proteinaceous infectious agent that consists of a misfolded, self-replicating state of a sialoglycoprotein called the prion protein, or PrPC. Sialylation of the prion protein N-linked glycans was discovered more than 30 years ago, yet the role of sialylation in prion pathogenesis remains poorly understood. Recent years have witnessed extraordinary growth in interest in sialylation and established a critical role for sialic acids in host invasion and host-pathogen interactions. This review article summarizes current knowledge on the role of sialylation of the prion protein in prion diseases. First, we discuss the correlation between sialylation of PrPSc glycans and prion infectivity and describe the factors that control sialylation of PrPSc. Second, we explain how glycan sialylation contributes to the prion replication barrier, defines strain-specific glycoform ratios, and imposes constraints for PrPSc structure. Third, several topics, including a possible role for sialylation in animal-to-human prion transmission, prion lymphotropism, toxicity, strain interference, and normal function of PrPC, are critically reviewed. Finally, a metabolic hypothesis on the role of sialylation in the etiology of sporadic prion diseases is proposed.

Role of sialylation in 3D multicellular tumor spheroid formation and in vivo tumors

Role of sialylation in 3D multicellular tumor spheroid formation and in vivo tumors

Galactose supplementation enhance sialylation of recombinant Fc-fusion protein in CHO cell: an insight into the role of galactosylation in sialylation.

Sialic acid levels of therapeutic glycoprotein play an important role in plasma half-life. An undesirable decrease of sialic acid content was observed when we increased Fc-fusion protein productivity fourfold in a GS-CHO cell line by bioprocess optimization. We investigated the potential mechanism for the sialic acid content reduction. We found that limited nucleotide sugar precursor and the extracellular sialidase were not responsible for the reduction of the sialic acid content after titer improvement. Oligosaccharide analysis revealed that the lack of protein galactosylation was the potential cause for the reduction of sialic acid content. Thus we validated this notion by evaluated galactose supplementation in 2 L bioreactors. Cell culture performance was not impacted by addition of up to 40 mM galactose except for the glucose consumption rate. Addition of 20 mM galactose to the bioreactor resulted in the increase of 44 % for total sialic acid content and 20.3 % for sialylated glycans. These data were further validated when the process was run on 200 L scaled bioreactor. These data together show that the galactosylation plays an apparent role in sialylation in our current system.

NEU3 activity enhances EGFR activation without affecting EGFR expression and acts on its sialylation levels.

Several studies performed over the last decade have focused on the role of sialylation in the progression of cancer and, in particular, on the association between deregulation of sialidases and tumorigenic transformation. The plasma membrane-associated sialidase NEU3 is often deregulated in colorectal cancer (CRC), and it was shown that this enzyme co-immunoprecipitates in HeLa cells with epidermal growth factor receptor (EGFR), the molecular target of most recent monoclonal antibody-based therapies against CRC. To investigate the role of NEU3 sialidase on EGFR deregulation in CRC, we first collected data on NEU3 gene expression levels from a library of commercial colon cell lines, demonstrating that NEU3 transcription is upregulated in these cell lines. We also found EGFR to be hyperphosphorylated in all cell lines, with the exception of SW620 cells and the CCD841 normal intestinal cell line. By comparing the effects induced by overexpression of either the wild-type or the inactive mutant form of NEU3 on EGFR, we demonstrated that the active form of NEU3 enhanced receptor activation without affecting EGFR mRNA or protein expression. Moreover, through western blots and mass spectrometry analysis, we found that EGFR immunoprecipitated from cells overexpressing active NEU3, unlike the receptor from mock cells and cells overexpressing inactive NEU3, is desialylated. On the whole, our data demonstrate that, besides the already reported indirect EGFR activation through GM3, sialidase NEU3 could also play a role on EGFR activation through its desialylation.