Single N-glycan Research Articles

N-glycosylation of immunoglobulin A in children and adults with type 1 diabetes mellitus

AimsTo identify N-glycan structures on immunoglobulin A related to type 1 diabetes mellitus among children at the disease onset and adults with type 1 diabetes mellitus. MethodsHuman polyclonal IgA N-glycans were profiled using hydrophilic interaction ultra performance liquid chromatography in two cohorts. The first cohort consisted of 62 children at the onset of type 1 diabetes mellitus and 86 of their healthy siblings. The second cohort contained 84 adults with the disease and 84 controls. Associations between N-glycans and type 1 diabetes mellitus were tested using linear mixed model for the paediatric cohort, or general linear model for the adult cohort. False discovery rate was controlled by Benjamini-Hochberg method modified by Li and Ji. ResultsIn children, an increase in a single oligomannose N-glycan was associated with type 1 diabetes mellitus (B = 0.529, p = 0.0067). N-glycome of the adults displayed increased branching (B = 0.466, p = 0.0052), trigalactosylation (B = 0.466, p = 0.0052), trisialylation (B = 0.629, p < 0.001), and mannosylation (B = 0.604, p < 0.001). The strongest association with the disease was a decrease in immunoglobulin A core fucosylation (B = −0.900, p < 0.001). ConclusionsChanges in immunoglobulin N-glycosylation patterns in type 1 diabetes point to disruptions in immunoglobulin A catabolism and dysregulated inflammatory capabilities of the antibody, potentially impacting immune responses and inflammation.

N-glycan on N262 of FGFR3 regulates the intracellular localization and phosphorylation of the receptor

N-glycosylation and proper processing of N-glycans are required for the function of membrane proteins including cell surface receptors. Fibroblast growth factor receptor (FGFR) is involved in a wide variety of biological processes including embryonic development, osteogenesis, angiogenesis, and cell proliferation. Human FGFR3 contains six potential N-glycosylation sites, however, the roles of glycosylation have not been elucidated. The site-specific profiles of N-glycans of the FGFR3 extracellular domain expressed and secreted by CHO-K1 cells were examined, and glycan occupancies and structures of four sites were determined. The results indicated that most sites were fully occupied by glycans, and the dominant populations were the complex type. By examining single N-glycan deletion mutants of FGFR3, it was found that N262Q mutation significantly increased the population with oligomannose-type N-glycans, which was localized in the endoplasmic reticulum. Protein stability assay suggested that fraction with oligomannose-type N-glycans in the N262Q mutant is more stable than those in the wild type and other mutants. Furthermore, it was found that ligand-independent phosphorylation was significantly upregulated in N262Q mutants with complex type N-glycans. The findings suggest that N-glycans on N262 of FGFR3 affect the intracellular localization and phosphorylation status of the receptor.

Impact of N-glycan mediated shielding of ADAMTS-13 on the binding of pathogenic antibodies in immune thrombotic thrombocytopenic purpura

BackgroundThrombotic thrombocytopenic purpura (TTP) is a rare thrombotic disorder, with 1.5 to 6.0 cases per million per year. The majority of patients with TTP develop inhibitory autoantibodies that predominantly target the spacer domain of ADAMTS-13. ADAMTS-13 is responsible for cleaving von Willebrand factor (VWF) multimers, thereby regulating platelet adhesion at sites of high-vascular shear stress. Inhibition and/or clearance of ADAMTS-13 by pathogenic autoantibodies results in accumulation of VWF multimers that promotes the formation of platelet-rich microthrombi. Previously, we have shown that insertion of a single N-glycan (NGLY) in the spacer domain prevents the binding of antispacer domain antibodies. ObjectivesTo explore whether NGLY mediated shielding of the ADAMTS-13 spacer domain effectively prevents binding of pathogenic antispacer autoantibodies in patients with immune-mediated TTP (iTTP). MethodsWe screened 5 NGLY-ADAMTS-13 variants (NGLY3, NGLY7, NGLY8, NGLY3+7, and NGLY3+8) for binding of autoantibodies and for their activity in the presence and absence of 50 samples derived from patients with iTTP. ResultsNGLY variants showed greatly reduced antibody binding, down to 27% of wild-type (wt) ADAMTS-13 binding. Moreover, NGLY variants of ADAMTS-13 remained more active in FRETS-VWF73 assay in the presence of the plasma samples from these 50 patients with acute phase iTTP when compared with wtADAMTS-13. On average, wtADAMTS-13 activity was reduced to 37% of regular levels in the presence of plasma, while NGLY3 and NGLY3+7 remained 69% and 81% active, respectively. ConclusionThese results reinforce our previous findings that NGLYs shield ADAMTS-13 from antibody binding and hence restore ADAMTS-13 activity in the presence of autoantibodies.

Mannosylated glycans impair normal T-cell development by reprogramming commitment and repertoire diversity.

T-cell development ensures the formation of diverse repertoires of T-cell receptors (TCRs) that recognize a variety of antigens. Glycosylation is a major posttranslational modification present in virtually all cells, including T-lymphocytes, that regulates activity/functions. Although these structures are known to be involved in TCR-selection in DP thymocytes, it is unclear how glycans regulate other thymic development processes and how they influence susceptibility to disease. Here, we discovered stage-specific glycome compositions during T-cell development in human and murine thymocytes, as well as dynamic alterations. After restricting the N-glycosylation profile of thymocytes to high-mannose structures, using specific glycoengineered mice (Rag1CreMgat1fl/fl), we showed remarkable defects in key developmental checkpoints, including ß-selection, regulatory T-cell generation and γδT-cell development, associated with increased susceptibility to colon and kidney inflammation and infection. We further demonstrated that a single N-glycan antenna (modeled in Rag1CreMgat2fl/fl mice) is the sine-qua-non condition to ensure normal development. In conclusion, we revealed that mannosylated thymocytes lead to a dysregulation in T-cell development that is associated with inflammation susceptibility.

Glyco engineered pentameric SARS-CoV-2 IgMs show superior activities compared to IgG1 orthologues.

Immunoglobulin M (IgM) is the largest antibody isotype with unique features like extensive glycosylation and oligomerization. Major hurdles in characterizing its properties are difficulties in the production of well-defined multimers. Here we report the expression of two SARS-CoV-2 neutralizing monoclonal antibodies in glycoengineered plants. Isotype switch from IgG1 to IgM resulted in the production of IgMs, composed of 21 human protein subunits correctly assembled into pentamers. All four recombinant monoclonal antibodies carried a highly reproducible human-type N-glycosylation profile, with a single dominant N-glycan species at each glycosite. Both pentameric IgMs exhibited increased antigen binding and virus neutralization potency, up to 390-fold, compared to the parental IgG1. Collectively, the results may impact on the future design of vaccines, diagnostics and antibody-based therapies and emphasize the versatile use of plants for the expression of highly complex human proteins with targeted posttranslational modifications.

Core Fucosylation Is Required for the Secretion of and the Enzymatic Activity of SOD3 in Nonsmall-Cell Lung Cancer Cells.

Aims: The anticancer function of superoxide dismutases (SODs) is still controversial. SOD3 is an extracellular superoxide dismutase and contains a single N-glycan chain. The role played by the N-glycosylation of SOD3, as it relates to lung cancer, is poorly understood. For this, we performed the structural and functional analyses of the N-glycan of SOD3 in lung cancer. Results: We report herein that the fucose structure of the N-glycan in SOD3 was increased in the sera of patients with lung cancer. In cell lines of non-small lung cancer cell (NSCLC), we also found a high level of the core fucose structure in the N-glycan of SOD3, as determined by lectin blotting and mass spectrometry analysis. To address the roles of the core fucose structure of SOD3, we generated FUT8 (α1,6-fucosyltransferase) gene knockout A549 cells. Using these cells, we found that the core fucose structure of SOD3 was required for its secretion and enzymatic activity, which contributes to the suppression of cell growth of NSCLC cells. Innovation: The core fucosylation is required for the secretion and enzymatic activity of SOD3, which contributes to anti-tumor functions such as the suppression of cell growth of NSCLC. Conclusion: The N-glycans, especially those with core fucose structures, regulate the anti-tumor functions of SOD3 against NSCLC. Antioxid. Redox Signal. 38, 1201-1211.

Dissecting the essential role of N-glycosylation in catalytic performance of xanthan lyase

Modified xanthan produced by xanthan lyase has broad application prospects in the food industry. However, the catalytic performance of xanthan lyase still needs to be improved through rational design. To address this problem, in this work, the glycosylation and its influences on the catalytic performance of a xanthan lyase (EcXly), which was heterologously expressed in Escherichia coli, were reported. Liquid chromatography coupled to tandem mass spectrometry analysis revealed that the N599 site of EcXly was modified by a single N-glycan chain. Based on sequence alignment and three-dimensional structure prediction, it could be deduced that the N599 site was located in the catalytic domain of EcXly and in close proximity to the catalytic residues. After site-directed mutagenesis of N599 with alanine, aspartic acid and glycine, respectively, the EcXly and its mutants were characterized and compared. The results demonstrated that elimination of the N-glycosylation had diminished the specific activity, pH stability, and substrate affinity of EcXly. Fluorescence spectra further revealed that the glycosylation could significantly affect the overall tertiary structure of EcXly. Therefore, in prokaryotic hosts, the N-glycosylation could influence the catalytic performance of the enzyme by changing its structure. To the best of our knowledge, this is the first report about the post-translational modification of xanthan lyase in prokaryotes. Overall, our work enriched research on the role of glycan chains in the functional performance of proteins expressed in prokaryotes and should be valuable for the rational design of xanthan lyase to produce modified xanthan for industrial application.Graphical

Serum N-Glycomics Stratifies Bacteremic Patients Infected with Different Pathogens.

Bacteremia—i.e., the presence of pathogens in the blood stream—is associated with long-term morbidity and is a potential precursor condition to life-threatening sepsis. Timely detection of bacteremia is therefore critical to reduce patient mortality, but existing methods lack precision, speed, and sensitivity to effectively stratify bacteremic patients. Herein, we tested the potential of quantitative serum N-glycomics performed using porous graphitized carbon liquid chromatography tandem mass spectrometry to stratify bacteremic patients infected with Escherichia coli (n = 11), Staphylococcus aureus (n = 11), Pseudomonas aeruginosa (n = 5), and Streptococcus viridans (n = 5) from healthy donors (n = 39). In total, 62 N-glycan isomers spanning 41 glycan compositions primarily comprising complex-type core fucosylated, bisecting N-acetylglucosamine (GlcNAc), and α2,3-/α2,6-sialylated structures were profiled across all samples using label-free quantitation. Excitingly, unsupervised hierarchical clustering and principal component analysis of the serum N-glycome data accurately separated the patient groups. P. aeruginosa-infected patients displayed prominent N-glycome aberrations involving elevated levels of fucosylation and bisecting GlcNAcylation and reduced sialylation relative to other bacteremic patients. Notably, receiver operating characteristic analyses demonstrated that a single N-glycan isomer could effectively stratify each of the four bacteremic patient groups from the healthy donors (area under the curve 0.93–1.00). Thus, the serum N-glycome represents a new hitherto unexplored class of potential diagnostic markers for bloodstream infections.

Multiple N-glycans cooperate in balancing misfolded BRI1 secretion and ER retention.

N-glycans play a protective or monitoring role according to the folding state of associated protein or the distance from structural defects. Asparagine-linked (Asn/N-) glycosylation is one of the most prevalent and complex protein modifications and the associated N-glycans play crucial roles on protein folding and secretion. The studies have shown that many glycoproteins hold multiple N-glycans, yet little is known about the redundancy of N-glycans on a protein. In this study, we used BRI1 to decipher the roles of N-glycans on protein secretion and function. We found that all 14 potential N-glycosylation sites on BRI1 were occupied with oligosaccharides. The elimination of single N-glycan had no obvious effect on BRI1 secretion or function except N154-glycan, which resulted in the retention of BRI1 in the endoplasmic reticulum (ER), similar to the loss of multiple highly conserved N-glycans. To misfolded bri1, the absence of N-glycans next to local structural defects enhanced the ER retention and the artificial addition of N-glycan could help the misfolded bri1-GFPs exiting from the ER, indicating that the N-glycans might serve as steric hindrance to protect the structure defects from ER recognition. We also found that the retention of misfolded bri1-9 by lectins and chaperones in the ER relied on the presence of multiple N-glycans distal to the local defects. Our findings revealed that the N-glycans might play a protective or monitoring role according to the folding state of associated protein or the distance from structural defects.

Novel Insights Into N-Glycan Fucosylation and Core Xylosylation in C. reinhardtii.

Chlamydomonas reinhardtii (C. reinhardtii) N-glycans carry plant typical β1,2-core xylose, α1,3-fucose residues, as well as plant atypical terminal β1,4-xylose and methylated mannoses. In a recent study, XylT1A was shown to act as core xylosyltransferase, whereby its action was of importance for an inhibition of excessive Man1A dependent trimming. N-Glycans found in a XylT1A/Man1A double mutant carried core xylose residues, suggesting the existence of a second core xylosyltransferase in C. reinhardtii. To further elucidate enzymes important for N-glycosylation, novel single knockdown mutants of candidate genes involved in the N-glycosylation pathway were characterized. In addition, double, triple, and quadruple mutants affecting already known N-glycosylation pathway genes were generated. By characterizing N-glycan compositions of intact N-glycopeptides from these mutant strains by mass spectrometry, a candidate gene encoding for a second putative core xylosyltransferase (XylT1B) was identified. Additionally, the role of a putative fucosyltransferase was revealed. Mutant strains with knockdown of both xylosyltransferases and the fucosyltransferase resulted in the formation of N-glycans with strongly diminished core modifications. Thus, the mutant strains generated will pave the way for further investigations on how single N-glycan core epitopes modulate protein function in C. reinhardtii.

Core-Fucosylated Tetra-Antennary N-Glycan Containing A Single N-Acetyllactosamine Branch Is Associated with Poor Survival Outcome in Breast Cancer.

(1) Glycoproteins account for ~80% of proteins located at the cell surface and in the extracellular matrix. A growing body of evidence indicates that α-L-fucose protein modifications contribute to breast cancer progression and metastatic disease. (2) Using a combination of techniques, including matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) based in cell and on tissue imaging and glycan sequencing using exoglycosidase analysis coupled to hydrophilic interaction ultra-high performance liquid chromatography (HILIC UPLC), we establish that a core-fucosylated tetra-antennary glycan containing a single N-acetyllactosamine (F(6)A4G4Lac1) is associated with poor clinical outcomes in breast cancer, including lymph node metastasis, recurrent disease, and reduced survival. (3) This study is the first to identify a single N-glycan, F(6)A4G4Lac1, as having a correlation with poor clinical outcomes in breast cancer.

Binding of the Bacterial Adhesin FimH to Its Natural, Multivalent High-Mannose Type Glycan Targets.

Multivalent carbohydrate-lectin interactions at host-pathogen interfaces play a crucial role in the establishment of infections. Although competitive antagonists that prevent pathogen adhesion are promising antimicrobial drugs, the molecular mechanisms underlying these complex adhesion processes are still poorly understood. Here, we characterize the interactions between the fimbrial adhesin FimH from uropathogenic Escherichia coli strains and its natural high-mannose type N-glycan binding epitopes on uroepithelial glycoproteins. Crystal structures and a detailed kinetic characterization of ligand-binding and dissociation revealed that the binding pocket of FimH evolved such that it recognizes the terminal α(1-2)-, α(1-3)-, and α(1-6)-linked mannosides of natural high-mannose type N-glycans with similar affinity. We demonstrate that the 2000-fold higher affinity of the domain-separated state of FimH compared to its domain-associated state is ligand-independent and consistent with a thermodynamic cycle in which ligand-binding shifts the association equilibrium between the FimH lectin and the FimH pilin domain. Moreover, we show that a single N-glycan can bind up to three molecules of FimH, albeit with negative cooperativity, so that a molar excess of accessible N-glycans over FimH on the cell surface favors monovalent FimH binding. Our data provide pivotal insights into the adhesion properties of uropathogenic Escherichia coli strains to their target receptors and a solid basis for the development of effective FimH antagonists.

Genetically engineered cell factories produce glycoengineered vaccines that target antigen-presenting cells and reduce antigen-specific T-cell reactivity

Genetically engineered cell factories produce glycoengineered vaccines that target antigen-presenting cells and reduce antigen-specific T-cell reactivity

Serum N-glycans outperform CA19-9 in diagnosis of extrahepatic cholangiocarcinoma.

Extensive efforts have been devoted to improve the diagnosis of extrahepatic cholangiocarcinoma (ECCA) due to its silent clinical character and lack of effective diagnostic biomarkers. Specific alterations in N-glycosylation of glycoproteins are considered a key component in cancer progression, which can serve as a distinct molecular signature for cancer detection. This study aims to find potential serum N-glycan markers for ECCA. In total, 255 serum samples from patients with ECCA (n=106), benign bile tract disease (BBD, n=60) and healthy controls (HC, n=89) were recruited. Only 2μL of serum from individual patients was used in this assay where the N-glycome of serum glycoproteins was profiled by DNA sequencer-assisted fluorophore-assisted capillary electrophoresis (DSA-FACE) technology. Multi-parameter models were constructed by combining the N-glycans and carbohydrate antigen 19-9 (CA19-9) which is currently used clinically. Quantitative analyses showed that among 13 N-glycan structures, the bifucosylated triantennary N-glycan (peak10, NA3F2) presented the best diagnostic performance for distinguishing ECCA from BBD and HC. Two diagnostic models (Glycotest1 and Glycotest2) performed better than single N-glycan or CA19-9. Additionally, two N-glycan structures (peak9, NA3Fb; peak12, NA4Fb) were tightly related to lymph node metastasis in ECCA patients. In conclusion, sera of ECCA showed relatively specific N-glycome profiling patterns. Serum N-glycan markers and models are novel, valuable and noninvasive alternatives in ECCA diagnosis and progression monitoring.

Isolation and characterization of renin-like aspartic-proteases from Echis ocellatus venom

Three aspartic proteases (SVAPs) have been isolated from venom of the saw-scaled viper, Echis ocellatus. In confirmation of prior transcriptomic predictions, all three forms match to sequences of either of the two SVAP transcripts (EOC00051 and EOC00123), have a molecular weight of 42 kDa and possess a single N-glycan. The SVAPs act in a renin-like manner, specifically cleaving human and porcine angiotensinogen into angiotensin-1 and possess no general protease activity. Their activity is completely inhibited by the aspartyl protease inhibitor Pepstatin A.

Glycosylation-deficient mutations in tissue-nonspecific alkaline phosphatase impair its structure and function and are linked to infantile hypophosphatasia.

Tissue-nonspecific alkaline phosphatase (TNSALP) is a membrane glycoprotein with a proposed role in bone mineralization. Indeed, mutations in TNSALP have been identified in patients with hypophosphatasia (HPP), a genetic disease characterized by hypomineralization of bone and teeth and a deficiency in serum ALP activity. TNSALP has five potential N-glycosylation sites at N140, N230, N271, N303 and N430 by standard nomenclature. A mutation at one of these sites, N430, was recently detected in a patient with infantile HPP. Using site-directed mutagenesis, we demonstrated that TNSALP has five N-glycans in transfected COS-1 cells and that individual single N-glycan deletion mutants of TNSALP retain the dimeric structure required for ALP activity, excluding the possibility that any single N-glycan plays a vital role in the structure and function of TNSALP. However, we found that TNSALP (N430Q) and TNSALP (N430E) mutants, but not a TNSALP (N430D) mutant, failed to form dimers. The TNSALP (N430S) mutant linked to infantile HPP was glycosylation-defective and unable to dimerise, similar to TNSALP (N430Q) and TNSALP (N430E) mutants; therefore, TNSALP (N430S) was established as a severe allele without strong ALP activity. By contrast to individual single N-glycan deletion mutants, TNSALP devoid of all five N-glycans was present to a much lesser extent than wild-type TNSALP in transfected cells, possibly reflecting its instability. A comprehensive analysis of a series of multiple N-glycan depletion mutants in TNSALP revealed that three N-glycans on N230, N271 and N303 were the minimal requirement for the structure and function of TNSALP and a prerequisite for its stable expression in a cell.

Microfluidic Chip-LC/MS-based Glycomic Analysis Revealed Distinct N-glycan Profile of Rat Serum.

The rat is an important alternative for studying human pathology owing to certain similarities to humans. Glycomic studies on rat serum have revealed that variations in the N-glycans of glycoproteins correlated with disease progression, which is consistent with the findings in human serum. Therefore, we comprehensively characterized the rat serum N-glycome using microfluidic chip-LC-ESI-QTOF MS and MS/MS techniques. In total, 282 N-glycans, including isomers, were identified. This study is the first to present comprehensive profiling of N-glycans containing O-acetylated sialic acid, among which 27 N-glycans are novel. In addition, the co-existence of N-acetylneuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuGc) in a single N-glycan (‘mixed’ N-glycan) was detected and represents a new type of N-glycan in rat serum. The existence of O-acetylated sialic acid is the characteristic feature of rat serum that distinguishes it from mouse and human sera. Comparisons between the rat, mouse, and human serum glycomes revealed that the rat glycome is more similar to that of human sera than to that of mouse sera. Our findings highlight the similarities between the glycomic profile of rat and human sera and provided important selection criteria for choosing an appropriate animal model for pathological and pharmacological studies.

N-glycosylation controls the function of junctional adhesion molecule-A.

Junctional adhesion molecule-A (JAM-A) is an adherens and tight junction protein expressed by endothelial and epithelial cells. JAM-A serves many roles and contributes to barrier function and cell migration and motility, and it also acts as a ligand for the leukocyte receptor LFA-1. JAM-A is reported to contain N-glycans, but the extent of this modification and its contribution to the protein's functions are unknown. We show that human JAM-A contains a single N-glycan at N185 and that this residue is conserved across multiple mammalian species. A glycomutant lacking all N-glycans, N185Q, is able to reach the cell surface but exhibits decreased protein half-life compared with the wild- type protein. N-glycosylation of JAM-A is required for the protein's ability to reinforce barrier function and contributes to Rap1 activity. We further show that glycosylation of N185 is required for JAM-A-mediated reduction of cell migration. Finally, we show that N-glycosylation of JAM-A regulates leukocyte adhesion and LFA-1 binding. These findings identify N-glycosylation as critical for JAM-A's many functions.

Critical role of evolutionarily conserved glycosylation at Asn211 in the intracellular trafficking and activity of sialyltransferase ST3Gal-II.

ST3Gal-II, a typeII transmembrane protein, is the main mammalian sialyltransferase responsible for GD1a and GT1b ganglioside biosynthesis in brain. It contains two putative N-glycosylation sites (Asn(92) and Asn(211)). Whereas Asn(92) is only conserved in mammalian species, Asn(211) is highly conserved in mammals, birds and fish. The present study explores the occupancy and relevance for intracellular trafficking and enzyme activity of these potential N-glycosylations in human ST3Gal-II. We found that ST3Gal-II distributes along the Golgi complex, mainly in proximal compartments. By pharmacological, biochemical and site-directed mutagenesis, we observed that ST3Gal-II is mostly N-glycosylated at Asn(211) and that this co-translational modification is critical for its exit from the endoplasmic reticulum and proper Golgi localization. The individual N-glycosylation sites had different effects on ST3Gal-II enzymatic activity. Whereas the N-glycan at position Asn(211) seems to negatively influence the activity of the enzyme using both glycolipid and glycoprotein as acceptor substrates, the single N-glycan mutant at Asn(92) had only a moderate effect. Lastly, we demonstrated that the N-terminal ST3Gal-II domain containing the cytosolic, transmembrane and stem region (amino acids 1-51) is able to drive a protein reporter out of the endoplasmic reticulum and to retain it in the Golgi complex. This suggests that the C-terminal domain of ST3Gal-II depends on N-glycosylation to attain an optimum conformation for proper exit from the endoplasmic reticulum, but it does not represent an absolute requirement for Golgi complex retention of the enzyme.

The single N-glycan deletion mutant of soluble ErbB3 protein attenuates heregulin β1-induced tumor progression by blocking of the HIF-1 and Nrf2 pathway

It has been well documented that activation of the ErbB3–PI3K–Akt pathway is implicated in tumor survival and progression. We previously demonstrated that the single N-glycan deletion mutant of soluble ErbB3 protein (sErbB3 N418Q) attenuates heregulin β1-induced ErbB3 signaling. The active PI3K–Akt pathway augments the nuclear accumulation of hypoxia inducible factor (HIF)-1α, which activates the transcription of many target genes and drives cancer progression. In this study, we focused on the effects of sErbB3 N418Q mutant on nuclear accumulation of HIF-1α. Pretreatment with the sErbB3 N418Q mutant suppressed heregulin β1-induced HIF-1α activation in MCF7 cells. Similar results were also obtained in other breast cancer cell lines, T47D and BT474. Interestingly, these suppressive effects were not observed with the sErbB3 wild type. In addition, pretreatment with the sErbB3 N418Q mutant suppressed the cell migration of MCF7 cells induced by heregulin β1. Furthermore, incubation with heregulin β1 also induced the nuclear accumulation of Nrf2, and this effect was also reduced by the sErbB3 N418Q mutant, but not the sErbB3 wild type. These findings indicated that the sErbB3 N418Q mutant suppressed malignant formation of cancer cells by blocking of the HIF-1α and Nrf2 pathways.