Changes In Glycosylation Pattern Research Articles

O-GlycoIsoQuant: A Novel O-Glycome Quantitative Approach through Superbase Release and Isotopic Girard's P Labeling.

Quantitative glycosylation analysis serves as an effective tool for detecting changes in glycosylation patterns in cancer and various diseases. However, compared with N-glycans, O-glycans present challenges in both qualitative and quantitative mass spectrometry analysis due to their low abundance, ease of peeling, lack of a universal enzyme, and difficult accessibility. To address this challenge, we developed O-GlycoIsoQuant, a novel O-glycome quantitative approach utilizing superbase release and isotopic Girard's P labeling. This method facilitates rapid and efficient nonreducing β-elimination to dissociate O-glycans from proteins using the organic superbase, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), combined with light and heavy isotopic Girard's reagent P (GP) labeling for relative quantification of O-glycans by mass spectrometry. Employing this method, labeled O-glycans exhibit a double peak with a mass difference of 5 Da, suitable for stable relative quantification. The O-GlycoIsoQuant method is characterized by its high labeling efficiency, excellent reproducibility (CV < 20%), and good linearity (R2 > 0.99), across a dynamic range spanning a 100-fold range. This method was applied to various complex sample types, including human serum, porcine spermatozoa, human saliva, and urinary extracellular vesicles, detecting 33, 39, 49, and 37 O-glycans, respectively, thereby demonstrating its broad applicability.

Structural Analysis and Characterization of an Antiproliferative Lectin from Canavalia villosa Seeds.

Cells use glycans to encode information that modulates processes ranging from cell-cell recognition to programmed cell death. This information is encoded within a glycocode, and its decoding is performed by carbohydrate-binding proteins. Among these, lectins stand out due to their specific and reversible interaction with carbohydrates. Changes in glycosylation patterns are observed in several pathologies, including cancer, where abnormal glycans are found on the surfaces of affected tissues. Given the importance of the bioprospection of promising biomolecules, the current work aimed to determine the structural properties and anticancer potential of the mannose-specific lectin from seeds of Canavalia villosa (Cvill). Experimental elucidation of the primary and 3D structures of the lectin, along with glycan array and molecular docking, facilitated the determination of its fine carbohydrate-binding specificity. These structural insights, coupled with the lectin's specificity, have been combined to explain the antiproliferative effect of Cvill against cancer cell lines. This effect is dependent on the carbohydrate-binding activity of Cvill and its uptake in the cells, with concomitant activation of autophagic and apoptotic pathways.

SARS-CoV-2 induced changes in the glycosylation pattern in the respiratory tract of Golden Syrian hamsters

Even after more than two years of intensive research, not all of the pathophysiological processes of Coronavirus Disease 2019 (COVID-19), induced by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection, have been fully elucidated. The initial virus-host interaction at the respiratory epithelium plays a crucial role in the course and progression of the infection, and is highly dependent on the glycosylation pattern of the host cell and of the secreted mucins. Glycans are polysaccharides that can be attached to proteins and thereby add to their stability and functionality. Lectins are glycan-binding proteins that recognize specific glycan motifs, and lectin histochemistry is a suitable tool to visualize and examine glycosylation pattern changes in tissues. In this study we used lectins with different glycan-specificities for the visualization of glycosylation pattern changes in the respiratory tract of SARS-CoV-2 infected Golden Syrian hamsters. While some lectins (LEL, STL) enable the visualization of the damage to alveolar type 1 pneumocytes, other lectins, e.g., GSLI, visualized the loss and subsequent hyperplasia of type 2 pneumocytes. UEAI staining was co-localized with KI67, a proliferation marker. Double staining of lectins LEL, STL and WGA with specific immune cell markers (Iba1, CD68) showed co-localization and the dominant infiltration of monocyte-derived macrophages into infected alveolar tissue. The elucidation of the glycosylation pattern of the respiratory tract cells in uninfected and infected Golden Syrian hamsters revealed physiological and pathological aspects of the disease that may open new possibilities for therapeutic development.

Analysis of the glyco-code in pancreatic ductal adenocarcinoma identifies glycan-mediated immune regulatory circuits

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most aggressive malignancies with a 5-year survival rate of only 9%. Despite the fact that changes in glycosylation patterns during tumour progression have been reported, no systematic approach has been conducted to evaluate its potential for patient stratification. By analysing publicly available transcriptomic data of patient samples and cell lines, we identified here two specific glycan profiles in PDAC that correlated with progression, clinical outcome and epithelial to mesenchymal transition (EMT) status. These different glycan profiles, confirmed by glycomics, can be distinguished by the expression of O-glycan fucosylated structures, present only in epithelial cells and regulated by the expression of GALNT3. Moreover, these fucosylated glycans can serve as ligands for DC-SIGN positive tumour-associated macrophages, modulating their activation and inducing the production of IL-10. Our results show mechanisms by which the glyco-code contributes to the tolerogenic microenvironment in PDAC.

Replication of 15 loci involved in human plasma protein N-glycosylation in 4802 samples from four cohorts.

Human protein glycosylation is a complex process, and its in vivo regulation is poorly understood. Changes in glycosylation patterns are associated with many human diseases and conditions. Understanding the biological determinants of protein glycome provides a basis for future diagnostic and therapeutic applications. Genome-wide association studies (GWAS) allow to study biology via a hypothesis-free search of loci and genetic variants associated with a trait of interest. Sixteen loci were identified by three previous GWAS of human plasma proteome N-glycosylation. However, the possibility that some of these loci are false positives needs to be eliminated by replication studies, which have been limited so far. Here, we use the largest set of samples so far (4802 individuals) to replicate the previously identified loci. For all but one locus, the expected replication power exceeded 95%. Of the 16 loci reported previously, 15 were replicated in our study. For the remaining locus (near the KREMEN1 gene), the replication power was low, and hence, replication results were inconclusive. The very high replication rate highlights the general robustness of the GWAS findings as well as the high standards adopted by the community that studies genetic regulation of protein glycosylation. The 15 replicated loci present a good target for further functional studies. Among these, eight loci contain genes encoding glycosyltransferases: MGAT5, B3GAT1, FUT8, FUT6, ST6GAL1, B4GALT1, ST3GAL4 and MGAT3. The remaining seven loci offer starting points for further functional follow-up investigation into molecules and mechanisms that regulate human protein N-glycosylation in vivo.

Changes in glycosylation patterns of monoterpenes during grape berry maturation in six cultivars of Vitis vinifera

Plants conjugate monoterpenoids to sugars, rendering them non-volatile. Hydrolysis of these glycosidic precursors frees the volatile aroma compounds. Here, we profile intact monoterpenyl glycosides in six Vitis vinifera grape berry cultivars. Relative concentrations of twenty-six monoterpenyl glycosides, including nine new putatively identified compounds, were analyzed by UHPLC–QTOF MS/MS at three times during grape maturation (pre-véraison, véraison, and post-véraison). Total glycoside content reached a maximum in Muscat cultivars post-véraison but remained relatively constant in all other cultivars. Three types of monoterpenyl glycosides predominated in all samples: malonylated monoterpenol glucosides, monoterpenol hexose-pentoses, and monoterpendiol hexose-pentoses. The two Muscat cultivars were not differentiated at the earlier developmental stages but could be differentiated post-véraison. In contrast, similarities between Chardonnay and Pinot noir glycoside profiles developed post-véraison. Overall monoterpene glycoconjugation patterns may align with underlying genetic relationships among cultivars. By understanding monoterpene glycoconjugation in wine grapes, scientists and winemakers can better understand grape and wine aromas.

Filter Beacon: A Gating-Free Architecture for Protein-Specific Glycoform Imaging on Cell Surface.

The imaging characterization of spatial proximity of covalently linked structural motifs (e.g., protein-specific glycoform) is essential for thorough understanding of cellular chemistry and biology. The current imaging formats rely on gating-based mechanisms for generating correct closed-loop signaling topology, and they can suffer from low signal intensity, restricted applicability, and complicated design. We report herein the development of a mechanistically distinct filter beacon architecture for protein-specific glycoform imaging on the cell surface. The elaborate structuring of molecular beacon segment, nicking restriction site, and docking moiety lays out a general nongated design principle for passing through intended closed-loop signaling topology and sifting out false-positive open-loop leakage topology, furnishing a straightforward imaging format with high signal intensity and broad applicability. Proof-of-concept protocols have been developed for the imaging of MUC1-bound terminal sialic acid and fucose. The versatile adaptability of the protocols also enables dynamic monitoring of protein-specific glycosylation pattern changes in response to the alteration of cellular physiological states. Given the convenience for achieving multiplexed encoding and decoding, through fluorescence signals alone or together with filter beacon sequences, the filter beacon architecture should permit comprehensive imaging of diverse-structured carbohydrates on a given glycoprotein.

Fighting the Cause of Alzheimer's and GNE Myopathy.

Age is the common risk factor for both neurodegenerative and neuromuscular diseases. Alzheimer disease (AD), a neurodegenerative disorder, causes dementia with age progression while GNE myopathy (GNEM), a neuromuscular disorder, causes muscle degeneration and loss of muscle motor movement with age. Individuals with mutations in presenilin or amyloid precursor protein (APP) gene develop AD while mutations in GNE (UDP N-acetylglucosamine 2 epimerase/N-acetyl Mannosamine kinase), key sialic acid biosynthesis enzyme, cause GNEM. Although GNEM is characterized with degeneration of muscle cells, it is shown to have similar disease hallmarks like aggregation of Aβ and accumulation of phosphorylated tau and other misfolded proteins in muscle cell similar to AD. Similar impairment in cellular functions have been reported in both disorders such as disruption of cytoskeletal network, changes in glycosylation pattern, mitochondrial dysfunction, oxidative stress, upregulation of chaperones, unfolded protein response in ER, autophagic vacuoles, cell death, and apoptosis. Interestingly, AD and GNEM are the two diseases with similar phenotypic condition affecting neuron and muscle, respectively, resulting in entirely different pathology. This review represents a comparative outlook of AD and GNEM that could lead to target common mechanism to find a plausible therapeutic for both the diseases.

Glycopeptides as a Tool for Early Detection of Cancer.

Protein glycosylation, as one of the most common and complex posttranslational modifications, plays an important role in many biological processes. Along with the intensive progress in MS techniques and development of glycan search tools and databases, glycoproteomics has become a popular subject of studies. The possibility of simultaneous identification of amino acid sequence, glycosylation sites, and glycan composition enabled the monitoring of changes in glycosylation patterns in various pathological states. In this issue, Saraswat etal. describe MS-based investigations of glycopeptide changes in oral cavity squamous cell carcinoma (OSCC). Their findings indicate glycopeptides with changed expression levels and the presence of altered glycan forms observed in four proteins derived from OSCC patients' sera. Proteins carrying this distinctive pattern are in the group of the most abundant components of serum, IgG1, IgG4, HPT, and TRFE, which makes their identification more accessible. Described changes, characteristic for cancer serum samples, may be considered as potential diagnostic indicators of OSCC; however, there is still a need to establish a universal glycopeptide-based biomarkers database, where all glycoproteomic data can be collected from all types of cancer studies and evaluated using meta-analyses. Only then, early diagnosis of patients using MS-based approach will make sense, as those investigations are very convoluted, and all efforts made during sample preparation and analysis will pay off when comprehensive anticancer prevention will be achieved during single measurement.

Density functional theory based probe of the affinity interaction of saccharide ligands with extra-cellular sialic acid residues

Changes in glycosylation pattern leads to malignant transformations among the cells. In combination with upregulated actions of sialyltransferases, it ultimately leads to differential expression of sialic acid (SA) at cell surface. Given its negative charge and localization to extracellular domain, SA has been exploited for the development of targeted theranostics using approaches, such as, cationization and appending recognition saccharides on carrier surface. In this study, we have performed quantum mechanical calculations based on density functional theory (DFT) to study the interaction of saccharides with extracellular SA. Gradient-corrected DFT with the three parameter function (B3) was utilized for the calculation of Lee–Yang–Parr (LYP) correlation function. Atomic charge, vibrational frequencies and energy of the optimized structures were calculated through B3LYP. Our calculations demonstrate a stronger galactose–sialic acid interaction at tumour-relevant low pH and hyperthermic condition. These results support the application of pH responsive delivery vehicles and targeted hyperthermic chemotherapy for eradicating solid tumour deposits. These studies, conducted a priori, can guide the formulation scientists over appropriate choice of ligands and their applications in the design of ‘smart’ theranostic tools.

GlycoSense™: A Rapid Method for Monitoring In Vitro Glycoengineering

The use of enzymes to modify glycans, in order to generate uniform protein glycosylation for optimal bioactivity, or to test glycan‐structure function hypotheses, or in chemo‐enzymatic synthesis, has emerged as a transformative technology. However, there are no convenient cost‐effective methods for monitoring the progress of the enzyme reactions during in vitro glycoengineering.Lectenz® Bio has developed an innovative technology called GlycoSense™ that will permit in vitro glycoenzyme reactions to be monitored in near real‐time using flow cytometry and multiplex microspheres (beads). The GlycoSense™ method detects binding between glycans and glycan‐specific reagents that are conjugated to spectrally‐unique beads. By combining the individual bead‐based reagents into a multiplexed array, a profile of key glycan features can be obtained in less than a minute on a basic cytometer.We illustrate the performance of the GlycoSense™ method using glycans and glycoprotein standards, and demonstrate its utility by monitoring changes in glycosylation patterns upon treatment with glycoenzymes. The GlycoSense™ approach will address the unmet need for a rapid tool to monitor protein glycosylation during in vitro glycoengineering.Support or Funding InformationNational Institutes of Health (U01CA221213, R43GM123863)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Glycan analysis of colorectal cancer samples reveals stage-dependent changes in CEA glycosylation patterns

BackgroundCarcinoembryonic antigen (CEA) is a glycoprotein associated with colorectal cancer (CRC). While the functions of its gene and protein have been fully characterized, its post-translational modifications in the context of CRC development remain undefined.MethodsTo show the correlation between the different stages of CRC development and changes in the glycosylation patterns of CEA, we analyzed CEA in tumor tissues (CEA-T) and paired tumor-adjacent normal tissues (CEA-A) from 53 colorectal cancer patients using a high-density lectin microarray containing 56 plant lectins.ResultsWe detected higher expression levels of fucose, mannose and Thomsen–Friedenreich antigen, and lower expression levels of N-acetylgalactosamine, N-acetylglucosamine, galactose, branched and bisecting N-glycans on CEA in the tumor tissues relative to the tumor-adjacent normal tissues. Furthermore, a combinatorial assessment of 9 lectins is sufficient to distinguish CRC tumor tissues from tumor-adjacent normal tissues with 83% sensitivity and ~ 90% specificity. Moreover, the levels of N-acetylgalactosamine, mannose, galactose, N-acetylglucosamine on CEA showed a downward trend after first experiencing an increase at Stage II with the stages of CRC.ConclusionsOur insights into the changing CEA glycosylation patterns and their role in the development of CRC highlight the importance of glycan variants on CEA for early clinical detection and staging of CRC.

Changes in glycosylation pattern and the emerging pathology of Zika Virus

Changes in glycosylation pattern and the emerging pathology of Zika Virus

Structural analysis of Dioclea lasiocarpa lectin: A C6 cells apoptosis-inducing protein.

Lectins are multidomain proteins that specifically recognize various carbohydrates. The structural characterization of these molecules is crucial in understanding their function and activity in systems and organisms. Most cancer cells exhibit changes in glycosylation patterns, and lectins may be able to recognize these changes. In this work, Dioclea lasiocarpa seed lectin (DLL) was structurally characterized. The lectin presented a high degree of similarity with other lectins isolated from legumes, presenting a jelly roll motif and a metal-binding site stabilizing the carbohydrate-recognition domain. DLL demonstrated differential interactions with carbohydrates, depending on type of glycosidic linkage present in ligands. As observed by the reduction of cell viability in C6 cells, DLL showed strong antiglioma activity by mechanisms involving activation of caspase 3.

IgG subclass and vaccination stimulus determine changes in antigen specific antibody glycosylation in mice.

Immunoglobulin G (IgG) glycosylation can modulate antibody effector functions. Depending on the precise composition of the sugar moiety attached to individual IgG glycovariants either pro- or anti-inflammatory effector pathways can be initiated via differential binding to type I or type II Fc-receptors. However, an in depth understanding of how individual IgG subclasses are glycosylated during the steady state and how their glycosylation pattern changes during vaccination is missing. To monitor IgG subclass glycosylation during the steady state and upon vaccination of mice with different T-cell dependent and independent antigens, tryptic digests of serum, and antigen-specific IgG preparations were analyzed by reversed phase-liquid chromatography-mass spectrometry. We show that there is a remarkable difference with respect to how individual IgG subclasses are glycosylated during the steady state. More importantly, upon T-cell dependent and independent vaccinations, individual antigen-specific IgG subclasses reacted differently with respect to changes in individual glycoforms, suggesting that the IgG subclass itself is a major determinant of restricting or allowing alterations in specific IgG glycovariants.

Integrated glycomic analysis of ovarian cancer side population cells.

BackgroundOvarian cancer is the most lethal gynecological malignancy due to its frequent recurrence and drug resistance even after successful initial treatment. Accumulating scientific evidence indicates that subpopulations of cancer cells with stem cell-like properties, such as so-called side population (SP) cells, are primarily responsible for these recurrences. A better understanding of SP cells may provide new clues for detecting and targeting these cancer-initiating cells and ultimately help to eradicate cancer. Changes in glycosylation patterns are remarkable features of SP cells. Here, we isolated SP cells from ovarian cancer cell lines and analyzed their glycosylation patterns using multiple glycomic strategies.MethodsSix high-grade serous ovarian cancer cell lines were used for SP cell isolation. Among them, HO8910 pm, which contained the highest proportion of SP cells, was used for glycomic analysis of SP cells. Cell lysate of SP cells and main population cells was applied to lectin microarray and mass spectrometry for glycan profiling. Differently expressed glycan structures were further verified by lectin blot, flow cytometry, and real-time PCR analysis of their relevant enzymes.ResultsExpression of core fucosylated N-glycan and tumor-associated Tn, T and sT antigens were increased in SP cells. By contrast, SP cells exhibited decreased hybrid glycan, α2,3-linked sialic glycan and multivalent sialyl-glycan.ConclusionsGlycan structures, such as Tn, T, sT antigens, and core fucosylation may serve as biomarkers of ovarian cancer stem cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12014-016-9131-z) contains supplementary material, which is available to authorized users.

Effects of HA and NA glycosylation pattern changes on the transmission of avian influenza A(H7N9) virus in guinea pigs

Avian influenza H7N9 virus has posed a concern of potential human-to-human transmission by resulting in seasonal virus-like human infection cases. To address the issue of sustained human infection with the H7N9 virus, here we investigated the effects of hemagglutinin (HA) and neuraminidase (NA) N-linked glycosylation (NLG) patterns on influenza virus transmission in a guinea pig model. Based on the NLG signatures identified in the HA and NA genetic sequences of H7N9 viruses, we generated NLG mutant viruses using either HA or NA gene of a H7N9 virus, A/Anhui/01/2013, by reverse genetics on the 2009 pandemic H1N1 virus backbone. For the H7 HA NLG mutant viruses, NLG pattern changes appeared to reduce viral transmissibility in guinea pigs. Intriguingly, however, the NLG changes in the N9 NA protein, such as a removal from residue 42 or 66 or an addition at residue 266, increased transmissibility of the mutant viruses by more than 33%, 50%, and 16%, respectively, compared with a parental N9 virus. Given the effects of HA-NA NLG changes with regard to viral transmission, we then generated the HA-NA NLG mutant viruses harboring the H7 HA of double NLG addition and the N9 NA of various NLG patterns. As seen in the HA NLG mutants above, the double NLG-added H7 HA decreased viral transmissibility. However, when the NA NLG changes occurred by a removal of residue 66 and an addition at 266 were additionally accompanied, the HA-NA NLG mutant virus recovered the transmissibility of its parental virus. These demonstrate the effects of specific HA-NA NLG changes on the H7N9 virus transmission by highlighting the importance of a HA-NA functional balance.

Aberrant Glycosylation in the Left Ventricle and Plasma of Rats with Cardiac Hypertrophy and Heart Failure.

Targeted proteomics focusing on post-translational modifications, including glycosylation, is a useful strategy for discovering novel biomarkers. To apply this strategy effectively to cardiac hypertrophy and resultant heart failure, we aimed to characterize glycosylation profiles in the left ventricle and plasma of rats with cardiac hypertrophy. Dahl salt-sensitive hypertensive rats, a model of hypertension-induced cardiac hypertrophy, were fed a high-salt (8% NaCl) diet starting at 6 weeks. As a result, they exhibited cardiac hypertrophy at 12 weeks and partially impaired cardiac function at 16 weeks compared with control rats fed a low-salt (0.3% NaCl) diet. Gene expression analysis revealed significant changes in the expression of genes encoding glycosyltransferases and glycosidases. Glycoproteome profiling using lectin microarrays indicated upregulation of mucin-type O-glycosylation, especially disialyl-T, and downregulation of core fucosylation on N-glycans, detected by specific interactions with Amaranthus caudatus and Aspergillus oryzae lectins, respectively. Upregulation of plasma α-l-fucosidase activity was identified as a biomarker candidate for cardiac hypertrophy, which is expected to support the existing marker, atrial natriuretic peptide and its related peptides. Proteomic analysis identified cysteine and glycine-rich protein 3, a master regulator of cardiac muscle function, as an O-glycosylated protein with altered glycosylation in the rats with cardiac hypertrophy, suggesting that alternations in O-glycosylation affect its oligomerization and function. In conclusion, our data provide evidence of significant changes in glycosylation pattern, specifically mucin-type O-glycosylation and core defucosylation, in the pathogenesis of cardiac hypertrophy and heart failure, suggesting that they are potential biomarkers for these diseases.

Altered glycosylation, expression of serum haptoglobin and alpha-1-antitrypsin in chronic hepatitis C, hepatitis C induced liver cirrhosis and hepatocellular carcinoma patients.

Liver cirrhosis with hepatitis C viral infection (HCV-LC) causes high risk to develop hepatocellular carcinoma (HCC). Besides diagnosis of liver cirrhosis by biochemical test, imaging techniques, assessment of structural liver damage by biopsy shows several disadvantages. Our aim was to monitor the changes in the expression level of serum proteins and their glycosylation pattern among chronic hepatitis C (HCV-CH), HCV-LC and HCC patients with respect to controls. 2D gel electrophoresis of HCV-CH, HCV-LC and HCC patients' sera showed several protein spots, which were identified by LC-MS. The change in the expression of two prominent protein spots, haptoglobin (Hp) and alpha 1-antitrypsin (AAT) was evaluated by western blot and ELISA. The changes in glycosylation pattern of these serum proteins were assayed using different lectins. Increased level of Hp and AAT was observed in HCV-LC and HCC patients' group whereas those were found to be present less in HCV-CH patient groups with respect to control as determined by ELISA using monoclonal antibodies. Decreased level of sialylation in both Hp and AAT was observed in HCV-LC and HCV-CH patients' group whereas increased level of sialylation was observed in HCC patient groups by ELISA using Sambucus nigra agglutinin. On the other hand increased level of fucosylation in two serum glycoproteins was observed in HCV-LC and HCC patients' group using Lens culinarris agglutinin. High glycan branching was found in HCV-LC and HCC patient groups in Hp but not in HCV-CH as determined by Datura stramonium agglutinin. However, there was no such change observed in glycan branching in AAT of HCV-CH and HCV-LC patients' groups, to the contrary high glycan branching was observed in HCC patients' group. Increased level of exposed galactose in both serum proteins was observed in both HCC patients' group as determined by Ricinus communis agglutinin. The present glycoproteomics study could predict the progression of HCV-CH, HCV-LC and HCC without the need of liver biopsy.

Role of N-acetylgalactosaminyltransferase 6 in early tumorigenesis and formation of metastasis.

Glycosylation is one of the most important posttranslational modifications of proteins and lipids that contributes to the structural diversity of cellular molecules. Enzymes of the glycosyltransferase class are responsible for altering glycosylation patterns by adding carbohydrate chains to the respective acceptor molecules. It is well known that glycosylation is commonly altered in cancerous tissue. Therefore, the present study aimed to determine the incidence of N‑acetylgalactosaminyltransferase 6 (GALNT6), a prominent member of the glycosyltransferase class, in breast cancer tissue of different developmental stages by immunohistochemistry. Although no correlation was identified between tumour characteristics and GALNT6 staining intensity, to the best of our knowledge, this is the first study to demonstrate that tissue from carcinoma in situ‑tumours and metastases were more heavily stained than late‑stage breast cancers. This may indicate an important role of glycosylation aberration in escaping the immune system at early phases of tumour development. The present study also hypothesised that nascent or early metastasizing tumours are normally recognized by the immune system of the patient, but glycosylation pattern changes may facilitate tumor escape from immune recognition. In follow‑up studies, our group will aim to confirm and consolidate these results in a larger patient cohort that may give greater insight into breast cancer characterization as well as tumour treatment.