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. 2026 Mar 5;6(3):1481–1487. doi: 10.1021/jacsau.5c01745

A Chemoenzymatic Labeling Strategy for Site-Specific Analysis of Tumor-Associated Sialyl Thomsen–Friedenreich Antigen

Yinping Tian †, Yuqiu Wang ‡,§, Jin Zhang ⊥, Wenjie Wang ⊥, Binbin Li ▽, Hu Zhou ‡,∥,*, Liuqing Wen †,∥,⊥,*
PMCID: PMC13014245  PMID: 41889732

Abstract

Sialyl Thomsen–Friedenreich (ST) antigen, the attachment of trisaccharide Neu5Acα2-3Galβ1-3GalNAc on a serine or threonine residue, is a well-known tumor-associated carbohydrate antigen. There is a growing body of studies indicating that aberrant expression of ST antigen has a strong relationship with tumor invasion and metastasis. However, details about the biological functions of ST antigen are still poorly understood, as there is a lack of an effective method for glycosylation site analysis of the ST antigen. In this study, we developed a chemoenzymatic labeling strategy for the efficient site-specific analysis of ST antigen. A temperature-sensitive probe carried by the CMP-Neu5Ac derivative was designed and synthesized. The probe could be specifically introduced to ST antigen through a one-pot reaction mediated by sialyltransferase ST6GalNAc4. Controlled increase–reduction temperature cycling enabled the effective enrichment of the labeled glycopeptides from complex biological samples. Subsequently, the enriched glycopeptides were released via UV356 irradiation for mass spectrometry analysis, providing the details of where ST antigen attaches to proteins. Finally, this approach was successfully applied to globally profile ST antigen-modified proteins in three breast cancer cell lines: MCF7, MDA-MB-231, and MDA-MB-468.

Keywords: glycosylation, glycoproteome, ST antigen, chemoenzymatic labeling, tumor-associated antigen, breast cancer

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Introduction

Protein glycosylation is one of the most important and complex protein posttranslational modifications. It is well established that abnormal protein glycosylation is associated with a variety of diseases, especially cancer. , Mucin-type O-glycosylation is a major class of protein O-glycosylation initiated by a family of polypeptide N-acetylgalactosaminyltransferases (ppGalNAcTs), which catalyze the transfer of N-acetylgalactosamine (GalNAc) to serine or threonine residues on target proteins, thereby forming the Tn antigen. Subsequently, core 1 β-3-galactosyltransferase (C1GalT1) adds galactose to GalNAc to generate the Thomsen–Friedenreich (T) antigen. Further sialylation of the T antigencatalyzed by human α2,3-sialyltransferase (ST3Gal1)yields the sialyl-T (ST) antigen (Neu5Acα2-3Galβ1-3GalNAcα-O-Ser/Thr). Tn, T, and ST antigens are well-known truncated mucin-type tumor-associated antigens. The aberrant expression of these truncated O-glycan forms is frequently observed during malignant transformation. Emerging evidence indicates that ST3Gal1 expression is tightly regulated. − And, ST antigen synthesized by this enzyme plays a critical role in tumor immune evasion across multiple cancer types. , ST antigen on mucin 1 could modulate the tumor immunological microenvironment through engagement of the sialic acid-binding immunoglobulin-type lectins-9 (Siglec-9). Collectively, these findings suggest that ST antigens can be a potential biomarker for use in immunotherapy and clinical diagnosis. However, the biological roles of ST antigen in a variety of physiological and pathological processes remain unclear, mainly due to the lack of an effective method for site-specific analysis.

Glycoproteomics is a powerful mass spectrometry (MS)-based technology for profiling of protein glycosylation sites. While substantial efforts have been devoted to O-glycoproteomics, − the occurrence of densely clustered O-glycosylation sites continues to hinder efficient proteolytic digestion and confident site localization by MS. Moreover, although numerous studies have enabled global profiling of Tn and T antigens, ,,, site-specific analysis of ST antigen remains limited. In 2018, a chemoenzymatic labeling method was reported for probing of ST antigen using human α2,6 sialyltransferases ST6GaLNAc4. However, the probe is not suitable for glycosylation site analysis. As glycosylation site identification is the prerequisite for the functional study of ST antigen, there is an urgent need for developing an effective probe that could enable the site-specific analysis of ST antigen-modified proteins.

Here, a simple one-step chemoenzymatic labeling method for enrichment and site-specific analysis of the ST antigen was reported (Scheme ). Glycopeptides from cellular proteins were treated with O-glycoprotease from Pseudomonas aeruginosa (IMPA) to generate O-glycopeptides with glycosylation sites at the N-termini. Then, a temperature-sensitive probe carried by a CMP-Neu5Ac derivative is specifically introduced to ST antigen through a one-pot reaction by taking advantage of the excellent relaxed donor specificity of ST6GalNAc4. After enrichment through the temperature changing cycle, the enriched peptides were released with UV light and analyzed using MS. By the described method, we profiled the ST antigen-modified glycoproteome in three breast cancer cell lines including luminal type with low aggressiveness (MCF7 cell line) and triple-negative type with high aggressiveness (MDA-MB-468 and MDA-MB-231 cell lines).

1. Chemoenzymatic Labeling Strategy for the Global Profiling of ST Antigen-Modified Glycoproteome.

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Results and Discussion

Chemoenzymatic Labeling and Enrichment of ST Antigen-Modified Glycopeptide

The chemoenzymatic labeling strategy is a very powerful method for glycoprotein analysis. Monosaccharides or sugar nucleotides bearing bioorthogonal reactive groups are incorporated into target glycans through in vivo metabolic pathways or in vitro enzymatic reactions. Subsequently, biotin- or fluorescent-based probes are conjugated to the labeled glycans via bioorthogonal reactions, enabling detection, visualization, or enrichment. This two-step labeling strategy is easy to operate and has a wide range of applications. − Nevertheless, several studies have demonstrated that one-step labeling approacheswhere biotin or fluorescent moieties are directly introduced via sugar nucleotidesexhibit significantly higher labeling efficiency compared to the two-step method. ,− However, these widely used one-step strategies often require additional enrichment procedures. In this work, a chemoenzymatic labeling strategy is employed to enrich an ST antigen-modified glycopeptide for MS analysis using ST6GalNAc4 (Figure S1) in a one-step manner. It has been reported that ST6GalNAc4 could selectively catalyze the incorporation of biotinylated probes onto the ST antigen.

Accordingly, compound 1 (Figure ), carrying a temperature-sensitive poly­(N-isopropylacrylamide) polymer (PNIPAM) group and a photocleavable linker, was designed to enable simultaneous labeling and enrichment of the ST antigen-modified glycopeptide (4) (Figure A). After intensive washing by changing the temperature as previously described, the enriched glycopeptide could be released with UV irradiation (365 nm). The supernatant of the labeling and enrichment process was collected and monitored using HPLC (Figure B) to detect the progress of the experiment. Besides, a two-step labeling strategy was carried out to detect the enrichment efficiency with probe 2 (Figure A). Glycopeptide 4 was reacted with 2 to produce azido-peptide (6) in the presence of ST6GalNAc4, and then 6 could be captured by 3 and released using UV light. HPLC analysis showed that the one-step enrichment strategy with 1 had a better enrichment efficiency than that of 2 (Figure B and C), which is beneficial for the subsequent steps in the process. The released glycopeptide (7) was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The total ion chromatogram showed a main peak (Figure D). MS indicated that the molecular weight was consistent with that of compound 7 (Figure E). Further MS/MS analysis could identify the potential glycosylation sites (Figure S2). A specific diagnostic ion with a molecular mass of 388.1463 Da was generated upon cleavage of the sialic acid analogues. These results demonstrated the feasibility of the designed strategy to analyze the ST antigen-modified glycopeptide.

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Probes and peptides used in this work.

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HPLC analysis of the enrichment process of the standard ST antigen-modified glycopeptide (4). (A) The chemoenzymatic labeling strategy for capture and release of the standard glycopeptide (4) with a one-step labeling strategy (Probe 1) and a two-step labeling strategy (Probe 2). (B) HPLC analysis of the capture and release process of the standard glycopeptide (4) by a one-step labeling strategy. (C) HPLC analysis of the capture and release process of the standard glycopeptide (4) by a two-step labeling strategy. (D) The total ion chromatogram of the released glycopeptide (7) from the one-step labeling strategy. (E) MS spectrum of the released glycopeptide (7) from the one-step labeling strategy, *the released glycopeptide.

Chemoenzymatic Labeling of ST Antigen-Modified Proteins on Cell Surfaces

Sialylation is a critical property of cancer cells and is involved in several pathophysiological processes. , T antigen, which is highly expressed in many human cancers, , could serve as a substrate for biosynthesis of ST antigen catalyzed by ST3Gal1. To investigate the glycan form on the cell surfaces, three cell lines were labeled using ST3Gal1 and biotinylated probe 8. Sialidase from Streptococcus pneumoniae (NanA), a broad specificity sialidase, was used to remove terminal sialic acid (Neu5Ac) from cell surfaces. The labeling signals were analyzed using SDS-PAGE and Western blot (Figure S3). The experimental group with NanA treatment showed an obvious biotin signal, while the experimental group without NanA had almost no signal. Consistent results were observed across all three cell lines. These results indicated that sialylated-T antigen, not T antigen, was the main glycoform on cell surfaces. These results are consistent with the previous reports, as glycoproteins were highly sialylated on cancer cells. ,

In addition, the expression of ST antigen on different cell lines, including MDA-MB-468, HCT116, HepG2, and H1299, was detected using ST6GalNAc4 and 8. The experimental groups from four cell lines showed strong biotin signals, and the breast cancer cell line MDA-MB-468 showed the best labeling signal (Figure S4). Furthermore, three breast cancer cell lines, including luminal type (MCF7) with low aggressiveness and triple-negative type with high aggressiveness (MDA-MB-468 and MDA-MB-231), were chosen to analyze the expression levels of ST antigen. Western blot results showed that the relative experimental groups from three cell lines showed obvious labeling signals compared to the control groups (Figure A). The expression level of ST antigen follows the order MDA-MB-468 > MCF7 > MDA-MB-231. The expression level was further quantified using flow cytometry (Figure S5). The relative intensity was determined as the ratio of the fluorescence value in the experimental group to that in the control group for each cell line. The results demonstrated that the expression level of ST antigen in MDA-MB-468 cells was 6- to 18-fold higher than those in the other two cell lines (Figure B). Furthermore, fluorescence imaging experiments were performed, revealing membrane-associated fluorescence signals in the enzymatic treatment groups across all three cell lines (Figure C). No fluorescence intensity was detected in the control groups without ST6GalNAc4, confirming the specificity of the chemoenzymatic labeling of ST antigen on the cell surfaces.

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Analysis of ST antigen in three breast cancer cell lines. (A) Western blot analysis of ST antigen. (B) Flow cytometry analysis of the expression levels of the ST antigen. The relative intensity was calculated as the ratio of the fluorescence intensity in the experimental group to that in the control group for each cell line. (C) Imaging of ST antigen on cell surfaces by fluorescence microscopy. Scale bar: 20 μm.

ST Antigen-Modified Glycoproteome Analysis with the Chemoenzymatic Labeling Strategy

Proteins from MDA-MB-468, MDA-MB-231, and MCF7 cells were collected and digested with trypsin. The resulting peptides were treated with peptide-N-glycosidase F from Flavobacterium meningosepticum (PNGF) to remove N-glycans. Recently, it was reported that an O-glycoprotease IMPA could hydrolyzes Tn, T, and ST antigen-modified structures. This hydrolysis generates simplified O-glycopeptides with glycosylation sites positioned at their N-termini, thereby facilitating the site-specific annotation analysis of such clustered modifications. Therefore, IMPA was added to simple mucin-type glycan cluster. The treated peptides were labeled using ST6GalNAc4 and 1. After washing, the enriched glycopeptides were irradiated with UV365 light for 20 min in an ice bath. The released peptides were collected, purified, and analyzed using LC-MS/MS (Scheme ) with a higher-energy collisional dissociation (HCD) fragmentation method.

After the raw data were searched with MaxQuant, a total of 321 O-glycosylated peptide matches (O-PSMs) were identified. As IMPA was used to assist site annotation, glycosites assigned to the first amino acid residue and supported by the presence of the specific diagnostic ion at 388.1463 Da were regarded as high-confidence glycosites. In total, 33, 10, and 56 ST antigen-modified sites were identified in MCF7, MDA-MB-231, and MDA-MB-468 cell lines, respectively (Figure A and Table S1). This result is consistent with the findings from Western blot and flow cytometry. In our work, ST antigen-modified CD44, an E/L-selectin ligand, , was detected exclusively in MDA-MB-468 cells, suggesting a potential role in promoting their proliferation. However, further investigation is needed to clarify the precise biological functions of the ST antigen on CD44. To better understand the potential biological functions of ST antigens, we further analyzed the motif and function of ST antigen-modified sites. Combined with the data sets of MCF7, MDA-MB-231, and MDA-MB-468, a total of 78 ST antigen-modified sites corresponding to 61 proteins were identified. The identified ST antigen-modified sites lacked a consensus motif, consistent with previous reports (Figure B). , Moreover, proline residues were highly enriched in the vicinity of the identified ST antigen-modified glycopeptides. This result is consistent with the previous reports that proline residues near glycosylation sites can enhance the activity of ppGalNAcTs. Among the identified 61 ST antigen-modified proteins, 18% had more than one glycosite (Figure C). Gene ontology analysis results showed that the identified ST antigen-modified proteins were mainly enriched in the membrane and plasma membrane, the biological process of protein glycosylation and receptor-mediated endocytosis, and the molecular function of calcium ion binding and low-density lipoprotein particle receptor activity (Figure D). Reactome pathway analysis indicated that they were mainly associated with trans-golgi network vesicle budding and regulation of insulin-like growth factor (IGF) transport and uptake by insulin-like growth factor binding proteins (IGFBPs) (Figure S6).

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ST antigen-modified glycoproteome identified in MCF7, MDA-MB-231, and MDA-MB-468 cells. (A) The identified ST antigen-modified sites in the three cell lines. (B) Motif alignment of amino acids adjacent to ST antigen-modified sites by WebLogo 3. (C) The distribution of the ST antigen-modified sites. (D) The clustering of the identified ST antigen-modified proteins based on the biological process, cellular component, and molecular function.

As electron-transfer/higher-energy collisional dissociation (EThcD) has been reported to provide high accuracy for the localization of the O-glycosylation site, its performance was evaluated and compared with that of HCD. Accordingly, the enriched and released peptides from MDA-MB-468 were equally divided and analyzed by LC-MS/MS using HCD and EThcD, respectively. Under identical search parameters, a total of 269 and 161 O-PSMs were identified using HCD and EThcD. Among these, glycosites at the first amino acid with a specific diagnostic ion were regarded to be high-confidence glycosites (78 sites for HCD, 41 sites for EThcD, Tables S2, S3). O-Glycoprotease IMPA digestion typically generates relatively short glycopeptides, and the ETD-based fragmentation method is generally more effective for longer peptides with higher charge states. Thus, the lower number of sites identified by EThcD in our data set is consistent with these technical considerations. These findings indicate that EThcD-MS/MS can be applied for profiling ST antigen-modified glycopeptides. However, it did not demonstrate superior performance under the current experimental conditions.

Conclusion

It is well-known that truncated mucin-type O-glycosylation, including Tn, T, and ST antigens, has a strong relationship with many diseases, especially cancers. Although extensive reports have been devoted to global profiling of the Tn and T antigens, site-specific analysis of the ST antigen remains limited due to the lack of effective analytical tools. Therefore, the biological functions that the ST antigen played in living cells are still poorly understood in comparison with Tn an T antigens. In this work, we found that human sialyltransferase ST6GalNAc4 possesses excellent relaxed donor specificity and could label ST antigen specifically with a polymer-modified probe. The labeled glycopeptides could be directly enriched through increased–reduced temperature cycling without the requirement of additional enrichment support. The enriched glycopeptides were released by UV light and analyzed using MS technology, providing details of where the ST antigen attaches to proteins. We demonstrated the feasibility of the strategy in many means, and the strategy was employed to profile ST antigen glycoproteomes from three breast cancer cell lines with low and high aggressiveness. We found that MDA-MB-468 with high aggressiveness possesses a high expression level of ST antigen, and more ST antigen-modified sites were identified in this cell line. Overall, this work provides a powerful tool for site-specific profiling of ST antigen. We believe that this work will accelerate the investigation of the biological functions of ST antigens in living cells.

Supplementary Material

au5c01745_si_001.pdf (647.3KB, pdf)
au5c01745_si_002.xlsx (28KB, xlsx)

Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (22307127, 22577143, 22425703), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB1060000, XDB0830000), the Talent Plan of Shanghai Branch, Chinese Academy of Sciences (CASSHB-QNPD-2023-018), State Key Laboratory of Chemical Biology (SKLCB-2025-04), and the Shanghai Municipal Science and Technology Major Project.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacsau.5c01745.

  • Figures, material, cell culture and enzyme preparation, experimental methods, spectra, tables (PDF)

  • Tables of high-confidence ST antigen-modified sites identified in MCF7, MDA-MB-231, and MDA-MB-468; MDA-MB-468 with HCD; and MDA-MB-468 with EThcD (XLSX)

#.

Y.T. and Y.W. contributed equally to this work.

The authors declare no competing financial interest.

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au5c01745_si_001.pdf (647.3KB, pdf)
au5c01745_si_002.xlsx (28KB, xlsx)

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