Synthesis of Type-I and Type-II LacNAc-Repeating Oligosaccharides as the Backbones of Tumor-Associated Lewis Antigens

Phang, Riping; Lin, Chun‐Hung

doi:10.3389/fimmu.2022.858894

Cited by 9 publications

(8 citation statements)

References 80 publications

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“…Structurally, SLeX is a terminal tetrasaccharide attached to proteins and/or lipids present at the cell surfaces or secreted. This tetrasaccharide is composed of a fucose α1,3 linked to N-Acetylglucosamine and a sialic acid α2,3 linked to a galactose, both in type II LacNac chains [30]. The terminal addition of sialic acid in SLeX glycans is described to be performed by members of the ST3Gal family, speci cally by ST3GalIII, ST3GalIV, and ST3GalVI.…”

Section: Discussionmentioning

confidence: 99%

ST3GalIV drives SLeX biosynthesis in gastrointestinal cancer cells and associates with cancer cell motility

Costa

Senra

Teixeira

et al. 2022

Preprint

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Expression of sialyl Lewis X (SLeX) is a well-documented event during malignant transformation of cancer cells, and largely associates with their invasive and metastatic properties. Glycoproteins and glycolipids are the main carriers of SLeX, whose biosynthesis is known to be performed by different glycosyltransferases, namely by the family of β-galactoside-α2,3-sialyltransferases (ST3Gals). In this study, we sought to elucidate the role of ST3GalIV in the biosynthesis of SLeX and in malignant properties of gastrointestinal (GI) cancer cells. By immunofluorescent screening, we selected SLeX-positive GI cancer cell lines and silenced ST3GalIV expression via CRISPR/Cas9. Immunofluorescence and western blot analysis showed that ST3GalIV KO efficiently impaired SLeX expression in most cancer cell lines, with the exception of the colon cancer cell line LS174T. The impact of ST3GalIV KO in the biosynthesis of SLeX isomer SLeA and non sialylated Lewis X and A were also evaluated and overall, ST3GalIV KO led to a decreased expression of SLeA and an increased expression in both LeX and LeA. In addition, the abrogation of SLeX on GI cancer cells led to a reduction in cell motility. Furthermore, ST3GalVI KO was performed in LS174T ST3GalIV KO cells, resulting in the complete abolishment of SLeX expression and consequent reduced motility capacity of those cells. Overall, these findings portray ST3GalIV as the main, but not the only, enzyme driving the biosynthesis of SLeX in GI cancer cells, with a functional impact on cancer cell motility.

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Section: Discussionmentioning

confidence: 99%

ST3GalIV drives SLeX biosynthesis in gastrointestinal cancer cells and associates with cancer cell motility

Costa

Senra

Teixeira

et al. 2022

Preprint

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show abstract

“…Furthermore, all sialylated glycans were found to be fucosylated. Glycan forms with more than six N-Acetylglucosamines and more than four galactoses are indicated as lactosamin-repeats (LacNAc-repeats) [ 25 , 26 , 27 , 28 ], which were exclusively detected at position N119 and slightly more present at cells cultured at 32 °C. The corresponding mass spectra ( Supplementary Figure S4 ) and a complete list of all found glycans ( Supplementary Table S1 ), including glycans with N-glycolylneuraminic acid (Neu5Gc), which showed a prevalence <4.5%, are represented in the supplements.…”

Section: Resultsmentioning

confidence: 99%

Recombinant Human CD19 in CHO-K1 Cells: Glycosylation Patterns as a Quality Attribute of High Yield Processes

Billerhart,

Hunjadi,

Hawlin

et al. 2023

IJMS

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CD19 is an essential protein in personalized CD19-targeting chimeric antigen receptor (CAR)-T cell-based cancer immunotherapies and CAR-T cell functionality evaluation. However, the recombinant expression of this “difficult to-express” (DTE) protein is challenging, and therefore, commercial access to the protein is limited. We have previously described the successful stable expression of our soluble CD19-AD2 fusion protein of the CD19 extracellular part fused with human serum albumin domain 2 (AD2) in CHO-K1 cells. The function, stability, and secretion rate of DTE proteins can be improved by culture conditions, such as reduced temperature and a shorter residence time. Moreover, glycosylation, as one of the most important post-translational modifications, represents a critical quality attribute potentially affecting CAR-T cell effector function and thus impacting therapy’s success. In this study, we increased the production rate of CD19-AD2 by 3.5-fold through applying hypothermic culture conditions. We efficiently improved the purification of our his-tagged CD19-AD2 fusion protein via a Ni-NTA-based affinity column using a stepwise increase in the imidazole concentration. The binding affinity to commercially available anti-CD19 antibodies was evaluated via Bio-Layer Interferometry (BLI). Furthermore, we revealed glycosylation patterns via Electrospray Ionization Mass Spectrometry (ESI–MS), and five highly sialylated and multi-antennary N-glycosylation sites were identified. In summary, we optimized the CD19-AD2 production and purification process and were the first to characterize five highly complex N-glycosylation sites.

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“…Structurally, SLeX is a terminal tetrasaccharide attached to proteins and/or lipids present at the cell surfaces or secreted ones. This tetrasaccharide is composed of a fucose α1,3 linked to N-Acetylglucosamine and a sialic acid α2,3 linked to a galactose, both in type II LacNac chains [ 30 ]. The terminal addition of sialic acid in SLeX glycans is described to be performed by members of the ST3Gal family, specifically by ST3GalIII, ST3GalIV, and ST3GalVI.…”

Section: Discussionmentioning

confidence: 99%

ST3GalIV drives SLeX biosynthesis in gastrointestinal cancer cells and associates with cancer cell motility

et al. 2023

View full text Add to dashboard Cite

Expression of sialyl Lewis X (SLeX) is a well-documented event during malignant transformation of cancer cells, and largely associates with their invasive and metastatic properties. Glycoproteins and glycolipids are the main carriers of SLeX, whose biosynthesis is known to be performed by different glycosyltransferases, namely by the family of β-galactoside-α2,3-sialyltransferases (ST3Gals). In this study, we sought to elucidate the role of ST3GalIV in the biosynthesis of SLeX and in malignant properties of gastrointestinal (GI) cancer cells. By immunofluorescent screening, we selected SLeX-positive GI cancer cell lines and silenced ST3GalIV expression via CRISPR/Cas9. Flow cytometry, immunofluorescence and western blot analysis showed that ST3GalIV KO efficiently impaired SLeX expression in most cancer cell lines, with the exception of the colon cancer cell line LS174T. The impact of ST3GalIV KO in the biosynthesis of SLeX isomer SLeA and non sialylated Lewis X and A were also evaluated and overall, ST3GalIV KO led to a decreased expression of SLeA and an increased expression in both LeX and LeA. In addition, the abrogation of SLeX on GI cancer cells led to a reduction in cell motility. Furthermore, ST3GalVI KO was performed in LS174T ST3GalIV KO cells, resulting in the complete abolishment of SLeX expression and consequent reduced motility capacity of those cells. Overall, these findings portray ST3GalIV as the main, but not the only, enzyme driving the biosynthesis of SLeX in GI cancer cells, with a functional impact on cancer cell motility.

show abstract

Synthesis of Type-I and Type-II LacNAc-Repeating Oligosaccharides as the Backbones of Tumor-Associated Lewis Antigens

Cited by 9 publications

References 80 publications

ST3GalIV drives SLeX biosynthesis in gastrointestinal cancer cells and associates with cancer cell motility

ST3GalIV drives SLeX biosynthesis in gastrointestinal cancer cells and associates with cancer cell motility

Recombinant Human CD19 in CHO-K1 Cells: Glycosylation Patterns as a Quality Attribute of High Yield Processes

ST3GalIV drives SLeX biosynthesis in gastrointestinal cancer cells and associates with cancer cell motility

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