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Published on: October 20, 2023
Identification and functional characterization of a human GalNAc [alpha]2,6-sialyltransferase with altered expression
Georgia Sotiropoulou1, Mari Kono, Anthony Anisowicz
1Department of Pharmacy, School of Health Sciences, university of Patras, Greece. G.Sotiropoulou@upatras.gr
Background:
We sought to identify genes with altered expression during human breast cancer progression by applying mRNA comparisons of normal and tumor mammary cell lines with increasingly malignant phenotypes. The gene encoding a new sialyltransferase (STM) was found to be down-regulated in tumor cells. Abnormal expression and enzymatic activities of sialyltransferases in tumor cells result in the formation of tumor-associated carbohydrate antigens that can be used for the better understanding of the disease process and are applied for tumor diagnosis and immunotherapy. Altered glycosylation patterns of the MUC1 mucin, in particular, is a target antigen for immunotherapy of breast and other cancers.
Materials And Methods:
Total RNAs from multiple normal mammary epithelial cell strains and tumor cell lines were compared by differential display and the differential expression of selected cDNAs was confirmed by Northern analyses. Recombinant STM was expressed in COS-7 cells. The substrate and linkage specificity of STM was examined using various oligosaccharides and O-glycosylated proteins as acceptor substrates. The chromosomal localization of the SIATL1 gene was assigned by somatic cell hybrid analysis.
Results:
A human sialyltransferase gene was identified by differential display as being down-regulated in breast tumor cell lines as compared to normal mammary epithelial cell strains, and the corresponding full-length cDNA (stm) was cloned. The encoded protein of 374 amino acid residues contained the L- and S-sialylmotifs, two catalytic regions conserved in all functional sialyltransferases. Recombinant STM is an active GalNAc alpha2,6-sialyltransferase with Gal beta 1,3 GalNAc-O-Ser/Thr and (+/- Neu5Ac alpha 2,3) Gal beta 1,3GalNAc-O-Ser/Thr acceptor specificity. The SIATL1 gene, encoding STM, was mapped to the long arm of human chromosome 17 at q23-qter, a region that is nonrandomly deleted in human breast cancers. However, Southern analyses indicated that SIATL1 is usually not grossly rearranged in breast tumors. Northern analyses showed that the gene was widely expressed in normal human tissues, as well as in normal breast and prostate epithelial cell lines, but significantly down-regulated or absent in corresponding tumor cell lines.
Conclusions:
Our findings suggest that aberrant expression of STM sialyltransferase in tumors could be a feature of the malignant phenotype. In breast cancers, the MUC1 mucin is overexpressed and contains shorter O-glycans as compared to the normal mucin. Because STM catalyzes the synthesis of O-glycans, cloning and characterization of its substrate specificity will contribute to the understanding of the molecular mechanisms underlying the aberrant glycosylation patterns of O-glycans and the formation of mucin-related antigens in human breast cancers.
Insights
A novel sialyltransferase (STM) gene was found to be down-regulated in breast tumor cells, impacting O-glycan synthesis. This discovery aids understanding of aberrant glycosylation in breast cancer progression and MUC1 antigen formation.
Area of Science:
- Molecular Biology
- Cancer Research
- Glycobiology
Background:
- Breast cancer progression involves altered gene expression in mammary cells.
- Sialyltransferases (STM) play a role in forming tumor-associated carbohydrate antigens.
- Aberrant glycosylation of MUC1 mucin is a target for breast cancer immunotherapy.
Purpose of the Study:
- To identify genes with altered expression during human breast cancer progression.
- To clone and characterize a novel sialyltransferase (STM) gene and its encoded protein.
- To investigate the role of STM in aberrant glycosylation patterns in breast cancer.
Main Methods:
- Differential display mRNA analysis comparing normal and tumor mammary cell lines.
- Northern blot analysis for gene expression confirmation.
- Recombinant protein expression and enzymatic activity assays.
- Somatic cell hybrid analysis for gene localization.
Main Results:
- A novel human sialyltransferase gene (SIATL1), encoding STM, was identified and cloned.
- STM was found to be significantly down-regulated or absent in breast tumor cell lines compared to normal cells.
- Recombinant STM functions as a GalNAc alpha2,6-sialyltransferase with specific acceptor substrate preferences.
- The SIATL1 gene was mapped to chromosome 17q23-qter, a region frequently deleted in breast cancers.
Conclusions:
- Aberrant expression of STM sialyltransferase may be a feature of the malignant phenotype in breast cancer.
- STM's role in O-glycan synthesis contributes to understanding aberrant glycosylation of MUC1 mucin.
- Characterization of STM provides insights into molecular mechanisms of tumor-associated antigen formation for diagnosis and immunotherapy.