A cancer-unique glycan: de-N-acetyl polysialic acid (dPSA) linked to cell surface nucleolin depends on re-expression of the fetal polysialyltransferase ST8SIA2 gene

Gregory R Moe,Lindsay M Steirer,Alejandro D Bolanos,Joshua A Lee,Adarsha Shivakumar

doi:10.1186/s13046-021-02099-y

Gregory R Moe, Lindsay M Steirer + Show 3 more

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https://doi.org/10.1186/s13046-021-02099-y

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Abstract

BackgroundPolysialic acid (polySia) modifies six cell surface proteins in humans mainly during fetal development and some blood cells in adults. Two genes in humans, ST8SIA2 and ST8SIA4, code for polysialyltransferases that synthesize polySia. ST8SIA2 is highly expressed during fetal development and in cancer but not in adult normal human cells. ST8SIA4 is expressed in fetal and adult brain, spleen, thymus, and peripheral blood leukocytes and in cancer. We identified a derivative of polySia containing de-N-acetyl neuraminic acid residues (dPSA), which is expressed on the cell surface of human cancer cell lines and tumors but not normal cells.MethodsdPSA-modified proteins in several human cancer cell lines and normal blood cells were identified using co-immunoprecipitation with anti-dPSA antibodies, mass spectroscopy and Western blot. RNAi and CRISPR were used to knockdown and knockout, respectively, the polysialyltransferase genes in human melanoma SK-MEL-28 and neuroblastoma CHP-134 cell lines, respectively, to determine the effect on production of cell surface dPSA measured by flow cytometry and fluorescence microscopy.ResultsWe found that dPSA is linked to or associated with nucleolin, a nuclear protein reported to be on the cell surface of cancer but not normal cells. Knocking down expression of ST8SIA2 with RNAi or knocking out each gene individually and in combination using CRISPR showed that cell surface dPSA depended on expression of ST8SIA2.ConclusionsThe presence of dPSA specifically in a broad range of human cancers but not human adult normal cells offers novel possibilities for diagnosis, prevention and treatment targeting the dPSA antigen that appears to be cancer-specific, consistent across not only human cancers but also species, and may be an unrecognized mechanism of immune shielding.

Highlights

Polysialic acid modifies six cell surface proteins in humans mainly during fetal development and some blood cells in adults
SEAM 2 has more than 100 times greater avidity for the N-Pr Polysialic acid (polySia) de-Nacetylated form of polySia (dPSA) antigen than dPSA containing N-acetyl sialic acid residues based on an inhibition ELISA [19]
Membrane-associated dPSA-nucleolin is distinct from nuclear nucleolin we looked at staining patterns inside cells by treating fixed SK-MEL-28 cells with the detergent Triton X100 to permeabilize them and allow entry of primary and secondary antibodies. dPSA inside cells exhibited a Effect on dPSA and cell surface nucleolin of knocking down expression of ST8SIA2 with small interfering RNA (siRNA) in human SKMEL-28 cells SK-MEL-28 cells express both ST8SIA2 and ST8SIA4 (Fig. 3a)

Summary

Introduction

Polysialic acid (polySia) modifies six cell surface proteins in humans mainly during fetal development and some blood cells in adults. A number of human cancers are reported to express polySia-NCAM abnormally [11,12,13,14] where its role in mediating interactions among cells and between cells and the extracellular matrix is associated with metastasis and poor clinical prognosis [11, 14]. Both polysialyltransferases ST8SIA2 and ST8SIA4 appear to synthesize the same polysaccharide, there may be differences in substrate specificity [15, 16] or functional activity.

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