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Differences in the sialylation patterns of membrane stress proteins in chemical carcinogen-induced tumors developed in BALB/c and IL-1α deficient mice

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Abstract

We evaluated the patterns of sialylation on fibrosarcoma cell lines arising following 3-methylcholanthrene treatments of wild-type and IL-1α-deficient mice; the former induced progressive tumors, whereas the latter cell lines induced regressing tumors or failed to develop into tumors in mice due to immune rejection. In regressing tumors, terminating α2-6-Neu5Ac residues were present at lower levels than in progressively growing tumors. In both tumor cells, the amount of α2-6-Neu5Ac residues was higher by an order of magnitude relative to the amount expressed in primary fibroblasts harvested from IL-1α-deficient and wild-type mice. We focused on membrane proteins, which may interact with the immune system. Interestingly, HSP65, grp75, and gp96 were found on the surfaces of malignant cells and were shown to possess sialylated N-glycans. The amount of trisialylated glycans on gp96 and HSP65 and monosialylated glycans on grp75 of regressing cells was significantly lower than in progressively growing cells, suggesting a dependency of these specific glycoforms on anti-tumor immunity.

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Abbreviations

DDW:

double distilled water

Gal:

galactose

GlcNAc:

N-acetylglucosamine

gp96:

96 kDa glycoprotein

grp:

glucose regulated protein

HPLC:

high performance liquid chromatography

HSP:

heat shock protein

IFN:

interferon

IL-1:

interleukin 1

Man:

mannose

3-MCA:

3-methylcholanthrene

MHC:

major histocompatibility class

Mw:

molecular weight

Neu5Ac:

N-acetylneuraminic acid

NK:

natural killer

NKT:

natural killer T

pI:

isoelectric point

PNGase-F:

N-glycosidase F

WT:

wild-type

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Acknowledgements

The authors thank Dr. Uri Abdu for his help in carrying out fluorescent staining and microscopic imaging and Dr. Yoram Tekoah for helpful advice on glycan analysis by HPLC. This work was supported in part by the Israel Cancer Association and by the Israeli Glycobiology Center of Ben-Gurion University of the Negev, Israel.

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Authors and Affiliations

  1. Department of Biotechnology Engineering, Faculty of Engineering and the Israeli Center for Glycobiology, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel

    Avi Avidan, Michal Perlmutter, Smadar Tal, Omer Oraki, Tsachi Kapp & Rachel G. Lichtenstein

  2. Department of Microbiology and Immunology, Faculty of Health Sciences and The Cancer Research Center, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel

    Yacov Krelin, Moshe Elkabets, Shahar Dotan & Ron N. Apte

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  1. Avi Avidan
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Correspondence to Rachel G. Lichtenstein.

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This study is dedicated to the memory of Professor Shraga Segal

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Supplementary Table 1

List of proteins from membrane fraction of regressive and progressive fibrosarcoma cell lines developed in IL1α-/- and WT mice identified by MS-Tag search. (PDF 103 kb)

Supplementary Figure 1

Flow cytometry of regressive immunogenic and progressive fibrosarcoma cell lines derived from 3-MCA-injected IL-1α-/- and BALB/c mice before (left column) and after neuraminidase treatment (right column). Cells (6 × 106) were incubated with 10 mU of Arthrobacter ureafaciens neuraminidase, (EC 3.2.1.18, Calbiochem, San Diego, CA) in sodium phosphate buffer, pH = 6.0, for 1 h at 37°C, washed, divided into tubes, and then incubated similar to the untreated cells with varying fluorescent lectins, as described in Materials and Methods. (PDF 280 kb)

Supplementary Figure 2

Immunoblotting of EGFR using anti-mouse EGFR antibody at a concentration of 1 μg/ml per blot. EGFR was extracted from regressive immunogenic and progressive fibrosarcoma cell membranes using a ProteoExtract kit (Calbiochem, San Diego, CA). The immunoblotting of EGFR was performed as described for the immunoblotting of HSPs in the Materials and Methods section. (PDF 91 kb)

Supplementary Figure 3

Immunocytometry of HSP65 (a), grp75 (b), grp78 (c), and fluorescent staining for nuclei and actin in each set is shown. Cells (1 × 104) derived from either 3-MCA-induced fibrosarcoma cells injected into IL-1α-/- and BALB/c mice or from primary fibroblast cells derived from the skin of IL-1α-/- and BALB/c. (PDF 342 kb)

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Avidan, A., Perlmutter, M., Tal, S. et al. Differences in the sialylation patterns of membrane stress proteins in chemical carcinogen-induced tumors developed in BALB/c and IL-1α deficient mice. Glycoconj J 26, 1181–1195 (2009). https://doi.org/10.1007/s10719-009-9238-9

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