Paucimannosidic glycoepitopes inhibit tumorigenic processes in glioblastoma multiforme

Abstract

Glioblastoma multiforme is an aggressive cancer type with poor patient outcomes. Interestingly, we reported previously a novel association between the little studied paucimannosidic N -linked glycoepitope and glioblastoma. Paucimannose has only recently been detected in vertebrates where it exhibits a very restricted tumor-specific expression. Herein, we demonstrate for the first time a very high protein paucimannosylation in human grade IV glioblastoma and U-87MG and U-138MG glioblastoma cells. Furthermore, we revealed the involvement of paucimannosidic epitopes in tumorigenic processes including cell proliferation, migration, invasion and adhesion. Finally, we identified AHNAK which is discussed as a tumor suppressor as the first paucimannose-carrying protein in glioblastoma and show the involvement of AHNAK in the observed paucimannose-dependent effects. This study is the first to provide evidence of a protective role of paucimannosylation in glioblastoma, a relationship that with further in vivo support may have far reaching benefits for patients suffering from this often fatal disease.

Protein Paucimannosylation Is an Enriched N‐Glycosylation Signature of Human Cancers

“…Similar cancer cell surface expression of PMGs was detected for a related human glioblastoma cell line (U138MG) (data not shown). Immunofluorescence experiments conducted with these and other human cancer cell lines using membrane permeabilization prior to Mannitou staining have previously indicated that paucimannosidic glycoepitopes are also located intracellularly …”

“…To assess the cancer cell surface expression of paucimannosidic glyco‐epitopes, cultured non‐permeabilized human glioblastoma cells (U87MG and U138MG) were fixed on 0.002% w/v poly‐ l ‐lysine‐coated microscopy glass slides with 4% v/v paraformaldehyde for 20 min. To prevent nonspecific antibody recognition, the fixed cells were treated with a blocking solution containing 5% v/v horse serum in PBS for 20 min, incubated with the PMG‐recognizing mouse IgM “Mannitou” (undiluted supernatant from hybridoma cells for 30 min at room temperature, and then incubated with a goat anti‐mouse IgM Cy2‐conjugated IgG antibody for 30 min in the dark (1:500 dilution, Dianova, Hamburg, Germany) . The immunofluorescence of the cells was determined using a Zeiss Axioplan2 Apotome system equipped with a Plan Apochromat 63×/1.40 oil immersion objective (Zeiss, Oberkochen, Germany) as recently described …”

“…Glycoproteomics and intact glycoprotein analysis are examples of analytical methods required to adequately address this question. However, several lines of evidence indicate that the PMGs reported herein are, at least in part, carried by fully processed non‐degraded proteins, that is, i) the presence of paucimannosidic glycoepitopes on the surface of cancer cells, ii) the PMGs were identified from the proteinaceous fractions obtained using classical protein extraction methods (MF, WCL, S) from biological specimens, iii) the PMGs were detected after PNGase F treatment following immobilization of the proteinaceous fractions on hydrophobic PVDF membranes, and iv) our recent detection of PMGs decorating a non‐degraded form of the very large (628 kDa) neuroblast differentiation‐associated protein (UniProtKB: Q09666) from human glioblastoma cells . Further, intact glycopeptides carrying PMGs have previously been identified for several cancer cell lines (e.g., THP‐1) and tumor tissues (e.g., PCa, unpublished observations) building further support for a protein‐conjugation of the reported PMGs.…”

Human protein paucimannosylation: cues from the eukaryotic kingdoms

“…Recent glycomics and glycoproteomics‐centric studies have demonstrated that PMPs are also elevated glycosylation signatures associated with various human cancers including those of ovarian (Everest‐Dass et al ., ; Chen et al ., ), colorectal (Joosten et al ., ; Balog et al ., ; Sethi et al ., ; Kaprio et al ., ; Holst et al ., ), breast (Lee et al ., ; Chen et al ., ), lung (Hua et al ., ; Ruhaak et al ., ; Wang et al ., ), paraganglioma (Leijon et al ., ), glioblastoma multiforme (Becker et al ., ), skin (Moginger et al ., ) and prostate (Shah et al ., ) origin. By systematically interrogating a very large collection of nearly 500 N ‐glycomics LC‐MS/MS data sets obtained from 11 cancer types and subtypes, we have obtained solid evidence that PMGs are significant features of human cancers and, at least in part, expressed on cancer cell surfaces (S. Chatterjee, L. Y. Lee, R. Kawahara, J. L. Abrahams, B. Adamczyk, M. Anugraham, C. Ashwood, Z. Sumer‐Bayraktar, M. T. Briggs, J. H. L. Chik, A. Everest‐Dass, S. Förster, H. Hinneburg, K. R. M. Leite, I. Loke, U. Möginger, E. S. X. Moh, M. Nakano, S. Recuero, M. K. Sethi, M. Srougi, K. Stavenhagen, V. Venkatakrishnan, K. Wongtrakul‐Kish, S. Diestel, P. Hoffmann, N. G. Karlsson, D. Kolarich, M. P. Molloy, M. H. Muders, M. K. Oehler, N. H. Packer, G. Palmisano & M. Thaysen‐Andersen, in preparation).…”

Increased Expression of Immature Mannose-Containing Glycoproteins and Sialic Acid in Aged Mouse Brains

“…Paucimannose represents a relatively simple structure containing N-acetylglucosamines and mannoses (see Figure 1). This epitope per se is involved in immune function [22] and has recently been shown to interfere with brain tumor progression [23]. Since paucimannose was strongly associated with specific molecular weights in Western blot analysis, we tried to identify carrier proteins of the upregulated paucimannose in aged brains.…”