GalNAc glycoprotein expression by breast cell lines, primary breast cancer and normal breast epithelial membrane

Abstract

Summary Over-expression of N-acetylgalactosamine glycoproteins as detected by binding of the lectin from Helix pomatia (HPA), is associated with metastatic competence and poor patient prognosis in a range of human adenocarcinomas. These glycoproteins remain poorly characterised, and their functional role has yet to be elucidated. This study describes characterisation of a range of human breast/breast cancer cell lines for the expression of the N-acetylgalactosaminylated glycoproteins of interest, and their comparison with normal breast epithelium and a range of clinical breast carcinoma samples. Confocal and light microscopy studies revealed cytochemical HPA-binding patterns consistent with a fundamental disruption in normal glycobiosynthetic pathways attending increasing metastatic potential. We report the most complete comparative analysis of HPA-binding ligands from cultured breast cells, clinical breast carcinoma samples and normal breast epithelium to date. Lectin blotting identified 11 major HPA-binding glycoprotein bands common to both clinical tumour samples and breast cell lines and 6 of these bands were also expressed by samples of normal breast epithelium, albeit at much lower levels. Moreover, very marked quantitative but not qualitative differences in levels of expression consistent with metastatic capability were noted.

Cell surface protein glycosylation in cancer

“…Investigation of glycan expression can be targeted (lectin based or antibody based) or non-targeted (MS based) (reviewed in [25]). Plant lectins which recognise specific glycan motifs are the most commonly exploited tool (reviewed in [26]) and have been used in several ways to detect differential cell surface glycan expression; such as lectin histochemical staining [27][28][29][30][31], lectin affinity chromatography [32][33][34], lectin blotting [33,[35][36][37][38], and lectin array [39]. The utility [42, 84, 195-197, 199, 211] Pauci-mannose type [19,20,23,192,193] Other LacdiNAc of lectins has been clearly demonstrated by their ability to identify the proteins that carry a tumour-specific glycan change.…”

Primary Breast Cancer Tumours Contain High Amounts of IgA1 Immunoglobulin: An Immunohistochemical Analysis of a Possible Carrier of the Tumour-Associated Tn Antigen

“…The majority of all carcinomas, 80–90%, are positive for the Tn antigen as defined by the lectin HPA. Furthermore, up-regulation of the Tn antigen in tumours is associated with poor prognosis [3], [5], [6], [7]. Previously HPA affinity chromatography of a number of solubilised breast cancer tumours followed by SDS-PAGE and peptide sequencing have identified a major Tn-carrying 55 kDa protein in breast cancer metastatic tissue lysate as the heavy chain of IgA1 [8].…”

Differential Expression of Sialic Acid and N-acetylgalactosamine Residues on the Cell Surface of Intestinal Epithelial Cells According to Normal or Metastatic Potential

“…Despite these observations, it was possible to determine that IEC-6 cells quantitatively expressed the lowest number of HPA-binding glycoproteins compared with Caco-2 and HCT-116 cells, which is consistent with the results obtained by cytochemistry at both light and electron microscopy. Previous studies using lysates of various cell lines derived from normal and cancerous breast epithelium have reported 11 major glycoprotein bands detected by HPA-lectin blotting (Brooks et al 2001). These bands ranged in molecular weight from 20 to 200 kD.…”