The lectin Helix pomatia agglutinin recognizes O-GlcNAc containing glycoproteins in human breast cancer

Abstract

There has been considerable interest in understanding the epitopes that bind the lectin Helix pomatia agglutinin (HPA) in breast cancer as the lectin has been shown to identify glycosylation changes associated with the development of metastatic disease. HPA has previously been shown to recognize aberrant O-linked α-N-acetylgalactosamine (GalNAcα)/mucin glycosylation in cancer, including exposed Tn epitopes. However, recent glycan-array analysis reported that diverse epitopes are also recognized by the lectin, e.g. consortium for functional glycomics (CFG) data: GalNAcα1,3Gal; β-GalNAc; GlcNAcβ1,4Gal. The intriguing observations from the CFG array led to this study, in which HPA-binding epitopes were localized and characterized in an in vitro model of breast cancer metastasis. HMT3522 (benign disease), BT474 (primary cancer) and T47D/MCF7 (metastatic cancer) cells were assessed in confocal microscopy-based co-localization studies and a glycoproteomic analysis based on 2-dimensional electrophoresis (2DE), western blotting and mass spectrometry was adopted. HPA binding correlated with levels of integrin α6, transcription factors heterogeneous nuclear ribonuclear protein (HnRNP) H1, HnRNP D-like, HnRNP A2/B1 as well as heat shock protein 27 (Hsp27), glial fibrillary acidic protein and enolase 1 (ENO1). These glycoproteins were non-detectable in the non-metastatic breast cancer cell lines. The recognition of HnRNPs, Hsp27 and ENO1 by HPA correlated with O-GlcNAcylation of these proteins. Integrin α6 was the most abundant HPA glycoprotein in the breast cancer cells with a metastatic phenotype; this concurred with previous findings in colorectal cancer. This is the first report in which HPA has been shown to bind O-GlcNAcylated transcription factors. This class of proteins represents a new means by which HPA differentiates cancer cells with an aggressive metastatic phenotype.

Aberrant O-GlcNAcylated Proteins: New Perspectives in Breast and Colorectal Cancer

“…As mentioned above, the O -GlcNAc cycling enzymes OGT and OGA tightly regulate the level of O -GlcNAcylation. Enhanced O -GlcNAcylation level corresponding to increased OGT and decreased OGA expression is commonly observed in various cancers including bone (41), bladder (42), breast (26, 43–45), bile duct (46), colon (47–49), leukemia (50), liver (51), lung (47), ovary (36), pancreas (30), and prostate (52, 53). Alteration of this glycosylation in thyroid cancer, however, occurs in the opposite way (54).…”

Aberrant Presentation of HPA-Reactive Carbohydrates Implies Selectin-Independent Metastasis Formation in Human Prostate Cancer

“…4,11) are common intermediates for the synthesis of selectin ligands (7,21), extravasation of CTCs is not crucially dependent on selectin-selectin ligand interactions in prostate cancer. Again, this is a peculiarity of prostate cancer and contrary to different other human malignancies (13)(14)(15)(16).…”

“…Hence, HPA has been shown to be a marker of metastatic competence in human breast and colorectal xenograft primary tumors in severe combined immunodeficient (SCID) mice (2) and of a poor patient prognosis in these malignancies (6,7) as well as in adenocarcinoma of the lung (8), gastric cancer (9), and malignant melanoma (10). HPA-reactive carbohydrates are typical for two of the most prominent O-glycans in cancer, namely Tn antigen and core 2 O-glycans (4,11), which are synthesized through the sequential activity of polypeptide GalNAc-transferases (pp-GalNac-T's), core 1 synthase (C1GALT1) and core 2 synthases (C2GNT1,2,3), respectively (Fig. 1A).…”

Lectin from Agrocybe aegerita as a Glycophenotype Probe for Evaluation of Progression and Survival in Colorectal Cancer

“…At present, lectin-based strategies are the popular techniques to observe glyco-alteration in various cancers (Indramanee et al, 2012;Rambaruth et al, 2012). The binding preference of AAL can be summarized as follows:…”