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.