Identification of O-Linked Glycoproteins Binding to the Lectin Helix pomatia Agglutinin as Markers of Metastatic Colorectal Cancer

Background Protein glycosylation is an important post-translational modification shown to be altered in all tumour types studied to date. Mucin glycoproteins have been established as important carriers of O-linked glycans but other glycoproteins exhibiting altered glycosylation repertoires have yet to be identified but offer potential as biomarkers for metastatic cancer. Methodology In this study a glycoproteomic approach was used to identify glycoproteins exhibiting alterations in glycosylation in colorectal cancer and to evaluate the changes in O-linked glycosylation in the context of the p53 and KRAS (codon 12/13) mutation status. Affinity purification with the carbohydrate binding protein from Helix pomatia agglutinin (HPA) was coupled to 2-dimensional gel electrophoresis with mass spectrometry to enable the identification of low abundance O-linked glycoproteins from human colorectal cancer specimens. Results Aberrant O-linked glycosylation was observed to be an early event that occurred irrespective of the p53 and KRAS status and correlating with metastatic colorectal cancer. Affinity purification using the lectin HPA followed by proteomic analysis revealed annexin 4, annexin 5 and CLCA1 to be increased in the metastatic colorectal cancer specimens. The results were validated using a further independent set of specimens and this showed a significant association between the staining score for annexin 4 and HPA and the time to metastasis; independently (annexin A4: Chi square 11.45, P = 0.0007; HPA: Chi square 9.065, P = 0.0026) and in combination (annexin 4 and HPA combined: Chi square 13.47; P = 0.0002). Conclusion Glycoproteins showing changes in O-linked glycosylation in metastatic colorectal cancer have been identified. The glycosylation changes were independent of p53 and KRAS status. These proteins offer potential for further exploration as biomarkers and potential targets for metastatic colorectal cancer

HPA lectin and P53 immunohistochemistry of CRC tissue specimens.

<p>Left: light microscopy images of colorectal tissue sections (5 μm) incubated with HPA (10 μg/ml) and streptavidin-HRP (5 μg/ml) or incubated with the anti-P53 antibody (1:200) and biotinylated horse rabbit anti-mouse IgG (1:1000) as indicated. The brown colouration indicates the peroxidase reaction with DAB/H₂O₂, the nuclei were counterstained with haematoxylin (blue). Magnification X400. Right: Table showing results for HPA, P53 and <i>KRAS</i> analysis.</p

2-DE analysis of <i>O-</i>linked glycoproteins affinity purified using HPA.

<p>Proteins pooled from LN+ve CRC tissue specimens were affinity purified with HPA and separated by isoelectric focussing on a pH 4–7 IPG strip and then by SDS-PAGE (12%) and visualised using colloidal Coomassie Blue. Inset: annexin 4/5 as indicated in LN+ve compared with LN-ve CRC tissue specimen protein pools. The proteins identified in the MALDI-MS analysis are indicated.</p