Abstract
BackgroundProtein 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.MethodologyIn 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.ResultsAberrant 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).ConclusionGlycoproteins 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.
Highlights
Protein glycosylation is an important post-translational modification shown to be altered in all tumour types studied to date
Affinity purification using the lectin Helix pomatia agglutinin (HPA) followed by proteomic analysis revealed annexin 4, annexin 5 and chloride channel protein 1 (CLCA1) to be increased in the metastatic colorectal cancer specimens
The glycoproteins recognized by HPA have been investigated in colorectal cancer (CRC) derived cell lines and this revealed that the utility of HPA lies in its ability to simultaneously bind to many glycoproteins involved in a range of pro-metastatic functions including cell migration, anti-apoptosis and cell signalling [12]
Summary
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. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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