The Prevalence and Nature of Glycan Alterations on Specific Proteins in Pancreatic Cancer Patients Revealed Using Antibody-Lectin Sandwich Arrays

Tingting Yue,Irwin J Goldstein,Michael A Hollingsworth,Karen Kaul,Randall E Brand,Brian B Haab

doi:10.1074/mcp.m900135-mcp200

Abstract

Changes to the glycan structures of proteins secreted by cancer cells are known to be functionally important and to have potential diagnostic value. However, an exploration of the population variation and prevalence of glycan alterations on specific proteins has been lacking because of limitations in conventional glycobiology methods. Here we report the use of a previously developed antibody-lectin sandwich array method to characterize both the protein and glycan levels of specific mucins and carcinoembryonic antigen-related proteins captured from the sera of pancreatic cancer patients (n = 23) and control subjects (n = 23). The MUC16 protein was frequently elevated in the cancer patients (65% of the patients) but showed no glycan alterations, whereas the MUC1 and MUC5AC proteins were less frequently elevated (30 and 35%, respectively) and showed highly prevalent (up to 65%) and distinct glycan alterations. The most frequent glycan elevations involved the Thomsen-Friedenreich antigen, fucose, and Lewis antigens. An unexpected increase in the exposure of alpha-linked mannose also was observed on MUC1 and MUC5ac, indicating possible N-glycan modifications. Because glycan alterations occurred independently from the protein levels, improved identification of the cancer samples was achieved using glycan measurements on specific proteins relative to using the core protein measurements. The most significant elevation was the cancer antigen 19-9 on MUC1, occurring in 19 of 23 (87%) of the cancer patients and one of 23 (4%) of the control subjects. This work gives insight into the prevalence and protein carriers of glycan alterations in pancreatic cancer and points to the potential of using glycan measurements on specific proteins for highly effective biomarkers.

Highlights

Changes to the glycan structures of proteins secreted by cancer cells are known to be functionally important and to have potential diagnostic value
Profiling Cancer-associated Glycans on Specific Proteins— Using a variant of standard sandwich methods to detect core protein levels (Fig. 1a), the glycans on selected mucins and carcinoembryonic antigen-related cell adhesion molecule (CEACAM) captured by antibody arrays were probed with a variety of lectins or glycan-binding antibodies (Fig. 1b)
A set of 46 serum samples (n ϭ 23 from pancreatic cancer patients and 23 from healthy control subjects) was incubated on the arrays of one microscope slide, and each array was probed with a detection lectin or antibody

Summary

Introduction

Changes to the glycan structures of proteins secreted by cancer cells are known to be functionally important and to have potential diagnostic value. We report the use of a previously developed antibody-lectin sandwich array method to characterize both the protein and glycan levels of specific mucins and carcinoembryonic antigen-related proteins captured from the sera of pancreatic cancer patients (n ‫ ؍‬23) and control subjects (n ‫ ؍‬23). This work gives insight into the prevalence and protein carriers of glycan alterations in pancreatic cancer and points to the potential of using glycan measurements on specific proteins for highly effective biomarkers. A potential advantage of using glycans for diagnostics is that carbohydrate modifications of particular proteins may be altered more frequently or in certain disease states than their underlying core protein concentrations. Affinity-based glycosylation studies do not provide the structural detail provided by mass spectrometry and enzymatic methods, they can provide information about the biological variation of a particular motif

Methods

Results

Conclusion