Gastroenterology

Gastroenterology

Volume 127, Issue 3, September 2004, Pages 741-748
Gastroenterology

Clinical-alimentary tract
A circulating ligand for galectin-3 is a haptoglobin-related glycoprotein elevated in individuals with colon cancer1

https://doi.org/10.1053/j.gastro.2004.06.016Get rights and content

Background & Aims: Galectin-3 is a β-galactoside-binding protein implicated in tumor progression and metastasis of colorectal cancers. To determine whether circulating galectin-3 ligands are related to the presence of colon cancer, we sought to identify and quantify ligands in serum that bind to galectin-3. Methods: Sera from patients with colon cancer, adenomas, and normal individuals were desialylated, reduced, and separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and blots probed with biotinylated galectin-3. Results: In colon cancer sera, the major galectin-3 ligand was a 40-kilodalton band distinct from mucin, carcinoembryonic antigen, and Mac-2 binding protein. Serum 40-kilodalton ligand was 10- to 30-fold higher in patients with colon cancer than in healthy subjects. Ligand was purified by gel filtration, affinity precipitation on galectin-3/agarose, and SDS-PAGE. When tryptic peptides were analyzed by matrix-assisted laser-desorption ionization mass spectrometry and protein database searching, the 40-kilodalton ligand was identified as haptoglobin β subunit. In confirmation of this finding, depletion of haptoglobin by immunoprecipitation also eliminated the 40-kilodalton ligand. Colon cancer sera had only a modest increase in total haptoglobin as compared with healthy subjects, suggesting that the structure rather than the amount of haptoglobin is altered in patients with colon cancer. Immunohistochemical staining confirmed the absence of haptoglobin in normal colon and the ectopic expression of haptoglobin in colon cancers and adenomatous polyps. Conclusions: A major circulating ligand for galectin-3, which is elevated in the sera of patients with colon cancer, is a cancer-associated glycoform of haptoglobin.

Section snippets

Materials

Galectin-3 (human recombinant protein) was purified by affinity chromatography on a column of asialofetuin/agarose and further purified by size-exclusion high-performance liquid chromatography on a column of Superdex 75.7 For the affinity purification of galectin-3 ligands, galectin-3 (2 mg, 1 mg/mL) in 0.1 mol/L sodium citrate and 0.05 mol/L sodium carbonate, pH 10.0, was coupled to 2 mL 4% activated beaded agarose for 4 hours at 4°C using the Aminolink Plus Immobilization kit (Pierce,

Cancer-associated alterations in serum levels of 40-kilodalton galectin-3 ligand

To identify and quantify potential galectin-3 ligands in serum, ligand blot analyses were performed. When colon cancer sera were desialylated, reduced, separated by SDS-PAGE, and analyzed for binding of biotinylated galectin-3, the major galectin-3 ligand was found to be a band of about 40 kilodaltons (Figure 1, inset). This band is distinct in size from previously described galectin-3 ligands, including mucin, carcinoembryonic antigen, and Mac-2 binding protein.4, 5, 6 This band was also

Discussion

The goal of this study was to identify cancer-associated alterations in circulating galectin-3 ligands. The main finding was the identification of a major galectin-3 ligand in colon cancer serum as a cancer-associated form of haptoglobin.

The possibility should be considered that the 40-kilodalton ligand is a haptoglobin-related protein distinct from haptoglobin. Haptoglobin-related protein is the product of the haptoglobin-related protein gene, which is adjacent to the HP gene encoding

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    Expression of an endogenous galactose-binding lectin correlates with neoplastic progression in the colon

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    • Cited by (0)

    Supported by National Cancer Institute grants R01CA69480 and U01CA86400.

    1

    Investigators of the Great Lakes-New England Clinical and Epidemiology Center of the Early Detection Research Network are Dean Brenner, Daniel Normalle, and Kim Turgeon (University of Michigan), Sapna Syngal (Dana Farber Cancer Institute), Robert S. Bresalier (University of Texas M.D. Anderson Cancer Center), John Barron (Dartmouth/Hitchcock Medical Center), and Norman Marcon (University of Toronto).

    2

    J.C.B. and N.M. contributed equally to this work.

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    Copyright © 2004 American Gastroenterological Association. Published by Elsevier Inc. All rights reserved.