Abstract
Sera from 25 metastatic breast cancer patients and 25 healthy controls were subjected to affinity chromatography using immobilized galectin-1. Serum from the healthy subjects contained on average 1.2 mg per ml (range 0.7–2.2) galectin-1 binding glycoproteins, whereas serum from the breast cancer patients contained on average 2.2 mg/ml (range 0.8–3.9), with a higher average for large primary tumours. The major bound glycoproteins were α-2-macroglobulin, IgM and haptoglobin. Both the IgM and haptoglobin concentrations were similar in cancer compared to control sera, but the percentage bound to galectin-1 was lower for IgM and higher for haptoglobin: about 50% (range 20–80) in cancer sera and about 30% (range 25–50) in healthy sera. Galectin-1 binding and non-binding fractions were separated by affinity chromatography from pooled haptoglobin from healthy sera. The N-glycans of each fraction were analyzed by mass spectrometry, and the structural differences and galectin-1 mutants were used to identify possible galectin-1 binding sites. Galectin-1 binding and non-binding fractions were also analyzed regarding their haptoglobin function. Both were similar in forming complex with haemoglobin and mediate its uptake into alternatively activated macrophages. However, after uptake there was a dramatic difference in intracellular targeting, with the galectin-1 non-binding fraction going to a LAMP-2 positive compartment (lysosomes), while the galectin-1 binding fraction went to larger galectin-1 positive granules. In conclusion, galectin-1 detects a new type of functional biomarker for cancer: a specific type of glycoform of haptoglobin, and possibly other serum glycoproteins, with a different function after uptake into tissue cells.
Highlights
A glycoprotein occurs in multiple glycoforms depending on which glycans are attached at each particular site
We show that in sera from patients with metastatic breast cancer, galectin-1 binds on average almost twice as much glycoprotein compared with healthy individuals, including a fraction of haptoglobin containing N-linked glycans with less terminal sialic acids, and increased proportion of additional antenna
A C3S mutant of human galectin-1 was used, which is less sensitive to inactivation by oxidation than wild type galectin-1 [31], and previously shown to have the same affinity for carbohydrates and glycoproteins, including human serum glycoproteins, as wild type galectin-1 [28]
Summary
A glycoprotein occurs in multiple glycoforms depending on which glycans are attached at each particular site. The glycan structures, and thereby the profile of glycoforms of different glycoproteins, have been known for a long time to be altered in cancer [6,7] This has stimulated an increasing effort to use particular glycoforms as biomarkers for cancer in serum, as detected by combinations of plant lectins, antibodies and structural analysis by mass spectrometry, summarized as glycoproteomics [8,9]. These may be derived from the cancer itself [8,10,11,12], and some of the most commonly used cancer biomarkers are carbohydrate based and detection of specific glycoforms of other commonly used cancer associated proteins, such as PSA, have been proposed to sharpen the diagnosis. Specific cancer induced forms of common serum glycoproteins, such as transferrin or haptoglobin that are synthesized mainly in the liver, have been observed and may serve as markers of the physiological effects of the cancer [2,3,13,14,15]
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