Human recombinant galectin-1 as a potential growth modulator

Abstract

Human galectin-1 is a soluble form of lectin known to play a role in various cellular processes by mediating recognition events in which glycoconjugates are implicated. As a number of studies have shown that galectin-1 is a growth inhibitor (Wells and Mallucci, 1991; Manilal et al., 1993), the starting hypothesis for this thesis was that galectin-1 might be a substrate for a growth-related proteinase (GRP). The intention was to identify the mechanism of action responsible for this growth-inhibitory property by looking at the effect of galectin treatment on the expression of the c-fos proto-oncogene. The structure of the recombinant galectin-1 molecule was investigated in mass spectrometry determinations. Galectin-1 was further characterised in hemagglutination, cellular growth, cytotoxicity, proteolysis and cellular degradation experiments. Even though the recombinant galectin-1 was not identical with the natural protein because it contained two pGEX-linker amino acid residues, and had an apparently 933 Da bigger molecular weight, it fully retained the carbohydrate binding and mitogenicity properties and was still a biphasic growth modulator. Repeated DNA sequencing and mass spectrometry determinations of the tryptic peptides have accounted for all the galectin molecule and have not detected an insertion. We concluded that the abnormal size was the result of a calibration error in the mass spectrometer. Even though the recombinant galectin was proved to be very susceptible to soluble proteinase action, there was no evidence for its active degradation when incubated with cells, and this disproved the original hypothesis. We showed for the first time that $/alpha/sb1$-antitrypsin inhibitor (which inhibits the GRP) had a down-regulatory effect on c-fos expression. Galectin-1 treatment of U2OS and HELA cells had a downregulatory effect on c-fos expression, which confirmed the hypothesis that this proto-oncogene is affected by the signal transduction pathway through which galectin-1 inhibits cell growth. C-fos expression is affected in HELA cells even though they do not undergo growth inhibition, indicating that this process is not as simple as we initially believed. Galectin-1 treatment also downregulated galectin-1 gene expression. This meant that feedback inhibition could take place in these tumour cells.