Functional and structural bases of a cysteine-less mutant as a long-lasting substitute for galectin-1

Abstract

Galectin-1 (Gal-1), a member of the beta-galactoside-binding animal lectin family, has a wide range of biological activities, which makes it an attractive target for medical applications. Unlike other galectins, Gal-1 is susceptible to oxidation at cysteine residues, which is troublesome for in vitro/vivo studies. To overcome this problem, we prepared a cysteine-less mutant of Gal-1 (CSGal-1) by substituting all cysteine residues with serine residues. In the case of wild-type Gal-1, the formation of covalent dimers/oligomers was evident after 10 days of storage in the absence of a reducing agent with a concomitant decrease in hemagglutination activity, while CSGal-1 did not form multimers and retained full hemagglutination activity after 400 days of storage. Frontal affinity chromatography showed that the sugar-binding specificity and affinity of Gal-1 for model glycans were barely affected by the mutagenesis. Gal-1 is known to induce cell signaling leading to an increase in the intracytoplasmic calcium concentration and to cell death. CSGal-1 is also capable of inducing calcium flux and growth inhibition in Jurkat cells, which are comparable to or more potent than those induced by Gal-1. The X-ray structure of the CSGal-1/lactose complex has been determined. The structure of CSGal-1 is almost identical to that of wild-type human Gal-1, showing that the amino acid substitutions do not affect the overall structure or carbohydrate-binding site structure of the protein. These results indicate that CSGal-1 can serve as a stable substitute for Gal-1.

Melanoma Cell Galectin-1 Ligands Functionally Correlate with Malignant Potential

“…While efforts using native Gal-1 do exist (Andre et al , 1999; Kaltner et al , 1997; Plzak et al , 2000), native human Gal-1 is problematic for use in bioassays due to rapid oxidative deactivation and the need for reducing chemicals, which complicates interpretation of ligand-binding data. Previous studies using alkylation-induced stabilization or cysteine-less Gal-1 mutants illustrate other methods used to circumvent drawbacks of probing with native Gal-1 (Inagaki et al , 2000; Nishi et al , 2008; Powell and Whitney, 1984; Stowell et al , 2009). …”

Engineering galectin–glycan interactions for immunotherapy and immunomodulation

“…60 Notably, the cysteine-less mutant retained hemagluttination activity during storage for more than 400 days, while activity of the wild-type protein was greatly diminished by 10 days in the absence of reducing agents. 60 Alkylation of cysteine residues with iodoacetamide or maleimide can also inhibit oxidative dimerization, thereby eliminating the need to modify the primary structure of galectin-1. 61 Similar to the cysteineless mutant, alkylated galectin-1 did not undergo oxidative dimerization over prolonged periods of time and retained the glycan-binding and biological properties of the wildtype protein.…”

The Lectin Frontier Database (LfDB), and Data Generation Based on Frontal Affinity Chromatography

“…Griffonia simplifolia (GSL-II) [53] Wisteria floribunda (WFA) [59] VIP36 [60] Fungal galectin (ACG) [62] Mutants of nematode galectin LEC-6 [63] Conger eel (congerin P) [64] Marine sponge Halichondria okadai [65] Nematode gene DC2.3 [66] Mutants of ACG [67] Two lectin domains of nematode galectin LEC-1 [68] Human galectins 1-9 and other lectins [38] Nematode galectins [69,70] American bullfrog Rna catesbeiana [71] Cysteine-less mutant of human galectin-1 [72] Nematode galectin (LEC-6) [73] Nematode galectins (LEC-1~LEC-11) [74] Human galectins [75] Marine sponge Halichondria okadai [76] Mutants of nematode galectin (LEC-1) [77] Human galectin-9 N-terminal CRD [78] The nematode galectin LEC-1 and its mutants [79] Argasid tick Ornithodoros moubata.…”