Abstract
The 32-kDa galectin (LEC-1 or N32) of the nematode Caenorhabditis elegans is the first example of a tandem repeat-type galectin and is composed of two domains, each of which is homologous to typical vertebrate 14-kDa-type galectins. To elucidate the biological meaning of this unique structure containing two probable sugar binding sites in one molecule, we analyzed in detail the sugar binding properties of the two domains by using a newly improved frontal affinity chromatography system. The whole molecule (LEC-1), the N-terminal lectin domain (Nh), and the C-terminal lectin domain (Ch) were expressed in Escherichia coli, purified, and immobilized on HiTrap gel agarose columns, and the extent of retardation of various sugars by the columns was measured. To raise the sensitivity of the system, we used 35 different fluorescence-labeled oligosaccharides (pyridylaminated (PA) sugars). All immobilized proteins showed affinity for N-acetyllactosamine-containing N-linked complex-type sugar chains, and the binding was stronger for more branched sugars. Ch showed 2-5-fold stronger binding toward all complex-type sugars compared with Nh. Both Nh and Ch preferred Galbeta1-3GlcNAc to Galbeta1-4GlcNAc. Because the Fucalpha1-2Galbeta1-3GlcNAc (H antigen) structure was found to interact with all immobilized protein columns significantly, the K(d) value of pentasaccharide Fucalpha1-2Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc-PA for each column was determined by analyzing the concentration dependence. Obtained values for immobilized LEC-1, Nh, and Ch were 6.0 x 10(-5), 1.3 x 10(-4), and 6.5 x 10(-5) m, respectively. The most significant difference between Nh and Ch was in their affinity for GalNAcalpha1-3(Fucalpha1-2)Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc-PA, which contains the blood group A antigen; the K(d) value for immobilized Nh was 4.8 x 10(-5) m, and that for Ch was 8.1 x 10(-4) m. The present results clearly indicate that the two sugar binding sites of LEC-1 have different sugar binding properties.
Highlights
The 32-kDa galectin (LEC-1 or N32) of the nematode Caenorhabditis elegans is the first example of a tandem repeat-type galectin and is composed of two domains, each of which is homologous to typical vertebrate 14kDa-type galectins
Comparison of the Kd values for 35 different sugar chains obtained for LEC-1, N-terminal half-domain (Nh), and C-terminal half-domain (Ch) clearly showed that LEC-1 has two comparably potent sugar binding sites with different affinities
Analysis of Binding Properties of the Two Domains of LEC1—The whole molecule (LEC-1), the N-terminal lectin domain (Nh), and the C-terminal lectin domain (Ch) of the 32-kDa galectin of the nematode C. elegans were expressed in E. coli by using the expression vector pET21a
Summary
N-terminal lectin domain of LEC-1; Ch, C-terminal lectin domain of LEC-1; PA, pyridylaminated; PBS, phosphate-buffered saline. Because we keenly believed in the importance of reinforcing this method, we sought to improve it in the following ways: (i) use of mechanically stable chromatographic media for packing; for this purpose, HiTrap N-hydroxysuccinimide-activated Sepharose was used; (ii) adoption of a fluorescence-based detection for increasing the sensitivity; in the present study, fluorescence-labeled sugars (PA-oligosaccharides; 320 nm for excitation and 400 nm for emission; Ref. 14) were used; (iii) design of a semiautomated system; for this purpose, an ordinary high performance liquid chromatography system was used, and the analyte solution was loaded into a relatively large sample loop (1–2 ml) and injected into a miniature column (4.0 mm inner diameter ϫ 10 mm; bed volume, 0.126 ml); and (iv) development of a simple and efficient data-processing procedure that enables the determination of very small differences in retardation with precision These reinforcements were successful, and reliable data for binding ability were obtained. Comparison of the Kd values for 35 different sugar chains obtained for LEC-1, Nh, and Ch clearly showed that LEC-1 has two comparably potent sugar binding sites with different affinities
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