Differential structure and activity between human and mouse intelectin-1: Human intelectin-1 is a disulfide-linked trimer, whereas mouse homologue is a monomer

Abstract

Human intelectin-1 (hITLN-1) is a 120-kDa lectin recognizing galactofuranosyl residues found in cell walls of various microorganisms but not in mammalian tissues. Although mouse intelectin-1 (mITLN-1) has been identified previously, its biochemical properties and functional characteristics have not been studied. Therefore, we have compared structures and saccharide-binding specificities of hITLN-1 and mITLN-1 using recombinant proteins produced by mammalian cells. Recombinant hITLN-1 is a trimer, disulfide-linked through Cys-31 and Cys-48, and Nglycosylated at Asn-163. Despite 84.9% amino acid identity to hITLN-1, recombinant and intestinal mITLN-1 are unglycosylated 30-kDa monomers. Recombinant hITLN-1, as well as recombinant and intestinal mITLN-1 were purified by Ca 2+ -dependent adsorption to galactose-Sepharose. In competitive binding studies, hITLN-1 was eluted from galactose-Sepharose by 100 mM 2-deoxygalactose, a galactofuranosyl disaccharide, D-xylose, and both D-and L-ribose. In contrast, mITLN-1 was partially eluted by the galactofuranosyl disaccharide, and only minimally by the other saccharides indicating that the two intelectins have different saccharide-binding specificities. When the N-and Cterminal regions of hITLN-1 were replaced, respectively, with those of mITLN-1, galactose-Sepharose binding was associated with the C-terminal regions. Finally, hITLN-1 binding to galactose-Sepharose was not affected by the substitution of the Cys residues in the N-terminal region that are necessary for oligomer formation, nor was it affected by the removal of the N-linked oligosaccharide at Asn-163. Although both hITLN-1 and mITLN-1 recognize galactofuranosyl residues, our comparative studies, taken together, demonstrate that these intelectins have different quaternary structures and saccharide-binding specificities.

Recombinant Human Intelectin Binds Bovine Lactoferrin and Its Peptides

“…In previous reports, both hInlL in fetal small intestine 5) and rhIntL expressed in cultured animal cells 2) were isolated as a glycosylated homotrimer with a molecular mass of about 120 kDa. Recently, mouse IntL has been purified from small intestine in a monomeric form with no glycan chains, 22) suggesting that IntL possesses ligand-binding capacity both in the forms of oligomer and monomer, and that its glycan chain is not necessary for the binding. The refolded rhIntL in the present study is likely to have been a monomer, for the reason that the refolding was performed in the presence of an excess amount of reducing agent.…”

Soluble Human Intestinal Lactoferrin Receptor: Ca²⁺-Dependent Binding to Sepharose-Based Matrices

“…3,6,7) Fifty microliters of NHS-activated Sepharose 4 FF, Epoxy-activated Sepharose 6B blocked with ethanolamine or Glutathione Sepharose 4B were incubated with 300 µL of the culture supernatant from transfectants in the presence or absence of 5 mM ethylene glycol bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) at room temperature for 5h. In case of competitive binding with Dgalactose or D-glucose, each monosaccharide at the indicated concentrations was added to the incubation step above.…”

“…Of note, the elution of bound shLFR at low pH (pH 2.5) was not adequate to obtain reproducible results, probably because concentrated shLFR is prone to aggregation in the presence of Ca 2+ . 3,6,7) The eluate was centrifuged and filtered through 0.22 µm membrane filters to completely remove the Sepharose beads. The eluate was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, followed by Western blotting (10 µL per lane was used in Figs.…”

“…1), in accordance with previously published data. 4,7) Faint bands of about 35-and 64-kDa under nonreducing conditions represented monomeric and dimeric forms, respectively. Three minor bands of >70-kDa under reducing conditions represented partially reduced shLFRs.…”

“…Recombinant shLFR (hITLN-1) is a disulfidelinked, 120-kDa, homotrimeric polypeptide under nonreducing conditions, but is a 40-kDa, monomeric polypeptide under reducing conditions. 4,7) Interactions between shLFR and human lactoferrin (hLF) are partially Ca 2+ dependent, with an apparent K d of 360 nM. 4) hITLN-1 is considered a new type lectin that recognizes galactofuranose and binds to various mono/disaccharides, including D-galactose, in the presence of Ca 2+ .…”