Isolectins from Solanum tuberosum with different detailed carbohydrate binding specificities:: Unexpected recognition of lactosylceramide by N-acetyllactosamine-binding lectins

Glycosphingolipid recognition by two isolectins from Solanum tuberosum was compared by the chromatogram binding assay. One lectin (PL-I) was isolated from potato tubers by affinity chromatography, and identified by MALDI-TOF mass spectrometry as a homodimer with a subunit molecular mass of 63,000, The other (PL-II) was a commercial lectin, characterized as two homodimeric isolectins with subunit molecular masses of 52,000 and 55,000, respectively. Both lectins recognized N-acetyllactosamine-containing glycosphingolipids, but the fine details of their carbohydrate binding specificities differed. PL-II preferentially bound to glycosphingolipids with N-acetyllactosamine branches, as Gal beta 4GlcNAc beta6(Gal beta 4GlcNAc beta3)Gal beta 4Glc beta 1Cer. PL-I also recognized this glycosphingolipid, but bound equally well to the linear glycosphingolipid Gal beta 4GlcNAc beta 3Gal beta 4GlcNAc beta 3Gal beta 4Glc beta 1Cer. Neolactotetraosylceramide and the B5 pentaglycosyl-ceramide were also bound by PL-I, while other glycosphingolipids with only one N-acetyllactosamine unit were non-binding. Surprisingly both lectins also bound to lactosylceramide, with an absolute requirement for sphingosine and non-hydroxy fatty acids. The inhibition of binding to both lactosylceramide and N-acetyllactosamine-containing glycosphingolipids by N-acetylchitotetraose suggests that lactosylceramide is also accomodated within the N-acetylchitotetraose/N-acetyllactosamine-binding sites of the lectins, Through docking of glycosphingolipids onto a three-dimensional model, of the PL-I hevein binding domain, a Gal beta 4GlcNAc beta 3Gal beta4 binding epitope was defined. Furthermore, direct involvement of the ceramide in the binding of lactosylceramide was suggested.