title: Interaction of human macrophage C-type lectin with O-linked N-acetylgalactosamine residues on mucin glycopeptides.
abstract: A fluorescein-labeled synthetic peptide, PTTTPITTTTK, was converted into O-glycosylated glycopeptides with various numbers of attached N-acetyl-D-galactosamines (GalNAcs) by in vitro glycosylation with UDP-GalNAc and a microsomal fraction of LS174T human colon carcinoma cells. Glycopeptides with 1, 3, 5, and 6 GalNAc residues (G1, G3, G5, and G6) were obtained, and their sizes were confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Their sequences were determined by a peptide sequencer to be PTTTGalNAcPITTTTK for G1, PTGalNAcTTPITGalNAcTGalNAcTTK for G3, PTTGalNAcTGalNAcPITGalNAcTGalNAcTGalNAcTK for G5, and PTGalNAcTGalNAcTGalNAcPITGalNAcTGalNAcTGalNAcTK for G6. A calcium-type human macrophage lectin (HML) was prepared in a recombinant form, and its interaction with these glycopeptides was investigated by surface plasmon resonance (SPR) spectroscopy and fluorescence polarization. The affinity of recombinant HML (rHML) for immobilized glycopeptides increased, as revealed by SPR, in parallel with the number of GalNAc. The highest affinity was obtained when the G6-peptide was immobilized at high density. Fluorescence polarization equilibrium-binding assays also revealed that the affinity of rHML for soluble gly-copeptides increased, depending on the number of attached GalNAcs. Carbohydrate recognition domain (CRD) fragments of HML were prepared, and their affinity for these four glycopeptides was also determined, this affinity was apparently lower than that of rHML. Affinity constants of rHML for the G3- and G5-peptides were 11- and 38-fold higher, respectively, than for the G1-peptide, whereas those of CRD fragments were only 2- and 6-fold higher, respectively. A chemical cross-linking study revealed that rHML but not recombinant CRD forms trimers in an aqueous solution. Thus, preferential binding of densely glycosylated O-linked glycopeptides should be due to the trimer formation of rHML.
authors: Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors: S
authors: S
authors: Iida
authors: K
authors: K
authors: Yamamoto
authors: T
authors: T
authors: Irimura
mesh_terms: D000116:Acetylgalactosamine / Q000378:metabolism*; D000595:Amino Acid Sequence; D002474:Cell-Free System; D005454:Fluorescence Polarization; D006020:Glycopeptides / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D037102:Lectins / Q000378:metabolism*; D037181:Lectins, C-Type; D008565:Membrane Proteins / Q000378:metabolism*; D009077:Mucins / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism; D011485:Protein Binding; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D020349:Surface Plasmon Resonance
chemical_list: C113583:Clecsf8 protein, mouse; D006020:Glycopeptides; D037102:Lectins; D037181:Lectins, C-Type; D008565:Membrane Proteins; D009077:Mucins; D009844:Oligosaccharides; D000116:Acetylgalactosamine