Subject(s)
Placenta Accreta/therapy , Postpartum Hemorrhage/etiology , Uterine Inversion/diagnostic imaging , Administration, Intravenous , Adult , Female , Humans , Hysterectomy , Oxytocin/administration & dosage , Placenta Accreta/diagnosis , Postpartum Hemorrhage/therapy , Pregnancy , Tomography, X-Ray ComputedABSTRACT
Jacalin, a lectin from the jackfruit Artocarpus integrifolia, has been known as a valuable tool for specific capturing of O-glycoproteins such as mucins and IgA1. Though its sugar-binding preference for T/Tn-antigens is well established, its detailed specificity has not been elucidated. In this study, we prepared a series of mucin-type glycopeptides using human glycosyltransferases, that is, ST6GalNAc1, Core1Gal-T1 and -T2, beta3Gn-T6, and Core2GnT1, and investigated their binding to immobilized Jacalin by frontal affinity chromatography (FAC). As a result, consistent with the previous observation, Jacalin showed high affinity for T-antigen (Core1) and Tn-antigen (alpha N-acetylgalactosamine)-attached peptides. Furthermore, we here show as novel findings that (1) Jacalin also showed significant affinity for Core3 and sialyl-T (ST)-attached peptides, but (2) Jacalin could not bind to Core2, Core6, and sialyl-Tn (STn)-attached peptides. The results were also confirmed by FAC using p-nitrophenyl (pNP)-derivatized saccharides. In conclusion, Jacalin binds to a GalNAcalpha1-peptide, in which C6-OH of alphaGalNAc is free (i.e., Core1, Tn, Core3, and ST), whereas it cannot recognize a GalNAcalpha1-peptide with a substitution at the C6 position (i.e., Core2, Core6, and STn). These findings provide useful information when applying jacalin for functional analysis of mucin-type glycoproteins and glycopeptides.
Subject(s)
Antigens, Tumor-Associated, Carbohydrate/chemistry , Glucosyltransferases/chemistry , Mucins/chemistry , Oligopeptides/chemistry , Plant Lectins/chemistry , Antigens, Tumor-Associated, Carbohydrate/metabolism , Chromatography, Affinity , Humans , Mucins/metabolism , Oligopeptides/metabolism , Plant Lectins/metabolism , Protein Binding , Substrate SpecificityABSTRACT
We have cloned, expressed and characterized a novel member of the human UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (pp-GalNAc-T) family, pp-GalNAc-T15. The pp-GalNAc-T15 transcript was ubiquitously expressed in human tissues. Recombinant pp-GalNAc-T15 transferred N-acetylgalactosamine (GalNAc) toward a panel of mucin-derived peptide substrates in vitro. Although pp-GalNAc-T15 showed significantly less catalytic activity than pp-GalNAc-T2, T15 transferred up to seven GalNAcs to the Muc5AC peptide, while T2 transferred up to five GalNAcs. These results clearly indicated that pp-GalNAc-T15 is a novel member of the human pp-GalNAc-T family with unique catalytic activity.
Subject(s)
N-Acetylgalactosaminyltransferases/genetics , N-Acetylgalactosaminyltransferases/metabolism , Amino Acid Sequence , Base Sequence , Catalysis , Glycosylation , Humans , Isoenzymes , Molecular Sequence Data , Oligopeptides/chemistry , Oligopeptides/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Substrate Specificity , Tissue Distribution , Transcription, Genetic , Uridine Diphosphate N-Acetylgalactosamine/metabolism , Polypeptide N-acetylgalactosaminyltransferaseABSTRACT
The hinge region of human immunoglobulin A1 (*IgA1) possesses multiple O-glycans, of which synthesis is initiated by the addition of GalNAc to serine or threonine through the activity of UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferases (pp-GalNAc-Ts). We found that six pp-GalNAc-Ts, pp-GalNAc-T1, -T2, -T3, -T4, -T6, and -T9, were expressed in B cells, IgA-bearing B cells, and NCI-H929 IgA myeloma cells. pp-GalNAc-T activities of these six enzymes for a synthetic IgA hinge peptide, which has nine possible O-glycosylation sites, were examined using a reversed phase-high performance liquid chromatography, a matrix-assisted laser desorption ionization time of flight mass spectrometry, and peptide sequencing analysis. pp-GalNAc-T2 showed the strongest activity transferring GalNAc to a maximum of eight positions. Other pp-GalNAc-Ts exhibited different substrate specificities from pp-GalNAc-T2; however, their activities were extremely weak. It was reported that the IgA1 hinge region possesses a maximum of five O-glycans, and their amino acid positions have been determined. We found that pp-GalNAc-T2 selectively transferred GalNAc residues to the same five positions. These results strongly suggested that pp-GalNAc-T2 is an essential enzyme for initiation of O-linked glycosylation of the IgA1 hinge region.
Subject(s)
Immunoglobulin A/biosynthesis , Immunoglobulin A/chemistry , Lymphocytes/immunology , N-Acetylgalactosaminyltransferases/metabolism , Polysaccharides/biosynthesis , Uridine Diphosphate N-Acetylgalactosamine/metabolism , Amino Acid Sequence , B-Lymphocytes/enzymology , B-Lymphocytes/immunology , Base Sequence , Cell Line , Humans , Kinetics , Lymphocytes/enzymology , Molecular Sequence Data , Multiple Myeloma/immunology , Reference Values , Reverse Transcriptase Polymerase Chain Reaction , Substrate Specificity , Transcription, Genetic , Tumor Cells, Cultured , Polypeptide N-acetylgalactosaminyltransferaseABSTRACT
A novel member of the human UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase (pp-GalNAc-T) gene family was cloned as a homolog of human pp-GalNAc-T7, and designated pp-GalNAc-T10. pp-GalNAc-T10 transcript was found in the small intestine, stomach, pancreas, ovary, thyroid gland and spleen. In a polypeptide GalNAc-transfer assay, recombinant pp-GalNAc-T10 transferred GalNAc onto a panel of mucin-derived peptide substrates. Furthermore, pp-GalNAc-T10 demonstrated strong transferase activity with glycopeptide substrates.
