title: Identification of glycans on plasma-derived ADAMTS13.
abstract: Patients suffering from acquired thrombotic thrombocytopenic purpura develop autoantibodies directed toward the plasma glycoprotein ADAMTS13. Here, we studied the glycan composition of plasma-derived ADAMTS13. Purified ADAMTS13 was reduced, alkylated, and processed into peptides with either trypsin or chymotrypsin. Glycopeptides were enriched using zwitterionic HILIC zip-tips and analyzed by tandem mass spectrometry employing higher-energy collision dissociation fragmentation. Upon detection of a diagnostic ion of a glycan fragment, electron transfer dissociation fragmentation was performed on the same precursor ion. The majority of N-linked glycans were of the complex type containing terminal sialic acids and fucose residues. A high mannose-containing glycan was attached to Asn614 in the spacer domain. Six O-linked glycans mostly terminating in sialic acid were found dispersed over ADAMTS13. Five O-linked glycans were attached to a Ser and one to Thr. All 6 O-linked glycans contained a terminal sialic acid. O-fucosylation is a common posttranslational modification of thrombospondin type 1 repeats. We identified 7 O-fucosylation sites in the thrombospondin (TSP) type 1 repeats. Unexpectedly, one additional O-fucosylation site was found in the disintegrin domain. This O-fucosylation site did not meet the proposed consensus sequence CSX(S/T)CG. C-mannosylation sites were identified in TSP1, linker TSP4-TSP5, and TSP8. Overall, our findings highlight the complexity of glycan modifications on ADAMTS13, which may have implications for its interaction with immune- or clearance receptors containing carbohydrate recognition domains.
authors: Department of Plasma Proteins, Sanquin-Academic Medical Center Landsteiner Laboratory, Amsterdam, The Netherlands.
authors: Fabian C
authors: FC
authors: Verbij
authors: Department of Plasma Proteins, Sanquin-Academic Medical Center Landsteiner Laboratory, Amsterdam, The Netherlands.|Emma Children's Hospital, Academic Medical Center, Amsterdam, The Netherlands; and.
authors: Eva
authors: E
authors: Stokhuijzen
authors: Department of Plasma Proteins, Sanquin-Academic Medical Center Landsteiner Laboratory, Amsterdam, The Netherlands.
authors: Paul H P
authors: PH
authors: Kaijen
authors: Department of Plasma Proteins, Sanquin-Academic Medical Center Landsteiner Laboratory, Amsterdam, The Netherlands.
authors: Floris
authors: F
authors: van Alphen
authors: Department of Plasma Proteins, Sanquin-Academic Medical Center Landsteiner Laboratory, Amsterdam, The Netherlands.|Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands.
authors: Alexander B
authors: AB
authors: Meijer
authors: Department of Plasma Proteins, Sanquin-Academic Medical Center Landsteiner Laboratory, Amsterdam, The Netherlands.
authors: Jan
authors: J
authors: Voorberg
mesh_terms: D000071120:ADAMTS13 Protein / Q000737:chemistry* / Q000302:isolation & purification; D002236:Carbohydrate Conformation; D006031:Glycosylation; D006801:Humans; D011134:Polysaccharides / Q000737:chemistry*
chemical_list: D011134:Polysaccharides; D000071120:ADAMTS13 Protein; C099604:ADAMTS13 protein, human