TY - JOUR
T1 - Identification of glycans on plasma-derived ADAMTS13
AU - Verbij, Fabian C.
AU - Stokhuijzen, Eva
AU - Kaijen, Paul H. P.
AU - van Alphen, Floris
AU - Meijer, Alexander B.
AU - Voorberg, Jan
PY - 2016
Y1 - 2016
N2 - 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
AB - 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
U2 - https://doi.org/10.1182/blood-2016-06-720912
DO - https://doi.org/10.1182/blood-2016-06-720912
M3 - Article
C2 - 27574189
SN - 0006-4971
VL - 128
SP - e51-e58
JO - Blood
JF - Blood
IS - 21
ER -