Tyrosine sulfation and N-glycosylation of human heparin cofactor II from plasma and recombinant Chinese hamster ovary cells and their effects on heparin binding.
The structure of post-translational modifications of human heparin cofactor II isolated from human serum and from recombinant Chinese hamster ovary cells and their effects on heparin binding have been characterized. Oligosaccharide chains were found attached to all three potential N-glycosylation sites in both protein preparations. The carbohydrate structures of heparin cofactor II circulating in blood are complex-type diantennary and triantennary chains in a ratio of 6 : 1 with the galactose being > 90% sialylated with alpha 2-->6 linked N-acetylneuraminic acid. About 50% of the triantennary structures contain one sLe(x) motif. Proximal alpha 1-->6 fucosylation of oligosacharides from Chinese hamster ovary cell-derived HCII was detected in > 90% of the diantennary and triantennary glycans, the latter being slightly less sialylated with exclusively alpha 2-->3-linked N-acetylneuraminic acid units. Applying the ESI-MS/ MS-MS technique, we demonstrate that the tryptic peptides comprising tyrosine residues in positions 60 and 73 were almost completely sulfated irrespective of the protein's origin. Treatment of transfected Chinese hamster ovary cells with chlorate or tunicamycin resulted in the production of heparin cofactor II molecules that eluted with higher ionic strength from heparin-Sepharose, indicating that tyrosine sulfation and N-linked glycans may affect the inhibitor's interaction with glycosaminoglycans.
Böhme
Christoph
C
Faculty of Technology, University of Bielefeld, Germany.
Nimtz
Manfred
M
Grabenhorst
Eckart
E
Conradt
Harald S
HS
Strathmann
Annemarie
A
Ragg
Hermann
H
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D006031:Glycosylation; D006493:Heparin / Q000378:metabolism*; D015844:Heparin Cofactor II / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011134:Polysaccharides / Q000032:analysis / Q000097:blood / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D013431:Sulfates / Q000737:chemistry; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism; D014415:Tunicamycin / Q000494:pharmacology; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism*
D010446:Peptide Fragments; D011134:Polysaccharides; D011994:Recombinant Proteins; D013431:Sulfates; D014415:Tunicamycin; D014443:Tyrosine; D015844:Heparin Cofactor II; D006493:Heparin; D014357:Trypsin