title: Partial glycosylation of antithrombin III asparagine-135 is caused by the serine in the third position of its N-glycosylation consensus sequence and is responsible for production of the beta-antithrombin III isoform with enhanced heparin affinity.
abstract: Two antithrombin III (ATIII) isoforms occur naturally in human plasma. The alpha-ATIII isoform has four N-linked oligosaccharides attached to asparagines 96, 135, 155, and 192. The beta-ATIII isoform lacks carbohydrate on asparagine-135 (N135), which is near the heparin binding site, and binds heparin with higher affinity than does alpha-ATIII. Two isoforms are also produced when the normal human ATIII cDNA sequence is expressed in baculovirus-infected insect cells, and the recombinant beta' isoform similarly binds heparin with higher affinity than the recombinant alpha' isoform. Consensus sequences (CSs) of the ATIII N-glycosylation sites are N-X-S for 135 and N-X-T for 96, 155, and 192. On the basis of database and in vitro glycosylation studies suggesting that N-X-S CSs are utilized less efficiently than N-X-T CSs, we hypothesized that the beta-ATIII isoform might result from inefficient core glycosylation of the N135 N-X-S CS due to the presence of a serine, rather than a threonine, in the third position. ATIIIs with N-X-S, N-X-T, and N-X-A consensus sequences were expressed in baculovirus-infected insect cells. In contrast to the N-X-S sequence, which expressed a mixture of alpha' and beta' molecules, the N-X-T variant produced alpha' exclusively, while the N-X-A variant produced beta' exclusively. Thus, serine in the third position of the N135 CS is responsible for its "partial" glycosylation and leads to production of beta-ATIII.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Microbiology & Immunology Department, Temple University Medical School, Philadelphia, Pennsylvania 19140, USA.
authors.forename: V
authors.initials: V
authors.lastname: Picard
authors.forename: E
authors.initials: E
authors.lastname: Ersdal-Badju
authors.forename: S C
authors.initials: SC
authors.lastname: Bock
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000990:Antithrombin III / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D001216:Asparagine; D016367:Baculoviridae; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D016384:Consensus Sequence; D017931:DNA Primers; D015870:Gene Expression; D006031:Glycosylation; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D012694:Serine; D018411:Spodoptera; D014162:Transfection
chemical_list: D017931:DNA Primers; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D012694:Serine; D001216:Asparagine; D000990:Antithrombin III; D006493:Heparin