Mutation of any site of N-linked glycosylation accelerates the in vivo clearance of recombinant rabbit antithrombin.
Antithrombin (AT) is a plasma protein with four sites of N-linked glycosylation. Asn 135 is incompletely glycosylated, and the resulting 3-glycan AT is cleared more rapidly in vivo than the 4-glycan form. The Asn codons in each of the four sites of glycosylation were altered in turn, to create four mutant rabbit AT cDNAs. Permanently transfected CHO cell lines were generated following transfection of the resulting constructs, encoding either the wild-type rabbit AT (AT-WT) or one of the four underglycosylated variants (AT-N96Q, AT-N135Q, AT-N155Q, and AT-N155Q). Comparison of the five resulting recombinant AT proteins revealed that the major AT species of each variant co-migrated on SDS gels, and migrated more rapidly than the major form of AT-WT. The shift in mobility, from 60 to 57 kDa, was consistent with the loss of one fully sialylated complex N-linked glycan. Neither the amount of AT secreted (range: 1.25 to 4.2 microg/10(6) cells/day) nor the kinetics of secretion differed significantly between cell lines expressing AT-WT or any of the AT variants. All forms of recombinant rabbit AT were capable of forming denaturation-resistant complexes with thrombin. Purification and radioiodination of each of the five recombinant AT proteins permitted pharmacokinetic analysis of their individual clearance in rabbits. While neither the equilibration half-life (t(0.5)alpha) nor the terminal catabolic half-life (t(0. 5)beta) differed significantly between plasma-derived rabbit AT and AT-WT, the t(0.5)beta of all the underglycosylated variants was decreased relative to that of AT-WT (maximum reduction in mean: from 70.1+/-3.2 h to 52.4+/-2.5 h). These results suggest that the overall extent of glycosylation, rather than the location within AT of the glycan chains, is a primary determinant of AT clearance.
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Departments of Pathology and Molecular Medicine and Biochemistry McMaster University, Hamilton, Ontario, Canada.
Blajchman
M A
MA
Ananthanarayanan
V S
VS
Smith
I J
IJ
Sheffield
W P
WP
D019943:Amino Acid Substitution; D000818:Animals; D000925:Anticoagulants / Q000737:chemistry / Q000493:pharmacokinetics; D000991:Antithrombins / Q000737:chemistry / Q000235:genetics / Q000493:pharmacokinetics*; D001216:Asparagine / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D016466:CHO Cells; D006224:Cricetinae; D004586:Electrophoresis; D006031:Glycosylation / Q000187:drug effects; D006207:Half-Life; D006801:Humans; D016297:Mutagenesis, Site-Directed / Q000502:physiology; D009154:Mutation / Q000502:physiology*; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000493:pharmacokinetics; D014162:Transfection
D000925:Anticoagulants; D000991:Antithrombins; D011994:Recombinant Proteins; D001216:Asparagine