Heparin cofactor II is more sensitive than antithrombin to secretory impairment arising from mutations introduced into its carboxy-terminal region

Thromb Res. 2004;113(2):163-73. doi: 10.1016/j.thromres.2004.02.015.

Abstract

Introduction: Antithrombin (AT) and heparin cofactor II (HCII) are plasma glycoproteins and serpins that inhibit thrombin. We showed [Blood 86 (1995) 3461] that recombinant rabbit AT containing the Utah mutation of AT, P407L, was inefficiently secreted by transfected primate and rodent cultured cells. In the current study, the effects of P407L and related substitutions in human AT and human HCII were investigated.

Materials and methods: Cultured cells were transfected transiently (COS-1) or permanently (CV-1) with AT and HCII expression vectors encoding the wild type or mutant serpins. The amount of protein secreted was determined immunologically, while RNA levels were assessed by reverse-transcription-PCR (RT-PCR). The kinetics of secretion were investigated by pulse-chase experiments, supplemented by endoglycosidase H or lactacystin treatment.

Results and conclusions: The F450L, P455L, P477L, P477*, and T446* (*=stop codon) mutations reduced HCII secretion 6.6- to 24-fold, while the F402L, A404T, and P407L mutations reduced AT secretion in COS-1 cells 1.7- to 5.2-fold. Homologous mutants HCII (P455L) and AT (P407L) were transcribed at similar levels in COS-1 cells, but were secreted less rapidly and less efficiently than their wild-type counterparts. HCII (P455L) exhibited intracellular proteasomal degradation in permanently transfected CV-1 cells, while AT (P407L) secretion was unaffected in this milieu. HCII secretion is thus more sensitive than that of AT to C-terminal mutations, as shown in two primate cell lines, likely reflecting a greater tendency to misfold during synthesis. We speculate that this difference may arise due to an interstrand s1C/s4B loop that is shorter in HCII than in AT.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Animals
  • Antithrombin III / genetics*
  • Antithrombin III / metabolism
  • Cell Line
  • Chlorocebus aethiops
  • Genetic Variation
  • Heparin Cofactor II / genetics*
  • Heparin Cofactor II / metabolism
  • Humans
  • Kinetics
  • Models, Molecular
  • Mutagenesis, Site-Directed*
  • Protein Folding
  • RNA, Messenger / analysis
  • Rabbits
  • Serpins / genetics
  • Serpins / metabolism
  • Transfection

Substances

  • RNA, Messenger
  • Serpins
  • Heparin Cofactor II
  • Antithrombin III