title: Type II antithrombin deficiency caused by a large in-frame insertion: structural, functional and pathological relevance.
abstract: BACKGROUND
  The metastable native conformation of serpins is required for their protease inhibition mechanism, but also renders them vulnerable to missense mutations that promote protein misfolding with pathological consequences.
  
  
  OBJECTIVE
  To characterize the first antithrombin deficiency caused by a large in-frame insertion.
  
  
  METHODS
  Functional, biochemical and molecular analysis of the proband and relatives was performed. Recombinant antithrombin was expressed in HEK-EBNA cells. Plasma and recombinant antithrombins were purified and sequenced by Edman degradation. The stability was evaluated by calorimetry. Reactive centre loop (RCL) exposure was determined by thrombin cleavage. Mutant antithrombin was crystallized as a dimer with latent plasma antithrombin.
  
  
  RESULTS
  The patient, with a spontaneous pulmonary embolism, belongs to a family with significant thrombotic history. We identified a complex heterozygous in-frame insertion of 24 bp in SERPINC1, affecting strand 3 of β-sheet A, a region highly conserved in serpins. Surprisingly, the insertion resulted in a type II antithrombin deficiency with heparin binding defect. The mutant antithrombin, with a molecular weight of 59 kDa, had a proteolytic cleavage at W49 but maintained the N-terminal disulphide bonds, and was conformationally sensitive. The variant was non-inhibitory. Analysis of the crystal structure of the hyperstable recombinant protein showed that the inserted sequence annealed into β-sheet A as the fourth strand, and maintained a native RCL.
  
  
  CONCLUSIONS
  This is the first case of a large in frame-insertion that allows correct folding, glycosylation, and secretion of a serpin, resulting in a conformationally sensitive non-inhibitory variant, which acquires a hyperstable conformation with a native RCL.
authors: Centro Regional de Hemodonación, University of Murcia, Regional Campus of International Excellence Campus Mare Nostrum, Murcia, Spain.
authors: I
authors: I
authors: Martínez-Martínez
authors: D J D
authors: DJ
authors: Johnson
authors: M
authors: M
authors: Yamasaki
authors: J
authors: J
authors: Navarro-Fernández
authors: A
authors: A
authors: Ordóñez
authors: V
authors: V
authors: Vicente
authors: J A
authors: JA
authors: Huntington
authors: J
authors: J
authors: Corral
mesh_terms: D000595:Amino Acid Sequence; D000991:Antithrombins / Q000737:chemistry / Q000378:metabolism*; D025861:Blood Coagulation Disorders, Inherited / Q000235:genetics* / Q000378:metabolism; D002152:Calorimetry, Differential Scanning; D002460:Cell Line; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013917:Thrombin / Q000378:metabolism
chemical_list: D000991:Antithrombins; D013917:Thrombin