Biochemistry of Factor IX and Molecular Biology of Hemophilia B, страница 3

Profactor IX, including the propeptide and factor IX sequence, undergoes carboxylation in the endoplasmic reticulum. Profactor IX, even in its fully carboxylated form and in contradistinction to factor IX, is not capable of binding to membrane surfaces. 51 Presumably, this feature prevents factor IX from being hung up inside the cell, where high levels of calcium ions within the endoplasmic reticulum could support factor IX-membrane interaction. Profactor IX undergoes additional post-translational processing, including disulfide bond formation and glycosylation, characteristic of other secreted plasma proteins. The propeptide is cleaved as a late processing event. MLID83065193 MLID86287341 MLID86189947  7,52,53 Furin/PACE cleaves peptide bonds at a site adjacent to the sequence Arg –4:X:Arg –2:Arg –1, where X can be any amino acid. MLID93231990  54 Bristol and colleagues MLID93216710  55 used site-directed mutagenesis to define a hierarchy of the efficiency of cleavage given different paired amino acids at the P1 and P2 positions: Lys-Arg > Arg-Arg > Thr-Arg > Arg-Lys > Lys-Lys Е Lys-Thr. g-Carboxylation precedes propeptide cleavage. 51

Structure of Factor IX

Human factor IX is a single-chain glycoprotein composed of 415 amino acids (Fig. 107-4). MLID82272386 MLID83065193 MLID80056619  6,7,56 It has a molecular weight of 56,000. MLID74051884  57,58

Propeptide

The propeptide directs g-carboxylation of profactor IX and then is cleaved to yield factor IX. The propeptide includes an amphipathic a-helix, with a hydrophobic and hydrophilic face. MLID92002030  59 The carboxylation recognition site is located proximal to this helix. Mutations in the propeptide have been documented as the cause of some forms of hemophilia B. Diuguid et al. MLID86287341  52 determined the size of the propeptide of factor IX by analysis of a mutant factor IX, factor IX Cambridge. This mutant has an 18-residue N-terminal extension due to the mutation of Arg -1 to a serine, precluding propeptide cleavage by a propeptidase with trypsin-like specificity. Concurrently Bent- ley et al. MLID86189947  53 evaluated factor IX Oxford 3, a mutant factor IX in which Arg -4 is mutated to glutamine; this mutation also prevents propeptide cleavage. MLID86189947  53 Similar mutations have been described: factor IX San Dimas (Arg -4 B Gln), MLID89291896  60 factor IX Troed-Y-Rhiw (Arg -4 B Gln), MLID89335613  61 factor IX London-3 (Arg -4 B Gln), factor IX London-4 (Arg -4 B Gln), MLID89305505  62 factor IX Malmo-6 (Arg -4 B Trp), MLID89305505  62 and factor IX Kawachinagano (Arg -4 B Gln). MLID89335614  63 Each of these mutants is characterized by the complete absence of factor IX activity and the presence of the 18-residue propeptide extension. Of the several proteins studied, MLID86287341 MLID89291896 MLID88067772  52,60,64 there is a partial defect in g-carboxylation of factor IX. Because of this defect in g-carboxylation and because of the failure of propeptide cleavage, these profactor IX mutants cannot bind to phospholipid vesicles, nor can they be activated by factor XIa. These data and the marked sequence homology of this domain in g-carboxyglutamic acid-containing proteins led to the proposal of a role for the propeptide in designating protein precursors containing this specific propeptide for subsequent g-carboxylation. MLID85298305  65 Using site-specific mutagenesis and an heterologous mammalian expression system, Jorgensen et al. MLID87102866 MLID88033058  26,41 demonstrated that protein-engineered factor IX lacking the 18-residue propeptide was not carboxylated in vivo. Similarly, point mutations at –16 (Phe B Ala) or –10 (Ala B Glu), both positions highly conserved in the propeptides of the vitamin K-dependent proteins, inhibited g-carboxylation. These results demonstrated that the propeptide contains a recognition element, designated the g-carboxylation recognition site, which signals for the g-carboxylation of the vitamin K-dependent proteins during hepatic biosynthesis.