Cell
Volume 48, Issue 2, 30 January 1987, Pages 185-191
Journal home page for Cell

Article
Recognition site directing vitamin K-dependent γ-carboxylation resides on the propeptide of factor IX

https://doi.org/10.1016/0092-8674(87)90422-3Get rights and content

Abstract

Posttranslational processing of vitamin K-dependent proteins includes γ-carboxylation of specific glutamic acid residues to form γ-carboxyglutamic acids. To determine whether carboxylation is directed by the propeptide sequence, homologous among the precursors of these proteins, alterations were made in the Factor IX propeptide cDNA. The extent of γ-carboxylation of recombinant Factor IX was assessed using conformation-specific antibodies directed against the γ-carboxyglutamic acid-dependent, metal-stabilized structure. Deletion of the propeptide (residues −18 to −1) abolished carboxylation, but not secretion, of Factor IX. Substitution of alanine for phenylalanine −16 or glutamic acid for alanine −10 also impaired carboxylation. These results indicate that the Factor IX propeptide participates in defining a recognition site that designates an adjacent glutamic acid-rich domain for γ-carboxylation. The association of the propeptide with the γ-carboxylation recognition site provides the first demonstration of a specific function served by a propetide in posttranslational protein processing.

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      However, FLEEL is a poor substrate analog for GGCX for the following reasons: (1) of the two glutamic acids, only the first glutamic acid of FLEEL is carboxylated during in vitro activity assay (Burgess, Esnouf, Rose, & Offord, 1983; Decottignies-Le Marechal, Rikong-Aide, Azerad, & Gaudry, 1979); (2) only small amounts of substrate are converted to product (Soute, Ulrich, & Vermeer, 1987); and (3) FLEEL binds poorly to GGCX, with a Km of ~ 4 mM (Suttie et al., 1979). Knowing that GGCX interacts with clotting-factor precursors, mainly through an 18-amino acid propeptide (Jorgensen et al., 1987; Pan & Price, 1985), and since propeptide significantly increases small peptide substrate carboxylation (Knobloch & Suttie, 1987), Ulrich et al. used a 28-residue peptide, based on residues − 18 to + 10 in prothrombin (proPT28), as the peptide substrate of GGCX (Ulrich, Furie, Jacobs, Vermeer, & Furie, 1988). This 28-residue peptide is efficiently carboxylated, with a Km that is three orders of magnitude lower than FLEELs.

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