Vitamin K-dependent carboxylation of the carboxylase

Abstract

Vitamin K-dependent (VKD) proteins require modification by the VKD-gamma-glutamyl carboxylase, an enzyme that converts clusters of glus to glas in a reaction that requires vitamin K hydroquinone, for their activity. We have discovered that the carboxylase also carboxylates itself in a reaction dependent on vitamin K. When pure human recombinant carboxylase was incubated in vitro with 14CO2 and then analyzed after SDS/PAGE, a radiolabeled band corresponding to the size of the carboxylase was observed. Subsequent gla analysis of in vitro-modified carboxylase by base hydrolysis and HPLC showed that all of the radioactivity could be attributed to gla residues. Quantitation of gla, asp, and glu residues indicated 3 mol gla/mol carboxylase. Radiolabeled gla was acid-labile, confirming its identity, and was not observed if vitamin K was not included in the in vitro reaction. Carboxylase carboxylation also was detected in baculovirus-(carboxylase)-infected insect cells but not in mock-infected insect cells, which do not express endogenous VKD proteins or carboxylase. Finally, we showed that the carboxylase was carboxylated in vivo. Carboxylase was purified from recombinant carboxylase BHK cells cultured in the presence or absence of vitamin K and analyzed for gla residues. Carboxylation of the carboxylase only was observed with carboxylase isolated from BHK cells cultured in vitamin K, and 3 mol gla/mol carboxylase were detected. Analyses of carboxylase and factor IX carboxylation in vitro suggest a possible role for carboxylase carboxylation in factor IX turnover, and in vivo studies suggest a potential role in carboxylase stability. The discovery of carboxylase carboxylation has broad implications for the mechanism of VKD protein carboxylation and Warfarin-based anti-coagulant therapies that need to be considered both retrospectively and in the future.

Figure 1

In vitro modification of the carboxylase. Carboxylase was purified from a r-fIX, r-carboxylase BHK cell line. Two independent preparations (lanes 1 and 2, 4 × 10⁶ cpm/h of peptide activity or ≈6 μg) were analyzed by SDS/PAGE and silver staining (A) and by incubation in a protein carboxylation reaction followed by SDS/PAGE and PhosphorImager analysis (B). (C) Solubilized microsomes (100 μg) from baculovirus(carboxylase)-infected or mock-infected SF-21 cells were incubated in a protein carboxylation reaction for 2 min and then analyzed by SDS/PAGE and PhosphorImager analysis. In parallel experiments performed in a reaction lacking vitamin K, no radiolabeled bands were detected even with incubations up to 2 h (data not shown).

Figure 2

Gla analysis of in vitro carboxylated carboxylase. The propeptide eluant isolated from r-fIX, r-carboxylase BHK cells (100 pmol, Fig. 1) was chromatographed on Q-Sepharose, in vitro carboxylated, and then TCA precipitated and base hydrolyzed. Samples with or without vitamin K in the in vitro protein carboxylation reaction were processed in parallel. After the amino acid hydrolysate was resuspended in water (25 μl), duplicate aliquots (2 μl) were quantitated for radioactivity and duplicate samples (10 μl) were analyzed by HPLC. Peaks corresponding to gla (0.8 min), asp (1.6 min), and glu (3.5 min) were collected and counted, and the radioactivity (in cpm) is indicated at the bottom of the chromatogram.

Figure 3

Analysis of carboxylase isolated from a r-carboxylase BHK cell line. (A) Solubilized microsomes were prepared from r-carboxylase BHK cells cultured in the presence or absence of vitamin K (vit K). Duplicate samples (50 μg) were incubated in an in vitro protein carboxylation reaction for 1 h and then processed by using SDS/PAGE and PhosphorImager analysis. (B) Carboxylase (≈200 ng) purified from the solubilized microsomes by chromatography on an α-C-terminal carboxylase peptide Ab column was analyzed by SDS/PAGE and silver staining.

Figure 4

Gla analysis of carboxylase purified from r-carboxylase BHK cells. Carboxylase purified from r-carboxylase BHK cells cultured with or without vitamin K (20 pmol each, Fig. 3B) was TCA precipitated and base hydrolyzed. The amino acid hydrolysates were resuspended in 100 μl of water, and triplicate aliquots were analyzed by HPLC. The numbers above each peak indicate the retention times.

Figure 5

Kinetics of carboxylation of factor IX and carboxylase. (A and B) Pure carboxylase (Fig. 1, 14 pmol, equivalent to 10⁶ cpm/h of peptide activity) was incubated (A) alone or (B) with profIX (340 pmol). (C) Carboxylase–fIX complex (equivalent to 2 × 10⁶ cpm/h of peptide activity or 28 pmol carboxylase and presumably an equimolar amount of fIX) was isolated by using α-C-terminal carboxylase peptide Ab resin, as described in Materials and Methods. The fIX in the complex is the profIX form of fIX. All three assays were performed in a final volume of 400 μl of protein carboxylation mix, and at timed intervals after the addition of vitamin K hydroquinone (20 μg), aliquots (40 μl) were withdrawn and the reaction was quenched by the addition of 40 μl of SDS/PAGE loading buffer. The samples were then analyzed by SDS/PAGE and PhosphorImager analysis by using ¹⁴C-methylated BSA standards to convert the PhosphorImager signal to cpm. The 50- and 60-kDa profIX forms in B are indicated by two lines on the left.

Figure 6

New questions raised by the discovery of carboxylase carboxylation. The carboxylation of fIX and the carboxylase are depicted. The enzymatic properties and fate of the carboxylated carboxylase are open questions, which include whether carboxylase carboxylation affects the stability of the carboxylase (3?) and whether carboxylase carboxylation and decarboxylation occur during the turnover of each VKD substrate (2?). The existence of a decarboxylase activity, either by reaction reversibility (1?) or by an unidentified decarboxylase (2?), is also unknown.