(Received for publication, February 6, 1997)
From the Biology Department, University of North
Carolina, Chapel Hill, North Carolina 27599-3280 and
§ Sloan-Kettering Institute for Cancer Research, Rockefeller
Research Institute, New York, New York 10021
-Glutamyl carboxylase is an integral membrane
protein required for the posttranslational modification of vitamin
K-dependent proteins. The main recognition between the
enzyme and its substrates is through an 18-amino acid propeptide. It
has been reported that this binding site resides in the
amino-terminal third of the
-glutamyl carboxylase molecule
(Yamada, M., Kuliopulos, A., Nelson, N. P., Roth, D. A., Furie,
B., Furie, B. C., and Walsh, C. T. (1995) Biochemistry
34, 481-489). In contrast, we found the binding site in the carboxyl
half of the
-glutamyl carboxylase. We show that the carboxylase may
be cleaved by trypsin into an amino-terminal 30-kDa and a
carboxyl-terminal 60-kDa fragment joined by a disulfide bond(s), and
the propeptide binds to the 60-kDa fragment. The sequence of the amino
terminus of the 60-kDa fragment reveals that the primary
trypsin-sensitive sites are at residues 349 and 351. Furthermore, the
tryptic fragment that cross-links to the propeptide also reacts with an
antibody specific to the carboxyl portion of the
-glutamyl
carboxylase. In addition, cyanogen bromide cleavage of bovine
-glutamyl carboxylase cross-linked to the peptide comprising
residues TVFLDHENANKILNRPKRY of human factor IX yields a cross-linked
fragment of 16 kDa from the carboxyl half of the molecule, the
amino-terminal sequence of which begins at residue 438. Thus, the
propeptide binding site lies carboxyl-terminal to residue 438 and is
predicted to be in the lumen of the endoplasmic reticulum.
-Glutamyl carboxylation, accomplished by the enzyme
-glutamyl carboxylase, is a posttranslational modification essential for the biological activities of a number of vitamin
K-dependent proteins. The importance of
-glutamyl
carboxylation is demonstrated by the various functions of the vitamin
K-dependent proteins. The best characterized functions are
related to coagulation and are exemplified by proteins such as
prothrombin, factor VII, factor IX, factor X, protein C, and protein S. In addition, there are two bone proteins, osteocalcin and matrix Gla
protein (1), and the newly discovered growth arrest-specific protein
Gas 6 (2).
The -glutamyl carboxylation reaction occurs in the endoplasmic
reticulum (3-5), where the enzyme
-glutamyl carboxylase uses the
small substrates carbon dioxide, oxygen, and vitamin K hydroquinone to
convert specific glutamic acid residues of the vitamin
K-dependent protein into the
-carboxyl glutamic acid Gla. During the process of
-glutamyl carboxylation, the vitamin K
hydroquinone is converted to vitamin K epoxide, which must be recycled
to vitamin K hydroquinone by the enzyme epoxide reductase for the
reaction to continue.
The enzymatic activity of the -glutamyl carboxylase was first
discovered in the postmitochondrial supernatant of hepatocytes (6).
Numerous early studies used microsomes as the crude enzyme sources and
the pentapeptide FLEEL as the in vitro substrate to establish a basic understanding of
-glutamyl carboxylation (7). By
comparing the cDNA-deduced amino acid sequences of different vitamin K-dependent proteins, Pan and Price (8) proposed
that the highly conserved propeptide sequences of the vitamin
K-dependent proteins function as a recognition site for the
enzyme
-glutamyl carboxylase. This hypothesis was confirmed by
Knobloch and Suttie (9), who demonstrated that the factor X propeptide
stimulated the incorporation of 14CO2 into the
substrate FLEEL. The recognition site was further elucidated by
Jorgensen et al. (10). Subsequently, propeptide-containing peptides were demonstrated to have a Km that was
three orders of magnitude lower than the pentapeptide substrate FLEEL for the enzyme
-glutamyl carboxylase (11, 12).
Further elucidation of the mechanisms of action of the carboxylase was
hindered by the lack of a purified enzyme. To begin a systematic study
we made a recombinant peptide, FIXQ/S,1
which contains the propeptide and the complete Gla domain sequence of
human factor IX (12), and successfully used it as an affinity ligand to
purify -glutamyl carboxylase to near homogeneity in a single step
purification (13). We were then able to clone the complete cDNA
sequence of
-glutamyl carboxylase using the deduced amino acid
sequence of a tryptic fragment (14).
The interaction between -glutamyl carboxylase and its substrate is
one of the most interesting aspects of
-glutamyl carboxylation, and
the major factor determining this interaction is the propeptide of the
various vitamin K-dependent proteins. Not only does the
-glutamyl carboxylase recognize the propeptide sequence, but it also
processes multiple modification sites in a single substrate molecule by
a processive mechanism (15).
We report here that the major propeptide binding site is in the hydrophilic, soybean seed lipoxygenase-like domain of the carboxylase molecule, which is predicted to reside in the lumen of the endoplasmic reticulum. This is in contradiction to the site proposed by Yamada et al. (16) and Sugiura et al. (17).
All chemicals were reagent grade. Disuccinimidyl suberate (DSS) and Iodobeads were purchased from Pierce. Na125I was from DuPont NEN. CentriPrep-30 was from Amicon. Immobilon-P was from Millipore. PVDF membranes were purchased from Bio-Rad. The ECL Western blotting detection reagent was from Amersham Corp. SP-Sepharose was from Pharmacia Biotech Inc. Protease inhibitors H-D-Phe-Phe-Arg-chloromethylketone and H-D-Phe-Pro-Arg chloromethylketone were from Bachem. Aprotinin, pepstatin A, and trypsin were purchased from Boehringer Mannheim. Leupeptin, phenylmethylsulfonyl fluoride, and CHAPS were obtained from Sigma. Peptides pro-FIX19 (AVFLDHENANKILNRPKRY), pro-FIX19-16BPA (TVBLDHENANKILNRPKRY; B is p-benzoylphenylalanine (BPA)), hbGC220-234 (CDADWVEGYSMEYLSR), and hbGC709-723 (CGRPSLEQLAQEVTYA) were obtained from Chiron (an amino-terminal cysteine was added to hbGC220-234 and hbGC709-723 for conjugation to keyhole limpet hemocyanin). Recombinant peptide FIXQ/S was prepared as described (12). Endoglycosidase H (Endo H) was purchased from New England Biolabs.
Cleavage of-Glutamyl carboxylase was affinity-purified by
elution method II as described (13). pro-FIX19 and protease inhibitors
(when used) were removed before trypsin cleavage by ultrafiltration using a CentriPrep-30 unit with deCIP buffer (25 mM MOPS,
pH 7.5, 500 mM NaCl, and 0.3% CHAPS). A pilot reaction was
run for each batch of
-glutamyl carboxylase and trypsin to determine
the optimal condition for limited trypsin cleavage. Because limited
trypsin cleavage could only be obtained with intact, enzymatically
active
-glutamyl carboxylase, the cleavage was carried out at
4 °C. Trypsin cleavage was stopped by adding
H-D-Phe-Pro-Arg chloromethylketone to a final
concentration of 1.6 µM.
Peptides pro-FIX19 and
FIXQ/S were radiolabeled with Na125I using Iodobeads
according to the manufacturer. To increase cross-linking efficiency,
the enzymatically active, intact or the limited trypsinized -glutamyl carboxylase (in elution buffer) was preincubated with propeptide-containing peptides (e.g. 0.5-2 µM
125I-peptide or 0.5-2 µM
125I-peptide with 40-60-fold excess unlabeled competitor)
at 4 °C for 1 h before DSS was added to a final concentration
of 50-200 µM. Cross-linking was carried out at 4 °C
and at times varying from 20 min to 3 h before it was stopped with
50 mM Tris-HCl, pH 8.8.
pro-FIX19-16BPA (16) was cross-linked to the carboxylase by exposure to a 365-nm UV lamp (Cole-Parmer E-97600) for 15 min at a distance of 15 cm. During photo cross-linking the sample was maintained on ice.
Generation of Antibodies against Synthetic Peptides Derived fromSynthetic peptides hbGC220-234 and hbGC709-723 were conjugated to keyhole limpet hemocyanin through an amino-terminal cysteine. One µg of conjugated peptide in Freund's complete adjuvant was subcutaneously inoculated into New Zealand White rabbits. One microgram of each conjugated peptide in Freund's incomplete adjuvant was used to boost the immunity 2 weeks after the first inoculation.
Identification of Tryptic Fragments by ImmunoblottingForty ng of the limited trypsinized carboxylase was analyzed by reducing SDS-PAGE (18) and electroblotted onto Immobilon-P (Millipore). The membrane was probed with 200-fold diluted anti-hbGC-amino acids antiserum and followed by a horseradish peroxidase-coupled anti-rabbit antibody. ECL Western blotting detection reagent was used as recommended by the manufacturer to reveal the reactive fragment.
Determination of the Propeptide Binding Region by Amino Acid Sequencing-Glutamyl carboxylase was purified as described
(13), except that 125I-pro-FIX19 or FIXQ/S was used in the
elution. Ten to 100 µg of affinity-purified
-glutamyl carboxylase
was concentrated 6-8-fold on CentriPrep-30 units. Cross-linking
reactions were carried out at 4 °C in 10-15 ml of 25 mM
MOPS, pH 7.5, 500 mM NaCl, 15% glycerol, 0.4%
phosphatidylcholine, and 0.25% CHAPS with 50-200 µM
DSS. Sixty-five percent ammonium sulfate was used to precipitate the proteins from the pro-FIX19 cross-linking mixture. Proteins were dissolved in 5 ml of 25 mM MOPS, pH 6.5, 0.5% CHAPS, and
10% glycerol and sonicated before being batch-adsorbed onto
SP-Sepharose. The
-glutamyl carboxylase and the cross-linked
-glutamyl carboxylase were co-eluted from an SP-Sepharose column
with a 0-1 M NaCl gradient in 25 mM MOPS, pH
7.5, 10% glycerol, 0.02% phosphatidylcholine, and 0.02% CHAPS. The
-glutamyl carboxylase-containing fractions were further concentrated
on CentriPrep-30, subjected to SDS-PAGE, electroblotted onto a
nitrocellulose membrane, cleaved with cyanogen bromide, concentrated,
refractionated by SDS-PAGE, and then analyzed by amino acid
sequencing.
The determination of the amino termini of the limited tryptic fragments was done at the Harvard Microchemistry Facility.
Quantitation of Radioactive Bands in PAGEThe radioactive bands were imaged with a Molecular Dynamics Storm 840 PhosphorImager, and quantitation was achieved with ImageQuant software.
The -glutamyl carboxylase is a 758-amino acid protein that
migrates on reducing SDS-PAGE as a 94-kDa protein. The enzymatically active
-glutamyl carboxylase is cleaved by limited trypsin digestion into two major fragments with electrophoretic mobilities of
approximately 60 and 30 kDa (Fig. 1, lane 3).
The 60-kDa fragment reacts with an antibody made to a 15-amino acid
peptide corresponding to residues 709-723, whereas the 30-kDa fragment
reacts with an antibody made to residues 220-234 near the amino
terminus of the
-glutamyl carboxylase (Fig. 2). In
the absence of the propeptide or during prolonged trypsin cleavage, the
60-kDa fragment is converted into a 50-kDa fragment. Because the 50-kDa
fragment, which is identified with the cross-linked fragment, failed to
react with an antibody made to residues near the carboxyl terminus of
the 60-kDa fragment (Fig. 2), we postulated that the 50-kDa fragment
arises by cleavage near the carboxyl terminus of the 60-kDa fragment.
From the results of the immunoblot, we predicted that both the 60- and
50-kDa fragments were derived from the carboxyl half and the 30-kDa
fragment from the amino-terminal half of the
-glutamyl carboxylase.
We also predicted that, although the 50- and 60-kDa fragments have
different carboxyl-terminal sequences, they would have the same
amino-terminal sequence.
To identify the primary trypsin-sensitive sites in the carboxylase,
affinity-purified bovine -glutamyl carboxylase was cleaved by
limited trypsin digestion, fractionated by reducing SDS-PAGE, and
transferred to PVDF membranes for amino acid sequence analysis. Sequence analysis revealed that the 50- and 60-kDa fragments arise from
cleavage following arginines 349 and 351 (Fig. 3). We
also attempted to determine the sequence of the 30-kDa fragment.
Analysis of amino acid composition predicted 15 pmol of the 30-kDa
fragment compared with 7 pmol each of the 60- and 50-kDa fragments in
the sample that was used for sequence analysis. This result indicated that the blotting efficiency for each fragment was similar and predicts
that, at each step in amino acid sequence analysis, the sum of the
amino acid yield of the 50- and 60-kDa fragments should equal the yield
at each step for the 30-kDa fragment. Because we had previously
determined that the amino terminus was blocked (14), however, we
expected sequence from the amino terminus only if trypsin cleavage had
occurred.
We obtained sequence from the amino terminus of the 30-kDa fragment,
indicating a trypsin cleavage site at lysine 19 of the bovine
carboxylase. Because the yield from each sequencing cycle of the 30-kDa
fragment is lower than the sum of the yields of the 50- and 60-kDa
fragments, this is consistent with a preparation in which the 30-kDa
band consists of about 50% of a -glutamyl carboxylase fragment with
a blocked amino terminus and about 50% with its amino terminus cleaved
at lysine 19 (Fig. 3).
Thus, if no other accessible tryptic cleavage sites occur before
residue 349, the band that migrates on SDS-PAGE at 30 kDa would have
actual molecular masses (excluding any modifications) of 40.2 (uncleaved form) and 38.2 (cleaved form) kDa. The larger carboxyl-terminal fragment, which migrates at 60 kDa on SDS-PAGE, would
have an actual mass of 47.4 kDa, again excluding modifications. The
difference in the mobility and expected mass of the 30-kDa fragment
could be due to anomalous migration on SDS-PAGE or to an additional
trypsin cleavage before residue 349. However, the difference in the
relative molecular mass of the 60-kDa fragments determined by mobility
on reducing SDS-PAGE and that predicted by the amino acid sequence
appears to be due to glycosylation of the potential glycosylation sites
in the luminal, carboxyl portion of the -glutamyl carboxylase. Fig.
1, lane 5, shows that removal of sugars from the carboxylase
with Endo H results in a change in the migration of the 60-kDa fragment
to that expected of an approximately 50-kDa fragment. In contrast,
there is no apparent change in the relative mobility of the 30-kDa
fragment following treatment with Endo H. A further insight gained from limited trypsin cleavage is that the 30- and 60-kDa fragments are
linked by one or more disulfide bonds, as they are not resolved on
SDS-PAGE except when the reducing agent is present (Fig.
4).
Before attempting to determine the site on the -glutamyl carboxylase
where the propeptide of factor IX binds, we cross-linked FIXQ/S or
pro-FIX19 to the
-glutamyl carboxylase with different chemical
cross-linkers to find one that gave the most efficient cross-linking.
We tried 1-ethyl-3-3(3-dimethylaminopropyl)-carbodiimide hydrochloride, dithiobis-(succinimidyl propionate), and DSS. The free
amine-specific, nonreversible cross-linker disuccinimidyl suberate gave
the highest cross-linking efficiency. Fig. 5
demonstrates that cross-linking of FIXQ/S to
-glutamyl carboxylase
results in a shift of approximately 40% of the
-glutamyl
carboxylase to a mobility consistent with a mass of about 100 kDa.
To determine which tryptic fragment contains the propeptide binding
site, we digested the -glutamyl carboxylase with trypsin using
conditions known to generate the 30- and 60-kDa fragments. The still
enzymatically active
-glutamyl carboxylase was then cross-linked to
125I-pro-FIX19 or FIXQ/S. Fig. 6 shows that
cross-linking is observed when either pro-FIX19 or FIXQ/S is used for
cross-linking and that cold pro-FIX19 effectively competes with either
radioactive peptide. Fig. 6 also clearly demonstrates that the
propeptide of factor IX binds to the carboxyl-terminal 60-kDa fragments
as well as the 50-kDa fragment of the
-glutamyl carboxylase
molecule. This observation has been repeated many times with different
preparations of
-glutamyl carboxylase and with several different
propeptides, including pro-FIX19, FIXQ/S, and biotin-pro-FIX19-biotin.
Identical results were obtained whether the cross-linking was done
before or after the limited trypsin digestion. The only requirement
appears to be that the
-glutamyl carboxylase is enzymatically
active. The fact that only active carboxylase will cross-link to the
propeptide is further evidence for the specificity of the cross-linking
reaction.
During the course of this work Yamada et al. (16) published
that the propeptide binding site was found within the amino-terminal 259 amino acids of the recombinant -glutamyl carboxylase. Because our results were different, we attempted to resolve the cause of the
difference. A photochemical peptide, pro-FIX19-16BPA, with sequence
identical to that used by Yamada et al. (16) was obtained and used for photochemical cross-linking. Fig. 7
demonstrates that pro-FIX19-16BPA predominantly cross-linked to the
60-kDa fragment no matter whether the cross-linking was accomplished photochemically or was DSS-mediated. Fig. 7A depicts a
silver-stained gel of the autoradiograph shown in Fig. 7B
and demonstrates that both the 60- and 30-kDa fragments are present in
our preparation. To confirm further that binding was predominately in
the 60-kDa fragment, we trypsin-cleaved another carboxylase sample and
photochemically cross-linked it to iodinated pro-FIX19-16BPA. We then
subjected the product to reducing and nonreducing SDS-PAGE and
quantitated the amount of radioactivity in each band with a
PhosphorImager. Fig. 8 shows a radioactive image of
photochemically cross-linked
-glutamyl carboxylase fractionated by
nonreducing and reducing PAGE. The 60-kDa band of the reduced sample
contained 87% of the radioactivity found in the 94-kDa unreduced
-glutamyl carboxylase. Approximately 5% appeared in the 30-kDa
band, and the remainder was in the uncleaved 94-kDa band. Thus, there
was no selective loss of a cleaved fragment in our preparation.
We have made numerous attempts to narrow further the region to which
the propeptide binds. These experiments were hampered by the
difficulties in obtaining complete cleavage of the carboxylase by
either enzymatic or chemical methods. Nevertheless, we were able to
obtain an amino-terminal sequence from a small cyanogen bromide
fragment of -glutamyl carboxylase cross-linked to pro-FIX19. The
-glutamyl carboxylase was chemically cross-linked to
125I-labeled pro-FIX19 by disuccinimidyl suberate. The
cross-linked
-glutamyl carboxylase was then fractionated by reducing
SDS-PAGE and transferred to a PVDF membrane. The location of the
cross-linked carboxylase was determined by autoradiography, and it was
excised from the membrane and cleaved in situ with cyanogen
bromide. The resulting cyanogen bromide fragments were again
fractionated by reducing SDS-PAGE and transferred to a PVDF membrane
for sequence analysis. The primary sequence obtained from the amino
terminus of this radioactive cyanogen bromide fragment was
KDHADMLKQYATC, which corresponds to residues 438-450 of the
-glutamyl carboxylase and is shown boxed in Fig. 3. In
this case, cyanogen bromide cleavage occurred at a tryptophan rather
than the expected methionine residue. Although not common, other
examples of cleavage at tryptophan residues by cyanogen bromide have
been reported (19). Secondary and tertiary sequences were also present
in the sequence analysis. The secondary sequence resulted from cleavage
after the methionine at 443, whereas the tertiary sequence was
apparently the result of unexplained cleavage following histidine
440.
The radioactive band migrated as 16 kDa (Fig. 9);
however, because it is cross-linked to pro-FIX19, which is 2.3 kDa, and there is a potential N-glycosylation site, we estimate that
the size is considerably less than 16 kDa. The most reasonable
assumption is that it terminates at residue 507, the next methionine in
the carboxylase. Cleavage at the next methionine beyond methionine 507 (methionine 579) would be expected to give a 17.7-kDa fragment, which,
if cross-linked, would certainly migrate as a larger fragment. This
size would probably be even larger, because four of the nine potential
N-glycosylation sites of the -glutamyl carboxylase are
located between residues 438 and 579. The cyanogen bromide fragment
that we isolated and sequenced appears to be the major species of
labeled fragment, because there are no other radioactive bands visible
on the gel. The 125I-proFIX19 used for cross-linking was
also used for a size marker, making it very unlikely that any
cross-linked peptides could have run off the end of the gel during PAGE
analysis. Furthermore, three overlapping sequences obtained from the
radioactive cyanogen bromide band are all found within the larger
tryptic fragment that we identified by immunoblotting and amino acid
sequencing.
Sugiura et al. (17) recently published a mutagenesis study
on bovine carboxylase expressed in Chinese hamster ovary cells (17).
They concluded from their studies that "lysine 217 or lysine 218 may
be key for substrate and/or propeptide recognition or for catalysis."
Chinese hamster ovary cells contain endogenous carboxylase activity,
and it is therefore difficult to interpret their kinetic data.
Interpretation is especially difficult in the relevant mutants in which
low activity levels of bovine carboxylase were encountered. It is of
note, however, that they observed that higher propeptide concentrations
were required to stimulate the incorporation of
14CO2 into FLEEL in the -glutamyl
carboxylase mutated at positions 513 and 515, which would support the
data presented in this article.
The differences in the results reported here and those reported by
Yamada et al. (16) and confirmed by Sugiura et
al. (17) are probably due to their implicit assumption that the
migration of proteins on gels is directly proportional to their
molecular mass. However, it is true that some regions of the two
fragments are in very close proximity, as shown by the fact that the
60- and 30-kDa fragments are joined by a disulfide bond(s). Therefore, it is possible that lysines 217 and 218 are in close proximity to
residues in the carboxyl half of the carboxylase molecule and that
their conditions were sufficiently different from ours that, in their
hands, the propeptide reacted with amino-terminal residues rather than
the carboxyl-terminal residues that we saw. Although there is serious
difficulty in interpreting some of the data of Sugiura et
al. (17) because of the endogenous carboxylase activity, their
data suggest that mutations at residues 234-235, 359-361, 406-408,
and 513-515 all affect propeptide binding. It is difficult to draw
firm conclusions until there is a more extensive characterization of
their mutated enzymes in a system in which there is no endogenous -glutamyl carboxylase. But it appears that the data of Sugiura et al. (17) agree as well with our data as they do with the data of Yamada et al. (16).
Our data, derived from both cyanogen bromide and tryptic cleavage,
demonstrate clearly that the propeptide is cross-linked to the
carboxyl, luminal portion of the -glutamyl carboxylase. We conclude
that the propeptide is cross-linked to residues between lysine 438 and
methionine 507. Thus, this area is a prime candidate for further
mutational analysis.