(Received for publication, January 13, 1995)
From the
As substrate for protein-mono-ADP-ribosyltransferases, NAD has been shown to be the donor of ADP-ribose to many different nucleophiles found in proteins. This post-translational modification of proteins has been implicated in the regulation of membrane-associated processes including signal transduction, muscle cell differentiation, and protein trafficking and secretion. Described here is the preparation and chemical characterization of low molecular weight conjugates that were used as models for an acetal linkage between ADP-ribose and the hydroxyl group of a protein acceptor such as serine, threonine, tyrosine, hydroxyproline, or hydroxylysine residues. Model conjugates of ADP-ribose containing an acetal linkage were prepared, their structures were established by NMR, and the chemical stability of the linkage to ADP-ribose was studied and compared to the other known ADP-ribosyl-amino acid linkages. The rapid release of intact ADP-ribose from the acetal model conjugates in 44% formic acid distinguished them chemically from all the other known ADP-ribosyl-amino acid modifications. Rat liver proteins were shown to be modified by ADP-ribose in vivo by acid-labile linkages, providing evidence for a new class of endogenous ADP-ribose modification of animal cell proteins. The amount of modification was approximately 16 pmol of ADP-ribose per mg of total protein, and proteins modified by acid-labile linkages were detected in all subcellular fractions examined, suggesting that the scope of this modification in vivo is broad.
In eucaryotic cells, NAD is consumed by multiple classes of ADP-ribose transfer reactions involved in the regulation of a variety of metabolic processes(1, 2) . Many different cellular nucleophiles have been shown to serve as acceptors of ADP-ribose. Poly(ADP-ribose) polymerase catalyzes transfer of ADP-ribose to glutamate and aspartate (3, 4) residues in proteins and to other ADP-ribose residues to generate polymers of ADP-ribose(1) . The synthesis of ADP-ribose polymers is involved in chromatin structural changes necessary for the recovery of cells from DNA damage(1) . Endogenous protein-mono-ADP-ribosyltransferases that modify arginine(5, 6, 7) , cysteine(8, 9, 10) , asparagine(11) , and modified histidine (diphthamide) (12) residues in protein acceptors have been detected. These enzymes have been implicated in the regulation of a number of membrane-associated processes including modulation of adenylate cyclase (13, 14, 15, 16) , muscle cell differentiation(17, 18, 19) , and membrane trafficking and secretion(20) . NAD glycohydrolases catalyze intramolecular transfer of ADP-ribose to the adenine ring of NAD forming cyclic ADP-ribose and also catalyze hydrolysis of cyclic ADP-ribose to free ADP-ribose(21) . Cyclic ADP-ribose has been postulated to be a second messenger involved in the regulation of calcium signaling(22) . Free ADP-ribose, which is generated by removal of ADP-ribose from proteins and by the turnover of ADP-ribose polymers and cyclic ADP-ribose, can react nonenzymatically with protein lysine (23) and cysteine (24) residues, resulting in protein glycation.
The versatility of NAD as an ADP-ribose donor in biological chemistry raises the possibility that cellular nucleophiles that heretofore have not been detected may be modified by ADP-ribose. Protein hydroxyl groups have been shown to be the site of numerous post-translational modifications. For example, the regulation of numerous processes is achieved by reversible phosphorylation of protein serine, threonine, and tyrosine residues(25) . We describe here the preparation and chemical characterization of low molecular weight conjugates to serve as models for the linkage between ADP-ribose and the hydroxyl group of an acceptor nucleophile in protein. Such linkages are acetals; thus, we have used the term acetal conjugates to distinguish them from the other known classes of ADP-ribose modification of proteins. Information obtained from the characterization of the acetal conjugates has allowed the detection of a new class of ADP-ribose modification of proteins in vivo with chemical properties expected for the modification of protein hydroxyl groups. The results described here also suggest that a recently described protein-mono-ADP-ribosyltransferase involved in the regulation of membrane trafficking and secretion modifies protein hydroxyl groups (20) .
A Varian XL-300 NMR
spectrometer operating at 299.9 MHz for H and 75.4 MHz for
C was used to acquire spectral data. Samples were
lyophilized three times in D
O prior to NMR analysis.
H NMR spectral parameters were as follows: sweep width,
4400 Hz; data points, 32K; acquisition time, 3.6 s; acquisition delay,
1 s; 32 acquisitions in double precision mode.
C NMR
spectral parameters were as follows: sweep width, 18,000 Hz; data
points, 32K; acquisition time, 1 s; acquisition delay, 2 s;
20,000-25,000 acquisitions in double precision mode.
H spectra were referenced to HOD at 4.68 ppm, and
C spectra were referenced using the software of the
instrument.
The C spectrum of ADP-ribosylthreonine
methyl ester showed the following absorptions:
176.75, 174.91,
156.39, 152.96, 143.87, 136.83, 122.10, 111.81, 91.10, 87.62, 87.53,
85.18, 85.08, 78.14, 77.86, 74.70, 74.17, 70.71, 69.81, 69.20, 69.17,
64.24, 59.03, and 23.50 ppm. The
C spectrum of
ADP-ribosylserine ethyl ester showed the following absorptions:
176.30, 174.89, 172.01, 158.98, 156.37, 152.56, 143.54, 136.82, 122.29,
110.43, 91.11, 87.20, 84.92, 84.83, 78.01, 77.86, 74.71, 74.00, 70.85,
69.19, 67.90, 67.60, 66.57, 66.25, 65.68, 64.14, 64.01, 63.12, 61.28,
60.61, 60.11, 58.63, 58.48, 18.16, and 17.23 ppm. The
C
spectrum of ethoxy-ADP-ribose showed the following absorptions:
158.69, 158.66, 155.88, 144.01, 120.00, 109.59, 89.95, 87.01, 86.96,
84.33, 84.28, 77.37, 77.27, 73.90, 73.47, 69.72, 69.67, 68.29, 67.31,
and 17.20 ppm.
Figure 1: HPLC analysis of the reaction of snake venom NADase, NAD, and ethanol. A shows the incubation of NAD and ethanol. B shows the incubation of NAD and NADase. C shows the complete reaction mixture containing NADase, NAD, and ethanol. The arrow indicates the putative model conjugate formed in the reaction. The HPLC running buffer contained 7% methanol.
Figure 2:
C NMR of the putative
ADP-ribosylthreonine methyl ester. The sample was prepared as described
under ``Materials and Methods.'' NMR assignments for ribose
and adenine regions are based on published data of related
adenosine-containing compounds (32, 33) and on the
spectra of the parent compounds. The A designations refer to
the adenine ring; the R1` etc. designations refer to the
adenine proximal ribose; and the R1" etc. designations refer
to the second ribose moiety.
The NMR data also allowed
determination of the stereochemistry of the glycosidic linkage of the
model conjugates. The study of Miwa et al.(32) has
shown that C chemical shift values can unambiguously
differentiate the anomeric configuration of an acetal linkage. The
signals for
glycosidic linkages occur between 102 and 104 ppm,
while
linkages are observed at 109 ppm or higher. The values for
the conjugates obtained in this study ranged from 109 to 112 ppm,
indicating
configurations. The study of Ferro and Oppenheimer (33) of models of ribosyl acetals showed that the J
values from
H NMR also can
be used to assign anomeric configuration. The
configuration shows J
values between 4.0 and 4.6 Hz, while the
configuration shows values of 2.2 Hz or less. The J
for ADP-ribose acetal conjugates were
between 1.3 and 1.7 Hz, corroborating a
configuration for the
conjugates. The structures of the three conjugates characterized by NMR
are shown in Fig. 3. The chemical stability of the methoxy and
propoxy conjugates were also studied, but they were not exhaustively
characterized by NMR.
Figure 3: Structures of model acetal conjugates synthesized and characterized in this study.
Figure 4: Analysis by HPLC of the formic acid-catalyzed hydrolysis of ethoxy-ADP-ribose. Ethoxy-ADP-ribose was incubated in 44% formic acid at 37 °C. The release products are shown at zero time in A and at 30 min in B. The HPLC running buffer contained 5% methanol.
Figure 5:
A, stability studies of ethoxy-ADP-ribose.
Purified ethoxy-ADP-ribose was incubated in 44% formic acid (), 1 M hydroxylamine in 100 mM MOPS, pH 7.0 (
), 1 M NaOH at 37 °C (
), or 10 mM HgCl
at 25 °C (
). Aliquots were diluted to 1 ml with 50
mM potassium phosphate buffer, pH 6.0. Samples were subjected
to reversed-phase HPLC with detection at 254 nm. B, kinetics
of formic acid-catalyzed hydrolysis of acetal conjugates of ADP-ribose.
The purified conjugates were incubated in 44% formic acid at 37 °C.
Aliquots were diluted to 1 ml with 50 mM potassium phosphate,
pH 6.0. Samples were subjected to reversed-phase HPLC with detection at
254 nm.
, methoxy-ADP-ribose;
, ethoxy-ADP-ribose;
,
propoxy-ADP-ribose;
, ADP-ribosylserine ethyl ester;
,
ADP-ribosylthreonine methyl ester. A representative experiment is
shown.
Previously, we have characterized the chemical stability
of each of the other protein nucleophiles known to be modified by
ADP-ribose(23, 28, 29) . In contrast to the
acetal model conjugates, ADP-ribose attached to carboxylate (aspartate,
glutamate), guanidinium (arginine), sulfhydryl (cysteine), imidazolyl
(diphthamide), and amido (asparagine) groups are stable in 44% formic
acid at 37 °C, having t values of more than
10 h. Likewise, the ketoamine glycation products formed by ADP-ribose
modification of amino (lysine) groups are also stable in 44% formic
acid. Thus, the rapid release of intact ADP-ribose from an acetal
linkage in 44% formic acid distinguishes the acetal conjugates from all
of the other known ADP-ribosyl amino acids. It should be noted that,
while the conjugates were rapidly hydrolyzed at 37 °C in formic
acid, they were quite stable in ice-cold trichloroacetic acid, with a t
value of approximately 4 h. This stability
allowed ice-cold trichloroacetic acid to be used for the preparation of
tissues as described below.
Further characterizations of the acetal
model conjugates demonstrated other differences in chemical stability
between these conjugates and other ADP-ribosyl amino acids. Previous
studies have shown that the presence of 1 M neutral
hydroxylamine results in the release of intact ADP-ribose from
guanidinium and carboxylate groups (28) and that 10 mM HgCl results in the release of intact ADP-ribose from
sulfhydryl groups(29) . Fig. 5A shows that
ethoxy-ADP-ribose was stable to both of these treatments and that the
conjugate also was stable to treatment with 1 M NaOH. Similar
results were obtained with the other acetal model conjugates (data not
shown).
Figure 6:
Strong anion exchange HPLC analysis of
-ADP-ribose released by formic acid from alkali-treated rat liver
proteins as described under ``Materials and Methods.'' A, formic acid release; B, omission of formic acid
treatment; C, omission of chloroacetaldehyde, which forms the
fluorescent derivative of ADP-ribose; D, formic acid-treated
sample containing a spike of authentic
-ADP-ribose. The
sensitivity of the fluorometer was increased 10-fold at 8 min. Numbers on the ordinate represent fluorescence
intensity after the change in sensitivity. The arrow represents the the expected elution position of
-ADP-ribose.
To determine if the ADP-ribose bound by acid-labile linkages was attached to protein, the acid-insoluble fraction from rat liver was treated with proteases and subjected to molecular sieve chromatography using Sephadex G-100 prior to formic acid treatment and analysis for ADP-ribose. The elution profile of a control not subjected to protease treatment is shown in Fig. 7A. As expected, the release of ADP-ribose by formic acid was observed only in the fractions corresponding to high molecular weight material. B shows that, after protease treatment, only fractions corresponding to low molecular weight material contained ADP-ribose released by formic acid treatment. These data demonstrate that acid-labile ADP-ribose was protein-bound.
Figure 7: Sephadex G-100 molecular sieve chromatography of alkali-treated rat liver proteins. Fractions were analyzed for ADP-ribose released by acid (solid line) as described under ``Materials and Methods.'' The dashed lines represent absorption at 595 nm as a measure of protein. A, undigested material; B, protease-digested material.
The
acid-labile ADP-ribose conjugates of rat liver proteins were further
characterized by their sensitivity to alkali. For this, the fractions
were subjected to treatment with 1 M NaOH for 6 h followed by
dialysis prior to treatment with formic acid to release ADP-ribose.
Fractions prepared in this manner were compared to fractions that had
not been treated with NaOH. The total amount of ADP-ribose released
from fractions not subjected to NaOH treatment was approximately 16.0
pmol/mg of protein. Pretreatment with NaOH prior to analysis yielded
8.2 pmol of ADP-ribose/mg of protein, indicating that alkaline
treatment had released approximately one-half of the ADP-ribose. To
examine this further, the kinetics of release of ADP-ribose by formic
acid from the protein fractions was examined (Fig. 8). For
proteins not pretreated with NaOH, a biphasic release was observed with
a rapid phase having a t of approximately 1.5
min and a slower phase with a t
of approximately
20 min. Approximately one-half of the total ADP-ribose released was
accounted for in the rapid phase (8 pmol/mg). Alkali-treated proteins
showed only a single phase of ADP-ribose release (Fig. 8B) with a t
of
approximately 20 min, corresponding both in stability and amount to the
slower phase of acid release seen for proteins not previously treated
with NaOH. These data indicate the presence in rat liver proteins of
two classes of acid-labile ADP-ribose conjugates. Both classes of
ADP-ribose modification were shown to track with rat liver protein in
the experiments shown in Fig. 7.
Figure 8: Kinetic analysis of the release of ADP-ribose from rat liver proteins. Sample in B was pretreated with NaOH, and the sample in A was not treated with NaOH prior to formic acid release of ADP-ribose as described under ``Materials and Methods.''
One approach to the evaluation of possible biological functions of protein modification by monomeric ADP-ribose residues has been to search for and characterize endogenous protein-mono-ADP-ribosyltransferases. Since the identity of most of the endogenous ADP-ribose acceptor proteins is unknown, the ADP-ribosyltransferases have been categorized according to their specificity for the amino acid modified. Endogenous transferases specific for arginine(3, 4) , cysteine(8, 9, 10) , asparagine(11) , and diphthamide (12) residues have been described. Both arginine-specific and cysteine-specific transferases have been implicated in the regulation of adenylate cyclase activity via ADP-ribose modification of heterotrimeric G proteins(13, 14, 15, 16) . Arginine-specific transferases also have been implicated in the regulation of muscle cell differentiation (17, 18, 19) .
Our laboratory has addressed the scope of protein modification by ADP-ribose by examining for the presence of proteins modified by ADP-ribose in vivo. This has led to the identification of proteins modified at carboxylate(28) , arginine(28) , and cysteine (29) residues. The identification of arginine and cysteine residues as sites of ADP-ribose modification was facilitated by the availability of ADP-ribosylating bacterial toxins that allowed the preparation of standards for the development of analytical methods for their detection. The search for endogenous modification by ADP-ribose of protein amino acid residues for which ADP-ribosylating toxins have not been reported has been technically more challenging. For protein hydroxyl groups, we have approached this problem by synthesizing model conjugates to allow characterization of the chemical stability of these linkages. The possibility of an unreported class of ADP-ribose modification of proteins was suggested by an earlier study that identified protein cysteine residues as ADP-ribose acceptors in rat liver proteins(29) . In that study, formic acid was used to dissolve a trichloroacetic acid-insoluble fraction of rat liver followed by treatment with mercuric ion to release ADP-ribose from cysteine residues. In those experiments, ADP-ribose was detected following incubation of proteins with formic acid alone, although it was not clear whether this material was derived from a covalent modification or from noncovalently bound ADP-ribose trapped in the protein pellet during precipitation(28) . In the experiments described here, care has been taken to exhaustively remove noncovalently bound material prior to analysis.
Characterization of the acetal model conjugates demonstrated that the rapid release of intact ADP-ribose in 44% formic acid distinguishes this linkage to ADP-ribose from all of the other known ADP-ribosyl amino acid linkages(23, 28, 29) . This provided the opportunity to search for proteins modified at linkages characteristic of acetals. We initially detected the release of intact ADP-ribose from an acid-insoluble fraction of rat liver which was first subjected to molecular sieve chromatography to remove any ADP-ribose that may have been generated from nucleotides trapped in the acid-insoluble fraction (28) . The association of acid-labile ADP-ribose with protein was confirmed by the demonstration that protease treatment resulted in a shift in the elution profile to lower molecular weight fractions (Fig. 7). While the acid-labile ADP-ribose modifications detected have the properties expected for the modification of protein hydroxyl groups, the identification of the amino acid acceptor(s) of ADP-ribose will require further study. Sources of cellular protein hydroxyl groups that could form an acetal linkage with ADP-ribose include serine, threonine, tyrosine, hydroxyproline, and hydroxylysine residues.
Kinetic analysis of ADP-ribose released from protein by
formic acid revealed two chemical classes of acid-labile linkages based
on the rate of release in acid. One of these classes showed a rate of
release (t of approximately 20 min) within the
range of release kinetics observed for the acetal model conjugates. The
second class showed a rate of release more rapid than that of any of
the model conjugates. The differences in the rates of release between
these two classes in acid may be due to differences caused by the
presence of amino acids in proximity to the site of ADP-ribose
modification. Although all of the acetal model conjugates released
intact ADP-ribose in formic acid, the rate of release varied
considerably. The mechanism of hydrolysis of an acetal linkage in acid
involves protonation of the oxygen, formation of an oxocarbenium ion
intermediate, and hydrolysis of the oxocarbenium ion. The rate-limiting
step for hydrolysis may be protonation of the acetal linkage or
formation of the oxocarbenium ion intermediate. In the case of the
methoxy, ethoxy, and propoxy conjugates, the relative rates of
hydrolysis probably reflect the rate of formation of the oxocarbenium
ion as the relative rates of hydrolysis agree with the relative
stability expected for the respective oxocarbenium ion intermediates.
The relative rates of hydrolysis of the amino acid ester conjugates
cannot be explained in a similar manner. For these conjugates, the
effect of the free amino group on the rate of protonation may account
for differences in the rates of hydrolysis. In the proteins analyzed,
it seems likely that amino acids in the proximity of the site of
modification could increase the rate of protonation and thus result in
a more rapid rate of hydrolysis than we observed with the model
conjugates. However, in all cases, the hydrolysis results in the
release of intact ADP-ribose, demonstrating that the linkage between
ADP-ribose and protein is the site of hydrolysis. We have also
considered the possibility that the ADP-ribose released by formic acid
treatment represents one of the known modifications that is rendered
acid-labile by a unique chemistry caused by the proximity of amino
acids near the site of modification. This possibility can be ruled out
for carboxylate, guanidinium, and sulfhydryl groups modified by
ADP-ribose since these modifications are quantitatively removed by 1 M NaOH treatment(23) , yet the amount of acid-labile,
alkaline-stable modifications remained unchanged. Although this
possibility cannot be completely ruled out for imidazolyl and amido
linkages to ADP-ribose, it seems unlikely as these linkages are
completely stable under a wide range of conditions and we have not
detected any release of intact ADP-ribose in formic acid from model
conjugates for these linkages(23) .
A second possibility is
that the classes with different rates of release in formic acid
represent ADP-ribose modification of different amino acids. Consistent
with this possibility is the fact that these two classes also differed
in their sensitivity to alkaline conditions. Glycoproteins have been
shown to be glycosylated at serine and threonine residues(34) .
Also, glucose linkages to tyrosine residues appear to be involved in
the priming of glycogen(35, 36) . The sugar linkages
formed with serine or threonine are alkaline-labile, while the linkages
to tyrosine are alkaline-stable(34) . The mechanism proposed
for the alkaline release involves abstraction of an acidic proton
followed by elimination of the sugar moiety. In
ADP-ribose-modified proteins, serine and threonine sites of
modification would contain acidic protons making alkaline lability
possible while modification at tyrosine, hydroxyproline, or
hydroxylysine residues would not. Thus, the rat liver ADP-ribose
protein modifications with different rates of release in acid and
different stability to alkaline treatment may represent modifications
of different amino acids. Interestingly, a recently described
endogenous protein-mono-ADP-ribosyltransferase that is activated by the
fungal toxin brefeldin A (20) catalyzes a modification with
chemical characteristics similar to the class described here that is
released by either acid or alkaline conditions.
Although the acetal model conjugates were quite stable in ice-cold trichloroacetic acid, it is still likely that the amount of endogenous protein modification reported here represents an underestimate, particularly for the ADP-ribose class most rapidly released in acid (Fig. 8). The acid lability of this ADP-ribose modification also complicates the detection of protein-mono-ADP-ribosyltransferases and their substrates since acid precipitation prior to electrophoresis is routinely employed in studies designed to detect these enzymes and the proteins they modify.
The cellular distribution of the modification of proteins in vivo by acid-labile linkages was assessed by comparing the total amount of the modification in various subcellular fractions. The total amount of modification, approximately 16 pmol of ADP-ribose/mg of protein, is in a similar range observed for proteins modified by ADP-ribose on arginine (28) and cysteine (29) residues. Assuming an average molecular mass of 40 kDa and one ADP-ribose modification per molecule, this amount of modification would represent ADP-ribose modification of approximately 1 in 1500 protein molecules. When the proteins modified by ADP-ribose linked via acid-labile linkages were characterized as to their cellular location, a wide distribution was observed. This is in contrast to the distribution of ADP-ribosylcysteine linkages, which were found to be located exclusively in the plasma membrane fraction(29) . These results indicate that the scope of ADP-ribose modification of protein via acid-labile linkages is broad.