(Received for publication, October 1, 1996, and in revised form, January 16, 1997)
From the Pulmonary-Critical Care Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892
Transfection of NMU (rat mammary adenocarcinoma)
cells with NAD:arginine ADP-ribosyltransferase cDNAs from Yac-1
murine lymphoma cells or rabbit muscle increased NAD glycohydrolase and
ADP-ribosyltransferase activities. The ADP-ribosyltransferase activity
was released from transformed NMU cells by
phosphatidylinositol-specific phospholipase C (PI-PLC) and hence
glycosylphosphatidylinositol (GPI)-anchored, whereas the NAD
glycohydrolase (NADase) activity remained cell-associated. By gel
permeation chromatography, the size of the PI-PLC-released transferase
was ~40 kDa and that of the detergent-solubilized NADase was ~100
kDa. Using polyclonal antibodies against rabbit muscle transferase on
Western blots, ~18- and ~30-kDa band were visualized among proteins
from the NADase fractions and 38-40-kDa bands with protein from the
transferase fractions. Incubation of blots with
[32P]NAD led to the incorporation of radioactivity
into the immunoreactive transferase bands of 38 kDa and the
immunoreactive NADase band of ~18 kDa. These data suggest that
proteolysis of ADP-ribosyltransferase synthesized in transformed NMU
cells might result in the formation of aggregates of an 18-kDa NAD
glycohydrolase. A fusion protein with glutathione
S-transferase linked to the amino terminus of Yac-1
transferase, from which the amino-terminal 121 amino acids had been
deleted (GST-Yac-1-121), exhibited NADase, but not transferase, activity. The size of the recombinant fusion protein was similar to
that of the proteolytic fragment seen in NMU cells transformed with
transferase cDNA. These results are compatible with the conclusion that the NAD glycohydrolase activity was generated in NMU cells by
proteolysis of ADP-ribosyltransferase, with release of a
carboxyl-terminal fragment that possesses glycohydrolase but not
transferase activity, i.e. the carboxyl-terminal portion of
the transferase can exist as a catalytically active NADase.
Mono-ADP-ribosylation is a post-translational modification of
proteins catalyzed by ADP-ribosyltransferases, which transfer the
ADP-ribose moiety of NAD to specific amino acids in protein acceptors
(1). ADP-ribosyltransferase activity has been detected in viruses,
bacteria, and eukaryotic cells (1-6). ADP-ribosylation of target
proteins by bacterial toxin transferases such as cholera (4),
diphtheria (5), and pertussis (6) toxins alters critical metabolic
pathways. For example, cholera toxin ADP-ribosylates an arginine in the
-subunit of the stimulatory heterotrimeric guanine
nucleotide-binding (G) protein, resulting in the activation of adenylyl
cyclase and an increase in intracellular cyclic AMP (4).
Eukaryotic ADP-ribosyltransferase activity has been detected in several
tissues (1, 7-14), and cDNAs have been cloned from rabbit (8) and
human (9) skeletal muscle, chicken polymorphonuclear granulocytes (10)
and nucleoblasts (11), and mouse lymphoma (Yac-1)1 cells (13, 14). Based on studies in
which the transferase cDNAs were transfected into mammalian cells,
it appears that skeletal muscle and mouse lymphocyte enzymes are
extracellular glycosylphosphatidylinositol (GPI)-anchored proteins (9,
13). Consistent with its extracellular location, the GPI-linked muscle
transferase ADP-ribosylates integrin 7 on cultured mouse myotubes
(15); inhibitor studies suggest that the muscle transferase may
participate in the regulation of myogenesis (16).
In murine cytotoxic T cells, ADP-ribosylation of a 40-kDa surface protein (p40) was associated with the inhibition of p56lck tyrosine kinase, which exists in a complex with p40, and suppression of cytotoxic T cell proliferation and cytotoxic activity (17, 18). The ADP-ribosyltransferase involved in this reaction has not been identified although candidate genes have been identified, including Yac-1. A second ADP-ribosyltransferase (Yac-2) was cloned and characterized from mouse lymphoma cells (14). In contrast to the muscle and Yac-1 transferases, the Yac-2 enzyme although membrane-bound is apparently not GPI-anchored and exhibits significant basal NAD glycohydrolase activity (14). Based on the PI-PLC sensitivity of the cytotoxic T cell transferase, Yac-2 may not be responsible for the activity.
Structural analysis of bacterial toxin ADP-ribosyltransferases has demonstrated three regions of similarity (regions I, II, and III) that are believed to form part of the active site (19). For some of the toxins, region I contains a critical histidine or arginine (20-22); region II is composed of closely spaced aromatic and hydrophobic amino acids (23, 24); region III contains a catalytic glutamic acid (20, 25-30). In other toxins, a serine replaces the region II domain (24). Similar to findings with the bacterial toxins, site-directed mutagenesis of the rabbit muscle transferase identified glutamates 238 and 240 in the putative region III as essential for enzyme activity (31). These data, along with the alignment of the deduced amino acid sequences of the cloned vertebrate transferases, are consistent with the hypothesis that the enzymes have a common mechanism of NAD binding and catalysis.
The muscle and lymphocyte ADP-ribosyltransferases catalyze the ADP-ribosylation of arginine, agmatine, and other simple guanidino compounds (8, 13). The NAD glycohydrolase activity of these enzymes is only a small percentage of maximal transferase activity in the presence of optimal concentrations of guanidino compounds as ADP-ribose acceptors. We report here that rat mammary adenocarcinoma (NMU) cells transformed with the rabbit skeletal muscle ADP-ribosyltransferase cDNA expressed significant NAD glycohydrolase as well as transferase activity. Membrane-bound NAD glycohydrolase activity was associated with a 100-kDa protein, an apparent oligomer of a proteolytic fragment of the transferase, and a truncated form of the transferase, synthesized in Escherichia coli, exhibited NADase activity.
Materials
NMU (rat mammary adenocarcinoma) cells were obtained from
American Type Culture Collection; Eagle's minimal essential medium and
Dulbecco's phosphate-buffered saline were from BioWhittaker; Geneticin
(G418) was from Life Technologies, Inc.; dexamethasone sodium phosphate
was from MG Scientific; NheI, XhoI, and
KspI restriction endonucleases and PCR Master kit were from
Boehringer Mannheim; Qiagen plasmid extraction kit from Qiagen;
-NAD, agmatine, phosphatidylinositol-specific phospholipase C,
phenylmethanesulfonyl fluoride, aprotinin, leupeptin, and pepstatin
were from Sigma; [adenine-U-14C]NAD (241 mCi/mmol), [carbonyl-14C]NAD (53 mCi/mmol),
and enhanced chemiluminescence Western blotting detection reagents were
from Amersham; [adenylate-32P]NAD (30 Ci/mmol)
was from DuPont NEN; nitrocellulose membrane and Dowex AG 1-X2 resin
were from Bio-Rad; Ultrogel AcA 44 was from Biosepra; bicinchoninic
acid protein reagent was from Pierce; and glutathione-Sepharose 4B was
from Pharmacia Biotech Inc.
Methods
Construction of Yac-1 and Rabbit Muscle Transferase Expression VectorsWild-type mouse lymphocyte (Yac-1) and rabbit muscle
transferase cDNAs were subcloned into the pMAMneo mammalian
expression vector as described (13, 31). Truncated rabbit muscle
transferase cDNAs were generated using polymerase chain reaction
(PCR)-based techniques (32). The pM53
T clone, lacking 23 and 24 amino acids from the amino and carboxyl termini, respectively, was amplified from the wild-type rabbit muscle transferase cDNA (100 ng) in a
100-µl reaction containing dNTPs (each 0.2 mM),
Taq DNA polymerase (2.5 units), and forward
(5
-ACGTACGTACGTGCTAGCATGAGCCACCTGGTCACACGTCGAGAC-3
) and
reverse (5
-ACGTACGTACGTCTCGAGTCAGGAGAGGCGCTCCTGAGCCGAGGC-3
) primers
(100 pmol each). Amplification was performed for 30 cycles of 94 °C,
1 min/55 °C, 1 min/72 °C, 1.5 min, followed by a 7-min extension
at 72 °C. The pM5
T clone, which lacks the amino-terminal 23 amino
acids, was amplified by PCR from the muscle transferase cDNA using
forward (5
-ACGTACGTACGTGCTAGCATGAGCCACCTGGTCACACGTCGAGAC-3
) and
reverse (5
-ACGTACGTACGTCTCGAGTCAGAAGAGGCCTGGGCTTCCTGG-3
) primers (100 pmol each) and conditions identical to those
described above. The truncated pM3
T clone, which lacks the
carboxyl-terminal 24 amino acids, was generated using forward
(5
-ACGTACGTACGTGCTAGCATGTGGGTTCCTGCCGTGGCGAAT-3
) and reverse
(5
-ACGTACGTACGTCTCGAGTCAGGAGAGGCGCTCCTGAGCCGAGGC-3
) primers (100 pmol each). PCR products were purified, digested with NheI and XhoI, and subcloned into the
pMAMneo vector.
NMU cells were transfected with 15 µg of the pMAMneo vector (control) or vector containing the full-length or truncated transferase cDNAs using the calcium phosphate precipitation method (33), and stable transformants were selected with Geneticin (0.5 mg/ml). Following induction of protein expression with dexamethasone (1 µM) for 24 h, cells were washed with Dulbecco's phosphate-buffered saline (PBS) and incubated for 60 min in PBS (0.7 ml) with or without phosphatidylinositol-specific phospholipase C (PI-PLC, 0.5 unit). After collecting the PBS fraction, the cells were washed with PBS, trypsinized, and lysed in 0.5 ml of lysis buffer (10 mM Tris, pH 8.0, 1 mM EDTA) by repeated freeze-thawing. After centrifugation (100,000 × g, 1 h) of the lysate, the supernatant (Supernatant, 0.5 ml) was collected and the membranes (Pellet) were suspended in 0.5 ml of PBS.
Gel Permeation Chromatography of ADP-Ribosyltransferase and NADase on Ultrogel AcA 44NMU cells transformed with wild-type
Yac-1 or rabbit muscle transferase cDNAs were incubated in PBS
without or with PI-PLC as described above. The PBS fraction was applied
to a column (2 × 120 cm) of Ultrogel AcA 44, equilibrated with
buffer A (20 mM Tris, pH 8.0, 1 mM EDTA, 100 mM NaCl, 1% Triton X-100). Samples of the eluate were
assayed for transferase activity (Fig. 1A). Membranes from
the transformed NMU cells were suspended in buffer A, stirred for
18 h at 4 °C, and centrifuged (100,000 × g,
1 h). The supernatant was applied to a column (2 × 120 cm)
of Ultrogel AcA 44, equilibrated, and eluted with buffer A. Samples of
fractions were assayed for NAD glycohydrolase activity (Fig.
1B). Fractions containing maximal activity were pooled and
used for activity measurements.
Immunoreactivity of ADP-Ribosyltransferase and NADase
Proteins in fractions from gel filtration that contained ADP-ribosyltransferase or NAD glycohydrolase activities were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in a 12% gel (Tris glycine) and transferred to nitrocellulose. The membranes were incubated first with polyclonal anti-rabbit muscle ADP-ribosyltransferase antibodies (9) and then with anti-rabbit IgG-horseradish peroxidase conjugate. Immunoreactive proteins were detected by enhanced chemiluminescence.
Labeling of ADP-Ribosyltransferase and NADase with [32P]NADADP-ribosyltransferases have been detected
by zymographic assays using overlays with [32P]NAD (34,
35). In this highly sensitive procedure, proteins released from
transformed NMU cells with PI-PLC, which contained transferase
activity, and residual membrane proteins containing NAD glycohydrolase
activity, were subjected to SDS-PAGE in a 14% gel and transferred to
nitrocellulose. The membrane was incubated in 50 ml of buffer
containing 25 mM Tris, pH 7.5, 100 mM NaCl, 0.3% bovine serum albumin, and 0.3% Tween 20 for 1 h at room
temperature before addition of [32P]NAD (100 µCi) to 25 ml of the buffer and further incubation for 5 h. The membrane was
washed five times in the same buffer without bovine serum albumin and
exposed to Kodak X-Omat film at 80 °C for 18 h.
Truncated-forms of Yac-1-GST fusion proteins were
synthesized in E. coli. GST-Yac-1-53
T, in which the
transferase cDNA lacks the amino- and carboxyl-terminal hydrophobic
signal sequences (23 and 37 amino acids from 5
and 3
ends,
respectively), was generated by PCR amplification of the Yac-1
transferase cDNA (100 ng) with forward
(5
-ACGTACGTACGTCCGCGGAGTTACTCCATCTCACAACTA-3
) and reverse
(5
-ACGTACGTACGTCCGCGGTCAACCCAGCCAGCAGGGCCCAGA-3
) primers (100 pmol
each) under PCR conditions described above. GST-Yac-1-
121, which
lacks 121 and 37 amino acids, respectively, from the hydrophobic amino-
and carboxyl termini, was generated using forward
(5
-ACGTACGTACGTCCGCGGCCCCTGCACAAGGAGTTCAACGCAGCT-3
) and reverse
(5
-ACGTACGTACGTCCGCGGTCAACCCAGCCAGCAGGGCCCAGA-3
) primers. PCR
products were gel-purified, subcloned into pGEX-5G/LIC (36), and
expressed in E. coli (DH5
). Transformed E. coli cells were grown to an absorbance at 600 nm of 0.4 in 1 liter
of LB medium with ampicillin, 100 µg/ml, before
isopropyl-
-D-thiogalactopyranoside (final concentration
0.3 mM) was added, and incubation was continued overnight
(15 h) at room temperature. Cells were pelleted by centrifugation, suspended in 20 ml of 10 mM Tris, pH 8.0, 1 mM
EDTA, 100 mM NaCl containing protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride, and leupeptin, aprotinin,
and pepstatin, each 0.5 µg/ml), and incubated with 10 mg of lysozyme
for 30 min on ice. After sonification for 1 min, the lysate was
centrifuged (5000 × g, 30 min) and the supernatant
mixed with glutathione-Sepharose beads on a rocking platform at
4 °C. After centrifugation (2000 × g, 5 min), beads were washed with PBS and suspended in two-bed volumes of PBS for assay
of ADP-ribosyltransferase and NAD glycohydrolase activities.
Assays, in a final volume of 300 µl, contained 50 mM potassium phosphate, pH 7.5, 20 mM agmatine, and 0.1 mM [adenine-U-14C]NAD (0.05 µCi). After incubation at 30 °C for 1 h, samples (100 µl) were applied to columns (0.5 × 4 cm) of Dowex AG 1-X2. [14C]ADP-ribosylagmatine was eluted with 5 ml of H2O for liquid scintillation counting.
NAD Glycohydrolase AssayThe NAD glycohydrolase assays were incubated at 30 °C for 1 h in a final volume of 300 µl containing 50 mM potassium phosphate, pH 7.5, and 0.1 mM [carbonyl-14C]NAD (0.05 µCi). Samples (100 µl) were applied to columns (0.5 × 4 cm) of Dowex AG 1-X2. [14C]Nicotinamide was eluted with 5 ml of H2O for liquid scintillation counting.
Protein AssayProtein concentration was determined by bicinchoninic acid assay or Bio-Rad assay with bovine serum albumin as standard.
NMU cells transformed with the Yac-1 and rabbit muscle transferase cDNAs expressed ADP-ribosyltransferase and NAD glycohydrolase activities (Table I). Since these activities were not detected in the vector-transformed cells, both were attributed to transformation of the cells with transferase cDNA. From the transformants, ~93 (Yac-1) or 86% (rabbit muscle) of the transferase activity was released into the medium (PBS fraction) by treatment with PI-PLC. In contrast, after treatment of cells with PI-PLC, most of the NAD glycohydrolase activity was associated with the membranes. Although the ADP-ribosyltransferase expressed in transformed NMU cells was a GPI-anchored protein as previously characterized (9), the NAD glycohydrolase activity retained in the membrane fraction appeared not to be GPI-anchored.
|
To elucidate the mechanisms responsible for the generation of both ADP-ribosyltransferase and NAD glycohydrolase activities in the transformed NMU cells, samples of PBS and pellet fractions, following treatment with PI-PLC, were applied to a gel permeation column. As shown in Fig. 1A, the transferase activity eluted from the column after ovalbumin (45 kDa), consistent with a protein of 38 kDa. The NAD glycohydrolase activity, solubilized with Triton X-100, eluted between thyroglobulin (670 kDa) and bovine serum albumin (68 kDa) and had an estimated molecular mass of ~100 kDa (Fig. 1B). The Yac-1 and rabbit muscle NAD glycohydrolase and ADP-ribosyltransferase activities, separated by column chromatography, were evaluated kinetically. Equivalent amounts of protein from vector-transformed NMU cells, subjected to gel filtration chromatography, did not demonstrate transferase or NAD glycohydrolase activities (data not shown). The apparent Km values for NAD in the ADP-ribosyltransferase reaction assayed with 20 mM agmatine were similar for the Yac-1 and rabbit muscle transferases, as were the Km values for NAD glycohydrolase activity, which were approximately one-third those of the respective transferases (Table II).
|
In contrast to NMU cells transformed with
the full-length rabbit muscle transferase, cells transformed with
truncated transferase cDNAs, lacking the amino- and
carboxyl-terminal signal sequences (pM53
T) or the amino-terminal
sequence (pM5
T), exhibited minimal transferase activity (Table
III). Cells transformed with a transferase cDNA
lacking the carboxyl terminus (pM3
T) released nearly all of the
transferase activity into the culture medium. The carboxyl-terminal truncation may prevent the PI-PLC-sensitive linkage from being formed,
thus leading to secretion. In contrast, cells transformed with the
full-length construct did not show an increased level of transferase in
the medium. The NAD glycohydrolase activity of pM5
T-transformed cells
was similar to that of control cells (vector alone), whereas activities
of pM5
3
T- and pM3
T-transformants were, respectively, ~8.5 and ~4
times those of cells transformed with vector alone. The inability to
detect a significant increase in NAD glycohydrolase activity with the
cells transformed with the pM5
T may reflect enhanced degradation due
to the presence of the carboxyl-terminal hydrophobic signal sequence,
as was shown with other GPI-linked proteins (37). Similar to activities
in cells transformed with the wild-type full-length transferase
cDNA, the NAD glycohydrolase activity of cells transformed with the carboxyl-terminal truncated cDNA was present in the membrane
fraction. These data suggest that proteins synthesized from cDNA
lacking the carboxyl-terminal signal sequence or having the full-length sequence may be retained by the cells; these intracellular proteins exhibit primarily NAD glycohydrolase activity.
|
Proteins
eluted from the Ultrogel AcA 44 column that possessed
ADP-ribosyltransferase and NAD glycohydrolase activities were separated
by SDS-PAGE under reducing (with DTT) or nonreducing (without DTT)
conditions (Fig. 2). In fractions containing
transferase, 38-40-kDa immunoreactive proteins were observed under
both nonreducing (lane 1) and reducing (lane 2)
conditions. In fractions containing NAD glycohydrolase activity, two
immunoreactive proteins (~18 and ~30 kDa) were observed without
(lane 3) or with (lane 4) DTT. Immunoreactive
proteins were not found in vector-transformed cells (lanes 7 and 8). These data are consistent with the hypothesis that
NAD glycohydrolase activity results from proteolysis of the transferase.
Radiolabeling of Transferase and NADase with [32P]NAD
The ADP-ribosyltransferase released from
transformed NMU cells following PI-PLC treatment and NAD glycohydrolase
solubilized from the pellet fraction with detergent were subjected to
SDS-PAGE, transferred to nitrocellulose, and incubated with
[32P]NAD (Fig. 3). 38-kDa proteins in the
PI-PLC-released fraction (lane 2) and ~18 kDa proteins
from the membrane fraction (lane 4) were detected by
autoradiography. Radiolabel was not detected with proteins from
vector-transformed cells (lanes 1 and 3).
Expression of GST-Yac-1 Fusion Proteins
Analyses of several bacterial toxin ADP-ribosyltransferases by x-ray crystallography, photoaffinity labeling with NAD+, and site-directed mutagenesis demonstrated three regions with amino acid sequence similarities that form the catalytic site (24). The mammalian ADP-ribosyltransferases (e.g. rabbit (8) and human (9) muscle and mouse lymphocyte (13, 14) transferases) also possess amino acid sequences similar to those in the active sites of the bacterial toxin transferases. In the bacterial toxin transferases, two structures have been proposed (24). In one, region I contains a nucleophilic arginine or histidine. Region II has closely spaced aromatic and hydrophobic amino acids, and region III is characterized by active-site glutamic acid, which is critical for enzyme activity. In the second, the region II domain is replaced with a serine (24). Amino acid sequence alignment and computer analysis of the mouse and rat RT6 proteins, which are T cell alloantigens that possess NAD glycohydrolase (rat) and ADP-ribosyltransferase (mouse) activities, suggest that, as in the bacterial toxin transferases (38), an analogous arginine or histidine (Arg-174 of Yac-1) and an acidic amino acid-containing region (Glu-233, 235 of Yac-1) are important for enzyme activity.
In the eukaryotic transferases, the catalytic site appears to be
located in the carboxyl-half of the protein. To determine whether an
~18-kDa NAD glycohydrolase, as found in the cDNA-transformed NMU
cells, could be generated from an ADP-ribosyltransferase, perhaps by
release of a stable catalytic domain, truncated GST-Yac-1 transferase
fusion proteins were synthesized in E. coli. Truncated fusion proteins, partially purified by glutathione-Sepharose 4B chromatography, were assayed for transferase and NAD glycohydrolase activities (Table IV). Neither enzyme activity was
detected in proteins from vector-transformed E. coli
separated by glutathione-Sepharose chromatography. The GST-Yac-1
construct that lacks the hydrophobic amino- and carboxyl-terminal
signal sequences (23 and 37 amino acids from amino- and
carboxyl-terminal ends, respectively) exhibited ADP-ribosyltransferase
activity; it had, however, negligible NADase activity. The
GST-Yac-1-121 protein, which lacks 121 amino acids from the amino
terminus, exhibited NAD glycohydrolase but negligible ADP-ribosyltransferase activity. The ratio of specific activities of
NAD glycohydrolase and ADP-ribosyltransferase was 0.004 for GST-Yac-1-5
3
T and 183 for GST-Yac-1-
121. The ratio of enzyme activities demonstrates that GST-Yac-1-
121 has NAD glycohydrolase but not ADP-ribosyltransferase activity. The estimated molecular mass
of a truncated Yac-1 enzyme corresponding to GST-Yac-1-
121 is
~18.8 kDa. These results are consistent with previous data that
NADase activity results from proteolysis of transferase and the
generation of a stable, enzymatically active carboxyl-terminal fragment.
|
Rat mammary adenocarcinoma (NMU) cells transformed with rabbit
muscle and Yac-1 ADP-ribosyltransferase cDNAs possessed GPI-linked ADP-ribosyltransferase activity that was released from intact cells by
PI-PLC. Transformed cells, however, also exhibited membrane-associated NAD glycohydrolase activity, not released by PI-PLC, which on solubilization and gel permeation chromatography appeared to be larger
than the transferase. Based on the data from denaturing gels, however,
it was concluded that this higher molecular weight species was an
aggregate of an ~18-kDa protein or, alternatively, an aggregate
containing the 18-kDa protein associated with other cellular
components. The ADP-ribosyltransferases generated from transformants
lacking amino- or and carboxyl-terminal signal sequences (pM5T and
pM5
3
T, respectively) should be unable to enter the endoplasmic
reticulum (ER). Despite low levels of cell-associated transferase
activity in cells expressing these truncated forms of the transferase,
significant levels of membrane-associated NAD glycohydrolase activity
resistant to release by PI-PLC were found. The transferase activity
from pM3
T-transformant, however, was secreted into the medium due to
the absence of the carboxyl-terminal hydrophobic signal sequence, which
is essential for GPI anchoring (39). This protein would be transported
into the ER but lacks the structure required for addition of the GPI
anchor and retention on the cell surface. These data are consistent
with the hypothesis that some of the ADP-ribosyltransferase produced by
transformed NMU cells is processed to an NAD glycohydrolase. This
observation was made with full-length transferase, a portion of which
was retained as an NADase, and with the amino-truncated forms, both of
which due to the absence of a signal sequence, were retained and
proteolyzed. Reactivity of the 18-kDa protein with anti-transferase antibodies and radiolabeling of these proteins with
[32P]NAD support the conclusion that the NAD
glycohydrolase arises from degradation of the
ADP-ribosyltransferase.
Proteins destined for GPI anchoring have characteristic hydrophobic amino- and carboxyl-terminal signal peptides. The amino-terminal sequence is required for insertion of the nascent protein into the ER, whereas the carboxyl-terminal sequence is important for attachment of the mature protein to a GPI anchor through which it can be retained in the membrane (39). Field et al. (37) suggested that nascent proteins with unprocessed carboxyl-terminal signal peptides are present in micelle-like aggregates, which are retained in the ER and are subject to proteolytic degradation. The same process may have led to proteolytic degradation of the transferase and generation of the fragment possessing NADase activity.
To establish that a stable, catalytically active carboxyl-terminal
fragment can be generated, a truncated transferase linked to GST was
synthesized. The truncated recombinant Yac-1 ADP-ribosyltransferase protein (GST-Yac-1-121) exhibited NAD glycohydrolase but not transferase activity. The proteolyzed transferase demonstrated NAD
glycohydrolase activity similar to that of the truncated
GST-Yac-1-
121 fusion protein. Because proteolysis generates an
~18-kDa fragment containing NADase activity and a GST-fusion protein
containing an ~18-kDa carboxyl-terminal region of the transferase
cDNA exhibits NADase activity, it appears that this portion of the
protein is capable of forming a stable, catalytically active domain.
This domain possesses NADase but not transferase activity, suggesting that the amino-terminal half of the protein is needed for effective interaction with the ADP-ribose acceptor. The fact that this domain is
active is consistent with the model proposed for the rat and mouse RT6
proteins, in which arginine 146 rather than histidine 91 is the
hydrogen-bonding amino acid involved in the formation of the catalytic
site (38). In the former model for the catalytic site, a much smaller
region of the protein is needed for activity than is the case with the
latter. The synthesis of an active truncated protein also supports the
hypothesis that the carboxyl-terminal half of the transferase can fold
to form a stable domain. Since proteolysis of the transferase generated
a stable NADase of a size similar to that of the recombinant truncated
protein, it may be that, even in the intact transferase, the NADase
domain is separated from the amino half of the protein and that
proteases cleave between the two regions.
We thank Dr. Martha Vaughan for helpful discussions and critical review of this manuscript and Carol Kosh for expert secretarial assistance.