(Received for publication, May 21, 1996, and in revised form, October 4, 1996)
From the Pulmonary-Critical Care Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-1590 and § Diabetes Division, University of Massachusetts Medical Center, Worcester, Massachusetts 01605
Rat RT6 proteins, and perhaps mouse Rt6, identify
a set of immunoregulatory T lymphocytes. Rat RT6.1 (RT6.1) and rat
RT6.2 (RT6.2) are NAD glycohydrolases, which catalyze
auto-ADP-ribosylation, but not ADP-ribosylation of exogenous proteins.
Mouse Rt6.1 (mRt6.1) also catalyzes auto-ADP-ribosylation. The activity
of mouse cytotoxic T lymphocytes is reportedly inhibited by
ADP-ribosylation of surface proteins, raising the possibility that mRt6
may participate in this process. The reactions catalyzed by mRt6,
would, however, need to be more diverse than those of the rat
homologues and include the ADP-ribosylation of acceptors other than
itself. To test this hypothesis, mRt6.1 and rat RT6.2 were synthesized
in Sf9 insect cells and rat mammary adenocarcinoma (NMU) cells. mRt6.1,
but not rat RT6.2, catalyzed the ADP-ribosylation of
guanidino-containing compounds (e.g. agmatine). Unlike
RT6.2, mRt6.1 was a weak NAD glycohydrolase. In the presence of
agmatine, however, the ratio of
[adenine-14C]ADP-ribosylagmatine formation
from [adenine-14C]NAD to
[carbonyl-14C]nicotinamide formation from
[carbonyl-14C]NAD was ~1.0, demonstrating
that mRt6.1 is primarily a transferase. ADP-ribosylarginine hydrolase,
which preferentially hydrolyzes the -anomer of ADP-ribosylarginine,
released [U-14C]arginine from
ADP-ribosyl[U-14C]arginine synthesized by mRT6.1,
consistent with the conclusion that mRt6.1 catalyzes a stereospecific
Sn2-like reaction. Thus, mRt6.1 is an
NAD:arginine ADP-ribosyltransferase capable of catalyzing a multiple
turnover, stereospecific Sn2-like reaction.
Rat RT6 proteins appear to identify a regulatory T cell population capable of modulating autoimmunity (1). Diabetes-prone BB rats are severely deficient in RT6+ T cells and spontaneously develop insulin-dependent diabetes mellitus (2, 3). Transfusion of spleen cells from histocompatible rats prevents diabetes provided that RT6+ T cells become engrafted (4, 5). Conversely, diabetes-resistant BB rats have normal numbers of RT6+ T cells and fail to manifest autoimmunity (1, 2). Depletion of RT6+ T cells induces insulitis, diabetes, and thyroiditis in these animals (6-8). RT6+ T cells have been suggested to be important also in the modulation of autoimmunity in the irradiated, thymectomized PVG model of insulin-dependent diabetes mellitus (9), and in the BN rat model of mercury chloride-induced autoimmune glomerulonephritis (10). The mechanism(s) by which RT6+ T cells regulate autoimmunity is uncertain, but may involve the RT6 protein itself.
The rat RT6 proteins are linked to the surface of T cells via a glycosylphosphatidylinositol (GPI)1-linkage (11, 12). Deduced amino acid sequences obtained from cDNA for two rat allotypes, RT6.1 and RT6.2, with molecular masses of 25-35 kDa and 24-26 kDa, respectively, contain hydrophobic amino- and carboxyl-terminal sequences, consistent with those in GPI-linked proteins (13, 14). The internal, coding region is remarkable for consensus sequences similar to those found in bacterial toxin ADP-ribosyltransferases and believed to be involved in catalytic activity (15). Consistent with this observation, the RT6 protein exhibited NAD glycohydrolase (15) and auto-ADP-ribosyltransferase activities (16, 17). These enzymatic activities of RT6 have recently been postulated to be important in the inhibition of T cell proliferation following incubation of cells in the presence of NAD (16).
Two mouse Rt6 genes have recently been identified and are transcribed (18-20). Expression of mouse Rt6 protein by immunoblotting and RT6 gene expression appear to be restricted to T cells (18, 21). Reduced levels of Rt6 gene expression have been reported in two models of autoimmunity in mice (18, 19). In the spontaneously diabetic nonobese diabetic mouse, levels of Rt6-specific mRNA in the spleens of 8-week-old mice were lower than those of control nonobese nondiabetic mice (18). In the NZW and (NZB + NZW)F1 mouse, which are models of systemic lupus erythematosus, defects in the structure and expression of the Rt6 gene were also observed (19). Similar to the rat homologue, mouse Rt6.1 had auto-ADP-ribosylation activity (16, 21). The immunomodulatory function of mRt6+ T-lymphocytes, or the role of mRt6 enzymatic activities in immune modulation, have not been determined. Recently, it was suggested that cytotoxic T cell (CTL) activity in mice may be regulated by GPI-linked ADP-ribosyltransferases that have many of the characteristics of the rat RT6 proteins (22, 23) and catalyze the modification of surface proteins (24).
Although various members of the rat RT6 and mouse Rt6 family of proteins possess NAD glycohydrolase and auto-ADP-ribosyltransferase activity, it was not evident that they could catalyze intermolecular reactions as described for CTL cells. In fact, the rat RT6.2 protein, which was shown to be an NAD glycohydrolase (15) and auto-ADP-ribosyltransferase, specifically did not catalyze the ADP-ribosylation of protein and simple guanidino compounds such as arginine and agmatine (16, 17). The present study was undertaken to determine whether the mRt6.1 protein, in contrast to the rat RT6 protein, could catalyze intermolecular reactions such as the ADP-ribosylation of free proteins and agmatine. These enzymatic activities would be consistent with a possible immunomodulatory function in mouse CTL and provide a potential mechanism by which mRt6+ T cells, and/or protein could influence autoimmunity.
Materials
pAcSG2 vector was purchased from PharMingen (San Diego, CA);
pMAMneo expression vector and ligation express kit from Clontech (Palo
Alto, CA); gene Amp PCR reagent kit and DNA thermal cycler from
Perkin-Elmer; ABI 394 DNA/RNA synthesizer from Applied Biosystems, Inc.
(Foster City, CA); NuSieve and GTG-agarose from FMC Corp. (Rockland,
ME); QIAEX gel extraction kit and QIAGEN plasmid purification kit from
QIAGEN (Chatsworth, CA); Tris acetate-EDTA buffer, LB broth, Eagle's
minimal essential medium with Earle's balanced salt solution
containing L-glutamine, Dulbecco's phosphate-buffered saline (DPBS), and fetal bovine serum, heat-inactivated, from BioWhittaker, Inc. (Walkersville, MD); XhoI and ampicillin,
sodium salt from Boehringer Mannheim; NheI, DH5-competent
cells, G418, buffer-saturated phenol, and phenol/chloroform/isoamyl
alcohol (25:24:1) from Life Technologies, Inc.; 3 M sodium
acetate from Quality Biological Inc. (Gaithersburg, MD); ethanol from
the Warner Graham Co. (Cockeyesville, MD); rat mammary adenocarcinoma
cells (NMU cells) from American Type Culture Collection (Rockville, MD); dexamethasone sodium phosphate from MG Scientific (Buffalo Grove, IL); trypsin and 7-deaza-dGTP sequencing kit from U. S. Biochemical Corp. (Cleveland, OH); Hoechst 33258 Dye and DyNa Quant 200 fluorometer from Hoefer Pharmacia Biotech, Inc. (San Francisco, CA);
-NAD, phosphatidylinositol-specific phospholipase C (PI-PLC), and
agmatine sulfate from Sigma;
[adenine-U-14C]NAD (243 mCi/mmol),
[carbonyl-14C]nicotinamide adenine
dinucleotide (53 mCi/mmol), and
L-[U-14C]arginine monohydrochloride (297 mCi/mmol) from Amersham Corp.; Dowex AG1-X2 resin, 200-400 mesh,
chloride form, from Bio-Rad; Long Ranger gel solution from J. T. Baker
Inc.; and BCA protein assay reagent from Pierce.
Methods
Subcloning of mRt6.1 cDNA and Transfection of NMU CellsmRt6.1 cDNA was subcloned into a pMAMneo expression
vector as described previously (25). The mRt6.1 insert was sequenced to
confirm its identity with that in GenBankTM (accession no.
X52991[GenBank]). The mRt6.1 cDNA was amplified from purified plasmid
cDNA (mRt6.1 cDNA in pAcSG2 vector) by PCR, using forward
(5-ACG ACG GCT AGC ATG CCA TCA AAT AAT TTC AAG-3
) and reverse (5
-CGT
CGT CTC GAG CTA CGG CTC AGC AAG AGT AAG-3
) primers (100 pmol each)
under the following conditions: 60 s at 95 °C, followed by 25 cycles at 95 °C for 60 s; 55 °C, 60 s; 72 °C, 90 s; and ending at 72 °C for 7 min. The PCR product was
isolated on a 3% NuSieve GTG-agarose gel followed by QIAEX
purification of the gel band. The purified PCR fragment was digested
with XhoI and NheI and ligated into the
NheI (5
) and XhoI (3
) sites of the pMAMneo
vector. DH5
competent cells were transformed with the ligated
product and grown on LB plates containing ampicillin (50 µg/ml). The
subcloned mRt6.1 cDNA in pMAMneo was amplified, and then purified
using the QIAGEN plasmid purification kit. The subclone was sequenced
using the 7-deaza-dGTP sequencing kit to verify orientation. NMU cells
were transfected with 30 µg of the mRt6.1 subclone by the calcium
phosphate/HEPES-buffered saline precipitation method. Transformed NMU
cells were selected with G418 (500 µg/ml) and maintained in Eagle's
minimal essential medium containing 10% fetal bovine serum with G418
(500 µg/ml).
Expression of ADP-ribosyltransferase was induced by incubating 2.5 × 106 transformed NMU cells with 1 µM dexamethasone sodium phosphate for the indicated times (6, 24, 48, and 72 h). Cells were washed with DPBS and incubated with or without 2 units of PI-PLC in 1.5 ml of DPBS at 37 °C. The media were collected. Cells were trypsinized, washed twice in DPBS, and lysed in 0.7 ml of hypotonic lysis buffer (10 mM potassium phosphate, pH 7.5, 1 mM EDTA) with three freeze/thaw cycles (dry ice/room temperature). Lysed cells were centrifuged for 1 h at 100,000 × g at 4 °C. The supernatant (0.7 ml) was collected; the membrane fraction was suspended in 0.7 ml of lysis buffer and sonified. ADP-ribosyltransferase activity was determined in samples (75 µl) of the medium, cell supernatant, and cell membranes. Data are expressed as total activity per fraction (picomoles of [14C]ADP-ribosylagmatine formed in 26 h in the transferase assay).
Assay of ADP-ribosyltransferase Activity from NMU CellsThe ADP-ribosyltransferase reaction was carried out for 26 h in 0.3 ml of reaction mix containing 50 mM potassium phosphate, pH 7.5, 0.1 mM nicotinamide [adenine-U-14C]NAD (0.05 µCi), with or without 20 mM agmatine. Duplicate samples were incubated at 30 °C, and at the end of the incubation, two 100-µl samples from each assay were applied to columns (1 ml bed volume) of Dowex AG1-X2 resin (26). [14C]ADP-ribosylagmatine was eluted with 5 ml of H2O for radioassay.
Protein AssayProtein was determined using BCA protein assay reagent (Pierce) with bovine serum albumin as a standard.
Preparation of Rat Brain ADP-ribosylarginine Hydrolase and Turkey Erythrocyte ADP-ribosyltransferaseADP-ribosylarginine hydrolase, purified from rat brain as described earlier, contained one major 39-kDa protein band on SDS-polyacrylamide gel electrophoresis (27). Turkey erythrocyte ADP-ribosyltransferase was purified as described previously (28).
Preparation of mRt6.1 and Rat RT6.2 in Sf9 CellsRecombinant mRt6.1 and RT6.2 were synthesized in Sf9 cells under the control of a baculovirus promoter, as described previously (16). Two rat RT6.2 genes were expressed in Sf9 cells, RT6.2-GB (accession nos. M85193[GenBank] and M30311[GenBank]) and RT6.2-LR. The latter gene, from Lewis.B (RT6.2) rats, differs from RT6.2-GB at position 29 (lysine in place of threonine), position 71 (arginine in place of lysine), position 96 (alanine in place of valine) and position 251 (a stop signal in place of arginine).
mRt6.1 was synthesized in Sf9 insect cell/baculovirus system and in transformed rat NMU cells. Transferase activity was found in the Sf9 cell pellet, cell supernatant, and the culture supernatant (data not shown). Using mRt6.1 isolated from Sf9 medium in assays containing either [adenine-U-14C]NAD or [carbonyl-14C]NAD to monitor the incorporation of ADP-ribose into ADP-ribosylagmatine and the release of nicotinamide from NAD, respectively, it was observed that the rate of formation of ADP-ribosylagmatine was closely correlated with the rate of release of nicotinamide, with a ratio of [adenine-U-14C]ADP-ribosylagmatine formation to [carbonyl-14C]nicotinamide release of 0.99 (Table I). These data are compatible with the conclusion that, in the presence of an ADP-ribose acceptor, the enzyme is primarily a transferase, with only minimal, uncoupled NAD glycohydrolase activity. In the absence of agmatine or other ADP-ribose acceptors, the rate of release of [carbonyl-14C]nicotinamide from [carbonyl-14C]NAD was significantly less than that observed in the presence of acceptor (Table I). Again, these data support the hypothesis that the enzyme is not as efficient as an NAD glycohydrolase as it is an ADP-ribosyltransferase. In contrast, the rat RT6.2, synthesized in the Sf9 system, did not appear to catalyze the formation of [adenine-U-14C]ADP-ribosylagmatine from [adenine-U-14C]NAD and agmatine, but did, as reported elsewhere, release [carbonyl-14C]nicotinamide from [carbonyl-14C]NAD (Table I). For NAD, the Km of RT6.2-LR was 23.4 µM ± 3.2; the Km of RT6.2-GB was 41.0 µM ± 9.4; both were significantly lower than that of mRt6.1 (1.9 mM ± 0.3) (Table II). The Km for NAD of mRt6.1 was similar to that of cholera toxin (29), a bacterial NAD:arginine ADP-ribosyltransferase. The mRt6.1 Km for agmatine of 4.8 mM ± 0.3 was significantly lower than that reported for the toxin (30).
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To verify that mRt6.1 was indeed catalyzing the formation of an
ADP-ribose-linkage to simple guanidino compounds and that the
ADP-ribose-guanidino linkage was identical to that formed by
erythrocyte and cardiac muscle NAD:arginine ADP-ribosyltransferases (25, 31), [14C]arginine and saturating concentrations of
unlabeled NAD were incubated with or without mRt6.1, and the products
resolved by SAX-HPLC (Fig. 1).
[14C]Arginine eluted at 2-4 min; with mRt6.1, a new
radiolabeled peak, corresponding to 6.4% of the total radiolabel, was
observed at 7-9 min (Fig. 1). To determine whether this peak
represented ADP-ribosyl-[14C]arginine, it was
concentrated and incubated with or without Mg2+ and DTT in
the presence of an ADP-ribosylarginine hydrolase, which cleaves the
ADP-ribose-arginine linkage (27, 32). ADP-ribosylarginine hydrolase is
dependent on Mg2+ and DTT for activity, and degrades
stereospecifically the -anomeric product of the erythrocyte
transferase-catalyzed reaction (27, 33). The reaction products were
again subjected to SAX-HPLC. In the presence of Mg2+, DTT,
and hydrolase a new peak, eluting at 2-4 min, corresponding to
[14C]arginine and representing 72% of the total
radiolabel, was formed (Fig. 2).
mRt6.1 utilized histones as ADP-ribose acceptors and, in the presence
of [32P]NAD, catalyzed the formation of
[32P]ADP-ribosylhistones (Fig. 3). Free
ADP-ribose did not inhibit [32P]ADP-ribosylation of
histone, suggesting that a nonenzymatic pathway resulting from the
initial generation of ADP-ribose by an NAD glycohydrolase, followed by
the nonenzymatic addition of ADP-ribose to protein, was not responsible
for the result. In addition, rat RT6.2 is more active as an NAD
glycohydrolase than is the mRt6.1 and it did not, under these
conditions, cause ADP-ribosylation of histone (Fig. 3). It is noted
also that auto-ADP-ribosylation of rat RT6.2 was not inhibited by
ADP-ribose and is, in all likelihood, enzymatic. For the avian
erythrocyte transferase A, histones serve as both ADP-ribose acceptors
and activators of the enzyme (26, 34). To determine whether histones
enhanced mRt6.1 activity, the enzyme was incubated with
[adenine-U-14C]NAD and agmatine, and
radiolabeled ADP-ribosylagmatine was isolated (Table
III). Histones inhibited ADP-ribosylagmatine formation, presumably, in part, by serving as an alternative ADP-ribose acceptor and competing for the catalytic site, but no enhancement of mRT6.1 activity was observed.
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Expression of mRt6.1 protein in lymphocytes has been demonstrated (21). It appeared, therefore, that the signal sequence at the carboxyl terminus was recognized by the enzymes involved in the formation of a GPI-linked protein (35). mRt6.1 was expressed under the control of a dexamethasone-regulated promoter in rat NMU cells. Under the assay conditions, NMU cells transfected with vector alone did not exhibit ADP-ribosyltransferase activity (data not shown). The cells, however, possessed significant activities that metabolized NAD, and caused release of nicotinamide (data not shown). For that reason, ADP-ribosyltransferase activity was measured directly with [adenine-U-14C]NAD and agmatine as substrates, and the formation of [adenine-U-14C]ADP-ribosylagmatine was monitored. As shown in Table IV, following induction with dexamethasone, NMU cells transfected with the mRt6.1 gene exhibited a time-dependent increase in ADP-ribosyltransferase activity. At 6 h following induction, activity was primarily associated with the cell pellet and was not released with PI-PLC. At 24 h, transferase activity was increased further and was released by PI-PLC. Although the total transferase activity declined after 48- and 72-h incubations with dexamethasone, the fraction susceptible to release by incubation with PI-PLC remained constant. These studies suggest that, following induction by dexamethasone, there is a delay in processing the mRt6.1 transferase to a PI-PLC-releasable form by NMU cells, although the carboxyl-terminal signal sequence in mRt6.1 is sufficient for the addition of the GPI anchor.
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The mouse Rt6 and rat RT6 families of proteins share both structural and functional properties (13-17, 21, 36). The deduced amino acid sequences of all four proteins contain signal sequences at their amino and carboxyl termini believed to be involved, respectively, in export to the endoplasmic reticulum and attachment of a GPI anchor (13, 14, 36). The internal regions of the coding domain contain the consensus sequences found in ADP-ribosyltransferases and NAD glycohydrolases, which are believed to be involved in the formation of the catalytic site (37, 38). Although the RT6 proteins, other mammalian and avian ADP-ribosyltransferases, and bacterial toxin ADP-ribosyltransferases contain the consensus sequences, their enzymatic activities appear clearly to differ (25, 31, 37-41). The rat RT6.1 and RT6.2 appear to be NAD glycohydrolases (15, 17); RT6.2 catalyzes auto-ADP-ribosylation, but there is some disagreement as to whether RT6.1 possesses this activity (17, 42). Unlike the avian erythrocyte and rabbit muscle ADP-ribosyltransferases (28, 31, 43), rat RT6.2 did not appear to use agmatine or similar, simple guanidino compounds as acceptors (15). A recent report on mouse Rt6.2 demonstrated that it had NAD glycohydrolase activity that was inhibited by agmatine; it was not established whether agmatine was used as an ADP-ribose acceptor (21). mRt6.1 protein was observed to have auto-ADP-ribosyltransferase activity (21); based on chemical stability of the ADP-ribose-protein bond, it was concluded that arginine was the ADP-ribose acceptor (21). It is not clear whether the auto-ADP-ribosylation reaction represents a single or multiple turnover reaction. Rat RT6.2, on the other hand, is an auto-ADP-ribosyltransferase but does not catalyze the ADP-ribosylation of arginine (15). The current report clearly demonstrates that mRt6.1 differs significantly in enzymatic properties from rat RT6.1 (17) and RT6.2 (15). Unlike the rat proteins, mRt6.1 catalyzes the stereospecific formation of ADP-ribosylagmatine, similar to cholera toxin and eukaryotic NAD:arginine ADP-ribosyltransferases (38, 44, 45). In the presence of agmatine, nicotinamide release parallels ADP-ribosylagmatine formation. As shown by the NMU cell transfection experiments, the putative carboxyl-terminal signal sequence for mRt6 is processed appropriately in mammalian cells leading to the addition of a GPI anchor. Thus, the mRt6.1 could serve a regulatory role in the ADP-ribosylation of cell surface proteins, suggesting a possible mechanism by which mRt6 protein on the surface of Rt6+ cells might regulate immune function.