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The Journal of Immunology, 1998, 161: 1212-1219.
Copyright © 1998 by The American Association of Immunologists

Characterization of High Density Lipoprotein-Bound and Soluble RT6 Released Following Administration of Anti-RT6.1 Monoclonal Antibody1

Elena Lesma*, Joel Moss2,*, H. Bryan Brewer{dagger}, Rita Bortell{ddagger}, Dale Greiner{ddagger}, John Mordes{ddagger} and Aldo A. Rossini{ddagger}

* Pulmonary-Critical Care Medicine Branch and {dagger} Molecular Disease Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892; and {ddagger} Diabetes Division, University of Massachusetts Medical Center, Worcester, MA 01605


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
RT6 is a rat lymphocyte glycosylphosphatidylinositol (GPI)-anchored alloantigen with nicotinamide adenine dinucleotide (NAD) glycohydrolase (NADase) and auto-ADP-ribosyltransferase activities. RT6 may have immunoregulatory properties based in part on the observation that injection of diabetes-resistant (DR)-BB rats with depleting doses of anti-RT6.1 mAb induced autoimmune diabetes and thyroiditis. We now report that injection of DR-BB rats with anti-RT6.1 mAb increased plasma NADase activity, which localized, by fluid phase liquid chromatography fractionation, to the high density lipoprotein (HDL) fraction. Following ultracentrifugation in high salt, however, RT6 was found in the nonlipoprotein fraction, where it existed, under nondenaturing conditions, as a 200-kDa complex and, by SDS-PAGE, as a 30- to 36-kDa species. Thy-1, another GPI-linked protein, and proteins that reacted with anti-GPI-oligosaccharide Abs also translocated from HDL to the nonlipoprotein fraction under similar conditions. Injection of anti-RT6.1 mAb into thymectomized DR and diabetes-prone-BB rats increased soluble RT6 to levels comparable to those observed in euthymic DR-BB rats, suggesting that HDL-bound RT6 is not derived from peripheral lymphocytes. In agreement, NADase activity in the plasma of eviscerated DR-BB rats did not increase following injection of anti-RT6 mAb. These data suggest that HDL is a carrier of plasma RT6 and other GPI-linked proteins, with equilibrium between the lipoprotein and nonlipoprotein fractions being salt dependent. Since GPI-linked proteins in HDL can transfer to cells in a functionally active form, the presence of RT6 in HDL is consistent with it having a role in signaling in nonlymphoid cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
RT6 (ART2)3 is a rat T cell glycosylphosphatidylinositol (GPI)4-anchored alloantigen whose expression is restricted to the final stages of post-thymic development (1-6). The gene has two known alleles, RT6a and RT6b (7). RT6.1 protein (product of the RT6a allele) occurs as both a 24- to 26-kDa nonglycosylated protein and variably glycosylated proteins with molecular masses of up to 36 kDa (8, 9). In contrast, RT6.2 is found only as a nonglycosylated 24- to 26-kDa protein (5, 8). RT6 has been reported to circulate in soluble form (10) and is also expressed on intestinal intraepithelial lymphocytes (IELs) (11).

Both RT6.1 and RT6.2 proteins exhibit NAD glycohydrolase (NADase) activity (12, 13), but there are conflicting reports on whether both proteins possess auto-ADP-ribosyltransferase activities (13-15). In the former enzymatic reaction, ADP-ribose and nicotinamide are generated from NAD, whereas in the latter reaction, ADP-ribose is transferred to the RT6 protein with the release of nicotinamide. Unlike the related family of NAD:arginine ADP-ribosyltransferases that use proteins and free arginine as ADP-ribose acceptors (16, 17), neither rat RT6.1 nor RT6.2 catalyzes the ADP-ribosylation of arginine or exogenous proteins (13-15). A single amino acid replacement at the active site, glutamine for glutamic acid at position 207, resulted in an RT6.1 protein that did ADP-ribosylate simple guanidino compounds (e.g., arginine) (18, 19). Two mouse RT6-like genes, Rt6.1 (Art2a) and Rt6.2 (Art2b), have been identified (20). Proteins encoded by these genes possess NAD:arginine ADP-ribosyltransferase activity (13, 21, 22). Both mouse Rt6 proteins have a glutamate at position 207 (20, 23). Replacement of glutamate 207 with glutamine, as found in the rat homologues, converted the ADP-ribosyltransferase to an NAD glycohydrolase (19).

The development and function of RT6-expressing cells have been studied extensively in the rat. A deficiency of RT6-expressing cells is associated with susceptibility to autoimmunity in the BB rat model of insulin-dependent diabetes mellitus (IDDM) (24, 25). Diabetes-prone (DP)-BB rats spontaneously develop IDDM and have a severe deficiency in the number of RT6-expressing lymphocytes (26). In contrast, the diabetes-resistant (DR)-BB rat remains disease free and has normal numbers of RT6-expressing lymphocytes (27). Injection of DR-BB rats with a mAb that results in depletion of the RT6.1 antigen leads to the rapid expression of autoimmune IDDM (28). Injection of anti-RT6.1 mAb also induces a transient decrease in soluble RT6, followed within 1 h by a rapid restoration of soluble RT6 levels (D. Waite, J. B. Whalen, R. Bortell, J. H. Leif, E. Lesma, J. Moss, J. P. Mordes, A. A. Rossini, and D. L. Greiner, manuscript in preparation).

Cell surface RT6 protein has both enzymatic (12-14) and signal transduction (29) capabilities, but the functional capability of soluble RT6 is unknown. Other soluble GPI-linked proteins, such as CD59, are known to circulate in the high density lipoprotein (HDL) fraction (30, 31). GPI-linked proteins may be transferred from HDL to cells and incorporated, following a delay, into functional signal transduction pathways. In the studies reported here, the tissue source, plasma localization, enzymatic activity, and molecular size of soluble RT6 have been determined. Soluble RT6 has NADase activity, appears to be derived from intestinal tissue, and is localized to the HDL fraction. Soluble RT6 can, however, also be recovered in the nonlipoprotein fraction, where it exists as a 200-kDa complex. The salt-dependent translocation of RT6 between lipoprotein and nonlipoprotein fractions also occurs with other GPI-linked proteins.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

DP- and DR-BB/Wor rats, 4 to 12 wk of age, of both sexes were obtained from the viral Ab-free colony maintained at the University of Massachusetts Medical Center (Worcester, MA). Animals from this colony express the RT6.1 T cell alloantigen.

All animals in the colony are certified to be serologically free of Sendai virus, pneumonia virus of mice, sialodacryoadenitis virus, rat corona virus, Kilham rat virus, H1 (Toolan’s virus), GD7, Reo-3, Mycoplasma pulmonis, lymphocytic choriomeningitis virus, mouse adenovirus, Hantaan virus, and Encephalitozoon cuniculi. All animals were maintained in accordance with recommendations in the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Research Council, National Academy of Science, 1996) and the guidelines of the institutional animal care and use committee of the University of Massachusetts Medical Center.

Ab treatment protocol

DS4.23 anti-RT6.1 or 6A5 anti-RT6.2 mAbs were administrated i.p. at a dose equivalent to approximately 50 µg/injection in 2 ml of hybridoma culture supernatant. The animals were killed 24 h after the last injection.

Antibodies

The DS4.23 anti-RT6.1 (rat IgG2b) and 6A5 anti-RT6.2 (rat IgG1) hybridomas were prepared from culture supernatant as previously described (28). Hybridoma culture supernatants were concentrated by precipitation with 50% saturated ammonium sulfate and dialyzed against PBS.

The rabbit polyclonal antiserum r1126 was developed using the peptide TGPVMLDTAPNAFD for immunization. The specificity of the r1126 antiserum for the RT6 alloantigens has been described (10).

Surgical procedures

Evisceration. DR-BB rats, 8 to 12 wk of age, were eviscerated under general inhalation anesthesia. Briefly, the animals were anesthetized, and the abdominal cavity was exposed from an area just below the stomach to just above the rectum. All the connecting blood supplies to the intestine were ligated, and the length of the intestine was removed. The control animals were placed under anesthesia but were not subjected to surgery. The surgically manipulated and control animals were then injected i.v. via the tail vein with approximately 50 µg of anti-RT6.1 or anti-RT6.2 mAb while still under anesthesia. All injected animals were maintained under general anesthesia for the duration of the 2-h experiment, at which time the blood was recovered, and the plasma was separated and frozen at -70°C until analysis. In preliminary experiments, we documented readily detectable increases in soluble RT6 levels within 30 min after i.v. anti-RT6.1 mAb administration.

Thymectomy. DR- and DP-BB rats, 5 to 6 wk of age, were thymectomized under general inhalation anesthesia as previously described (1). Thymectomized animals were maintained for at least 2 wk, and then injected with anti-RT6.1 mAb as described above to deplete circulating RT6+ T cells. The absence of the thymus was confirmed by gross examination at necropsy. Data from rats with incomplete thymectomy were excluded from analysis. Flow cytometric analysis revealed that anti-RT6.1 mAb treatment of DR-BB rats thymectomized 2 to 4 wk earlier reduced the percentage of RT6+ lymph node cells from 72 to 7%.

Lipoprotein analysis

Plasma fractions (very low/low density lipoproteins (VLDL/LDL), HDL, and nonlipoprotein fractions) were obtained by either fluid-phase liquid chromatography (FPLC) size fractionation or ultracentrifugation. In the first procedure, plasma (400 µl) was applied to an FPLC system consisting of two Superose 6 columns connected in series (Pharmacia-LKB Biotechnology, Piscataway, NJ). Lipoproteins were eluted at 0.3 ml/min with PBS containing EDTA. Sixty fractions (0.5 ml) were collected. The elution positions of VLDL, LDL, and HDL were established using lipoproteins separated by ultracentrifugation. To verify the location of the VLDL/LDL and HDL in the samples, total cholesterol was quantified in the individual effluent fractions. In the second procedure, lipoproteins were isolated by ultracentrifugation as previously described (32).

NAD glycohydrolase assay

NAD glycohydrolase was assayed in 50 mM potassium phosphate (pH 7.5) containing 0.1 mM [carbonyl-14C]NAD (5 x 104 cpm; 35.0 mCi/mmol, Amersham, Aylesbury, U.K.) at 30°C (final volume, 300 µl). Samples (100 µl) were applied to columns (0.5 x 4 cm) of Dowex AG1-X2 (Bio-Rad, Melville, NY), and [carbonyl-14C]nicotinamide was eluted with 5 ml of water for scintillation counting.

Molecular weight determination by electrophoresis

Samples of lipoprotein fractions were subjected to electrophoresis using 12% Tris-glycine gels under nonreducing conditions. Lanes were sliced into 2-mm pieces, which were incubated with 300 µl of 50 mM potassium phosphate (pH 7.5), 0.1 mM [carbonyl-14C]NAD (5 x 105 cpm), and 1% 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate (CHAPS; J. T. Baker, Phillipsburg, NJ) at 30°C for 14 h. The release of carbonyl-14C was determined as described above.

Affinity immunobeads

Immunoprecipitation of RT6.1 protein using anti-RT6.1 affinity immunobeads was performed as described previously (10).

Western blot analysis

Immunoprecipitated RT6 was prepared for electrophoresis by boiling the beads for 10 min in SDS-PAGE sample buffer before centrifugation at 10,000 x g for 2 min. Supernatants were loaded on 12% polyacrylamide gels containing 0.1% SDS. Following electrophoresis, proteins were transferred to Hybond-ECL nitrocellulose membranes (Amersham, Arlington Heights, IL). Blots were incubated overnight with diluted (1/5000) r1126 Ab (directed against an RT6-specific peptide) at 4°C, followed by incubation with a 1/2500 dilution of horseradish peroxidase-conjugated anti-rabbit IgG (Promega, Madison, WI) for 1 h at room temperature. Western analyses of plasma fractions separated by ultracentrifugation and FPLC were performed using anti-mouse and anti-rat CD90.1 (Thy-1.1; PharMingen, San Diego) overnight at 4°C, followed by incubation with a 1/2500 dilution of horseradish peroxidase-conjugated anti-rabbit IgG (Promega) for 1 h at room temperature. Blots were developed using enhanced chemiluminescence (Amersham, Aylesbury, U.K.).

High performance liquid chromatography

After fractionation of plasma from anti-RT6.1 mAb-treated DR-BB rats by ultracentrifugation, the nonlipoprotein fraction was subjected to chromatography on a 3000 SW HPLC column (7.5 mm inside diameter x 60 cm) (TosoHaas, Montgomeryville, PA) eluted with 150 mM NaCl containing 20 mM sodium phosphate, pH 7.5 (flow rate, 1 ml/min).

Isolation of IELs

IELs were isolated according to previously described procedures (33). Briefly, segments of rat small intestine were incubated in saline at 37°C with vigorous shaking for 20 min (275 rpm) and then vortexed for 15 s. Suspended cells were decanted and filtered through nylon wool. Viable cells were quantified in the presence of 0.1% trypan blue.

Phosphatidylinositol-specific phospholipase C (PI-PLC) treatment

Lymph node cells (1 x 106) or IELs were incubated with or without 0.5 U PI-PLC (Sigma, St. Louis, MO) for 1 h at 37°C.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of injection of anti-RT6.1 mAb on NADase activity in plasma

A large i.v. bolus (9 mg/rat) of anti-RT6.1 mAb clears soluble RT6.1 protein from the circulation of DR-BB rats within 2 min (28). The modulation of RT6 and other NADase activities in plasma following the injection of smaller doses (~50 µg) of anti-RT6.1, an amount known to deplete RT6.1+ T cells and induce diabetes in the BB rat (24, 25, 28) was determined. DR rats were injected i.p. on 3 consecutive days with 50 µg of anti-RT6.1 mAb. Plasma NADase activity increased following injection with DS4.23 anti-RT6.1 mAb, but not following injection of 6A5, an anti-RT6.2 mAb (Fig. 1GoA).



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  		FIGURE 1. NAD glycohydrolase activities and their relative molecular masses in plasma from DR-BB/Wor rats after injection of anti-RT6.1 mAb. DR-BB/Wor rats (10-12 wk old) were injected i.p. with anti-RT6.1 mAb (2 ml) for 3 consecutive days. A, Plasma from anti-RT6.1 mAb-treated, control anti-RT6.2 mAb-treated, and untreated rats was assayed for NAD glycohydrolase activity for 1 h at 30°C. B, Plasma was separated by electrophoresis using 12% gels, and NAD glycohydrolase activity was extracted in 1% CHAPS and quantified in gel slices for 14 h at 30°C as noted in Materials and Methods. The results shown are triplicate determinations (A) of a pool of plasma from three to six DR-BB rats in each group (A and B) and are representative of three separate experiments.

 
Since NADases are relatively ubiquitous, the molecular size of protein(s) with NADase activity was determined. Plasma from control and injected animals was subjected to gel electrophoresis under nonreducing conditions, and gel slices were assayed for NADase activity (Fig. 1GoB). Fractions with NADase activity had a mobility consistent with a protein of 30 to 36 kDa, in agreement with the size of glycosylated RT6.1 (6, 34).

Effect of injection of anti-RT6.1 mAb on the NADase activity in plasma lipoprotein and nonlipoprotein fractions

To determine the location of RT6 in plasma fractions, plasma from control and anti-RT6.1 mAb-treated rats was analyzed by gel permeation-FPLC under physiologic conditions. Three major peaks of NADase activity were detected in anti-RT6.1 mAb-treated rats (Fig. 2GoA), whereas only two were found in control plasma. Based on comparison with lipoprotein standards (Fig. 2GoB), the first peak corresponds to VLDL/LDL, the second peak to HDL, and the third to the nonlipoprotein fraction. Only the NADase activity in the second peak was significantly increased after injection of anti-RT6.1 mAb, suggesting that RT6 in plasma is associated with HDL. By SDS-PAGE, the size of the NADase in the active peak was 33 to 36 kDa, in agreement with the size of RT6 (Fig. 2GoC).



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  		FIGURE 2. NAD glycohydrolase activity in plasma fractions separated by FPLC. Plasma from pools obtained from three to six DR-BB control or anti-RT6.1 mAb-injected rats was analyzed to characterize the NADase activities and cholesterol content. A, Plasma was fractionated by FPLC in 60 fractions of 0.5 ml, and NADase activity was measured for 3 h at 30°C as noted in Materials and Methods. The results shown are representative of three separate experiments. B, By cholesterol analysis, HDL eluted in fractions 30 to 40. C, The approximate molecular mass of each FPLC fraction was plotted as a function of NADase activity as noted in Materials and Methods.

 
To characterize further the NADase activity in plasma from rats injected with anti-RT6.1 mAb, plasma from DR-BB rats treated or not with the anti-RT6.1 mAb was fractionated by ultracentrifugation into VLDL/LDL, HDL, and nonlipoprotein (density, >1.21 g/ml) fractions. In contrast to the FPLC finding, after Ab treatment a significant increase in NADase activity was found in the nonlipoprotein fraction (Fig. 3Go). The effects on NADase activities in the HDL and VLDL/LDL fractions were much less. It appeared that the high salt concentration (0.5 M KBr) during ultracentrifugation had released NADase from the HDL particles into the nonlipoprotein fraction.



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  		FIGURE 3. NAD glycohydrolase activity in plasma fractions separated by ultracentrifugation. Plasma from pools obtained from three to six DR-BB control and anti-RT6.1 mAb-injected animals was fractionated by ultracentrifugation into HDL, LDL/VLDL, and nonlipoprotein fractions (density, >1.21 g/ml; 1.21B). The samples were dialyzed for 24 h. NAD glycohydrolase activity was determined for 1 h at 30°C. Activity is expressed per milliliter of each fraction; volumes of the fractions were 1 ml for VLDL/LDL, 1 ml for HDL, and 4 ml for the nonlipoprotein fraction.

 
Effect of ultracentrifugation on the NADase activity in lipoproteins and nonlipoprotein fractions

To determine the molecular size of the NADase activity released by the treatment with anti-RT6.1 mAb, the nonlipoprotein fraction separated by ultracentrifugation was subjected to gel electrophoresis under nonreducing conditions, and gel slices were assayed for NADase activity. The size of NADase activity in the nonlipoprotein fraction was consistent with a protein of 30 to 36 kDa, in agreement with it being an isoform of RT6 (Fig. 4GoB). The soluble fraction after separation by ultracentrifugation was analyzed by gel permeation HPLC under nondenaturing conditions, and the fractions were assayed for NADase activity. The size of the RT6 NADase activity was about 200 kDa (Fig. 5Go), suggesting that it exists as a complex. SDS would be anticipated to disrupt intermolecular interactions in the aggregate, allowing the recovery of 30- to 36-kDa RT6.



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  		FIGURE 4. Separation by gel electrophoresis of NAD glycohydrolase activity in HDL and nonlipoprotein fractions in control and anti-RT6.1 mAb-treated rats. Three to six rats were injected with anti-RT6.1 mAb. The pooled plasma in each group was fractionated by ultracentrifugation into lipoprotein and nonlipoprotein fractions. A, The HDL fraction was separated by electrophoresis using 12% gels, and the NAD glycohydrolase activity was extracted in 1% CHAPS and measured in 50 mM potassium phosphate for 14 h at 30°C. B, NAD glycohydrolase activity from the nonlipoprotein fraction was quantified as described in A for HDL.

 


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  		FIGURE 5. Nonlipoprotein fraction analysis by HPLC after separation by ultracentrifugation. Plasma from DR rats treated with anti-RT6.1 mAb was fractionated by ultracentrifugation, and the soluble fraction was analyzed by HPLC. Forty fractions were assayed for NADase activity for 3 h at 30°C.

 
To characterize the non-RT6 NADase activity found in HDL, the same analysis was performed. By gel electrophoresis under nonreducing conditions, the NADase activity exhibited an apparent molecular mass of 40 to 50 kDa (Fig. 4GoA).

To determine whether the presence of high salt concentrations during ultracentrifugation resulted in the release of GPI-anchored proteins other than RT6 into the nonlipoprotein fraction, Thy-1.1 was quantified. By Western analysis, Thy-1.1, a GPI-anchored glycoprotein expressed in the rat on thymocytes, hemopoietic stem cells, recent thymic migrants, and immature B lymphocytes in bone marrow and in peripheral lymphoid organs (35-37), was detected in HDL after FPLC fractionation and in the nonlipoprotein fraction after ultracentrifugation (Fig. 6Go, A and B). In contrast to its effects on RT6.1, injection of DS4.23 anti-RT6.1 mAb did not alter the amount of Thy-1.1 in plasma. An Ab against the oligosaccharide portion of the GPI anchor reacted with two major proteins in HDL fractionated by FPLC; the same proteins were found in the nonlipoprotein fraction following ultracentrifugation (data not shown). These observations confirmed that the high salt concentration can cause GPI-anchored proteins to move from the HDL into the nonlipoprotein fraction during ultracentrifugation, presumably by affecting the GPI anchor.



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  		FIGURE 6. Plasma localization of Thy-1.1 following injection of anti-RT6.1 mAb. Three to six DR rats were injected with anti-RT6.1 mAb. The pooled plasma from control and Ab-treated animals was fractionated by FPLC (A) and by ultracentrifugation (B), and the localization of Thy-1.1 protein was determined by Western analysis using anti-rat CD90.1 (Thy-1.1) mAb.

 
Effect of thymectomy on the increase in plasma NADase activity induced by injection of anti-RT6.1 mAb

DR-BB rats were thymectomized, allowed to recover for 4 wk, and then treated with anti-RT6.1 mAb for 3 days. Following injection of anti-RT6.1 mAb, total NADase activity was increased in plasma (data not shown). Most of the NADase activity was recovered in the nonlipoprotein fraction after ultracentrifugation (Fig. 7GoA) and had a molecular mass of 30 to 36 kDa (Fig. 7GoC), similar to that observed in euthymic rats. The NADase activity released from HDL was estimated to be 40 to 50 kDa (Fig. 7GoB). Effects of DS4.23 anti-RT6.1 mAb on plasma NADase levels in thymectomized and control rats were similar.



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  		FIGURE 7. Effect of injection of anti-RT6.1 mAb on NAD glycohydrolase activity in plasma fractions of thymectomized and anti-RT6.1 mAb-treated thymectomized rats. Four- to five-week-old rats were thymectomized. Four weeks later, the animals were injected i.p. with anti-RT6.1 mAb for 3 consecutive days. Serum was obtained from all rats 24 h after the last injection of mAb. Plasma was fractionated by ultracentrifugation into VLDL/LDL, HDL, and nonlipoprotein fractions. A, NAD glycohydrolase activity was measured for 1 h at 30°C. B, After gel electrophoresis, the NAD glycohydrolase activity of HDL from anti-RT6.1 mAb-treated, thymectomized rats was assayed for 14 h at 30°C. C, NAD glycohydrolase activity from nonlipoprotein fraction of anti-RT6.1 mAb-treated, thymectomized rats was measured as described in B.

 
Persistent elevated levels of soluble RT6 protein in thymectomized BB rats treated with anti-RT6.1 mAb

We next excluded the possibility that elevated levels of soluble RT6.1 protein observed after mAb treatment resulted from the lysis of existing peripheral RT6.1+ T cells in thymectomized animals. DR- and DP-BB/Wor rats were thymectomized, allowed to recover for 4 wk, and then treated with one injection per day (50 µg) of either anti-RT6.1 or anti-RT6.2 mAb given on 9 of 11 days. Levels of soluble and lymph node cell RT6.1 protein were measured 24 h after the final injection. Age-matched DR- and DP-BB/Wor rats were used as nonthymectomized, untreated controls. Levels of both serum and lymph node cell RT6.1 protein in nonthymectomized, untreated DR and DP rats were comparable to those in thymectomized DR and DP rats, respectively, that were treated with anti-RT6.2 mAb (Fig. 8Go). This result is consistent with previous reports that thymectomy alone has little effect on RT6+ lymph node cell populations (1). Following treatment with anti-RT6.1 mAb, levels of lymph node cell RT6.1 protein in thymectomized DR-BB/Wor rats declined from intermediate to low levels, and those in thymectomized DP-BB/Wor rats declined from low to undetectable levels (Fig. 8Go). In contrast, serum RT6.1 protein in DR- and DP-BB/Wor thymectomized rats treated with anti-RT6.1 mAb increased to levels that were elevated compared with the basal levels characteristic of each strain.



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  		FIGURE 8. Immunoblot of RT6 protein from serum and lymph nodes of thymectomized BB rats treated with anti-RT6.1 or anti-RT6.2 mAb. DR-BB/Wor and DP-BB/Wor rats, 3 to 4 wk old, were either thymectomized (n = 12 of each strain) or not treated (n = 6 of each strain). Four weeks after surgery, thymectomized animals were injected i.p. with 50 µg of either anti-RT6.1 mAb (n = 6 of each strain) or anti-RT6.2 mAb (n = 6 of each strain) in a volume of 2 ml. One injection was given daily on 9 days during an 11-day period. Rats that were not thymectomized received no mAb treatment. Serum and lymph nodes were obtained from all rats 24 h after the last injection of mAb. Samples from each group of six rats were pooled. PI-PLC lymph node extracts (right lanes) and serum (left lanes) were subjected to immunoprecipitation and Western blot analysis using the r1126 rabbit anti-RT6 peptide antiserum as previously described (10). Upper panel, Reaction with r1126 antiserum. Lower panel, Reaction with the r1126 antiserum in the presence of excess immunizing peptide. The treatment status of each group is indicated at the bottom of the figure.

 
Identification of the NADase activity in nonlipoprotein fraction as RT6

After immunoprecipitation of VLDL/LDL, HDL, and nonlipoprotein fractions with anti-RT6.1 mAb followed by Western analysis of the immunoprecipitates with polyclonal rabbit anti-RT6 antiserum 1126, an immunoreactive protein of 33 kDa was detected in the nonlipoprotein fraction from Ab-treated euthymic (Fig. 9Go) and thymectomized (Fig. 10Go) rats. The apparent molecular size of recombinant RT6.1 synthesized in Sf9 cells was significantly less than that of the RT6.1 immunoreactive proteins detected in the nonlipoprotein fractions. These differences in apparent molecular size may reflect differences in protein glycosylation in the rat and the insect cells. To better understand the characteristics of soluble RT6, the source of RT6.1 and its immunoreactivity and physical properties were investigated.



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  		FIGURE 9. Immunoblot of RT6 protein in plasma fractions of untreated and anti-RT6.1 mAb-treated rats. Euthymic DR-BB rats were injected with anti-RT6.1 mAb for 3 days or left untreated. Plasma from three to six animals treated or not with mAb was collected, pooled within each group, and fractionated by ultracentrifugation into lipoprotein and nonlipoprotein fractions. RT6.1 protein was immunoprecipitated with DS4.23 anti-RT6.1 mAb-Sepharose immunobeads and subjected to SDS-PAGE and immunoblot analysis with the r1126 anti-RT6 peptide antiserum. A, Western blot of plasma fractions from control rats. B, Western blot of plasma fractions from mAb-treated rats and immunoprecipitated RT6 from Sf9 cells. The five arrows indicate bands identified by the r1126 antiserum. The lowest band is rat Ig light chain present in all fractions incubated with anti-RT6.1 affinity immunobeads (10). The middle arrows recognize variously glycosylated forms of RT6.1. The recombinant form (lane Rt6.1-IP) is a nonglycosylated form of RT6 with a lower molecular mass. The uppermost arrow indicates an uncharacterized RT6-like immunoreactivity at about 60 kDa.

 


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  		FIGURE 10. Immunoblot of RT6 protein in plasma fractions of thymectomized and anti-RT6.1 mAb-treated, thymectomized rats. Three to six DR-BB rats were thymectomized and injected with anti-RT6.1 mAb (B) or left untreated (A). Plasma was collected, pooled within each group, and fractionated by ultracentrifugation into lipoprotein and nonlipoprotein fractions. RT6 protein was immunoprecipitated and analyzed by Western blot as described in Figure 9Go. The arrows indicate the same bands as those described in Figure 9Go.

 
Identification of the source of NADase activity released after injection of anti-RT6.1 mAb

Since RT6+ lymphocytes are found in intestine, a surgical model was prepared in which the intestine was removed. Following injection of anti-RT6.1 mAb, the NADase activity in plasma and that in the nonlipoprotein fraction were not increased (Fig. 11Go, A and B), suggesting that the source of soluble RT6.1 was intestinal tissue. Injection of anti-RT6.2-specific mAb into RT6.1+ DR-BB rats did not affect NADase activity in control or surgically treated animals. An RT6+ lymphocyte-enriched fraction was isolated from intestinal tissue and analyzed by Western blotting (Fig. 12Go). Two immunoreactive bands of 33 to 36 kDa were detected, similar to those in plasma, and larger than the RT6 species synthesized in Sf9 cells. These results support the hypothesis that the intestine is the major source of plasma RT6-NADase activity.



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  		FIGURE 11. Injection of eviscerated DR-BB rats with anti-RT6.1 mAb and anti-RT6.2 mAb and determination of NAD glycohydrolase activity in total plasma and specific plasma fractions. DR-BB rats were eviscerated as described in Materials and Methods and injected with anti-RT6.1 (DS4.23) mAb or anti-RT6.2 (6A5) mAb. A, NAD glycohydrolase activity from total plasma was measured for 1 h at 30°C. B, Plasma from anti-RT6.1 mAb-injected rats was fractionated by ultracentrifugation, and NAD glycohydrolase activity was measured as described in A. Data shown are triplicate determinations of plasma from a single animal and are representative of results obtained in two animals.

 


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  		FIGURE 12. Western analysis of RT6 protein in IELs. Three or four DR-BB rats were injected with anti-RT6.1 mAb 2 h before tissue recovery. Cell suspension preparations enriched for IELs (~80%) were placed into culture in RPMI with 10% FBS at 37°C in an atmosphere of 5% CO2. After 3 days in culture the cells were collected and incubated with or without PI-PLC (0.5 U) for 1 h at 37°C; cell supernatants were prepared and subjected to 12% SDS-PAGE followed by immunoblotting analysis using r1126 anti-RT6 peptide antiserum.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study it was demonstrated that following injection of anti-RT6.1 mAb, there was an increase in a 33- to 36-kDa NADase activity in the plasma HDL fraction that colocalized with an RT6-immunoreactive protein. The localization of soluble RT6 in HDL was dependent on the lipoprotein fractionation procedure; RT6 appeared to be in a salt-dependent equilibrium between the lipoprotein and nonlipoprotein fractions. Based on studies with thymectomized and eviscerated animals, most of the soluble RT6 appeared to be derived from intestine, and not peripheral, RT6.1+ lymphocytes.

Fractionation by FPLC under physiologic conditions showed that RT6 present in the circulation under steady state conditions is predominately in the HDL fraction of plasma. Injection of anti-RT6.1-mAb increased NADase activity only in HDL, with the NADase activities in VLDL/LDL and the nonlipoprotein fraction remaining unchanged. The NADase activity released by injection of anti-RT6 mAb represented a significant fraction of total plasma NADase.

In contrast to the FPLC data, when plasma was fractionated by ultracentrifugation in the presence of 0.5 M KBr into VLDL/LDL, HDL, and nonlipoprotein fractions, RT6, as assayed by immunoreactivity, size, and enzymatic activity, was found in the nonlipoprotein fraction where it exhibited a molecular size of about 200 kDa, suggesting that it existed as an aggregate or complex with other proteins. Under denaturing conditions, it had a size of approximately 33 kDa, comparable to that of glycosylated RT6. Based on the difference from FPLC data, it appears that the salt used in the ultracentrifugation process may dissociate RT6 from the HDL particles. These data suggest that GPI-linked proteins bound to HDL may be in equilibrium with a soluble fraction. Thy-1.1, another GPI-linked protein, and proteins recognized by an Ab against the oligosaccharide moiety in the GPI anchor, behaved like RT6 after FPLC or ultracentrifugation, confirming the salt effect on the localization of GPI-linked proteins. After injection of RT6.1 mAb, however, the levels of Thy-1.1 and these other proteins were unchanged, suggesting that the effects of anti-RT6.1 mAb on RT6 were specific and did not involve other GPI-linked proteins.

In thymectomized and euthymic control rats, injection of anti-RT6.1 mAb increased, to the same extent, both NADase activity and an RT6.1 immunoreactive protein. These results suggested that the Ab-enhanced RT6 NADase activity was not derived from a peripheral lymphocyte population. A previous report (38) showed that anti-RT6.1 Ab treatment cleared RT6+ T cells within 24 h from the peripheral lymphoid tissues, but did not completely clear the RT6+ IEL population. With eviscerated animals, injection of Ab did not increase soluble RT6 levels and NADase. Although all animals were anesthetized, it is possible that the surgical trauma precluded an increase in soluble RT6 following anti-RT6 mAb administration. RT6.1 has been shown to be post-translationally modified, resulting in multiple species of different sizes. Plasma and intestinal RT6 were similar in their NADase activity, molecular size, and immunoreactivity with anti-RT6 Ab. Taken together with the results of thymectomy experiments and the data from DP-BB rats, these data are consistent with the conclusion that the intestine is the source of soluble RT6 and may have an important role in the regulation of its levels in the circulation.

Cell surface RT6 has signal transduction capabilities (29), although the cell-signaling activity of soluble RT6 has not been described. It has been shown that HDL acts as a carrier of GPI-anchored proteins such as CD59 (30). These HDL-linked proteins can be transferred to a number of types of cells. After uptake, GPI-linked proteins appear, following a delay, to be incorporated functionally into the acceptor cells, modifying the activity of intracellular signaling pathways. In light of our results, RT6 released after injection of anti-RT6 mAb appears to become incorporated into circulating HDL particles and may be transferred to other cells. The capability of HDL to be a vehicle for protein transfer has been found to have significant implications in diseases such as paroxymal nocturnal hemoglobinuria, in which the absence of specific GPI-linked proteins increases the susceptibility of red cells to complement-mediated lysis (39). Similarly, the association of RT6, a protein on lymphocytes with known immunoregulatory activity in the autoimmune BB rat, with the HDL fraction in plasma offers the possibility that its translocation may have important roles in signaling pathways in modified cells.


   Acknowledgments
 
We thank Dr. Martha Vaughan for helpful discussions and critical review of this manuscript and Carol Kosh for expert secretarial assistance.


   Footnotes
 
1 This work was supported in part by Grants DK41235, DK25306, and DK36024 from the National Institutes of Health and Program Project Grant DK53006, jointly funded by National Institutes of Health and the Juvenile Diabetes Foundation International. Back

2 Address correspondence and reprint requests to Dr. J. Moss, National Institutes of Health, 10 Center Dr., MSC 1590, Building 10, Room 6D03, Bethesda, MD 20892-159. E-mail address: Back

3 The nomenclature of the family of mono-ADP-ribosyltransferase is being revised. The provisional revised designations are as follows: rat RT6.1, ART2a; rat RT6.2, ART2b; mouse Rt6 locus 1, Art2a; and mouse Rt6 locus 2, Art2b. Back

4 Abbreviations used in this paper: ART, adenosine diphosphate-ribosyltransferase; GPI, glycosylphosphatidylinositol; IEL, intraepithelial lymphocytes; NAD, nicotinamide adenine dinucleotide; IDDM, insulin-dependent diabetes mellitus; DP-BB, diabetes-prone BB rat; DR-BB, diabetes-resistant BB rat; VLDL/LDL, very low/low density lipoproteins; HDL, high density lipoproteins; FPLC, fluid phase liquid chromatography; CHAPS, 3-(cholamidopropyl) dimethylammonio-1-propanesulfanate; PI-PLC, phosphatidylinositol-specific phospholipase C. Back

Received for publication January 12, 1998. Accepted for publication April 2, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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