Characterization of high density lipoprotein-bound and soluble RT6 released following administration of anti-RT6.1 monoclonal antibody.
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.
Lesma
E
E
Pulmonary-Critical Care Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Moss
J
J
Brewer
H B
HB
Bortell
R
R
Greiner
D
D
Mordes
J
J
Rossini
A A
AA
D036002:ADP Ribose Transferases / Q000097:blood / Q000737:chemistry* / Q000276:immunology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000008:administration & dosage*; D000945:Antigens, Differentiation, T-Lymphocyte; D002850:Chromatography, Gel; D002853:Chromatography, Liquid; D004789:Enzyme Activation / Q000276:immunology; D005260:Female; D006649:Histocompatibility Antigens / Q000276:immunology; D007274:Injections, Intraperitoneal; D007421:Intestine, Small / Q000201:enzymology / Q000276:immunology; D008075:Lipoproteins, HDL / Q000097:blood* / Q000276:immunology; D008297:Male; D008562:Membrane Glycoproteins / Q000097:blood / Q000737:chemistry* / Q000276:immunology*; D011065:Poly(ADP-ribose) Polymerases / Q000097:blood; D051381:Rats; D011913:Rats, Inbred BB; D012995:Solubility; D013934:Thymectomy; D014461:Ultracentrifugation
D000911:Antibodies, Monoclonal; D000945:Antigens, Differentiation, T-Lymphocyte; D006649:Histocompatibility Antigens; D008075:Lipoproteins, HDL; D008562:Membrane Glycoproteins; D036002:ADP Ribose Transferases; D011065:Poly(ADP-ribose) Polymerases; C090163:Art2b protein, rat