Genetic remodeling of protein glycosylation in vivo induces autoimmune disease Daniel Chui Gayathri Sellakumar Ryan S Green Mark Sutton Smith Tammie McQuistan Kurt W Marek Howard R Morris Anne Dell and Jamey D Marth Glycobiology Research and Training Center Howard Hughes Medical Institute Department of Cellular and Molecular Medicine University of California at San Diego La Jolla CA 92093 and Department of Biochemistry Imperial College of Science Technology and Medicine London SW7 2AY England Edited by Stuart A Kornfeld Washington University School of Medicine St Louis MO and approved December 1 2000 received for review August 9 2000 Autoimmune diseases are among the most prevalent of afflictions yet the genetic factors responsible are largely undefined Protein glycosylation in the Golgi apparatus produces structural variation at the cell surface and contributes to immune self recognition Altered protein glycosylation and antibodies that recognize endogenous glycans have been associated with various autoimmune syndromes with the possibility that such abnormalities may reflect genetic defects in glycan formation We show that mutation of a single gene encoding mannosidase II which regulates the hybrid to complex branching pattern of extracellular asparagine N linked oligosaccharide chains N glycans results in a systemic autoimmune disease similar to human systemic lupus erythematosus Mannosidase IIdeficient autoimmune disease is due to an incomplete overlap of two conjoined pathways in complex type N glycan production Lymphocyte development abundance and activation parameters are normal however serum immunoglobulins are increased and kidney function progressively falters as a disorder consistent with lupus nephritis develops Autoantibody reactivity and circulating immune complexes are induced and anti nuclear antibodies exhibit reactivity toward histone Sm antigen and DNA These findings reveal a genetic cause of autoimmune disease provoked by a defect in the pathway of protein N glycosylation autoimmunity genetics lupus glomerulonephritis A utoimmune diseases afflict an estimated 5 of the human population yet inherited genetic susceptibilities and causes are for the most part unknown 1 2 The immune system recognizes glycan dependent features in self non self discrimination and distinct changes in protein glycosylation have been reported in various autoimmune syndromes 3 7 The first autoantibodies to be discovered were the cold agglutinins that bind to glycan chains termed I i antigens and appear to be responsible for approximately 20 of human autoimmune hemolytic anemia cases 3 Elevated levels of autoantibodies to glycolipids are noted in various neurologic disorders including motor neuron disease 3 Altered glycosylation may also affect immune complex formation Immunoglobulins with affinity for the Fc region of IgG molecules are found in rheumatoid arthritis and the severity of the disease is associated with the extent of galactose deficient N glycans on Fc 8 Human IgA nephropathy has been associated with altered O glycosylation of the IgA1 hinge region and Ig deposition in the kidney 9 10 Another possible role for aberrant glycan production in autoimmune disease includes Tn syndrome in which reduced transcription of the core 1 O glycan 1 3 GalT enzyme occurs among hematopoietic compartments This reduced transcription results in exposure of the Tn antigen on cell surfaces and some patients suffer hemolytic anemia thrombopenia and leukopenia likely because of the presence of anti Tn antibodies found in normal serum 11 Glycan structures can clearly participate in pathogenic processes Yet determining whether glycan recognition and production abnormalities are a cause of autoimmune disease or are secondary events induced by lesions in other metabolic pathways has awaited studies involving in vivo genetic modifications of the glycosylation program itself Golgi resident glycosidase and glycosyltransferase 1142 1147 PNAS January 30 2001 vol 98 no 3 enzymes operating in the glycan synthesis pathways are thereby hypothetically promising targets of genetic studies aimed at gaining further insights into the pathogenesis of autoimmune disease The mannosidase II enzyme is encoded by a single gene in mammals and resides in the Golgi apparatus where it trims two mannose residues from hybrid N linked oligosaccharides This trimming of the mannose residues allows the subsequent addition of multiple glycan branches by glycosyltransferases as required for the generation of complex N glycans the most prevalent and diverse forms found on mammalian cell surfaces 12 15 Nonerythroid cells from mice lacking a functional mannosidase II gene were unexpectedly found to compensate for this defect by the activity of another mannosidase defining an alternative pathway Fig 1 and ref 14 In erythroid cells glycoproteins were expressed normally at the cell surface but their portfolio of attached carbohydrate structures was altered with a loss of complex N glycan branching concurrent with an induction of hybrid N glycan forms These animals exhibit a non life threatening dyserythropoiesis similar to human congenital dyserythropoietic anemia type II 14 We have since observed an increased morbidity of aged mice lacking mannosidase II and have therefore attempted to determine whether the loss of mannosidase II in some tissues is not fully compensated for by the alternative pathway and leads to physiologic defects among nonerythroid cell types Our findings herein have revealed that mannosidase II is essential for promoting complex N glycan branching to varying degrees in different tissues and cell types and on subsets of glycoproteins The resulting alteration of N glycan branching provokes a systemic autoimmune disease indicating that inheritance of an abnormal protein N glycosylation pathway is an etiologic factor in the pathogenesis of autoimmunity Materials and Methods Mice The null allele for mannosidase II 14 was bred into the C57BL 6 genetic background for more than eight generations before these studies The mice were maintained in a restrictedaccess barrier facility under specific pathogen free conditions Lectin Blotting Membrane and total cellular proteins were iso lated from various tissues and complex N glycans were visualized by binding to E phytohemagglutinin lectin as previously described 14 Mass Spectrometry N Glycans were isolated from protein by peptide N glycanase F PNGase F treatment and subjected to