Genetic remodeling of protein glycosylationin vivoinduces autoimmune diseaseDaniel 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 atSan Diego, La Jolla, CA 92093; and§Department of Biochemistry, Imperial College of Science, Technology and Medicine, London SW7 2AY, EnglandEdited 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. Proteinglycosylation in the Golgi apparatus produces structural variation atthe cell surface and contributes to immune self-recognition. Alteredprotein glycosylation and antibodies that recognize endogenousglycans have been associated with various autoimmune syndromes,with the possibility that such abnormalities may reflect geneticdefects in glycan formation. We show that mutation of a single gene,encoding␣-mannosidase II, which regulates the hybrid to complexbranching pattern of extracellular asparagine (N)-linked oligosaccha-ride chains (N-glycans), results in a systemic autoimmune diseasesimilar to human systemic lupus erythematosus.␣-Mannosidase II-deficient autoimmune disease is due to an incomplete overlap of twoconjoined pathways in complex-type N-glycan production. Lympho-cyte development, abundance, and activation parameters are normal;however, serum immunoglobulins are increased and kidney functionprogressively falters as a disorder consistent with lupus nephritisdevelops. Autoantibody reactivity and circulating immune complexesare induced, and anti-nuclear antibodies exhibit reactivity towardhistone, Sm antigen, and DNA. These findings reveal a genetic causeof autoimmune disease provoked by a defect in the pathway ofprotein N-glycosylation.autoimmunity 兩 genetics 兩 lupus 兩 glomerulonephritisAutoimmune diseases afflict an estimated 5% of the humanpopulation, yet inherited genetic susceptibilities and causes arefor the most part unknown (1, 2). The immune system recognizesglycan-dependent features in self-兾non-self-discrimination, and dis-tinct changes in protein glycosylation have been reported in variousautoimmune syndromes (3–7). The first autoantibodies to be dis-covered were the cold agglutinins that bind to glycan chains (termedI兾i antigens) and appear to be responsible for approximately 20%of human autoimmune hemolytic anemia cases (3). Elevated levelsof autoantibodies to glycolipids are noted in various neurologicdisorders, including motor neuron disease (3). Altered glycosyla-tion may also affect immune complex formation. Immunoglobulinswith affinity for the Fc region of IgG molecules are found inrheumatoid arthritis, and the severity of the disease is associatedwith the extent of galactose-deficient N-glycans on Fc (8). HumanIgA nephropathy has been associated with altered O-glycosylationof the IgA1 hinge region and Ig deposition in the kidney (9, 10).Another possible role for aberrant glycan production in autoim-mune disease includes Tn syndrome, in which reduced transcriptionof the core 1 O-glycan␤1–3 GalT enzyme occurs among hemato-poietic compartments. This reduced transcription results in expo-sure of the Tn antigen on cell surfaces, and some patients sufferhemolytic anemia, thrombopenia, and leukopenia, likely because ofthe 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 ab-normalities are a cause of autoimmune disease or are secondaryevents induced by lesions in other metabolic pathways has awaitedstudies involving in vivo genetic modifications of the glycosylationprogram itself. Golgi-resident glycosidase and glycosyltransferaseenzymes operating in the glycan synthesis pathways are therebyhypothetically promising targets of genetic studies aimed at gainingfurther insights into the pathogenesis of autoimmune disease.The␣-mannosidase II enzyme is encoded by a single gene inmammals and resides in the Golgi apparatus, where it trims twomannose residues from hybrid N-linked oligosaccharides. Thistrimming of the mannose residues allows the subsequent additionof multiple glycan branches by glycosyltransferases, as required forthe generation of complex N-glycans—the most prevalent anddiverse forms found on mammalian cell surfaces (12–15). Non-erythroid cells from mice lacking a functional␣-mannosidase IIgene were unexpectedly found to compensate for this defect by theactivity of another␣-mannosidase defining an alternative pathway(Fig. 1 and ref. 14). In erythroid cells, glycoproteins were expressednormally at the cell surface, but their portfolio of attached carbo-hydrate structures was altered with a loss of complex N-glycanbranching concurrent with an induction of hybrid N-glycan forms.These animals exhibit a non-life-threatening dyserythropoiesis sim-ilar to human congenital dyserythropoietic anemia type II (14).We have since observed an increased morbidity of aged micelacking␣-mannosidase II and have therefore attempted todetermine whether the loss of␣-mannosidase II in some tissuesis not fully compensated for by the alternative pathway and leadsto physiologic defects among nonerythroid cell types. Ourfindings herein have revealed that␣-mannosidase II is essentialfor promoting complex N-glycan branching to varying degrees indifferent tissues and cell types and on subsets of glycoproteins.The resulting alteration of N-glycan branching provokes a sys-temic autoimmune disease, indicating that inheritance of anabnormal protein N-glycosylation pathway is an etiologic factorin the pathogenesis of autoimmunity.Materials and MethodsMice. The null allele for␣-mannosidase II (14) was bred into theC57BL兾6 genetic background for more than eight generationsbefore these studies. The mice were maintained in a restricted-access 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 visual-ized by binding to E-phytohemagglutinin lectin as previouslydescribed (14).Mass