Cell, Vol. 90, 157–167, July 11, 1997, Copyright 1997 by Cell PressAlpha-Mannosidase-II Deficiency Results inDyserythropoiesis and Unveils an AlternatePathway in Oligosaccharide BiosynthesisDaniel Chui,* Masayoshi Oh-Eda,†Yung-Feng Liao,‡Swainsona (Elbein et al., 1981; Tulsiani et al., 1982).Krishnasamy Panneerselvam,†Anita Lal,‡Ingestion by vertebrates produces a disease known asKurt W. Marek,* Hudson H. Freeze,†“locoism,” biochemically similar to a-mannosidosis andKelley W. Moremen,‡Michiko N. Fukuda,†associated with aberrant behavior, male sterility, cyto-and Jamey D. Marth*plasmic vacuolation, and the accumulation of hybrid*Howard Hughes Medical InstituteN-glycans in the brain and other tissues (Dorling et al.,and Division of Cellular and Molecular Medicine1978; Colgate etal., 1979; Tulisaniet al.,1988). InanimalUniversity of California San Diegostudieswiththepurifiedalkaloid,theeffectsoflocoweedLa Jolla, California 92093ingestion appear to be due to swainsonine (Tulsiani et†The Burnham Instituteal., 1984, 1988). However, a role for aM-II deficiency in10901 North Torrey Pines Roadthese responses isnot clear,since swainsonine is alsoaLa Jolla, California 92037potent inhibitor ofthe lysosomal a-mannosidase, which‡Department of Biochemistryfunctions in N-glycan catabolism (Dorling et al., 1980;University of GeorgiaTulsiani et al., 1982; Tulsiani and Touster, 1987).Athens, Georgia 30602Human deficiency of aM-II has been reported in onecase of congenitaldyserythropoietic anemia (CDA) typeII, also known as HEMPAS (hereditary erythroblasticSummarymultinuclearity witha positive acidifiedserum-lysistest)disease (Crookston et al., 1969; Fukuda et al., 1990).Alpha-mannosidase-II (aM-II) catalyzes the first com-CDA typeII/HEMPAS isinherited inan autosomalreces-mitted stepin thebiosynthesisofcomplex asparagine-sive manner, with patients developing mild-to-severelinked (N-linked) oligosaccharides (N-glycans). Ge-anemia associated with splenomegaly and marrownetic deficiency of aM-II should abolish complexerythroplasia in the presence of multinucleated erythro-N-glycan production as reportedly does inhibition ofblasts (reviewed in Fukuda, 1993). Most patients live aaM-II by swainsonine. We find that mice lacking anormal lifespan without neurologic involvement, al-functional aM-II gene develop a dyserythropoieticthough complications, including hepatomegaly withcir-anemia concurrent with loss of erythrocyte complexrhosis, hemosiderosis, gallstones, and diabetes, fre-N-glycans.Unexpectedly, nonerythroidcelltypes con-quently develop. CDA type II erythrocytes commonlytinued to produce complex N-glycans by an alternateexhibit plasma membrane abnormalities with loss ofpathway comprising a distinct a-mannosidase. Thesecomplex N-linked oligosaccharides from glycoproteinsstudies reveal cell-type-specific variations in N-linkedband 3 (AE1) and band 4.5 (GLUT1) (Baines et al., 1982;oligosaccharide biosynthesis and an essential role forScartezzini et al., 1982; Fukuda et al., 1984,1992). How-aM-II in theformation oferythroid complexN-glycans.ever, the clinicaldiagnosis of CDA type II encompassesaM-II deficiencyelicits a phenotype inmice thatcorre-heterogenic symptomswithonly one reportedcasethuslateswith humancongenital dyserythropoietic anemiafarassociated withaM-II deficiency,inwhichthe patienttype II.retained only 10% of normal aM-II levels with an unre-solved genetic defect.IntroductionThe biologicalroleof aM-IIis furtherpuzzling, consid-ering itspivotalpositionincomplexN-glycanproductionVertebrate cell surfaces are covered with a diverse andand the relatively mild phenotype of CDA type II whendynamic repertoire of asparagine-linked (N-linked) oli-compared to GlcNAc-TII deficiency in the next biosyn-gosaccharides (also termed N-glycans, indicating link-thetic step. Studies have linked inactivating mutationsage to underlying protein). Complex N-glycans are thein the MGAT2 gene to human carbohydrate-deficientmost abundant type found on the cell surface, andglycoprotein syndrome (CDGS) type II(Tan et al., 1996).alpha-mannosidase-II (aM-II) acts as a key enzyme inThis autosomal recessive disease presents severetheirbiosynthesis by catalyzing thefirst committed stepsymptoms early withchildren exhibitingfailure to thrive,in the conversion of hybrid to complex forms (Kornfelddysmorphic features, severe mental retardation, andand Kornfeld, 1985; Schachter, 1991; Figure 1). In thesusceptibilty to multiple infections (Jaeken et al., 1994;Golgi apparatus, aM-II cleaves two mannose residuesCharuk et al., 1995). In order to understand how aM-attached ina3and a6linkages fromthe hybrid N-glycanII functions in mammalian physiology and in N-glycanGlcNAc1Man5GlcNAc2-Asn, thereby producing a pro-diversification, it was necessary to inactivate the aM-IIcessed hybrid that is also the specific substrate ofgene inthemouse germline. Wereportthat mice lackingGlcNAc-TII (Harpaz and Schachter, 1980; Tulsiani et al.,a functional aM-II allele develop a dyserythropoietic1982; Moremen et al., 1994; Figure 1). This roleis furtheranemia similarto CDA typeII, with productionof abnor-supported from studies of the Ric15 BHK cell line, inmal erythrocyteslackingcomplexN-glycans. Unexpect-whicha reductioninaM-IIactivity occurs withan attenu-edly, complex N-glycan production continued amongation of complex N-glycan production (Hughes andnonerythroid cell types in the absence of aM-II activityFeeney, 1986). An exogenous inhibitor of aM-II activityknown as swainsonine is found in plants of the genus by an alternate biosynthetic pathway.Cell158Figure 1. Asparagine-Linked Oligosaccharide BiosynthesisThe depicted oligosaccharide precursor is transferred from lipid to asparagines on nascent peptides in the endoplasmic reticulum prior toglucosidase and a1–2 mannosidase trimming. In the medial Golgi, Mgat1-encoded GlcNAc-TI initiates hybrid N-glycan synthesis. Conversionto complex N-glycans requires a-mannosidase-II to generatethe processed hybrid N-glycan that is also the substrate of GlcNAc-TII.Anomericlinkage types are denoted. Closed triangles represent glucose; open circles, mannose; closed squares, N-acetylglucosamine. Addition offucose (open triangle) may occur earlier than indicated. Multi-antennary complex N-glycans result from other branching reactions (arrows).Results GlcNAc1Man3GlcNAc-PA, but was absent from extractsderived from aMIID/aM-IIDmice or extracts treated withswainsonine(Figures 3A and3B;