Cell Vol 90 157 167 July 11 1997 Copyright 1997 by Cell Press Alpha Mannosidase II Deficiency Results in Dyserythropoiesis and Unveils an Alternate Pathway in Oligosaccharide Biosynthesis Daniel Chui Masayoshi Oh Eda Yung Feng Liao Krishnasamy Panneerselvam Anita Lal Kurt W Marek Hudson H Freeze Kelley W Moremen Michiko N Fukuda and Jamey D Marth Howard Hughes Medical Institute and Division of Cellular and Molecular Medicine University of California San Diego La Jolla California 92093 The Burnham Institute 10901 North Torrey Pines Road La Jolla California 92037 Department of Biochemistry University of Georgia Athens Georgia 30602 Summary Alpha mannosidase II aM II catalyzes the first committed step in the biosynthesis of complex asparaginelinked N linked oligosaccharides N glycans Genetic deficiency of aM II should abolish complex N glycan production as reportedly does inhibition of aM II by swainsonine We find that mice lacking a functional aM II gene develop a dyserythropoietic anemia concurrent with loss of erythrocyte complex N glycans Unexpectedly nonerythroid cell types continued to produce complex N glycans by an alternate pathway comprising a distinct a mannosidase These studies reveal cell type specific variations in N linked oligosaccharide biosynthesis and an essential role for aM II in the formation of erythroid complex N glycans aM II deficiency elicits a phenotype in mice that correlates with human congenital dyserythropoietic anemia type II Introduction Vertebrate cell surfaces are covered with a diverse and dynamic repertoire of asparagine linked N linked oligosaccharides also termed N glycans indicating linkage to underlying protein Complex N glycans are the most abundant type found on the cell surface and alpha mannosidase II aM II acts as a key enzyme in their biosynthesis by catalyzing the first committed step in the conversion of hybrid to complex forms Kornfeld and Kornfeld 1985 Schachter 1991 Figure 1 In the Golgi apparatus aM II cleaves two mannose residues attached in a3 and a6 linkages from the hybrid N glycan GlcNAc1Man5 GlcNAc2 Asn thereby producing a processed hybrid that is also the specific substrate of GlcNAc TII Harpaz and Schachter 1980 Tulsiani et al 1982 Moremen et al 1994 Figure 1 This role is further supported from studies of the Ric15 BHK cell line in which a reduction in aM II activity occurs with an attenuation of complex N glycan production Hughes and Feeney 1986 An exogenous inhibitor of aM II activity known as swainsonine is found in plants of the genus Swainsona Elbein et al 1981 Tulsiani et al 1982 Ingestion by vertebrates produces a disease known as locoism biochemically similar to a mannosidosis and associated with aberrant behavior male sterility cytoplasmic vacuolation and the accumulation of hybrid N glycans in the brain and other tissues Dorling et al 1978 Colgate et al 1979 Tulisani et al 1988 In animal studies with the purified alkaloid the effects of locoweed ingestion appear to be due to swainsonine Tulsiani et al 1984 1988 However a role for aM II deficiency in these responses is not clear since swainsonine is also a potent inhibitor of the lysosomal a mannosidase which functions in N glycan catabolism Dorling et al 1980 Tulsiani et al 1982 Tulsiani and Touster 1987 Human deficiency of aM II has been reported in one case of congenital dyserythropoietic anemia CDA type II also known as HEMPAS hereditary erythroblastic multinuclearity with a positive acidified serum lysis test disease Crookston et al 1969 Fukuda et al 1990 CDA type II HEMPAS is inherited in an autosomal recessive manner with patients developing mild to severe anemia associated with splenomegaly and marrow erythroplasia in the presence of multinucleated erythroblasts reviewed in Fukuda 1993 Most patients live a normal lifespan without neurologic involvement although complications including hepatomegaly with cirrhosis hemosiderosis gallstones and diabetes frequently develop CDA type II erythrocytes commonly exhibit plasma membrane abnormalities with loss of complex N linked oligosaccharides from glycoproteins band 3 AE1 and band 4 5 GLUT1 Baines et al 1982 Scartezzini et al 1982 Fukuda et al 1984 1992 However the clinical diagnosis of CDA type II encompasses heterogenic symptoms with only one reported case thus far associated with aM II deficiency in which the patient retained only 10 of normal aM II levels with an unresolved genetic defect The biological role of aM II is further puzzling considering its pivotal position in complex N glycan production and the relatively mild phenotype of CDA type II when compared to GlcNAc TII deficiency in the next biosynthetic step Studies have linked inactivating mutations in the MGAT2 gene to human carbohydrate deficient glycoprotein syndrome CDGS type II Tan et al 1996 This autosomal recessive disease presents severe symptoms early with children exhibiting failure to thrive dysmorphic features severe mental retardation and susceptibilty to multiple infections Jaeken et al 1994 Charuk et al 1995 In order to understand how aMII functions in mammalian physiology and in N glycan diversification it was necessary to inactivate the aM II gene in the mouse germline We report that mice lacking a functional aM II allele develop a dyserythropoietic anemia similar to CDA type II with production of abnormal erythrocytes lacking complex N glycans Unexpectedly complex N glycan production continued among nonerythroid cell types in the absence of aM II activity by an alternate biosynthetic pathway Cell 158 Figure 1 Asparagine Linked Oligosaccharide Biosynthesis The depicted oligosaccharide precursor is transferred from lipid to asparagines on nascent peptides in the endoplasmic reticulum prior to glucosidase and a1 2 mannosidase trimming In the medial Golgi Mgat1 encoded GlcNAc TI initiates hybrid N glycan synthesis Conversion to complex N glycans requires a mannosidase II to generate the processed hybrid N glycan that is also the substrate of GlcNAc TII Anomeric linkage types are denoted Closed triangles represent glucose open circles mannose closed squares N acetylglucosamine Addition of fucose open triangle may occur earlier than indicated Multi antennary complex N glycans result from other branching reactions arrows Results aM II Gene Inactivation in Embryonic Stem Cells and Mice aM II is a type II transmembrane protein of 1150 amino acids encoded by a single gene locus in studied vertebrates Moremen and Robbins 1991 Moremen et al