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Mannosidosis: Assignment of the Lysosomal A-Mannosidase B Gene to Chromosome 19 in Man (Gene Mapping/Cell Hybrids/Inherited Storage Disease) M

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Mannosidosis: Assignment of the Lysosomal A-Mannosidase B Gene to Chromosome 19 in Man (Gene Mapping/Cell Hybrids/Inherited Storage Disease) M Proc. Nati. Acad. Sci. USA Vol. 74, No. 7, pp. 2968-2972, July 1977 Genetics Mannosidosis: Assignment of the lysosomal a-mannosidase B gene to chromosome 19 in man (gene mapping/cell hybrids/inherited storage disease) M. J. CHAMPION AND T. B. SHOWS Biochemical Genetics Section, Roswell Park Memorial Institute, New York State Department of Health, Buffalo, New York 14263 Communicated by James V. Neel, April 28, 1977 ABSTRACT Human a-mannosidase activity (a-D-mannos- genetic control (12). Residual acidic a-mannosidase activity, ide mannohydrolase, EC 3.2.1.24) from tissues and cultured skin purified from mannosidosis tissues, shows abnormal metal ion fibroblasts was separated by gel electrophoresis into a neutral, cytoplasmic form (a-mannosidase A) and two closely related activation (13), altered thermostability, and increased Km (14), acidic, lysosomal components (a-mannosidase B). Human demonstrating a structural change in the enzyme. Such a mannosidosis, an inherited glycoprotein storage disorder, has structural change implicates a mutation in a structural gene been associated with severe deficiency of both lysosomal a- whose product is common to both forms of acidic a-mannos- mannosidase B molecular forms. Chromosome assignment of idase. A similar deficiency of acidic a-mannosidase has been the gene coding for human a-mannosidase B (MANB) has been seen in inherited mucolipodisis II, but this disorder, in contrast, determined in human-mouse and human-Chinese hamster somatic cell hybrids. The human a-mannosidase B phenotype appears to result from a defect in enzyme processing (15). showed concordant segregation with the human-enzyme glu- Human-rodent somatic cell hybridization has been used to cosephosphate isomerase (GPI) (D-glucose-6-phosp ate ketol- investigate the genetic, linkage, and structural relationships of isomerase, EC 5.3.1.9) but discordant segregation with 30 other several acid hydrolases involved in inherited lysosomal storage enzyme markers representing 20 linkage groups. The glucose- diseases (16, 17). We have investigated the expression of human phosphate isomerase gene has been assigned to chromosome a-mannosidase in somatic cell hybrids to assign the gene coding 19 in man. This MANB-GPI linkage and confirming chromo- some studies demonstrate assignment of the a-mannosidase B for the lysosomal a-mannosidase associated with mannosidosis structural gene to chromosome 19 in man. Since mannosidosis to a specific chromosome and to determine the genetic and is believed to result from a structural defect in a-mannosidase possible structural relationships of the a-mannosidase molecular B, these findings suggest that the mannosidosis mutation is lo- forms. A gel electrophoretic procedure was developed to sep- cated on chromosome 19 in man. arate human lysosomal a-mannosidase (MANB) in human- mouse and human-Chinese hamster somatic cell hybrids. Ev- Mannosidosis is a rare lysosomal storage disease associated with idence is reported for the linkage of the MANB gene to the gene deficient activity for the acid hydrolase a-mannosidase (a- coding for human glucosephosphate isomerase (GPI) (D-glu- D-mannoside mannohydrolase, EC 3.2.1.24). Clinical features cose-6-phosphate ketolisomerase, EC 5.3.1.9) and the assign- of this disorder include early onset, mild physical deformities, ment of the MANE structural locus to human chromosome 19. progressive mental and psychomotor retardation, and death These findings, together with previous biochemical evidence in early childhood (1, 2). Affected children show less than 10% demonstrating that the enzyme defect in mannosidosis is a a-mannosidase activity, leading to impaired glycoprotein ca- structural mutation of MANB, suggest that the mannosidosis tabolism and massive accumulations in nervous tissues and defect exists at the MANE locus assigned to chromosome 19. urinary excretion of mannose-rich oligosaccharides (3, 4). The genetics of this disorder remain unclear, although existing ev- MATERIALS AND METHODS idence suggests an autosomal mode of inheritance. The enzyme defect of mannosidosis in humans has been Human and Rodent Parental Cells. Human cells for fusion identified in tissues and cultured fibroblasts as deficient activity were GM 1006 fibroblasts (mucolipidosis II), JoVa fibroblasts, for acidic a-mannosidase (MANB), while neutral a-mannosidase DUV fibroblasts, AnLy fibroblasts, SH 421 fibroblasts, AlTr (MANA) is unaffected (5, 6). Acidic and neutral forms of a- leukocytes, PeLa leukocytes, and CaVa leukocytes (15, 17-20). mannosidase have been separated by DEAE-cellulose chro- Human mannosidosis fibroblasts (GM 654) and mucolipidosis matography (5) and cellulose acetate electrophoresis (7). The II fibroblasts (GM 1006) were obtained from the Human Ge- acidic MANB activity (optimum pH 4.0-4.5) consists of two netic Mutant Cell Repository, Camden, NJ. The human fi- closely related molecular forms which show similar thermo- broblasts were maintained on Eagle's basal medium (diploid) stability, enzyme kinetics, and molecular weight, while the (GIBCO), 10% fetal calf serum, and antibiotics. Rodent parental single neutral MANA enzyme (optimum pH 6.0-6.5) is ther- lines with selected markers were mouse A9, LM/TK-, and molabile and enzymatically distinct (8-10). The two acidic RAG; and Chinese hamster lines were CHW-1 102 and A3 a-mannosidase enzymes are distributed in the lysosome while (17). the neutral form shows highest activity in the cytoplasm (8, 9). Human-Rodent Cell Hybrids. Human and rodent cells were The two purified acidic, lysosomal forms interconvert sponta- fused in suspension or as monolayers with inactivated Sendai neously (9, 10) or with neuraminidase treatment (11); they are virus, and hybrid cells were cloned and maintained on HAT immunologically identical (12) and appear to be structurally (hypoxanthine/aminopterin/thymidine) selection medium and genetically interrelated. Neutral a-mannosidase activity consisting of Dulbecco's modified Eagle's medium (GIBCO), is immunologically distinct and is probably under separate HAT, 10% fetal calf serum, and antibiotics (18). Primary hybrid clones established from 14 separate fusion experiments with 8 Abbreviations: MANA and MANB, neutral and acidic a-mannosidase, different human parental cells were analyzed. Hybrid sets respectively; GPI, glucosephosphate isomerase. consisted of the human-mouse hybrids DUA (DUV X A9), ICL 2968 Downloaded by guest on September 28, 2021 Genetics: Champion and Shows Proc. Natl. Acad. Sci. USA 74 (1977) 2969 (GM 1006 X LM/TK-), and ICR (GM 1006 X RAG) qnd human-Chinese hamster hybrids ATC (AlTr)X CHW-116i), MANA- PLA (PeLa X A3), PLC (PeLa X CHW-1102), JVA (JoVa X A3), and JVC (JoVa X CHW-1102). Additional human-mouse primary hybrid lines were REX, RAS, ATR, ALR, ALA, and JWR hybrids (17-20). a-Mannosidase Electrophoresis. Cell homogenates from |-Origin confluent monolayers were prepared in 0.05 M Tris-HCl buffer |-Origin (pH 7.5) at concentrations of 0.75 to 1.0 X 108 cells per ml (21). Supernatant fractions of cell homogenates were examined by MANB - vertical starch-gel electrophoresis (Buchler Instruments). Electrophoresis of acidic a-mannosidase (MANB) was accom- plished in 15% starch gels (Connaught Laboratories) for 17 hr 1 2 3 4 5 6 7 8 at 120 V with a bridge buffer of 0.2 M sodium phosphate (pH FIG. 1. Starch-gel electrophoretic pattern of a-mannosidase 5.0) and a gel buffer consisting of a 1/20 dilution of this. Neutral cytoplasmic (MANA) and lysosomal (MANg) components stained a-mannosidase (MANA) was examined in 12% starch gels with the fluorescent substrate 4-methyllumbelliferyl a-mannopyra- noside. (1) Human liver; (2) human kidney; (3) cultured human fi- (Electrostarch Co.) with a Tris-citrate (pH 7.0) buffer system broblasts, WI-38; (4) human mannosidosis fibroblasts, GM 654; (5) as described (22). Acidic MANB activity was observed in a 0.1 human mucolipidosis II fibroblasts, GM 1006; (6) mannosidosis cell M-phosphate/citric acid pH 5.4 buffer and neutral MANA in extracts treated with neuraminidase; (7) mucolipidosis II cell extracts 0.01 M Tris-citrate (pH 7.0) buffer with 2 mM fluorescent 4- treated with neuraminidase; and (8) cultured mouse cells, LM/ methylumbelliferyl-a-D-mannopyranoside (Koch-Light) as TK-. substrate (6, 7). Human a-mannosidase has been reported to and mouse (26) tissues. The fast and slow components of MANB show activity towards this synthetic substrate similar to that with appear to correspond to peaks A and B, respectively, demon- naturally occurring mannose-rich oligosaccharides (10, 23). strated by DEAE-cellulose chromatography of human tissue a-Mannosidase Terminology. a-Mannosidase A (MANA) extracts (9-11). Neuraminidase treatment of liver and fibroblast has previously been referred to as the neutral or cytoplasmic extracts resulted in a partial conversion of the slow MAN' band enzyme and is identical to peak C separated by DEAE-cellulose to the fast form, consistent with previous reports on the inter- column chromatography (5, 7-11). a-Mannosidase B (MANB) conversion of these forms (11). Homologous neutral and acidic is the acidic or lysosomal enzyme and is composed of peaks A forms of a-mannosidase were resolved in extracts of mouse and and B demonstrated by chromatography on DEAE-cellulose Chinese hamster tissues and cultured cells with these same (5, 7-11). The MANA and MANB terminology for these enzyme electrophoretic systems (Fig. 1, channel 8). forms has been adopted to agree with previous terminology for Extracts from skin fibroblasts
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