Hum Genet (1992) 88:295-297
9 Springer-Verlag1992
Chromosomal localization of the human glycoasparaginase gene to 4q32-q33 Christine Morris 1, Nora Heisterkamp 2, John Groffen z, Julian C. Williams 3, and Ilkka Mononen 3. 1Cytogenetic and Molecular Oncology Unit, Department of Pathology, The Chirstchurch School of Medicine, Christchurch Hospital, Christchurch, New Zealand 2Section of Molecular Diagnosis, Department of Pathology, and 3Division of Medical Genetics, Children's Hospital of Los Angeles, Los Angeles, California, USA Received April 9, 1991
Summary. Glycoasparaginase cleaves the N-glycosidic linkage between asparagine and N-acetylglucosamine in the degradation of glycoproteins. In humans, a deficient activity of glycoasparaginase results in accumulation of glycoasparagines, causing the lysosomal storage disease aspartylglyeosaminuria. Recombinant plasmid containing the cDNA insert encoding human glycoasparaginase was used to localize the enzyme to chromosome 4q32-q33 by in situ hybridization to metaphase chromosomes prepared from normal human lymphocytes.
Introduction Glycoasparaginase (aspartylglucosaminidase, aspartylglycosylaminase, N4-([3-N-acetylglucosaminyl)-L-asparaginase, EC 3.5.1.26) is a hydrolytic enzyme that cleaves the linkage between asparagine and N-acetylglucosamine in the degradation of N-glycosidic glycoproteins (Beaudet and Thomas 1989; Kaartinen et al. 1991). A deficient activity of glycoasparaginase in humans results in the lysosomal storage disease aspartylglycosaminuria (AGU; McKusick 20840), which is an autosomal recessive inherited disorder of glycoprotein catabolism. Characteristically, a series of glycoasparagines with aspartylglucosamine (2-acetamido-l-L-[3-aspartamido-l,2-dideoxy-[3-Dglucose; GlcNAcAsn) as the main metabolite accumulate in tissues and body fluids (Beaudet and Thomas 1989). Patients affected with the disease suffer from severe psychomotor retardation, mild skeletal deformation, and coarse facial features. They are diagnosed by detection of 50- to 1,000-fold elevated excretion of aspartylglucosamine in urine (T. Mononen et al. 1986) and a deficiency of glycoasparaginase activity in leukocytes and fibroblasts (Aula et al. 1982; I. Mononen et al. 1988). To date, aspartylglycosaminuria is the most common dis* Present address and address for offprint requests: Department of Clinical Chemistry, Kuopio University Central Hospital, SF-70210 Kuopio, Finland
order of glycoprotein metabolism (Beaudet and Thomas 1989). Its prevalence in Finland (T. Mononen et al. 1991) is comparable to the incidence of the most common lysosomal storage diseases, Gaucher disease type I (Barranger and Ginns 1989) and Tay-Sachs disease (Sandhoff et al. 1989) among Ashkenazi Jews. To facilitate molecular studies of aspartylglycosaminuria, glycoasparaginase was isolated from human leukocytes (Kaartinen et al. 1991). It was characterized as a heterotetrameric protein of 88 kDaltons (kDa) composed of two light chains (19 kDa) and two heavy chains (25 kDa). Amino acid sequence data covering about 80% of the total protein sequence of glycoasparaginase was determined, a cDNA clone for the coding region of the enzyme was isolated and its nucleotide sequence determined (I. Mononen et al. 1991). The enzyme is synthesized as a single polypeptide chain that is posttranslationally cleaved to generate glycosylated subunits (I. Mononen et al. 1991; Fisher et al. 1990). The structural gene of glycoasparaginase was assigned to the long arm of chromosome 4 (4q21-4qter) by somatic cell hybridization (Aula et al. 1984). Recently it has been tentatively linked to the fibrinogen locus at 4q26-q28 and to that of the MNS blood group at 4q28-31 by linkage analysis (Gr6n et al. 1990). We now report the localization of the structural gene for glycoasparaginase to human chromosome 4q32-q33 by in situ hybridization to metaphase chromosomes prepared from normal human lymphocytes.
Materials and methods S o u t h e r n blot analysis
High-molecular-weight DNA isolated from blood cells of an individual not affected by aspartylglycosaminuria was digested with EcoRI and BgIII and subjected to electrophoresis through 0.6% agarose gels for 2-3 days at 5-18mA. After blotting to nitrocellulose, filters were hybridized to a 1-kb glycoasparaginase probe containing the coding region of the enzyme (I. Mononen et al. 1991) labeled by primer extension.
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Fig. 1. Combined data from two experiments showing localization of grains on 200 karyotypically normal, 46,XY,metaphase cells after in situ hybridization of the glycoasparaginase cDNA probe. The amount and distribution of grains on chromosome 4 are shown to the left
Chromosome preparations Metaphase cells were prepared from heparinized peripheral blood of two karyotypically normal male donors. Whole blood (0.5 ml) was cultured for 72 h at 37~ in Hams F10 with added phytohemagglutinin and harvested using standard colchicine, hypotonic, and fixation procedures.
Chromosome in situ hybridization A 1-kb cDNA clone containing the coding sequence of glycoasparaginase (I. Mononen et al. 1991) was 3H-labeled by nick translation to a specific activity of 1 • 10 7 tO 2 • 10 7 dprrdgg and hybridized in situ to metaphase chromosomes as previously described (Morris and Fitzgerald 1987; Morris et al. 1990). The location of all silver grains touching chromosomes was recorded from consecutive metaphase cells with good banding and minimal overlaps to determine the pattern of hybridization.
Results and discussion Southern blot analysis using the c D N A p r o b e and BglIIdigested h u m a n D N A of unaffected individuals gave bands of approximately 5, 8 and 9 kb (data not shown). Similarly, EcoRI hybridizing fragments of approximately 1, 3, and 6.5 kb were seen. We expected a minimum of two hybridizing EcoRI fragments, since the c D N A con-
tains an EcoRI site (I. Mononen et al. 1991). The relative intensity of the hybridizing bands remained the same upon successive more stringent posthybridization washings. In conclusion, the data strongly suggested that after m o d e r a t e posthybridization washing coditions (1 x saline citrate; 65~ only the gene for glycoasparaginase was detected. To define the chromosomal location of the structural gene of glycoasparaginase, a tritiated c D N A probe containing the coding region of the enzyme was hybridized in situ to normal metaphase (46,XY) cells prepared from peripheral blood lymphocytes of two male donors. Grains on 100 metaphase cells were scored from each donor and their distribution is shown in Fig. 1. In both experiments, the same specific labeling of chromosome 4 was observed. O f a combined total of 311 grains scored from 200 metaphases, 47 grains (15%) were located on c h r o m o s o m e 4 confirming the previous location of glycoasparaginase. Of these 47, 34 grains (11% of the total and 72% of those on chromosome 4) labeled across the region 4 @ 1 - 3 5 (Figs. 1, 2). The modal n u m b e r of grains was at band q33 on chromosomes 4, and we estimate that the structural gene of glycoasparaginase lies within this band, or in the distal part of band q32 (Fig. 1). The assigned site (q32-33) contained 20 grains or 6.4% of the total grains. The localization of grains over chromosomes 4 from 12 different metaphases is shown in Fig. 2. A previous report of Aula et al. (1984) localized glycoasparaginase activity to the long arm of c h r o m o s o m e 4 (4q21-4qter) using h u m a n - m o u s e somatic cell hybrids. Recently, a tentative linkage of glycoasparaginase gene to the fibrinogen locus at 4q26-q28 and to that of the MNS blood group at 4q28-q31 has been reported (Gr/3n
297
Fig. 2. Chromosomes 4 from 12 different metaphase cells showing labeling on the terminal long arm in bands 4q31-q35
et al. 1990). T h e s e studies a r e in a c c o r d a n c e w i t h t h e results o f t h e p r e s e n t s t u d y , w h i c h l o c a t e t h e s t r u c t u r a l gene for g l y c o a s p a r a g i n a s e distal to the M N S b l o o d g r o u p locus on c h r o m o s o m e 4 a n d refine t h e g e n e l o c a t i o n to b a n d q33 o r to the distal p a r t o f q32. T o d a t e , t h e g e n e s for f a c t o r X I ( C o x et al. 1989) a n d f a c i o s c a p u l o h u m e r a l m u s c u l a r d y s t r o p h y ( W i j m e n g a et al. 1991) a r e the o n l y g e n e s s h o w n to m a p distal to g l y c o a s p a r a g i n a s e o n t h e l o n g a r m o f c h r o m o s o m e 4.
References Aula P, Autio S, Raivio KO, Rapola J (1982) Aspartylglycosaminuria. In: Durand P, O'Brien JS (eds) Genetic errors of glycoprotein metabolism. Edi-Ermes, Milan; Springer, Berlin Heidelberg New York, pp 123-152 Aula P, Astrin KH, Francke U, Desnick RJ (1984) Assignment of the structural gene encoding aspartylglucosaminidase to the long arm of chromosome 4 (4q21-4qter). Am J Hum Genet 36:1215-1224 Barranger JA, Ginns EI (1989) Glucosylceramide lipidosis: Gaucher disease. In: Scriver CR, Beaudet AL, Sly WS, Valle D (eds) The metabolic basis of inherited disease, 6th edn. McGrawHill, New York, pp 1677-1698 Beaudet AL, Thomas GH (1989) Disorders of glycoprotein degradation: mannosidosis, fucosidosis, sialidosis, and aspartylglycosaminuria. In: Scriver CR, Beaudet AL, Sly WS, Valle D (eds) The metabolic basis of inherited disease, 6th edn. McGrawHill, New York, pp 1603-1622
Cox DR, Murray JE, Buetow KH (1989) Report of the committee on the genetic constitution of chromosome 4. Cytogenet Cell Genet 51:121-136 Fisher K, Tollersrud OK, Aronson NN (1990) Cloning and sequence analysis of a cDNA for human glycosylasparaginase. FEBS Lett 269:440-444 Gr6n K, Aula P, Peltonen L (1990) Linkage of aspartylglucosaminuria (AGU) to marker loci on the long arm of chromosome 4. Hum Genet 85 : 233-236 Kaartinen V, Williams J, Tomich J, Yates J, Hood L, Mononen I (1991) Glycoasparaginase from human leukocytes: inactivation and covalent modification with diazo-oxonorvaline. J Biol Chem 266 : 5860-5869 Mononen I, Kaartinen V, Mononen T (1988) Laboratory detection of aspartylglycosaminuria. Scand J Clin Lab Invest [Suppl 191] :7-11 Mononen I, Heisterkamp N, Kaartinen V, Williams J, Yates JR, Griffin PR, Hood LE, Groffen J (1991) Aspartylglycosamiuria in the Finnish population: characterization of two point mutations in the heavy chain of glycoasparaginase. Proc Natl Acad Sci USA 88 : 2941-2945 Mononen T, Parviainen M, Penttilfi I, Mononen I (1986) Liquidchromatographic detection of aspartylglycosaminuria. Clin Chem 32 : 501-502 Mononen T, Mononen I, Matilainen R, Airaksinen E (1991) High prevalence of aspartylglycosaminuria among school age children in eastern Finland. Hum Genet 87 : 266-268 Morris CM, Fitzgerald PH (1987) Complexity of an apparently simple variant Ph translocation in chronic myeloid leukemia. Leuk Res 11 : 163-169 Morris C, Heisterkamp N, Hao QL, Testa JR, Groffen J (1990) The human tyrosine kinase gene (FER) maps to chromosome 5 and is deleted in myeloid leukemias with a del(5q). Cytogenet Cell Genet 53 : 196-200 Sandhoff K, Conzelmann E, Neufeld EF, Kaback MM, Suzuki K (1989) The GM2 gangliosidosis. In: Scriver CR, Beaudet AL, Sly WS, Valle D (eds) The metabolic basis of inherited disease, 6th edn. McGraw-Hill, New York, pp 1807-1842 Wijmenga C, Padberg GW, Moerer P, Wiegant J, Liem L, Brouwer OF, Milner ECB, Weber JL, Ommen GB van, Sandkuyl LA, Frants RR (1991) Mapping of facioscapulohumeral muscular dystrophy gene to chromosome 4q35-qter by multipoint linkage analysis and in situ hybridization. Genomics 9 : 570-575
9 Springer-Verlag1992
Chromosomal localization of the human glycoasparaginase gene to 4q32-q33 Christine Morris 1, Nora Heisterkamp 2, John Groffen z, Julian C. Williams 3, and Ilkka Mononen 3. 1Cytogenetic and Molecular Oncology Unit, Department of Pathology, The Chirstchurch School of Medicine, Christchurch Hospital, Christchurch, New Zealand 2Section of Molecular Diagnosis, Department of Pathology, and 3Division of Medical Genetics, Children's Hospital of Los Angeles, Los Angeles, California, USA Received April 9, 1991
Summary. Glycoasparaginase cleaves the N-glycosidic linkage between asparagine and N-acetylglucosamine in the degradation of glycoproteins. In humans, a deficient activity of glycoasparaginase results in accumulation of glycoasparagines, causing the lysosomal storage disease aspartylglyeosaminuria. Recombinant plasmid containing the cDNA insert encoding human glycoasparaginase was used to localize the enzyme to chromosome 4q32-q33 by in situ hybridization to metaphase chromosomes prepared from normal human lymphocytes.
Introduction Glycoasparaginase (aspartylglucosaminidase, aspartylglycosylaminase, N4-([3-N-acetylglucosaminyl)-L-asparaginase, EC 3.5.1.26) is a hydrolytic enzyme that cleaves the linkage between asparagine and N-acetylglucosamine in the degradation of N-glycosidic glycoproteins (Beaudet and Thomas 1989; Kaartinen et al. 1991). A deficient activity of glycoasparaginase in humans results in the lysosomal storage disease aspartylglycosaminuria (AGU; McKusick 20840), which is an autosomal recessive inherited disorder of glycoprotein catabolism. Characteristically, a series of glycoasparagines with aspartylglucosamine (2-acetamido-l-L-[3-aspartamido-l,2-dideoxy-[3-Dglucose; GlcNAcAsn) as the main metabolite accumulate in tissues and body fluids (Beaudet and Thomas 1989). Patients affected with the disease suffer from severe psychomotor retardation, mild skeletal deformation, and coarse facial features. They are diagnosed by detection of 50- to 1,000-fold elevated excretion of aspartylglucosamine in urine (T. Mononen et al. 1986) and a deficiency of glycoasparaginase activity in leukocytes and fibroblasts (Aula et al. 1982; I. Mononen et al. 1988). To date, aspartylglycosaminuria is the most common dis* Present address and address for offprint requests: Department of Clinical Chemistry, Kuopio University Central Hospital, SF-70210 Kuopio, Finland
order of glycoprotein metabolism (Beaudet and Thomas 1989). Its prevalence in Finland (T. Mononen et al. 1991) is comparable to the incidence of the most common lysosomal storage diseases, Gaucher disease type I (Barranger and Ginns 1989) and Tay-Sachs disease (Sandhoff et al. 1989) among Ashkenazi Jews. To facilitate molecular studies of aspartylglycosaminuria, glycoasparaginase was isolated from human leukocytes (Kaartinen et al. 1991). It was characterized as a heterotetrameric protein of 88 kDaltons (kDa) composed of two light chains (19 kDa) and two heavy chains (25 kDa). Amino acid sequence data covering about 80% of the total protein sequence of glycoasparaginase was determined, a cDNA clone for the coding region of the enzyme was isolated and its nucleotide sequence determined (I. Mononen et al. 1991). The enzyme is synthesized as a single polypeptide chain that is posttranslationally cleaved to generate glycosylated subunits (I. Mononen et al. 1991; Fisher et al. 1990). The structural gene of glycoasparaginase was assigned to the long arm of chromosome 4 (4q21-4qter) by somatic cell hybridization (Aula et al. 1984). Recently it has been tentatively linked to the fibrinogen locus at 4q26-q28 and to that of the MNS blood group at 4q28-31 by linkage analysis (Gr6n et al. 1990). We now report the localization of the structural gene for glycoasparaginase to human chromosome 4q32-q33 by in situ hybridization to metaphase chromosomes prepared from normal human lymphocytes.
Materials and methods S o u t h e r n blot analysis
High-molecular-weight DNA isolated from blood cells of an individual not affected by aspartylglycosaminuria was digested with EcoRI and BgIII and subjected to electrophoresis through 0.6% agarose gels for 2-3 days at 5-18mA. After blotting to nitrocellulose, filters were hybridized to a 1-kb glycoasparaginase probe containing the coding region of the enzyme (I. Mononen et al. 1991) labeled by primer extension.
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Fig. 1. Combined data from two experiments showing localization of grains on 200 karyotypically normal, 46,XY,metaphase cells after in situ hybridization of the glycoasparaginase cDNA probe. The amount and distribution of grains on chromosome 4 are shown to the left
Chromosome preparations Metaphase cells were prepared from heparinized peripheral blood of two karyotypically normal male donors. Whole blood (0.5 ml) was cultured for 72 h at 37~ in Hams F10 with added phytohemagglutinin and harvested using standard colchicine, hypotonic, and fixation procedures.
Chromosome in situ hybridization A 1-kb cDNA clone containing the coding sequence of glycoasparaginase (I. Mononen et al. 1991) was 3H-labeled by nick translation to a specific activity of 1 • 10 7 tO 2 • 10 7 dprrdgg and hybridized in situ to metaphase chromosomes as previously described (Morris and Fitzgerald 1987; Morris et al. 1990). The location of all silver grains touching chromosomes was recorded from consecutive metaphase cells with good banding and minimal overlaps to determine the pattern of hybridization.
Results and discussion Southern blot analysis using the c D N A p r o b e and BglIIdigested h u m a n D N A of unaffected individuals gave bands of approximately 5, 8 and 9 kb (data not shown). Similarly, EcoRI hybridizing fragments of approximately 1, 3, and 6.5 kb were seen. We expected a minimum of two hybridizing EcoRI fragments, since the c D N A con-
tains an EcoRI site (I. Mononen et al. 1991). The relative intensity of the hybridizing bands remained the same upon successive more stringent posthybridization washings. In conclusion, the data strongly suggested that after m o d e r a t e posthybridization washing coditions (1 x saline citrate; 65~ only the gene for glycoasparaginase was detected. To define the chromosomal location of the structural gene of glycoasparaginase, a tritiated c D N A probe containing the coding region of the enzyme was hybridized in situ to normal metaphase (46,XY) cells prepared from peripheral blood lymphocytes of two male donors. Grains on 100 metaphase cells were scored from each donor and their distribution is shown in Fig. 1. In both experiments, the same specific labeling of chromosome 4 was observed. O f a combined total of 311 grains scored from 200 metaphases, 47 grains (15%) were located on c h r o m o s o m e 4 confirming the previous location of glycoasparaginase. Of these 47, 34 grains (11% of the total and 72% of those on chromosome 4) labeled across the region 4 @ 1 - 3 5 (Figs. 1, 2). The modal n u m b e r of grains was at band q33 on chromosomes 4, and we estimate that the structural gene of glycoasparaginase lies within this band, or in the distal part of band q32 (Fig. 1). The assigned site (q32-33) contained 20 grains or 6.4% of the total grains. The localization of grains over chromosomes 4 from 12 different metaphases is shown in Fig. 2. A previous report of Aula et al. (1984) localized glycoasparaginase activity to the long arm of c h r o m o s o m e 4 (4q21-4qter) using h u m a n - m o u s e somatic cell hybrids. Recently, a tentative linkage of glycoasparaginase gene to the fibrinogen locus at 4q26-q28 and to that of the MNS blood group at 4q28-q31 has been reported (Gr/3n
297
Fig. 2. Chromosomes 4 from 12 different metaphase cells showing labeling on the terminal long arm in bands 4q31-q35
et al. 1990). T h e s e studies a r e in a c c o r d a n c e w i t h t h e results o f t h e p r e s e n t s t u d y , w h i c h l o c a t e t h e s t r u c t u r a l gene for g l y c o a s p a r a g i n a s e distal to the M N S b l o o d g r o u p locus on c h r o m o s o m e 4 a n d refine t h e g e n e l o c a t i o n to b a n d q33 o r to the distal p a r t o f q32. T o d a t e , t h e g e n e s for f a c t o r X I ( C o x et al. 1989) a n d f a c i o s c a p u l o h u m e r a l m u s c u l a r d y s t r o p h y ( W i j m e n g a et al. 1991) a r e the o n l y g e n e s s h o w n to m a p distal to g l y c o a s p a r a g i n a s e o n t h e l o n g a r m o f c h r o m o s o m e 4.
References Aula P, Autio S, Raivio KO, Rapola J (1982) Aspartylglycosaminuria. In: Durand P, O'Brien JS (eds) Genetic errors of glycoprotein metabolism. Edi-Ermes, Milan; Springer, Berlin Heidelberg New York, pp 123-152 Aula P, Astrin KH, Francke U, Desnick RJ (1984) Assignment of the structural gene encoding aspartylglucosaminidase to the long arm of chromosome 4 (4q21-4qter). Am J Hum Genet 36:1215-1224 Barranger JA, Ginns EI (1989) Glucosylceramide lipidosis: Gaucher disease. In: Scriver CR, Beaudet AL, Sly WS, Valle D (eds) The metabolic basis of inherited disease, 6th edn. McGrawHill, New York, pp 1677-1698 Beaudet AL, Thomas GH (1989) Disorders of glycoprotein degradation: mannosidosis, fucosidosis, sialidosis, and aspartylglycosaminuria. In: Scriver CR, Beaudet AL, Sly WS, Valle D (eds) The metabolic basis of inherited disease, 6th edn. McGrawHill, New York, pp 1603-1622
Cox DR, Murray JE, Buetow KH (1989) Report of the committee on the genetic constitution of chromosome 4. Cytogenet Cell Genet 51:121-136 Fisher K, Tollersrud OK, Aronson NN (1990) Cloning and sequence analysis of a cDNA for human glycosylasparaginase. FEBS Lett 269:440-444 Gr6n K, Aula P, Peltonen L (1990) Linkage of aspartylglucosaminuria (AGU) to marker loci on the long arm of chromosome 4. Hum Genet 85 : 233-236 Kaartinen V, Williams J, Tomich J, Yates J, Hood L, Mononen I (1991) Glycoasparaginase from human leukocytes: inactivation and covalent modification with diazo-oxonorvaline. J Biol Chem 266 : 5860-5869 Mononen I, Kaartinen V, Mononen T (1988) Laboratory detection of aspartylglycosaminuria. Scand J Clin Lab Invest [Suppl 191] :7-11 Mononen I, Heisterkamp N, Kaartinen V, Williams J, Yates JR, Griffin PR, Hood LE, Groffen J (1991) Aspartylglycosamiuria in the Finnish population: characterization of two point mutations in the heavy chain of glycoasparaginase. Proc Natl Acad Sci USA 88 : 2941-2945 Mononen T, Parviainen M, Penttilfi I, Mononen I (1986) Liquidchromatographic detection of aspartylglycosaminuria. Clin Chem 32 : 501-502 Mononen T, Mononen I, Matilainen R, Airaksinen E (1991) High prevalence of aspartylglycosaminuria among school age children in eastern Finland. Hum Genet 87 : 266-268 Morris CM, Fitzgerald PH (1987) Complexity of an apparently simple variant Ph translocation in chronic myeloid leukemia. Leuk Res 11 : 163-169 Morris C, Heisterkamp N, Hao QL, Testa JR, Groffen J (1990) The human tyrosine kinase gene (FER) maps to chromosome 5 and is deleted in myeloid leukemias with a del(5q). Cytogenet Cell Genet 53 : 196-200 Sandhoff K, Conzelmann E, Neufeld EF, Kaback MM, Suzuki K (1989) The GM2 gangliosidosis. In: Scriver CR, Beaudet AL, Sly WS, Valle D (eds) The metabolic basis of inherited disease, 6th edn. McGraw-Hill, New York, pp 1807-1842 Wijmenga C, Padberg GW, Moerer P, Wiegant J, Liem L, Brouwer OF, Milner ECB, Weber JL, Ommen GB van, Sandkuyl LA, Frants RR (1991) Mapping of facioscapulohumeral muscular dystrophy gene to chromosome 4q35-qter by multipoint linkage analysis and in situ hybridization. Genomics 9 : 570-575
