Hum Genet (1992) 88:295-297

�9 Springer-Verlag 1992

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 2 Section of Molecular Diagnosis, Department of Pathology, and 3 Division 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 contain- ing the cDNA insert encoding human glycoasparaginase was used to localize the enzyme to chromosome 4q32-q33 by in situ hybridization to metaphase chromosomes pre- pared from normal human lymphocytes.

Introduction

Glycoasparaginase (aspartylglucosaminidase, aspartyl- glycosylaminase, N4-([3-N-acetylglucosaminyl)-L-aspara- ginase, 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 ac- tivity of glycoasparaginase in humans results in the lyso- somal storage disease aspartylglycosaminuria (AGU; McKusick 20840), which is an autosomal recessive inher- ited disorder of glycoprotein catabolism. Characteristi- cally, a series of glycoasparagines with aspartylglucos- amine (2-acetamido-l-L-[3-aspartamido-l,2-dideoxy-[3-D- glucose; GlcNAcAsn) as the main metabolite accumu- late in tissues and body fluids (Beaudet and Thomas 1989). Patients affected with the disease suffer from se- vere psychomotor retardation, mild skeletal deforma- tion, and coarse facial features. They are diagnosed by detection of 50- to 1,000-fold elevated excretion of as- partylglucosamine 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 ly- sosomal storage diseases, Gaucher disease type I (Bar- ranger and Ginns 1989) and Tay-Sachs disease (Sandhoff et al. 1989) among Ashkenazi Jews.

To facilitate molecular studies of aspartylglycosamin- uria, glycoasparaginase was isolated from human leuko- cytes (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 de- termined, a cDNA clone for the coding region of the en- zyme was isolated and its nucleotide sequence deter- mined (I. Mononen et al. 1991). The enzyme is synthe- sized as a single polypeptide chain that is posttransla- tionally 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 so- matic 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 hu- man lymphocytes.

Materials and methods

Southern blot analysis

High-molecular-weight DNA isolated from blood cells of an indi- vidual 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 nitrocel- lulose, 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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03

d Z

Chromosome no.

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 phytohemag- glutinin and harvested using standard colchicine, hypotonic, and fixation procedures.

Chromosome in situ hybridization

A 1-kb cDNA clone containing the coding sequence of glycoas- paraginase (I. Mononen et al. 1991) was 3H-labeled by nick trans- lation to a specific activity of 1 • 10 7 tO 2 • 10 7 dprrdgg and hy- bridized 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 con- secutive 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 probe and BglII- digested human 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 wash- ings. In conclusion, the data strongly suggested that after modera te posthybridization washing coditions (1 x sa- line 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 con- taining 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. Of a combined total of 311 grains scored from 200 meta- phases, 47 grains (15%) were located on chromosome 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@1-35 (Figs. 1, 2). The modal number 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 lo- calization of grains over chromosomes 4 from 12 differ- ent metaphases is shown in Fig. 2.

A previous report of Aula et al. (1984) localized glyco- asparaginase activity to the long arm of chromosome 4 (4q21-4qter) using human-mouse 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

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Fig. 2. Chromosomes 4 from 12 different metaphase cells showing labeling on the terminal long arm in bands 4q31-q35

et al. 1990). These s tudies a re in a cco rdance wi th the re- sults of the p r e sen t s tudy , which loca te the s t ruc tura l gene for g lycoasparag inase distal to the MNS b lood group locus on c h r o m o s o m e 4 and ref ine the gene loca t ion to b a n d q33 or to the d is ta l pa r t of q32. To da te , the genes for fac tor X I (Cox et al. 1989) and f a c i o s c a p u l o h u m e r a l muscu la r d y s t r o p h y ( W i j m e n g a et al. 1991) a re the on ly genes shown to m a p dis ta l to g lycoaspa rag inase on the long a rm of c h r o m o s o m e 4.

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