Ser72Pro active-site disease mutation in human lysosomal aspartylglucosaminidase: abnormal intracellular processing and evidence forextracellular activation
Ser72Pro active-site disease mutation in human lysosomal aspartylglucosaminidase: abnormal intracellular processing and evidence for extracellular activationMinna Peltola, Ritva Tikkanen, Leena Peltonen and Anu Jalanko*
Department of Human Molecular Genetics, National Public Health Institute, Mannerheimintie 166, FIN-00300 Helsinki, Finland
Received January 12, 1996;Revised and Accepted March 5, 1996
Aspartylglucosaminuria (AGU) is a lysosomal storage disease caused by deficient activity of aspartylglucos- aminidase (AGA). We report here a T214C mutation leading to a Ser72Pro substitution in four Arab families. This is the first naturally occurring AGU mutation involving an active-site amino acid of this recently crystallized hydrolase and it seems to represent the second most common AGU mutation worldwide. The intracellular consequences of the Ser72Pro mutation were analyzed by transient expression in COS-1 cells and we were able to demonstrate that this active-site mutation most probably does not destroy the enzyme activity per se, but specifically prevents the proteolytic activation cleavage of AGA in the endoplasmic reticulum (ER). The mutant enzyme is, however, folded correctly enough to allow mannose-6-phosphorylation and targeting to lysosomes. The overexpressed mutant enzyme remained inactive intracellularly, but the secreted mutant precursor was proteolytically activated extracellularly, resulting in a similar subunit composition to that in the wild-type AGA in the ER. The partially activated mutant enzyme was endocytosed further by the recipient cells. These data demonstrate that the proteolytic activation of AGA can also occur extracellularly and suggest that the driving mechanism of AGA precursor cleavage is autocatalytic.
The human aspartylglucosaminidase (AGA) gene encodes a lysosomal hydrolase catalyzing one of the final steps in the degradation of N-linked glycoproteins (1 ). The wild-type AGA enzyme is synthesized as an inactive precursor polypeptide which is activated by a cleavage in the endoplasmic reticulun (ER), resulting in two subunits, 27 kDa pro-[alpha] and 17 kDa [beta] (2 ), which have only intrachain disulfide bonds and are non-covalently bound to each other. The active ([alpha][beta])2 heterotetrameric enzyme complex is transported to the lysosomes, where the final maturation occurs in the form of proteolytic processing, which removes a short peptide from the carboxy-terminus of the [alpha] subunit (2 ). The C-terminus of the [beta] subunit can also be processed resulting in 14 kDa [beta]' in the lysosomes without affecting the enzyme activity (3 ). The mature AGA enzyme is heterogeneously glyco- sylated at two N-glycosylation sites, one in each subunit, but glycosylation is not needed for the activity and just one oligosaccharide in either subunit is sufficient for lysosomal targeting via the mannose-6-phosphate receptor-mediated pathway (3 ).
Several different mutations in the AGA gene are known to cause inactivation of the AGA enzyme and lead to a lysosomal accumulation disease, aspartylglucosaminuria (AGU) (4 ). AGUis enriched in the Finnish population, where one major mutation, AGUFin major, represents 98% (5 ) and AGUFin minor 2% (6 ) of AGU alleles. AGUFin major is a double point mutation, where the disease-causing Cys163Ser substitution prevents the formation of an intrachain disulfide bond, leading to misfolding and inhibition of the proteolytic cleavage of the AGA precursor (7 ,8 ). Analyses of mutations in non-Finnish AGU patients have revealed 13 different family-specific mutations distributed over the coding region of AGA (5 ,9 -12 ). In addition to six mutations leading to premature stop codons, four missense mutations, one splice defect, one deletion and one insertion have been found. The consequences of four different AGU mutations have been analyzed by expression studies in COS cells, and in all cases the mutation has most likely caused improper folding of the precursor polypeptide and the prevention of proteolytic cleavage in the ER (7 ,10 -12 ). In each case, the abnormal protein was not targeted to lysosomes. The recent crystallization (13 ) of AGA and determination of the three-dimensional structure and the putative active-site amino acids (14 ) confirmed that none of the previously identified AGU mutations directly interfere with the amino acids involved in substrate binding or catalysis, but rather influence the packing of the polypeptide chain.
Here we report a novel AGU mutation found in four Arab families, three from the region of Jerusalem and one from Chicago. This mutation in the second exon of the AGA gene results in a Ser72Pro substitution in the AGA polypeptide and is the first reported AGU mutation involving an active-site amino acid and not disturbing the folding of AGA. It is also the first AGU mutation found in several families of non-Finnish origin. We have also analyzed an artificial Arg234Gln active-site substitution (15 ), which proved to result in a processing defect similar to Ser72Pro. We analyzed the intracellular consequences of these mutations by transient expression of mutant polypeptides in COS-1 cells and demonstrate that these interesting mutations specifically prevent the cleavage of AGA into subunits. Furthermore, partial extracellular activation of overexpressed Ser72Pro mutant AGA could be demonstrated, providing new insight into proteolytic activation of the enzyme precursor.
To identify the mutation causing aspartylglucosaminuria in three Palestinian Arab families from the region of Jerusalem and one Arab family (of Jerusalemite origin) from North America, the complete coding region of AGA was amplified by PCR from genomic DNA by intron-specific primers. The nucleotide sequences of each exon and the exon-intron boundaries were determined by solid-phase sequencing (11 ,16 ) and, interestingly, all the 12 patients were found to be homozygous for a T214C mutation in the second exon of AGA leading to a Ser72Pro substitution in the AGA polypeptide (Fig. 1 ). No other mutations were detected. The T214C mutation was also confirmed by sequencing the PCR product from the other strand. When other members of the four families were analyzed, the parents of each were carriers of this mutation, as were two unaffected children and an unaffected brother of one mother.
The intracellular consequences of the Ser72Pro substitution in the AGA polypeptide were studied by transient expression of the cDNA constructs in COS-1 cells. The T214C mutation was introduced into AGA cDNA by oligonucleotide-directed site-specific mutagenesis. The mutated cDNA construct in SVpoly expression vector was transfected into COS-1 cells, which were metabolically labeled with [35S]cysteine and immunoprecipitated. Analysis of immunoprecipitated AGA by SDS-PAGE revealed that the Ser72Pro precursor polypeptide was cleaved into two subunits corresponding to the 24 kDa [alpha] and an abnormally large [beta] subunit, which migrated more slowly than the 17 kDa [beta] subunit of the wild-type enzyme (Fig. 2 ). Pulse-chase labeling with chase times of 1, 3, 5 (Fig. 2 ) and 24 h (not shown) was performed to analyze the maturation of the mutant AGA. No 27 kDa pro-[alpha] subunit was detected even in a short 1 h chase, where this pre-lysosomal pro-[alpha] subunit was more abundant in the wild-type enzyme than the lysosomal 24 kDa form (Fig. 2 ). A significant proportion of the Ser72Pro polypeptide was retained in the precursor form, which was also secreted into the medium. A similar processing defect was observed in the case of two artificial mutants: His204Ser, which locates one amino acid N-terminally from the [alpha]-[beta] subunit cleavage site and an active-site substitution, Arg234Gln (Fig. 2 ). As compared with the AGA activity in COS cells transfected with the wild-type AGA, enzymatic activity of the Ser72Pro, His204Ser and Arg234Gln mutants was <10%, and the COS cell background activity ~5%.
To identify in which intracellular compartment the abnormal cleavage takes place, we used a 20oC temperature block to selectively prevent the protein transport from the Golgi network. Metabolic labeling and immunoprecipitation of mutant polypeptides showed that all three mutants remained in their non-cleaved 42 kDa precursor form after 4 h pulse-labeling at 20oC, whereas the wild-type enzyme was cleaved to the pro-[alpha] and [beta] subunits (Fig. 3 ). A 16 h chase with a 20oC block and a 6 h chase with a 32oC block were also employed to slow down the intracellular processing, but the 27 kDa pro-[alpha] subunit was not observed with any of the mutants (data not shown). Proteinase inhibitors of different specificities were tested to block the proteolytic processing steps of labeled AGA polypeptides. A proteinase inhibitor, leupeptine, inhibiting serine and cysteine proteinases with trypsin-like specifity, inhibited the cleavage of all the mutant precursor polypeptides (not shown). The same proteinase inhibitors also prevented the lysosomal processing, but not the activation cleavage of the wild-type AGA in the ER. These results indicate that the cleavage in the ER is hampered in the Ser72Pro, His204Ser and Arg234Gln mutant polypeptides. Furthermore, these mutant polypeptides become proteolytically processed in a cellular compartment distal to the trans-Golgi, probably in late endosomal or lysosomal compartments (Fig. 4 ).
Enzymatic activation of human AGA occurs by a proteolytic cleavage very soon after synthesis of the polypeptide in the ER (2 ). We have shown previously that this cleavage, which generates the 27 kDa pre-lysosomal [alpha] and 17 kDa [beta] subunits, is dependent on correct folding of the precursor polypeptide (8 ,19 ) and the mechanism has been suggested to be autocatalytic (15 ). Accordingly, most of the naturally occurring AGU mutations cause aberrant folding of the precursor polypeptide and also prevent the activation cleavage (7 ,10 ,12 ).
Here we have identified the first naturally occurring AGU mutation that involves an active-site amino acid, Ser72, as deduced from our recent structure determination (14 ,15 ). The mutation is located in the mutation hotspot region in the second exon of AGA. Now altogether four different AGU mutations have been reported in this 36 bp region of 1 kb AGA cDNA (5 ,6 ,11 ), whereas all other AGU mutations are distributed over the entire coding region of AGA. Interestingly, this T214C mutation, leading to a Ser72Pro substitution, was detected in four unrelated Arab families. So far, enrichment of one AGU mutation, AGUFin, has only been shown in the isolated population of Finland (5 ) and the present mutation most probably reflects similar enrichment in an inbred population. On the basis of the crystal structure, Ser72 resides in the active site of AGA, its hydroxyl group being hydrogen bonded to the [alpha]-amino group of the catalytic Thr206, located at the amino-terminus of the [beta] subunit (14 ,15 ). Ser72 could thus modify the chemical properties of the Thr206 [alpha]-amino group, which acts as a base increasing the nucleophilicity of the hydroxyl group of Thr206. This was also suggested by the results obtained from our mutagenesis studies of the active-site amino acids. The Ser72Ala mutation showed normal processing and only partial reduction of AGA activity, thus demonstrating that the hydrogen bond to Thr206, however, is not essential for the catalysis(15 ).
Ser72 is conserved in the bacterial AGA of Flavobacterium meningosepticum, but in mouse it has been replaced by Thr, which evidently is able to serve the same function. The crystal structure of AGA suggests that Pro in place of Ser72 would not cause any major structural changes (J. Rouvinen, unpublished data). This mutant active-site variation should therefore retain at least part of the catalytic activity of AGA if the proteolytic processing took place normally. This also turned out to be the case for the mutant enzyme, which was cleaved with a delay extracellularly. Evidently, the rate of activation is very rapid for the native enzyme only coincidently occurring in the ER, and the mutation slows down this activation process. However, it is surprising that the Ser72Pro substitution causes a severe processing defect intracellularly, even though the polypeptide chain is evidently correctly folded. Evidence for proper folding is provided by the normal phosphorylation and lysosomal targeting of the mutant polypeptides. This indicates that the determinants required for the recognition of AGA by phosphotransferase, an enzyme generating the lysosomal targeting signal Man-6-P and recognizing a common three-dimensional structure in lysosomal enzymes, are properly presented. Thus, the mutant enzyme is capable of escaping from the ER.
We have shown previously that His204Ser substitution leads to a similar inhibition of the activation cleavage into subunits as Ser72Pro (2 ), but this is to be expected, since His204 is located only two amino acids N-terminally from the proteolytic cleavage site between Asp205 and Thr206 and is conserved in all three known AGAs. Interestingly, substitution of the active-site amino acid Arg234 by Gln, Ala and Lys and Thr257 by Ala also had similar consequences, while substitutions of other active-site amino acids as well as Thr257Ser allowed normal processing (15 ). The location of Ser72, far apart from Arg234, Thr257 or His204 in the primary sequence, but physically close in the higher-order structure of the enzyme, would suggest that the folding of the precursor polypeptide brings these four amino acids close to each other prior to cleavage and that they could participate in autocatalytic cleavage of wild-type AGA at Thr206. The active-site of the enzyme and autocatalysis would thus be partially related. Autocatalysis has also been suggested to be the activation mechanism of enzymes belonging to the recently described family of N-terminal nucleophile hydrolases, to which AGA also seems to belong (20 ).
The existence of an ER-specific protease performing the activation cleavage is now unlikely because, according to the recent crystallization data, these amino acids are located in a pocket in the active enzyme that cannot be reached by any protease (14 ) unless the still unknown conformation of the precursor polypeptide is very different from the mature one. Anyway, His204 could serve as part of the recognition sequence. The next 17 amino acids N-terminal to His204 are only very poorly conserved in human as compared with mouse AGA (21 ) and are not present in bacterial AGA (22 ). Thus, they probably do not carry any recognition function for the cleavage.
A previously unidentified activation cleavage of mutant AGA precursor polypeptide at Thr206 taking place after secretion was demonstrated here. This was observed with the Ser72Pro and His204Ser mutants, but the Arg234Gln mutant was not cleaved, suggesting that this amino acid is of more profound importance in the proteolytic cleavage of AGA. This extracellular activation would provide further evidence for autocatalytic processing of wild-type AGA. The existence of a secreted protease with the same specificity as an ER protease is unlikely. The amount of the cleaved mutant enzyme increased depending on the incubation time in the medium and could not be prevented by any basic protease inhibitor. Thus the mutant precursor had more time extracellularly for the processing to occur, and also the conditions may have been more favorable. COS cells were also able to endocytose this active polypeptide from the medium, suggesting that similar activation and endocytosis could also happen in vivo. However, it has to be noted that mutant precursor polypeptides are overexpressed and oversecreted in the COS expression system and may not fully reflect the in vivo situation. It has been reported that overexpression of mutant [beta]-glucuronidase leads to intracellular activation of the mutant enzyme (23 ). Previous expression data for the AGUFin mutant have not shown intra- or extracellular activation in COS-1 or CHO cells (8 ), but small amounts of active enzyme have been detected in AGUFin patient tissues (24 ) indicating that, in vivo, a small fraction of the AGUFin mutant polypeptides are also capable of activation. However, the site of this partial activation, whether intra- or extracellular, has not been shown due to difficulties in the detection of small amounts of this housekeeping enzyme in tissues.
The information acquired on the Ser72Pro novel AGU mutation implies, in contrast to the case with other reported mutations in AGU patients, correct packing, phosphorylation and lysosomal targeting of the mutant enzyme. The most unique finding was probably that the mutant precursor polypeptide, remaining unprocessed in the ER, could be partially processed extracellularly into an active enzyme and endocytosed by recipient cells. These results would suggest that the activation of AGA is autoproteolytic, which is also supported by our mutagenesis studies of the active-site amino acids (15 ), and encourage future determination of the three-dimensional structure of the AGA precursor polypeptide. The extracellular activation of a precursor polypeptide also gives new insight into the development of therapy for AGU, allowing, besides enzyme or gene therapy, studies of the activation of the mutant enzyme in vivo.
Samples of three AGU families were received from Israel and samples of one family from Chicago. All the families are Palestinian Arabs. In each of two families there were four affected children and in each of the two other families two affected children; altogether there were 12 AGU patients. Although the families are unrelated, the parents in each family are related. PCR amplification and solid-phase sequencing of the coding region of AGA were performed as described previously (11 ).
The plasmid constructs containing the wild-type (WT) AGA, His204Ser, Arg234Gln (15 ) and AGUFin coding region have been described previously (2 ,7 ). Ser72 was changed to Pro by oligonucleotide-directed site-specific mutagenesis of the WT AGA cDNA in SVpoly expression vector (25 ) using a Chameleon double-stranded site-directed mutagenesis kit (Stratagene). Mutant clones were identified by the minisequencing method (26 ), and the whole insert was sequenced to exclude the possibility of unwanted mutations.
COS-1 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and antibiotics. The cells were seeded 1 day before transfection at 4*105 cells/3 cm dish and 2.2*106 cells/10 cm dish. The cells were transfected with 3-20 [mu]g of the plasmid constructs using the DEAE-dextran transfection method (27 ) and analyzed 3 days after transfection. Lipofection (28 ) with Lipofectamin (Gibco BRL) was used for transfection only for immunofluorescence analyses and transfection was performed as suggested by the manufacturer.
The cells were incubated in Cys-free medium for 30-60 min before labeling with 150 [mu]Ci/ml [35S]Cys (Amersham) using 1 h pulse followed by 1-16 h chase, or 4-7 h pulse with no chase. Labeling with [32P]ortophosphate (Amersham) was carried out as described earlier (3 ) in phosphate-free medium with 400 [mu]Ci [32P]Pi/ml, with a 2.5 h pulse and a 4 h chase. The cells were harvested, lysed and the proteins immunoprecipitated and separated as described earlier (11 ). The media were incubated at 37oC for different time periods and thereafter concentrated to 100-200 [mu]l with Centricon-30 microconcentrators (Amicon) before immunoprecipitation.
The COS-1 cells were co-transfected by lipofection with [beta]-hexosaminidase B (HexB) cDNA (17 ) and the mutant Ser72Pro AGA cDNA or wild-type AGA construct. The transfected cells seeded onto coverslips were fixed and incubated with a 1:200 dilution of rabbit antiserum against AGA (29 ) and a 1:100 dilution of goat antiserum against HexB (30 ). The cells were co-stained with a 1:50 dilution of rhodamine-conjugated anti-rabbit antibody (Dako) to localize AGA and with a 1:25 dilution of fluorescein-conjugated anti-goat antibody (Sigma) to localize HexB, which is located in the lysosomes. Immunostaining was performed as described previously (8 ).
The AGA activity assay was based on the colorimetric measurement of liberated N-acetylglucosamine from the synthetic substrate 2-acetamido-1-[beta]-(L-aspartamido)-1,2-dideoxy-[beta]-D-glucose (AADG) (31 ). An appropriate amount (1-25 [mu]l) of cell lysate or concentrated medium was incubated with 100 nmol of AADG in 67 mM potassium phosphate buffer pH 6.0 in a final volume of 50 [mu]l at 37oC for 18 h. The reaction was stopped with 107 [mu]l of 0.8 M borate buffer pH 8.8 and boiling for 5 min. The liberated N-acetylglucosamine was measured as described earlier (32 ). One unit (U) was defined as the amount of enzyme liberating 1 [mu]mol of N-acetylglucosamine in 1 min at 37oC.
We thank Dr Joel Zlotogora and Professor Allen Horwitz for the DNA and blood samples of the members of the Arabic AGU families. This work was supported by the Academy of Finland, the Juselius Foundation and the Hjelt Foundation.
1 Makino, M., Kojima, T., Ohgushi, T. and Yamashina, I. (1968) Studies on enzymes acting on glycopeptides. J. Biochem., 63, 186-192.MEDLINE Abstract
2 Ikonen, E., Julkunen, I., Tollersrud, O.-K., Kalkkinen, N. and Peltonen, L. (1993) Lysosomal aspartylglucosaminidase is processed to the active subunit complex in the endoplasmic reticulum. EMBO J.,12, 295-302.MEDLINE Abstract
3 Tikkanen, R., Enomaa, N., Riikonen, A., Ikonen, E. and Peltonen L. (1995a) Intracellular sorting of aspartylglucosaminidase: the role of N-linked oligosaccharides and evidence of Man-6-P-independent lysosomal targeting. DNA Cell Biol.,14, 305-312.MEDLINE Abstract
4 Pollitt, R., Jenner, F. and Merskey, Y. (1968) Aspartylglucosaminuria. An inborn error of metabolism associated with mental defect. Lancet,2, 253-255.MEDLINE Abstract
5 Ikonen, E., Baumann, M., Grön, K., Syvänen, A.-C., Enomaa, N., Halila, R., Aula P. and Peltonen L. (1991a) Aspartylglucosaminuria: cDNA encoding human aspartylglucosaminidase and the missense mutation causing the disease. EMBO J.,10, 51-58.MEDLINE Abstract
6 Isoniemi, A., Hietala, M., Aula, P., Jalanko, A. and Peltonen, L. (1995) Identification of a novel mutation causing aspartylglucosaminuria reveals a mutation hotspot region in the aspartylglucosaminidase gene. Hum. Mutat., 5, 318-326.MEDLINE Abstract
7 Ikonen, E., Enomaa, N., Ulmanen, I. and Peltonen, L. (1991b) In vitro mutagenesis helps to unravel the biological consequences of aspartylglucosaminuria mutation. Genomics,11, 206-211.MEDLINE Abstract
8 Riikonen, A., Ikonen, E., Sormunen, R., Lehto, V.-P., Peltonen, L. and Jalanko, A. (1994) Dissection of the molecular consequences of a double mutation causing a human lysosomal disease. DNA Cell Biol.,13, 257-264.MEDLINE Abstract
9 Yoshida, K., Yanagisawa, N., Oshima, A., Sakuraba, H., Iida, Y. and Suzuki, Y. (1992) Splicing defect of the glycoasparaginase gene in 2 Japanese siblings with aspartylglucosaminuria. Hum. Genet.,90, 179-180.MEDLINE Abstract
10 Park, H., Vettese, M.B., Fensom, A.H., Fisher, K.J., and Aronson, N.N., Jr (1993) Characterization of three alleles causing aspartylglucosaminuria: two from a British family and one from an American patient. Biochem. J.,290, 735-741. MEDLINE Abstract
11 Peltola, M., Chitayat, D., Peltonen, L. and Jalanko, A. (1994) Characterization of a point mutation in aspartylglucosaminidase gene: evidence for a readthrough of a translational stop codon. Hum. Mol. Genet.,12, 2237-2242.
12 Jalanko, A., Manninen, T. and Peltonen, L. (1995) Deletion of the C-terminal end of aspartylglucosaminidase resulting in a lysosomal accumulation disease: evidence for a unique genomic rearrangement. Hum. Mol. Genet.,3, 435-441.
13 Tikkanen, R., Rouvinen, J., Törrönen, A., Kalkkinen, N. and Peltonen, L. (1996) Large-scale purification and preliminary X-ray diffraction studies of human aspartylglucosaminidase. Proteins, 24, 253-258.MEDLINE Abstract
14 Oinonen, C., Tikkanen, R., Rouvinen, J. and Peltonen, L. (1995) Three-dimensional structure of human lysosomal aspartylglucosaminidase. Nature Struct. Biol., 2, 1102-1108.
15 Tikkanen, R., Riikonen, A., Oinonen, C., Rouvinen, J. and Peltonen, L. (1996) Functional analyses of active site residues of human lysosomal aspartylglucosaminidase: implications for catalytic mechanism and autocatalytic activation. EMBO J., in press.MEDLINE Abstract
16 Syvänen, A.-C., Aalto-Setälä, K., Kontula, K. and Söderlund, H. (1989) Direct sequencing of affinity-captured amplified human DNA: application to the detection of apolipoprotein E polymorphism. FEBS Lett.,258, 71-74. MEDLINE Abstract
17 Sonderfeld-Fresco, S. and Proia, R.L. (1988) Analysis of the glycosylation and phosphorylation of the lysosomal enzyme, [beta]-hexosaminidase B, by site-directed mutagenesis. J. Biol. Chem.,263, 13463-13469.MEDLINE Abstract
18 Enomaa, N., Danos, O., Peltonen, L. and Jalanko, A. (1995) Correction of deficient enzyme activity in a lysosomal storage disease, aspartylglucosaminuria, by enzyme replacement and retroviral gene transfer. Hum. Gene Ther., 6, 723-731.MEDLINE Abstract
19 Riikonen, A., Tikkanen, R., Jalanko, A. and Peltonen, L. (1995) Immediate interaction between the nascent subunits and two conserved amino acids Trp34 and Thr206 are needed for the catalytic activity of aspartylglucosaminidase. J. Biol. Chem.,9, 4903-4907.
20 Brannigan, J., Dodson, G., Duggleby, H., Moody, P., Smith, J., Tomchick, D. and Murzin, A. (1995) A new catalytic framework acting through an N-terminal nucleophile and capable of autocatalytic processing. Nature,378, 416-419.MEDLINE Abstract
21 Tenhunen, K., Laan, M., Manninen, T., Palotie, A. Peltonen, L. and Jalanko, A. (1995) Molecular cloning, chromosomal assignment, and expression of the mouse aspartylglucosaminidase gene. Genomics,30, 244-250.MEDLINE Abstract
22 Tarentino, A.L., Quinones, G., Hauer, C.R., Changchien, L.-M. and Plummer, T.H., Jr (1995) Molecular cloning and sequencing analysis of Flavobacterium meningosepticum glucosylasparaginase: a single gene encodes the [alpha] and [beta] subunits. Arch. Biochem. Biophys.,316, 399-406.MEDLINE Abstract
23 Wu, B.M., Tomatsu, S., Fukuda, S., Sukegava, K., Orii, T. and Sly, W.S. (1994) Overexpression rescues the mutant phenotype of L176F mutation causing [beta]-glucuronidase deficiency mucopolysaccharidosis in two mennonite siblings. J. Biol. Chem., 38, 23681-23688.MEDLINE Abstract
24 Enomaa, N., Lukinmaa, P.-L., Ikonen, E.M., Waltimo, J.C., Palotie, A., Paetau, A.E. and Peltonen, L. (1993) Expression of aspartylglucosaminidase in human tissues from normal individuals and aspartylglucosaminuria patients. J. Histochem. Cytochem.,7, 981-989.
25 Stacey, A. and Schnieke, A. (1990) SVpoly: a versatile mammalian expression vector. Nucleic Acids Res.,18, 2829.MEDLINE Abstract
26 Syvänen, A.-C., Aalto-Setälä, K., Harju, L., Kontula, K. and Söderlund, H. (1990) A primer-guided nucleotide incorporation assay in genotyping of apolipoprotein E. Genomics,8, 684-692.MEDLINE Abstract
27 Luthman, H. and Magnusson, G. (1983) High efficiency polyoma DNA transfection of chloroquine treated cells. Proc. Natl Acad. Sci.USA,11, 1295-1307.
28 Felgner, P.L., Gadek, T.R., Holm, M., Roman, R., Chan, H.W., Wenz, M., Northrop, J.P., Ringold, G.M. and Danielsen, M. (1987) Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc. Natl Acad. Sci. USA, 84, 7413-7417.MEDLINE Abstract
29 Halila, R., Baumann, M., Ikonen, E., Enomaa, N. and Peltonen, L. (1991) Human leukocyte aspartylglucosaminidase. Evidence for two different subunits in a more complex native structure. Biochem. J.,276, 251-256.MEDLINE Abstract
30 Proia, R., d'Azzo, A. and Neufeld, E. (1984) Association of alpha- and beta-subunits during biosynthesis of beta-hexosaminidase in cultured fibroblasts. J. Biol. Chem.,259, 3350-3354.MEDLINE Abstract
31 Makino, M., Kojima, T. and Yamashina, I. (1966) Enzymatic cleavage of glycopeptides. Biochem. Biophys. Res. Commun., 24, 961-966.MEDLINE Abstract
32 Reissig, J.L., Strominger, J.L. and Leloir, L.F. (1955) A modified colorimetric method for the estimation of N-acetylaminosugars. J. Biol. Chem.,217, 959-966.
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