Department of Cell Biology, Albert Einstein College Medicine, New York, NY 10461, USA, 2Lysosomal Diseases Research Unit, The Womens and Childrens Hospital, Adelaide, South Australia, 5006, Australia, and 3Dipartimento di Scienze Biochimiche e Biotecnologie Molecolare Universita di Perugia, Perugia, Italy
Received on August 2, 2000; revised on September 14, 2000; accepted on September 14, 2000.
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Abstract |
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Key words: Sanfilippo syndrome/MPS III A/sulfamidase/point mutation
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Introduction |
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MPS III A has been reported in the dog (Fischer et al., 1998), and we previously identified a mouse with MPS III A that replicates many of the histopathological features found in MPS III A in children (Bhaumik et al., 1999). Mice of this strain die at about 10 months of age exhibiting a distended bladder and hepatosplenomegaly. In brain sections from these mice, distended lysosomes, some with typical zebra body morphology, and many containing periodic-acid Schiff positive storage material, are prevalent. Urinalysis revealed an accumulation of heparan sulfate that contained glucosamine-N-sulfate at the non-reducing end and was susceptible to digestion with recombinant sulfamidase. Assays of a variety of lysosomal hydrolases in brain, liver, and kidney extracts uncovered a specific defect in sulfamidase activity. Interestingly, the activity was reduced by about 97% but was not completely absent, suggesting a point mutation as the basis of the mouse MPS III A phenotype. In this paper, we identify a missense mutation in the coding region of the sulfamidase gene, and show that this provides the molecular basis of MPS III A in our spontaneous mouse mutant.
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Results |
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Origin of the MPS III A mouse
The MPS III A mouse was initially identified in a colony of mice generated from an embryonic stem cell clone, WW6.186, that carries a targeted mutation in one allele of the Mgat3 gene (Bhaumik et al., 1999). Thus, it was possible that either the original embryonic stem (ES) cell line, or the targeted clone derived from it, had acquired a mutation in the sulfamidase gene. However, this was not the case as the parent ES cell line, WW6, and the mutant clone, WW6.186, were both shown to carry the wild type sequence at nucleotide 91 of the sulfamidase gene coding region (Figure 1). In addition, CD1 mice to which WW6.186 chimeras were mated in deriving the colony, are also wild type at this position (Figure 1). Therefore, the sulfamidase mutation was not preexisting in either the ES cells or in the mouse strain used to generate the colony. The mutation may have arisen from a rare heterozygous CD1 mouse, or from a mutation that occurred during the first few generations of interbreeding of WW6.186 x CD1 progeny that derived from the chimera.
Recombinant sulfamidase (D31N) is inactive in CHO cells
To assay the sulfamidase activity of wild type and mutant cDNAs, they were cloned into the pCDNA3.1 expression vector and transfected into CHO-K1 cells. Sulfamidase activity of independent clones was determined with a radiolabeled tetrasaccharide (Hopwood and Elliott, 1982). Sulfamidase expressed from mutant cDNAs had markedly reduced N-sulfatase activity (
3%) compared to wild type cDNAs (Table I). Therefore, the D31N missense mutation severely cripples sulfamidase activity. As would be predicted, correction of the A at nucleotide 91 to G by site-directed mutagenesis restored sulfamidase activity to wild type levels (Table I).
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Discussion |
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The MPS III A mouse is an excellent model for the human disease which arises, in many cases, from a point mutation in the sulfamidase gene coding region as summarized in Figure 3. Targeted sulfamidase gene deletion would not so closely mimic the human disease. It is clear from the spread of inactivating mutations almost throughout the molecule, that reductions in sulfamidase activity easily arise from single amino acid changes in the sequence. Mutant sulfamidase molecules might therefore be induced to refold into an active form if an appropriate chemical conformerone could be selected from a combinatorial library. This type of strategy was successful in improving the transport and maturation of -galactosidase A in Fabry lymphoblasts (Fan et al., 1999
). The MPS III A mouse will be useful for evaluating pathogenic mechanisms arising from reduced sulfamidase activity and for testing a range of therapeutic strategies such as enzyme refolding or replacement, gene replacement and cellular transplantation therapies for the human disease. The MPS III B mouse that lacks
-N-acetylglucosaminidase due to a targeted mutation and cannot degrade heparan sulfate, has a similar phenotype to the MPS III A mouse (Li et al., 1999
) and also provides an excellent mouse model for testing therapies for Sanfilippo syndrome.
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Materials and methods |
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Cloning mouse sulfamidase cDNA
Poly(A+) RNA was isolated from mouse liver using Trizol (Gibco BRL) and oligo-dT cellulose (Pharmacia). Oligo(dT)1218 (Gibco BRL) and superscript II RT enzyme (Gibco BRL) were used for reverse transcription. Gene specific primers based on the C57BL/6 mouse cDNA sequence (Costanzi et al., 2000) were: 5' RESN (CGCTTGCGGCCGCGAGCCGGAACCGCTTACCCTGACC) from 33 to 10 from the ATG (+1); 3' RESH (GTCCCAAGCTTCTCCATGTCCAGGTGGGCTGGCAG) spanned nucleotide +1525 to +1548 in the 3' UTR just beyond the sulfamidase coding sequence. Expand PCR DNA polymerase (Boehringer Mannheim) was used to amplify the
1.6 kb coding region. NotI and HindIII restriction sites in the primers (underlined) were used to clone cDNAs into pCDNA3.1(-) (Stratagene). PCR products and plasmid DNA prepared by the QIAGEN kit were sequenced. Mutant cDNA was corrected to wild type using site-directed mutagenesis (QuickchangTM kit, Stratagene). with primers PS330 (GCTACTGATAGTTGCGGATGACGGAGGCTTTG) and PS331 (CAAAGCCTCCGTCATCCGCAACTATCAGTAGC).
Genotyping MPS III A mice
Genomic DNA from mouse tail was amplified with primers PS339(5' GGTCTGTCTTCCTCAGCG) and PS340 (5' GATAAGGCTGTGGCGGGACAGGG ) following 3 min at 94°C, 30 cycles of 45 sec at 94°C, 45 s at 60°C, 1 min at 72°C and 4 min final extension at 72°C. PCR products were purified with the QIAGEN PCR spin column and digested with MspA1I (New England BioLabs) at 37°C for 2 h before electrophoresis on a 3.5% agarose gel. MspA1I cuts between the G and C of the sequence CAGCGG.
Sulfamidase assay
To determine sulfamidase activity, cDNAs encoding wild type, mutant and corrected mutant sequences in pCDNA3.1(-) were transfected into CHO-K1 cells. G418 resistant transfectants were grown to confluence, lysates were prepared and assayed using a radiolabeled tetrasaccharide (glucosamine-N-sulfate-(1,4)-iduronic or glucuronic acid-1,4-glucosamine-N-sulphate-(1,4)-[3H]-idonic, gluconic or anhydroidonic acid as described previously (Hopwood and Elliott, 1982; Bhaumik et al., 1999).
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Acknowledgments |
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Footnotes |
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References |
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