Lysosomal acid phosphatase (LAP) is synthesized as a transmembrane protein with a short carboxy‐terminal cytoplasmic tail of 19 amino acids, and processed to a soluble protein after transport to lysosomes. Deletion of the membrane spanning domain and the cytoplasmic tail converts LAP to a secretory protein, while deletion of the cytoplasmic tail as well as substitution of tyrosine 413 within the cytoplasmic tail against phenylalanine causes accumulation at the cell surface. A chimeric polypeptide, in which the cytoplasmic tail of LAP was fused to the ectoplasmic and transmembrane domain of hemagglutinin is rapidly internalized and tyrosine 413 of the LAP tail is essential for internalization of the fusion protein. A chimeric polypeptide, in which the membrane spanning domain and cytoplasmic tail of LAP are fused to the ectoplasmic domain of the Mr 46 kd mannose 6‐phosphate receptor, is rapidly transported to lysosomes, whereas wild type receptor is not transported to lysosomes. We conclude that a tyrosine containing endocytosis signal in the cytoplasmic tail of LAP is necessary and sufficient for targeting to lysosomes.

Sanfilippo B syndrome (mucopolysaccharidosis IIIB, MPS IIIB) is caused by a deficiency ofα-N-acetylglucosaminidase, a lysosomal enzyme involved in the degradation of heparan sulphate. Accumulation of the substrate in lysosomes leads to degeneration of the central nervous system with progressive dementia often combined with hyperactivity and aggressive behaviour. Age of onset and rate of progression vary considerably, whilst diagnosis is often delayed due to the absence of the pronounced skeletal changes observed in other mucopolysaccharidoses. Cloning of the gene and cDNA encoding α-N-acetylglucosaminidase enabled a study of the molecular basis of this syndrome. We were able to identify 31 mutations, 25 of them novel, and two polymorphisms in the 40 patients mostly of Australasian and Dutch origin included in this study. The observed allellic heterogeneity reflects the wide spectrum of clinical phenotypes reported for MPS IIIB patients. The majority of changes are missense mutations; also four nonsense and nine frameshift mutations caused by insertions or deletions were identified. Only five mutations were found in more than one patient and the observed frequencies are well below those observed for the common mutations in MPS IIIA. R643C and R297X each account for around 20% of MPS IIIB alleles in the Dutch patient group, whilst R297X, P521L, R565W and R626X each have a frequency of about 6% in Australasian patients. R643C seems to be a Dutch MPS IIIB allele and clearly confers the attenuated phenotype. One region of the gene shows a higher concentration of mutations, probably reflecting the instability of this area which contains a direct repeat. Several arginine residues seem to be 'hot-spots' for mutations, being affected by two or three individual base pair exchanges.

Sanfilippo syndrome type A or mucopolysaccharidosis IIIA (MPS IIIA) is an autosomal recessive lysosomal storage disorder caused by the deficiency of sulfamidase. The resulting lysosomal storage of heparan sulfate may lead to severe neurodegeneration preceded by progressive dementia, often combined with aggressive and hyperactive behaviour. A total of 109 patients from four different geographic areas were screened for the common mutation R245H and two other previously identified mutations. SSCP analysis of exons was used to characterize the unknown alleles. We identified 16 novel sequence variants, 12 of them likely to be pathogenic. The majority of the pathogenic variants were single base pair changes leading to missense mutations. Several single base pair deletions/insertions and one nonsense mutation were also identified. Altogether, we were able to characterize 55% of the pathogenic alleles. Sequence homology between sulfamidase and N-acetylgalactosamine 4-sulfatase, the first sulfatase to have its tertiary structure defined, suggests that amino acid residues R74 and T79, which were found to be mutated, are likely to be involved in the formation of the active site of sulfamidase. R245H accounts for 31% of the Sanfilippo A alleles in Australasia, for 19.2% of the alleles in patients from the UK and has a high frequency of 57.8% in patients from The Netherlands. The identification of mutations common in certain geographic regions or ethnic groups will help in the diagnosis of MPS IIIA and allow carrier testing and improved genetic counselling.

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Sanfilippo syndrome type A or mucopolysaccharidosis IIIA (MPS IIIA) results from the deficiency of the enzyme heparan N-sulfatase (NS, EC 3.10.1.1), required for the degradation of heparan sulfate. Molecular defects of 24 Italian MPS IIIA patients were recently reported by our group. We report here two novel mutations: 1040insT and Q365X and the expression studies on 15 of the identified defects. Transient expression of COS cells by cDNA mutagenized to correspond to heparan N-sulfatase mutations Y40N, A44T, 166delG, G122R, P128L, L146P, R150Q, D179N, R182C, R206P, P227R, 1040insT, 1093insG, E369K, R377C did not yield active enzyme, demonstrating the deleterious nature of the mutations. Western blot analysis and metabolic labeling experiments revealed, for cells transfected with wild-type enzyme, a precursor 62-kDa form and a mature 56-kDa form. Western blot resulted, for 11 mutations, in the presence of both forms, indicating a normal maturation of the mutant enzyme. Western blot, metabolic labeling and immunofluorescence experiments suggested, for mutations 166delG, L146P, 1040insT and 1093insG, an increased degradation of the mutant enzymes.

Sanfilippo B syndrome is caused by a deficiency of alpha-N-acetylglucosaminidase, a lysosomal enzyme involved in the degradation of heparan sulphate. Accumulation of the substrate in lysosomes results in degeneration of the central nervous system with progressive dementia often combined with hyperactivity and aggressive behaviour. In order to clone the deficient gene, we purified the enzyme from human placenta and obtained amino acid sequence information. Alignment of one of the CNBr generated internal peptides to sequence from the database revealed the chromosomal location of the gene in the 5' upstream flanking region of the gene for 17-beta-hydroxysteroid-dehydrogenase at 17q21.1. The available DNA sequence was used to clone the cDNA coding for alpha-N-acetylglucosaminidase and analyse its gene structure. The gene is fully contained in the 5' upstream flanking region of the gene for 17-beta-hydroxysteroid-dehydrogenase and interrupted by five introns. The cDNA clone has a length of 2575 bp and encodes a protein of 743 amino acids. Chinese hamster ovary cells transfected with the cDNA construct show alpha-N-acetylglucosaminidase activity about 17-fold over background. This will allow correction studies with NAG deficient Sanfilippo B cell lines and facilitate the development of enzyme replacement therapy for these patients.

Mucopolysaccharidosis IIIA (MPS-IIIA) is an autosomal recessive lysosomal storage disorder caused by the deficiency of sulfamidase (NS; EC 3.10.1.1), resulting in defective degradation and storage of heparan sulfate. This paper reports the production and characterization of monoclonal and polyclonal antibodies against recombinant human sulfamidase (rhNS) to quantitate and characterize normal and mutant sulfamidase produced from the wild type NS expression vector. Glycosylation and phosphorylation studies of immunoprecipitated rhNS show that all five potential glycosylation sites are utilized, with three high mannose/hybrid oligosaccharides and two simpler chains, with at least one functional mannose 6-phosphate group. An NS quantification system was developed to determine the effect of the three most common and severe patient mutations: S66W (Italy), R74C (Poland), and R245H (The Netherlands). The quantity and specific activity of expressed mutant rhNS was significantly lower than expressed normal rhNS, with 0.3, 0.2, and 0.05% of normal rhNS produced and 15, 17, and 83% of normal specific activity for S66W, R74C, and R245H observed, respectively. The recent structural elucidation of N-acetylgalactosamine-4-sulfatase was utilized to postulate the effect on the structure-function relationship of NS. The characterization of normal and mutated rhNS has relevance for efficient diagnosis and therapeutic developments for MPS-IIIA patients.

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