Determination of acid alpha-glucosidase protein: evaluation as a screening marker for Pompe disease and other lysosomal storage disorders.

BACKGROUND: In recent years, there have been significant advances in the development of enzyme replacement and other therapies for lysosomal storage disorders (LSDs). Early diagnosis, before the onset of irreversible pathology, has been demonstrated to be critical for maximum efficacy of current and proposed therapies. In the absence of a family history, the presymptomatic detection of these disorders ideally can be achieved through a newborn screening program. One approach to the development of such a program is the identification of suitable screening markers. In this study, the acid alpha-glucosidase protein was evaluated as a marker protein for Pompe disease and potentially for other LSDs. METHODS: Two sensitive immunoquantification assays for the measurement of total (precursor and mature) and mature forms of acid alpha-glucosidase protein were used to determine the concentrations in plasma and dried blood spots from control and LSD-affected individuals. RESULTS: In the majority of LSDs, no significant increases above control values were observed. However, individuals with Pompe disease showed a marked decrease in acid alpha-glucosidase protein in both plasma and whole blood compared with unaffected controls. For plasma samples, this assay gave a sensitivity of 95% with a specificity of 100%. For blood spot samples, the sensitivity was 82% with a specificity of 100%. CONCLUSIONS: This study demonstrates that it is possible to screen for Pompe disease by screening the concentration of total acid alpha-glucosidase in plasma or dried blood spots.

Saposins A, B, C, and D in plasma of patients with lysosomal storage disorders.

BACKGROUND: Early diagnosis of lysosomal storage disorders (LSDs), before the onset of irreversible pathology, will be critical for maximum efficacy of many current and proposed therapies. To search for potential markers of LSDs, we measured saposins A, B, C, and D in patients with these disorders. METHODS: Four time-delayed fluorescence immunoquantification assays were used to measure each of the saposins in plasma from 111 unaffected individuals and 334 LSD-affected individuals, representing 28 different disorders. RESULTS: Saposin A was increased above the 95th centile of the control population in 59% of LSD patients; saposins B, C, and D were increased in 25%, 61%, and 57%, respectively. Saposins were increased in patients from several LSD groups that in previous studies did not show an increase of lysosome-associated membrane protein-1 (LAMP-1). CONCLUSION: Saposins may be useful markers for LSDs when used in conjunction with LAMP-1.

Two-dimensional mapping and microsequencing of lysosomal proteins from human placenta.

Lysosomes degrade a wide range of macromolecules to yield monomer products which are exported out of the lysosome by a series of transporters. In addition, lysosomes perform a range of other functions which are cell or tissue specific. In order to gain insight into the tissue specific role of lysosomes, carrier-ampholyte two-dimensional electrophoresis (2-DE) was used in combination with N-terminal sequencing to identify the major proteins present in both the membrane and luminal space of placental lysosomes. From the 45 N-terminal peptide sequences generated, 14 luminal and five membrane proteins were identified while three other sequences were novel. The sequenced proteins were a mixture of lysosomal and non-lysosomal proteins. The lysosomal proteins consisted of gamma-interferon-inducible protein (IP-30), Saposin D, cathepsins B and D, beta-hexosaminidase, palmitoyl protein thioesterase, alpha-glucosidase, and LAMP-1. The non-lysosomal proteins were serum albumin, serotransferrin, haemoglobin gamma G chain, alpha-1-antitrypsin, placental lactogen, endoplasmin, peptide binding protein 74, p60 lymphocyte protein, p450 side chain cleavage enzyme and placental alkaline phosphatase. The 2-DE maps obtained in this study are the first to identify the major proteins in both the lumen and membrane of placental lysosomes through sequence analysis, and thus provide the basis upon which to build a complete 2-DE database of the lysosome. Furthermore, the identities of the proteins sequenced from the placental lysosomes suggest a role for lysosomes in the transport of nutrients across the trophoblastic layer.

Development of a two-dimensional gel electrophoresis database of human lysosomal proteins.

Two-dimensional gel electrophoresis databases have been generated for a range of tissue cell and fluid types, from a number of species. A major difficulty in the development of such databases is the large number of proteins present in even a single cell type (> 10,000) and the low levels of many of these proteins. One approach to overcome these difficulties is to fractionate the cell into its organelles and generate individual databases for each subcellular component. This has the added advantage of assigning a cellular location to each protein identified. Here we report the development of a two-dimensional gel electrophoresis database of lysosomal proteins.

Two site-directed mutations abrogate enzyme activity but have different effects on the conformation and cellular content of the N-acetylgalactosamine 4-sulphatase protein.

The sulphatase family of enzymes have regions of sequence similarity, but relatively little is known about either the structure-function relationships of sulphatases, or the role of highly conserved amino acids. The sequence of amino acids CTPSR at position 91-95 of 4-sulphatase has been shown to be highly conserved in all of the sequenced sulphatase enzymes. The cysteine at amino acid 91 of 4-sulphatase was selected for mutation analysis due to its potential role in either the active site, substrate-binding site or part of a key structural domain of 4-sulphatase and due to the absence of naturally occurring mutations in this residue in mucopolysaccharidosis type VI (MPS VI) patients. Two mutations, C91S and C91T, altering amino acid 91 of 4-sulphatase were generated and expressed in Chinese hamster ovary cells. Biochemical analysis of protein from a C91S cell line demonstrated no detectable 4-sulphatase enzyme activity but a relatively normal level of 4-sulphatase polypeptide (180% of the wild-type control protein level). Epitope detection, using a panel of ten monoclonal antibodies, demonstrated that the C91S polypeptide had a similar immunoreactivity to wild-type 4-sulphatase, suggesting that the C91S substitution does not induce a major structural change in the protein. Reduced catalytic activity associated with normal levels of 4-sulphatase protein have not been observed in any of the MPS VI patients tested and all show evidence of structural modification of 4-sulphatase protein with the same panel of antibodies [Brooks, McCourt, Gibson, Ashton, Shutter and Hopwood (1991) Am. J. Hum. Genet. 48, 710-719]. The loss of enzyme activity without a detectable protein conformation change suggests that Cys-91 may be a critical residue in the catalytic process. In contrast, analysis of protein from a C91T cell line revealed low levels of catalytically inactive 4-sulphatase polypeptide (0.37% of the wild-type control protein level) which had missing or masked epitopes, suggesting an altered protein structure or conformation. Subcellular fractionation studies of the C91T cell line demonstrated a high proportion of 4-sulphatase polypeptide content in organelles characteristic of microsomes. The aberrant intracellular localization and the reduced cellular content of 4-sulphatase polypeptide was consistent with the observed structural modification leading to retention and degradation of the protein within an early vacuolar compartment.