Sulfonation and phosphorylation of regions of the dioxin receptor susceptible to methionine modifications.

Tagged murine dioxin receptor was purified from mammalian cells, digested with trypsin, and analyzed by capillary HPLC-MALDI-TOF/TOF-MS and -MS/MS. Several chromatographically distinct semitryptic peptides matching two regions spanning residues Glu(409)-Arg(424) and Ser(547)-Arg(555) of the dioxin receptor were revealed by de novo sequencing. Methionine residues at 418 and 548 were detected in these peptides as either unmodified or modified by moieties of 16 (oxidation) or 57 amu (S-carboxamidomethylation) or in a form corresponding to degradative removal of 105 amu from the S-carboxamidomethylated methionine. MS/MS spectra revealed that the peptides containing modified methionine residues also existed in forms with a modification of +80 amu on serine residues 411, 415, and 547. The MS/MS spectra of these peptide ions also revealed diagnostic neutral loss fragment ions of 64, 98, and/or 80 amu, and in some instances combinations of these neutral losses were apparent. Taken together, these data indicated that serines 411 and 547 of the dioxin receptor were sulfonated and serine 415 was phosphorylated. Separate digests of the dioxin receptor were prepared in H(2)(16)O and H(2)(18)O, and enzymatic dephosphorylation was subsequently performed on the H(2)(16)O digest only. The digests were mixed in equal proportions and analyzed by capillary HPLC-MALDI-TOF/TOF-MS and -MS/MS. This strategy confirmed assignment of sulfonation as the cause of the +80-amu modifications on serines 411 and 547 and phosphorylation as the predominant cause of the +80-amu modification of serine 415. The relative quantitation of phosphorylation and sulfonation enabled by this differential phosphatase strategy also suggested the presence of sulfonation on a serine other than residue 411 within the sequence spanning Glu(409)-Arg(424). This represents the first description of post-translational sulfonation sites and identification of a new phosphorylation site of the latent dioxin receptor. Furthermore this is only the second report of serine sulfonation of eukaryotic proteins. Mutagenesis studies are underway to assess the functional consequences of these modifications.

Determination of oligosaccharides and glycolipids in amniotic fluid by electrospray ionisation tandem mass spectrometry: in utero indicators of lysosomal storage diseases.

Prenatal diagnosis is available for many lysosomal storage disorders (LSD) using chorionic villus samples or amniocytes. Such diagnoses can be problematical if sample transport and culture are required prior to analysis. The purpose of this study was to identify useful biochemical markers for the diagnosis of lysosomal storage disorders from amniotic fluid. Amniotic fluid samples from control (n=49) and LSD affected (n=36) pregnancies were analysed for the protein markers LAMP-1 and saposin C by ELISA, and for oligosaccharide and lipid metabolite markers by electrospray ionisation-tandem mass spectrometry. Lysosomal storage disorder samples include; aspartylglucosaminuria, galactosialidosis, Gaucher disease, GM1 gangliosidosis, mucopolysaccharidosis types I, II, IIIC, IVA, VI, and VII, mucolipidosis type II, multiple sulfatase deficiency, and sialidosis type II. Each disorder produced a unique signature metabolic profile of protein, oligosaccharide, and glycolipid markers. Some metabolite elevations directly related to the disorder whilst others appeared unrelated to the primary defect. Many lysosomal storage disorders were clearly distinguishable from control populations by the second trimester and in one case in the first trimester. Samples from GM1 gangliosidosis and mucopolysaccharidosis type VII displayed a correlation between gestational age and amount of stored metabolite. These preliminary results provide proof of principal for the use of biomarkers contained in amniotic fluid as clinical tests for some of the more frequent lysosomal storage disorders causal for hydrops fetalis.

Correlation among genotype, phenotype, and biochemical markers in Gaucher disease: implications for the prediction of disease severity.

Gaucher disease is a lysosomal storage disorder characterized by a deficiency of the enzyme acid beta-glucosidase. The clinical manifestations of Gaucher disease are highly variable, and although certain genotypes are often associated with mild or severe symptoms, a defined correlation between genotype and phenotype does not exist. Identification of biochemical markers characteristic of pathology may be of use in predicting the progression of the disease state. In this study the relationship among genotype, glycolipid substrates, lysosomal proteins, and the clinical manifestations of Gaucher disease has been evaluated. Plasma glycolipids were analyzed using electrospray ionization-tandem mass spectrometry. Lysosomal-associated membrane protein-1 (LAMP-1) and saposin C were determined by immunoquantification. Patients with Gaucher disease were shown to have an increased 16:0-glucosylceramide/16:0-lactosylceramide ratio and elevated concentrations of LAMP-1 and saposin C in plasma. A general relationship was found to exist among the 16:0-glucosylceramide/16:0-lactosylceramide ratio, LAMP-1 and saposin C levels, and patient phenotype, providing a refinement of the genotype-phenotype correlation. These findings have major implications for the diagnosis, prediction of disease severity, and monitoring of therapy in patients with Gaucher disease.