Sulphatase activities are regulated by the interaction of sulphatase-modifying factor 1 with SUMF2.

Sulphatases undergo a unique post-translational modification that converts a highly conserved cysteine located within their active site into formylglycine. This modification is necessary for the catalytic activities of the sulphatases, and it is generated by the protein product of sulphatase-modifying factor 1 (SUMF1), the gene mutated in multiple sulphatase deficiency (MSD). A paralogous gene, SUMF2, was discovered through its sequence similarity to SUMF1. We present evidence that SUMF2 colocalizes with SUMF1 within the endoplasmic reticulum and that the two proteins form heterodimers. SUMF1 and SUMF2 also form homodimers. In addition, SUMF2 is able to associate with the sulphatases with and without SUMF1. We have previously shown that co-transfection of SUMF1 with the sulphatase complementary DNAs greatly enhances the activities of the overexpressed sulphatases. Here, we show that SUMF2 inhibits the enhancing effects of SUMF1 on sulphatases, suggesting that the SUMF1-SUMF2 interaction represents a further level of control of these sulphatase activities.

Influencia del a-Factor sobre la expresión de IDShr en Pichia pastoris: Revisión sistemática de literatura y análisis computacional / Influence of a-Factor over the expression of hrIDS in Pichia pastoris: Literature review and computational analysis

En la búsqueda de alternativas para mejorar la expresión de la enzima Iduronato Sulfatasa (IDSh) en la levaduraPichia pastoris, se realizó una Revisión Sistemática de la Literatura con el fin de recopilar información quepermitiera relacionar los niveles de expresión de proteínas humanas recombinantes con la señal de secreción y las características propias de la molécula a expresar. Se hallaron 349 publicaciones de las cuales sólo 7 (2)porciento reportaron la expresión de proteínas que cumplían con los criterios de inclusión manejados en el estudio. Con la información obtenida en los 7 artículos se realizó una prueba de hipótesis tomando como muestras los datos recopilados y un análisis cualitativo de la información, con los cuales se evidenció que al reemplazar la señal de secreción nativa por el a-Factor como péptido líder se incrementa el nivel de expresión de proteínas humanas recombinantes en P. pastoris (p=0.053). Se encontró que la eliminación de la secuencia que codifica para el péptido nativo heterólogo en el ADNc de la proteína, es imprescindible para que el a-Factor pueda favorecer la secreción de proteínas heterólogas y por consiguiente incrementar el nivel de expresión. En el caso de la IDShr se halló que en la construcción GS115/pPIC9-IDS, aparecen dos secuencias de péptido señal al mismo tiempo, la nativa de la IDSh y la putativa proveniente de Saccharomyces cerevisiae; sin embargo, se han obtenidos expresiones hasta de ~30mmol/h mg de proteína, lo que deja la incógnita de un posible conflicto en el reconocimiento erróneo de una u otra señal de secreción, teniendo en cuenta el grado de hidrofobicidad de ambas.

Prenatal diagnosis of the Hunter syndrome and the introduction of a new fluorimetric enzyme assay.

Prenatal diagnosis of the Hunter syndrome (mucopolysaccharidosis type II; MPS II) is preferably achieved by the assay of iduronate-2-sulphate sulphatase (IDS) in uncultured chorionic villi (CV) as this allows early (12th week), rapid (2-3 days) and reliable results. We summarize the results of 174 prenatal analyses in the past 30 years, using various methods such as radiolabelled sulphate incorporation in amniotic fluid (AF) cells, glycosaminoglycan (GAG)-electrophoresis in AF and IDS assay in CV, CV-cells, AF and AF-cells. Twenty-seven fetuses with MPS II were diagnosed after finding clearly abnormal results in pregnancies with a male fetus; very low IDS activity has also been measured in some pregnancies with a (heterozygous) female fetus, emphasizing the need to combine enzyme assay with fetal sex determination. IDS activity has until recently been assessed by a cumbersome radioactive enzyme assay. Here we describe the use of a novel fluorigenic 4-methylumbelliferyl substrate, which allows a sensitive, rapid and convenient assay of IDS activity and reliable early prenatal diagnosis. This novel IDS assay was validated in retrospective analyses of 14 CV, CV-cell, AF and AF-cell samples from affected pregnancies in addition to prospective prenatal diagnosis in eight pregnancies at risk with one MPS II-affected fetus.

Bone marrow transplantation in Hunter syndrome.

Hunter syndrome (mucopolysaccharidosis II) is a rare X-linked disorder of mucopolysaccharide metabolism that typically progresses to severe mental retardation and death by 18 years of age. A child with Hunter syndrome received an allogeneic bone marrow transplantation from an unaffected human leukocyte antigen-identical sibling at the age of 29 months without complications. Despite full and sustained engraftment now at 70 months after transplantation, the patient's neurocognitive abilities have continued to deteriorate. In this case, replacement of defective marrow-derived macrophages by bone marrow transplantation was not effective in preventing the neurologic progression of the disease in a child with the severe phenotype of Hunter syndrome.

DNA and enzyme studies on chorionic villi for use in antenatal diagnosis.

A study has been undertaken to determine the efficiency of current methods in providing an adequate amount of chorionic villus DNA for antenatal diagnosis using recombinant DNA techniques or enzyme assay. Chorionic biopsies were obtained from 40 women undergoing elective first trimester termination of pregnancy (8-12 weeks) under general anaesthesia. The villus tissue was isolated from maternal tissue under a dissection microscope and the presence of any remaining contamination was ascertained by conventional histology and immuno-cytochemical examinations. A high level of success was achieved in obtaining a pure fetal sample. In the first 20 samples the DNA yield obtained using the method of Williamson et al [1] was found to be 0.5 +/- 0.5 micrograms/mg wet weight of villus tissue (mean +/- 1 SD). In the subsequent 20 biopsies using a modified procedure, the yield was significantly improved to 1.0 +/- 0.65 (p less than 0.002). A normal range for the enzyme iduronate sulphatase, which is deficient in Hunter's syndrome (mucopolysaccharidosis II), is also reported. It is suggested that as little as 20 mg of chorionic villi may be used to provide sufficient material for a reliable study using recombinant DNA or biochemical methods.

A clinical trial of fibroblast transplantation for the treatment of mucopolysaccharidoses.

This paper reports the clinical and biochemical results in six patients with Hurler disease (Mucopolysaccharidosis IH; McKusick 25280), two patients with Hunter disease (Mucopolysaccharidosis II; McKusick 25285) and one patient with Sanfilippo B disease (Mucopolysaccharidosis IIIB; McKusick 25292) who were treated by fibroblast transplantation. Except for one patient who died for a coincidental reason, the patients have been studied for between 2.5 and 4.5 years. The clinical course of the disease was not materially altered. There was no evidence that the patients had developed immune responses against the transplanted fibroblasts. Transplantation did not produce measurable levels of either alpha-L-iduronidase (EC 3.2.1.76) in the leukocytes from patients with Hurler disease or of N-acetyl-alpha-D-glucosaminidase (EC 3.2.1.50) in the plasma of the patients with Sanfilippo B disease. Under the conditions used for the assay, leukocytes from the patients with Hunter disease had detectable levels of residual alpha-L-idurono-2-sulphate sulphatase activity which were increased after the transplants, although these changes were of inconstant size and their time course was not consistently related to the transplantations. Cytogenetic studies in cases where the donor was of the opposite sex detected only cells of the recipient's sex among the fibroblasts grown from biopsies of the transplantation sites. The technique used would have detected a donor to recipient cell ratio of 1:100. We found no consistent long-term trends in the excretion patterns of glycosaminoglycans and oligosaccharides from either a quantitative or qualitative point of view which could be specifically related to the transplantation. The combined administration of immunosuppressive doses of prednisolone and azathioprine was associated with an increased excretion of the lower molecular weight glycosaminoglycans. We conclude that fibroblast transplantation is not therapeutically useful in the diseases studied.