H Kresse

Metabolism of sulfated glycosaminoglycans in cultured endothelial cells and smooth muscle cells from bovine aorta.

The glycosaminoglycan metabolism of cultured endothelial cells and of cells grown from the intima and from the media layer of bovine aorta thoracia was investigated in a comparative study. The following results were obtained: 1. Endothelial cells have in common with intima and media cells the distribution of newly formed sulfated glycosaminoglycans into extracellular, pericellular and intracellular compartments. Endothelial cells, however, synthesize lower amounts of glycosaminoglycans and distribute them in a different ratio into the three pools. 2. Though all the various cell lines synthesize chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, heparan sulfate and small amounts of keratan sulfate, endothelial cells exhibit a unique distribution pattern of sulfated glycosaminoglycans in each of the three compartments. Generally, a high proportion of heparan sulfate and chondroitin 6-sulfate and a very low dermatan sulfate content was detected. 3. Heparan sulfate produced by endothelial cells has a higher N-sulfonate content when compared with that from other sources. The cell membrane-associated heparan sulfate, especially, exhibits some heparin-like features as judged by nitrous acid degradation and susceptibility towards heparitinase.

A sensitive procedure for the diagnosis of N-acetyl-galactosamine-6-sulfate sulfatase deficiency in classical Morquio's disease

The trisaccharide 6-sulfo-N-acetylgalactosamine-glucuronic acid-6-sulfo-N-acetyl-[1-3H]galactosaminitol was used as a substrate for the determination of N-acetylgalactosamine-6-sulfate sulfatase activity. The amount of liberated sulfate was measured indirectly by separating monosulfated reaction products from the substrate on Dowex 1 X 2 microcolumns in a simple two step procedure. Fibroblast homogenates from patients with various genotypes, except classical Morquios disease, released 410 +/- 90 pmol sulfate/h/mg cell protein. The enzyme exhibited a pH optimum of pH 4.8 and a KM of about 1 X 10(-4) mol/1. It was strongly inhibited by phosphate, sulfate and chloride ions. In three cell lines from patients with classical Morquios disease a residual activity between 1 and 2% of the mean normal activity was found. All cell lines tested released sulfate from 6-sulfo-N-acetylglucosamine-glucuronic acid-[1-3H]-anhydromannitol. Cell extracts from cultured amniotic fluid cells exhibited a N-acetylgalactosamine-6-sulfate sulfatase activity between 120 and 320 pmol/h/mg protein. An enzyme activity of 370 +/- 100 pmol sulfate/h/mg protein was found in peripheral leucocytes from healthy donors. The determination of N-acetyl-galactosamine-6-sulfate sulfatase activity in one family with an affected patient indicated that the enzyme deficiency is also expressed in leucocytes.

Quantitative Aspects of Pinocytosis and the Intracellular Fate of N-acetyl-α-D-glucosaminidase in Sanfilippo B Fibroblasts

The cellular uptake of N-acetyl-alpha-D-glucosaminidase, the deficient enzyme in Sanfilippo B disease, and the intracellular fate and metabolic effect of this enzyme have been investigated in Sanfilippo B and normal fibroblasts. For both genotypes the uptake is highly efficient (up to 0.025 mU/h/mg cell protein), specific and constant over a period of at least 6 days. It is probable that the enzyme protein is taken up by adsorptive pinocytosis. The enzymatic activity as well as the biological activity towards (35)SO(4)-labeled mucopolysaccharides persist in Sanfilippo B cells with a half-life of 34 h, indicating the intralysosomal localization of the pinocytosed enzyme. The data obtained are discussed with regard to a possible enzyme replacement therapy. For Sanfilippo B disease the doses used in the past are considered to be insufficient to cause measurable effects.

Multiple deficiency of mucopolysaccharide sulfatases in mucosulfatidosis.

Summary: Fibroblasts of four patients affected with mucosulfatidosis (multiple sulfatase deficiency, Austin variant of metachromatic leukodystrophy) were assayed for activities of the five sulfatases known to degrade mucopolysaccharides. These were iduronide 2-sulfate sulfatase, sulfamidase, N-acetyl-galactosamine 6-sulfate sulfatase, arylsulfatase B (N-acetylgalactosamine 4-sulfate sulfatase), and N-acetylglucosamine 6-sulfate sulfatase. The activities of these five sulfatases were severely depressed, thus confirming the known deficiency of arylsulfatase B and the absence of the Hunter and Sanfilippo III A corrective factors that have iduronide 2-sulfate sulfatase and sulfamidase activity, respectively.Together with earlier reports on the deficiencies of arylsulfatases A and C, cholesteryl sulfatase, and dehydroepiandrosterone sulfatae, mucosulfatidosis is now characterized by the deficiency of nine different sulfatases.Speculation: Mucosulfatidosis is a disease characterized by the deficiency of multiple sulfatases in cultured fibroblasts in contrast to the deficiencies of singe sulfatases that are known for the five mucopolysaccharide degrading sulfatases and arylsulfatase A. The genes responsible for the expression of the sulfatases are located both on autosomes and the X-chromosomes. Mucosulfatidosis fibroblasts should provide an experimental model for the study of a hitherto unknown common mechanism responsible for the expression of sulfatase activities.

Uptake of hyaluronate by cultured cells.

[14C]hyaluronate is internalized by adsorptive pinocytosis by cultured rat hepatocytes and human synovial cells, but not by human skin fibroblasts and smooth muscle cells. Hyaluronate oligosaccharides compete for the uptake of hyaluronate by hepatocytes without being internalized themselves at the doses used. It is suggested that for adsorptive pinocytosis a hyaluronate molecule has to bind to at least two receptors on the cell membrane.

Sanfilippo A disease in the fetus

A pregnancy at risk for the Sanfilippo syndrome has been studied in which clear evidence was obtained from the study of amniotic fluid and fetal organs that the fetus was affected. Increased levels of heparan sulphate were found in amniotic fluid and fetal liver, while electronmicroscopy of cultured fetal fibroblasts and fetal liver showed abnormal cytoplasmic inclusions. 35SO4 uptake studies of cultured fetal cells showed abnormal intracellular accumulation of mucopoly saccharide, while both cultured amniotic cells and fetal skin fibroblasts demonstrated deficiency of heparin sulphamidase, the enzyme responsible for the `A subtype of the disease. It is suggested that by use of a combination of these methods Sanfilippo A disease can now be diagnosed reliably in utero.

The mucopolysaccharidoses: Biochemistry and clinical symptoms

SummaryThe mucopolysaccharidoses are a group of genetic diseases which are characterized by an excessive intralysosomal accumulation of partially degraded mucopolysaccharides. This storage is caused by the inactivity of one of eleven enzymes that are required for the degradation of the different types of mucopolysaccharides. There is a rough correlation between phenotype and chemical nature of the storage material. Similar clinical pictures, however, may be caused by an inactivity of different enzymes. Conversely, different clinical expressions of the defect of a single enzyme may be attributed to allelic mutations.The recent development of specific assay procedures for the respective enzymes allows 1. an early genotype-specific diagnosis of affected patients, 2. prenatal diagnosis of the metabolic defect in families at risk, 3. to prognosticate the course of the disease at least in some instances, and 4. genetic counselling for members of affected families. At present, there is no specific therapy. Attempts of enzyme replacement therapy are still at an experimental stage.ZusammenfassungDie Mucopolysaccharidosen stellen eine Gruppe genetischer Krankheiten dar, die durch eine intralysosomale Speicherung partiell abgebauter Mucopolysaccharide charakterisiert sind. Ursache der Speicherung ist die Inaktivität eines von elf verschiedenen Enzymen, die für den Abbau der verschiedenen Mucopolysaccharidtypen benötigt werden. Die Art des Speichermaterials korreliert annähernd mit dem klinischen Phänotyp. Ähnliche klinische Erscheinungsbilder können auf dem Defekt verschiedener Enzyme beruhen. Umgekehrt kann der Defekt eines einzelnen Enzyms beim Auftreten alleler Mutationen eine unterschiedliche klinische Expression zur Folge haben. Die Entwicklung spezifischer Methoden zur Aktivitätsbestimmung der betreffenden Enzyme ermöglicht 1. eine frühzeitige Genotyp-spezifische Diagnose erkrankter Patienten, 2. in Risikofamilien eine pränatale Erkennung des Stoffwechseldefektes, 3. in einzelnen Fällen eine Prognose über den Krankheitsverlauf und 4. eine genetische Beratung von Mitgliedern betroffener Familien. Eine kausale Therapie existiert zur Zeit nicht. Versuche zur Enzymsubstitution befinden sich noch im experimentellen Stadium.

Sanfilippo's disease type a: sulfamidase activity in peripheral leukocytes of normal, heterozygous and homozygous individuals

Abstract Sulfamidase activity was determined in peripheral leukocytes from normal individuals and from members of four families with patients suffering from Sanfilippos disease type A. [ N-sulfonate - 35 S]Heparin was used as the substrate. The following results were obtained: 1. 1. In normal leukocytes a pH optimum of pH 4.5 and an apparent K m = 1 × 10 −4 mol N-sulfonyl groups/l was found. Upon standing at room temperature a two-fold increase of sulfamidase activity was found within two weeks. According to mixing experiments this was most likely due to the formation of an activator. 2. 2. After separation of leukocytes in granulocytes and a lymphocyte-rich fraction, sulfamidase activity was 6–15 times higher in lymphocytes than in granulocytes, depending on the pH of the incubation mixture. 3. 3. Under strictly standardized assay conditions a mean activity of 66.8 ± 24.0 pmol sulfate release/h/mg protein was found in normal leukocytes. No age- or sex-dependent changes of the activity could be observed. 4. 4. Very low levels between 0 and 3% of the normal mean sulfamidase activity were found in six patients with Sanfilippos disease type A. 5. 5. In the leukocytes of their parents sulfamidase activity was reduced to 43 ± 16% of normal activity. Although there was an overlap between the activities of the groups of normal and obligate heterozygous individuals the probability of an individual being a carrier of the Sanfilippo A gene at a given sulfamidase activity could be calculated. On that basis, other family members were considered to be heterozygous carriers of the disease.

Serum α-N-acetylglucosaminidase: Determination, Characterization, and Corrective Activity in Sanfilippo B Fibroblasts

: Assays for the determination of serum alpha-N-acetylglucosaminidase (EC 3.2.1.50) activity are described employing p-nitrophenyl-N-acetyl-alpha-D-glucosaminide, phenyl-N-acetyl-alpha-D-glucosaminide, and UDP-N-acetylglucosamine as substrates. A log normal distribution of the serum enzyme activity was found. The determination of serum alpha-N-acetylglucosaminidase activity proved to be a valuable tool for the recognition of homozygous and heterozygous carriers of the Sanfilippo B gene.

Morquio's disease type A: Absence of material cross reacting with antibodies against N-acetylgalactosamine-6-sulfate sulfatase

SummaryAn antiserum was raised in guinea pigs against purified normal human N-acetylgalactosamine-6-sulfate sulfatase, the enzyme affected in Morquios disease type A. The antiserum precipitated most of N-acetylgalactosamine-6-sulfate sulfatase from a concentrate of normal human urine. The antigen-antibody complex was enzymatically active. Urine concentrates from five patients with Morquios disease type A did not contain material competing with the normal enzyme for binding to soluble or Sepharose-bound antibodies. No precipitin arc was obtained on immunodiffusion of antiserum and urine from the single patient investigated by this method. From the sensitivity of the indirect immunoassay it was concluded that the urine of the five patients contained less than 5% of the normal amount of cross-reacting material.