Clara W. Hall

Hurler and Hunter Syndromes: Mutual Correction of the Defect in Cultured Fibroblasts

The biochemical defect of cultuired skin fibroblasts from Hurler or Hunter patients (faulty degradation of sulfated mucopolysaccharide, resulting in excessive intracellular accumulation) may be corrected if cells of these two genotypes are mixed with each other or with normal cells. The effect is mediated by substances released into the medium.

INHIBITION OF UDP-D-GLUCOSE DEHYDROGENASE BY UDP-D-XYLOSE: A POSSIBLE REGULATORY MECHANISM.

Abstract The formation of several sugar nucleotides has recently been shown to be subject to inhibition or activation by allosteric modifiers ( Kornfeld et , al , 1964 ; Melo and Glaser, 1965 ; Bernstein and Robbins, 1965 ; Preiss et , al , 1965 ; Ghosh and Preiss, 1965 ; Mayer and Ginsburg, 1965; Kornfeld, 1965; Kornfeld and Ginsburg, 1965). Data presented in this paper show that UDP- D -glucose dehydrogenases from pea cotyledons, calf liver and chick cartilage are strongly and specifically inhibited by UDP- D -xylose. As this sugar nucleotide has been found to arise in some organisms by decarboxylation of UDP- D -glucuronic acid ( Feingold et , al , 1960 ; Ankel and Feingold, 1964 ) the inhibition of UDPG ∗ dehydrogenases may represent another instance of regulation by feedback.

A β-glucuronidase deficiency mucopolysaccharidosis: studies in cultured fibroblasts

Cultured skin fibroblasts derived from a patient with an atypical mucopolysaccharidosis are severely deficient in β-glucuronidase (β- d -glucuronide glucuronohydrolase). These fibroblasts display an, excessive accumulation and lengthened turnover time of sulfated mucopolysaccharide. The abnormal mucopolysaccharide metabolism can be corrected by addition of bovine β-glucuronidase to the culture medium. Identity of corrective factor and β-glucuronidase was demonstrated by coelectrophoresis of the two activities in polyacrylamide gel. Cells are able to take up β-glucuronidase in amounts far exceeding the requirement for full correction.

α-l-Iduronidase activity in cultured skin fibroblasts and amniotic fluid cells

Abstract Using phenyl-α- l -iduronide as substrate, we have examined the level of α- l -iduronidase activity in homogenates of fibroblasts derived from normal individuals, from patients affected with α- l -iduronidase deficiency disorders (Hurler syndrome, Scheie syndrome, and a disease of intermediate severity presumed to be a Hurler/ Scheie compound) and from parents of such patients. Extracts derived from the affected individuals had no detectable α- l -iduronidase activity, whereas those derived from heterozygotes varied between 20% and 95% of the normal mean. Overlap between normal and heterozygous levels was reduced if the α- l -iduronidase activity was expressed on the basis of the β-galactosidase activity in the same homogenate. Cultured amniotic fluid cells from normal pregnancies had less than half as much α- l -iduronidase activity as fibroblasts from normal adults; this might cause problems in distinguishing a heterozygous fetus from an affected one by the enzyme assay alone.

Inhibition of receptor-mediated uptake of a lysosomal enzyme into fibroblasts by chloroquine, procaine and ammonia.

Abstract Cultured human skin fibroblasts take up α- l -iduronidase by receptor-mediated pinocytosis. Certain lysosomotropic amines such as chloroquine, ammonia and procaine inhibit this process, without affecting the fluid endocytosis of dextran. In contrast to the competitive inhibition by mannose 6-phosphate, the inhibition by amines is non-competitive and is therefore presumed not to affect binding of the enzyme to receptors. The dose response curves are very steep, and equations that best fit the data use a power of inhibitor concentration ( i 2 for procaine, i 4 for chloroquine), indicating interaction of several amine molecules at the inhibitory site(s). The inhibition is reversed by removal of the amine from the medium and does not result from accelerated efflux of endocytosed enzyme. We suggest that the amines interfere with delivery of receptor-bound enzyme to lysosomes.

Glycosylation of serine residues by a uridine diphosphate-xylose, protein xylosyltransferase from mouse mastocytoma.

Abstract A preparation from an ascitic mast cell tumor catalyzes the transfer of xylose from UDP-xylose-14C to serine residues of an endogenous protein acceptor. The formation of xylosyl-serine linkages, such as occur in native heparin, is based on the following evidence. Though not esterified, the radioactivity could readily be released by mild alkali; xylitol-14C was the only radioactive product obtained by treatment with 0.5 n NaOH in the presence of NaBH4. Pronase digestion of the transfer product released a radioactive glycosyl-amino acid identified as 14C-xylosyl-serine by coelectrophoresis with authentic material by co-chromatography on paper and on Dowex-50, and by co-chromatography of the DNP derivatives. The transfer takes place in the absence of protein synthesis, suggesting that glycosylation of proteins begins only after assembly of the polypeptide. The mastocytoma contains a second xylosyltransferase, which links xylose to an unidentified acceptor, to give a relatively alkali-stable xyloside.

Biochemical heterogeneity of the sanfilippo syndrome: Preliminary characterization of two deficient factors

Abstract Fibroblasts cultured from the skin of Sanfilippo patients show excessive accumulation and prolonged turnover time of sulfated mucopolysaccharide. This abnormality can be corrected by a macromolecular factor contained in the secretions of fibroblasts of differing genotype, as well as in normal human urine. By cross-correction tests, the Sanfilippo fibroblasts can be subdivided into two groups, each deficient in a different factor. Analytical polyacrylamide gel electrophoresis shows the two factors, which are probably proteins, to have a similar molecular weight (ca. 200,000) but to differ in charge at pH 8.5.

Incorporation of D-xylose-C14 into glycoprotein by particles from hen oviduct

Abstract D-Xylose, once thought to be restricted to polysaccharides of plants and microorganisms, has recently been found in a number of macromolecules of animal origin. This pentose occupies a unique position in chondroitin through a xylosyl-serine linkage ( Gregory et al ., 1964 ; Lindahl and Roden, 1965 ; Roden and Lindahl, 1965 ). Free xylosyl-serine has been isolated from human urine ( Tominaga et al ., 1965 ). Xylose ∗ has been reported to be a constituent of mucopolysaccharides of mammalian brain ( Wardi et al ., 1965 ), and of glycoproteins from human lung ( Masamune et al ., 1957 ), cancerous ascitic fluid ( Sugimoto, 1956 ), placenta ( Tomoda and Murayama, 1965 ) and aorta ( Klemer and Burbaum, 1965 ), and from cockroach plasma (Lipke et al ., 1965). UDP-Xylose, the probable precursor of such glycosidically-bound xylose, has been isolated from the milk of sows and goats, and from cow colostrum ( Kobata and Ziro, 1965 ), while its biosynthesis by decarboxylation of UDP-glucuronic acid has been demonstrated in preparations from hen oviduct ( Bdolah and Feingold, 1965 ). The present communication describes a particulate system from the oviduct of laying hens, which catalyzes the transfer of xylose from UDP-xylose into glycoprotein.

Transfer of N-acetylneuraminic acid to incomplete glycoproteins associated with microsomes

Abstract Rat-liver microsomes incubated with 14C -labeled CMP- N -acetylneuraminic acid (CMP-NANA) transfer N -acetylneuraminic acid (NANA) to an endogenous acceptor. The K m for CMP-NANA is 2·10 −6 M. The reaction stops when acceptor sites are filled (50 μμmoles/g liver). The transfer is reduced by 85% if the rats are treated with puromycin 45 min before removal of the liver, but not after exposure to puromucin in vitro . Transfer to a neuraminidase-treated glycoprotein is not affected by the puromycin injection indicating that the endogenous acceptor is depleted after treatment with the antibiotic. The product of endogenous transfer remains associated with microsomes, from which it can be solubilized by detergents or sonication. Sonic extracts give rise to radioactive precipitin lines with antiserum against rat plasma glycoprotein lacking NANA residues.

The relationship of α-l-iduronidase and Hurler corrective factor

Abstract Hurler corrective factor (the protein that normalizes the faulty mucopolysaccharide catabolism of fibroblasts derived from Hurler patients) was previously identified as the enzyme α- l -iduronidase. However, not all α- l -iduronidase functions as Hurler corrective factor. The corrective and noncorrective forms of the human urinary enzyme have been separated from each other by chromatography on Sepharose-bound heparin. They have similar catalytic properties, some difference in ability to bind to the galactose-specific lectin of castor bean, and a significant difference in molecular weight (87,000 and 67,000 for the corrective and noncorrective forms, respectively). Only the corrective form is efficiently internalized by Hurler fibroblasts.