Hyalobiuronic acid, a glucuronido glucosamine earlier isolated from hydrolysates of hyaluronic acid from umbilical cord, has been converted to its heptaacetyl methyl ester and its N-acetyl derivative. Esterification of the disaccharide, oxidation of the glucosamine residue to glucosaminic acid, and reduction of the uronic ester residue to a glucose residue yields a crystalline glucosidoglucosaminic acid. This is oxidatively deaminated to give a glucosidoarabinose, isolated as its crystalline heptaacetate, identical with the heptaacetate VI1 obtained by Zernplen degradation of laminaribiose (VIII). Hyalobiuronic acid is thus 3-O-(~-~-glucopyranosyluronic acid)-2-amino-2-deoxy-D-g~ucose (I). That its N-acetyl derivative I1 is the basic repeating unit of hyaluronic acid linked linearly in the polymer, probably by 3-0-(2-acetamido-2-deoxy-j3-~-g~ucopyranosyl) linkages (Fig. 2 ) , follows from earlier hydrolytic and enzymatic experiments, and from periodate oxidation data in the literature. A modification of the hydroxamic acid test suitable for sugar esters is described. (24) We are indebted to Prof. E. L. Hirst for seed material. (25) W. E. Trevelyan and J. S. Harrison, Biochcm. J . , 60, 298 (1 952). (26) W. hlcjhnrini. Z . p h y s i o l . Chmi., '268, 117 (193!)).

Skin fibroblasts cultured from patients affected with the Hurler or Scheie syndromes (mucopolysaccharidoses I or V, respectively) have a functional deficiency of a protein required for catabolism of sulfated mucopolysaccharide that has been designated the "Hurler corrective factor." We now show Hurler factor purified from normal human urine to be associated with a-Liduronidase activity. Cell lines deficient in Hurler corrective factor have no detectable activity of cv-L-iduronidase (less than 3% of that found in cells from individuals of other genotypes). Such correspondence indicates that Hurler corrective factor and a-L-iduronidase are the same entity. Correction of deficient cells is accompanied by an efficient uptake of a-L-iduronidase from the medium.The Hurler syndrome (mucropolysaccharidosis I) is the most striking and best known of the inborn errors of mucopolysaccharide metabolism. Lysosomal deposits of mucopolysaccharide, found in nearly all cells, are no doubt responsible for the severe clinical manifestations that include skeletal deformities, hepatosplenomegaly, cloudy corneas, stunting of physical and mental growth, and cardiovascular pathology (1, 2). The mucopolysaccharides stored in the lysosomes and excreted in the urine are fragments of dermatan sulfate and heparan sulfate (2).Fibroblasts cultured from the skin of Hurler patients likewise accumulate excessive mucopolysaccharide (3-5) because of the deficiency of a specific protein required for degradation (6). When this protein (designated "Hurler corrective factor") is supplied exogenously, mucopolysaccharide catabolism of Hurler fibroblasts is normalized.Hurler corrective factor, purified 1000-fold from normal human urine, has no effect on the mucopolysaccharide metabolism of fibroblasts cultured from normal individuals, nor from patients with several other mucopolysaccharidoses, namely, the Hunter, Sanfilippo, and Maroteaux-Lamy syndromes (7). However, cells from individuals affected with the Scheie syndrome (mucopolysaccharidosis V) and from some individuals with a phenotype intermediate between Hurler and Scheie, are likewise deficient in, and correctible by, the Hurler factor (7-9).Barton and Neufeld (7) reported that the Hurler factor assisted Hurler and Scheie cells in degrading intracellular mucopolysaccharide (primarily dermatan sulfate), but was distinct from the common lysosomal hydrolases such as f3-Dgalactosidase or ,3-D-glucuronidase. The recent chemical synthesis of phenyl a-riduronide has enabled us to test the factor for a-iiduronidase activity. a--Iduronidase (a-Liduronide iduronohydrolase) activity has been detected in homogenates of human liver and cultured skin fibroblasts (10), and a preliminary characterization has been presented for the enzyme extracted from rat-liver lysosomes (11). A deficit of a--iduronidase in cells from individuals with the Hurler syndrome has been reported by Matalon et al. (10).We now present evidence for the identity of Hurler corrective factor and a-i-iduronidase. The Hurler and Scheie...

Receptor-binding of "high-uptake" forms of lysosomal enzymes to human diploid skin fibroblasts had been predicted from the Michaelis-Menten kinetics' of uptake of these enzymes [e.g., Cell 12, [619][620][621][622][623][624][625][626][627]. We have now demonstrated such binding directly by using a sensitive assay for the bound enzyme. Cells deficient in a-L-iduronidase were detached from plastic dishes by mild trypsinization, allowed to'recover, and used in suspension. They were incubated with urinary a-L-iduronidase at 00C for 90 minutes and. theh washed by centrifugation through concentrated bovine serum albumin; the activity of the cell-associated enzyme was measured with 4-methylumbelliferyl a-L-iduronide as substrate. A Scatchard analysis showed 14,000 binding sites per cell and a Kd of 1 X 10-9 M for high-uptake a-L-iduronidase; binding of the low-uptake form was barely detectable. Mannose -phosphate, a known competitive inhibitor of uptake, inhibited the binding competitively, with Ki = 1 x 10-4 M. Unexpectedly, mannose 6-phosphate greatly accelerated the dissociation of bound enzyme. During uptake of a-L-iduronidase at 350C, the receptors were regenerated every few minutes, even in the absence of protein synthesis. Cultured human fibroblasts take up certain hydrolytic enzymes from the medium efficiently and selectively and incorporate them into lysosomes (1, 2). A receptor-binding step has been postulated from the Michaelis-Menten kinetics of the uptake (pinocytosis) process (3). Those isozymes that are pinocytosed most avidly, designated as the "high-uptake" forms, have a specific marker that is recognized by the cells (4-6). Kaplan et al. (7) first suggested the structure of the marker to be mannose 6-phosphate or a structural analog thereof; this proposal was based on the competitive inhibition of f3-glucuronidase uptake by mannose 6-phosphate and phosphomannans and on the decrease of uptake after the enzyme was treated with phosphatase. Similar data and hypotheses have been reported for the uptake of a-L-iduronidase (3), 3-N-acetylhexosaminidase (8,9), and several other hydrolytic enzymes (9).'Chemical and radioisotopic evidence for the presence of phosphorylated mannose in certain lysosomal enzymes has been presented in preliminary form (10, 11). The recognition of hydrolytic enzymes by specific receptors has been proposed as an essential step in the insertion of these enzymes into lysosomes by way of secretion and subsequent recapture (1, 2).In contrast to the rapid progress on the structure of the enzyme marker, studies of the receptor have been lagging, hampered by the lack of a suitable assay for bound enzyme. Our previous attempts to radiolabel a-L-iduronidase failed because the high-uptake form of the enzyme was impure as well as unstable to iodination. This problem has now been overcome by use of a fluorometric assay for a-L-iduronidase (12), sensitive enough to measure the activity of less than 1 fmol of bound enzyme. a-L-Iduronidase was purified from normal human urine on heparin-Se...

No abstract

No abstract

No abstract

No abstract

No abstract