Accumulation of sulfate-containing acid mucopolysaccharides in I-cell fibroblasts.

A rapid and sensitive papper electrophoretic assay for 35SO4-containing compounds was developed which allowed measurement of 35S-acid mucopolysaccharides synthesized by skin fibroblasts grown in the presence of inorganic 35S-sulfate. Fibroblasts from a skin explant of a patient with I-cell disease when grown in culture accumulated abnormal amounts of 35S-acid mucopolysaccharides and other, as yet unidentified, 35S-labeled compounds. Approximately 75% of the 35S-compounds accumulated by I-cell fibroblasts were not metabolized and remained in the cells after transfer to nonlabeled medium. I-cell fibroblasts differ from fibroblasts derived from classical mucopolysaccharidoses such as Hurler's and Hunter's syndromes in the amount and types of 35S-labeled acid mucopolysaccharides accumulated. I-cell fibroblasts accumulated chondroitin 4- and 6-sulfates (16 per cent), dermatan sulfate (32 per cent), heparan sulfate (32 per cent), and other unidentified 35S-compounds. The unidentified fraction was not hydrolyzed by microbial chondroitinase or heparinase. Attempts to correct the defect in I-cell fibroblasts by growth in the presence of extracts of normal cells resulted in release of only 10 per cent of the accumulated mucopolysaccharides. Under the same conditions, Hurler and Hunter fibroblasts lost over 90 per cent of accumulated mucopolysaccharides.

Biosynthesis of chondroitin sulfate proteoglycan. Subcellular distribution of glycosyltransferases in embryonic chick brain.

The subcellular distributions of five glycoslytransferases involved in the biosynthesis of the chondroitin sulfate proteoglycan and of a sixth glycosyltransferase, presumably involved in glycoprotein biosynthesis, were examined in 13-day chick enbryo brain. Fractionation studies performed by the procedure of Gray and Whittaker (Gray, E.G., and Whittaker, V.P. (1962) J. Anat. (London) 96, 79-88) revealed that three of the six enzymes were directly associated with the membrane fraction of synaptosome-enriched preparations; varying amounts of the remaining glycosyltransferases were distributed between the 100,000 times g supernatant and the synaptosome-enriched fraction after differential and density gradient centrifugation of crude chick brain homogenates. The time of appearance of three of the glycosyltransferases was examined in chick embryo brain tissue at several stages of development. The brain content of each glycosyltransferase increased rapidly between day 7 and hatching at day 21. A sharp decline in each of the glycosyltransferase activities occurred at hatching.

Isolation and characterization of mannose 6-phosphate/insulin-like growth factor II receptor from bovine serum.

A convenient means was devised for the purification of milligram quantities of a soluble form of the mannose 6-phosphate/insulin-like growth factor II receptor (Man-6-P/IGF II receptor). The receptor was purified to near homogeneity from bovine serum by affinity chromatography on agarose-pentamannosephosphate in the absence of detergent. Approximately 2.5 mg of receptor were obtained from 500 ml of fetal calf serum. The concentration of receptor in serum decreased sharply with development. Fetal calf serum Man-6-P/IGF II receptor was immunologically similar to detergent-solubilized, membrane-bound Man-6-P/IGF II receptor from bovine liver. N-Terminal sequence analysis revealed that the purified serum receptor, but not the solubilized, membrane-associated receptor, contains stoichiometric amounts of bound IGF II. The results of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel chromatography studies suggest that the fetal calf serum receptor (in contrast to the solubilized, membrane-bound bovine testis receptor) does not aggregate. The affinity of the fetal calf serum receptor for bovine testis beta-galactosidase approximated one-half that observed for solubilized, membrane-bound bovine testis receptor.

The aggregation and dissociation properties of a low molecular weight mannose 6-phosphate receptor from bovine testis.

The aggregation state of low molecular weight mannose 6-phosphate receptor from bovine testis was determined in membrane preparations and in purified soluble preparations. The effect of aggregation on binding of the receptor to immobilized pentamannose 6-phosphate was also examined. Nonreducing SDS-PAGE followed by immunoblotting revealed that interchain disulfide bonds exist in detergent-solubilized and purified receptor preparations, but not in membrane-associated receptor. Reduction of the receptor with dithiothreitol abolished its ligand binding activity and drastically altered its ability to bind antibodies. The results of receptor crosslinking and molecular sieving chromatography studies suggest that the receptor exists in membranes as a noncovalently linked dimer and in solution as oligomeric forms, largely as a tetramer. The formation of the tetramer is affected by the concentration of the receptor, but not by its solubilization from membranes with detergent, nor by the presence of mannose 6-phosphate. Mono-, di-, and tetramer forms of 125I-labeled receptor were separated by molecular sieving chromatography and examined for their ability to bind to immobilized ligand, agarose-pentamannose-phosphate. The order of binding observed was tetramer greater than dimer greater than monomer. Binding of the monomer and dimer to immobilized ligand was dependent on the presence of divalent cations while the tetramer had little requirement for divalent cations.

Immobilization and assay of low-molecular-weight phosphomannosyl receptor in multiwell plates.

A novel sensitive binding assay for quantitation of a low-molecular-weight phosphomannosyl receptor (41-46 K) was devised. The receptor was immobilized by immunochemical means in the wells of polystyrene multiwell plates. The lysosomal enzyme ligand, testicular beta-galactosidase, was added and receptor-bound beta-galactosidase was measured by conventional colorimetric analysis using p-nitrophenyl beta-galactoside as substrate. Inhibitors of the binding of beta-galactosidase to the receptor were removed prior to addition of beta-galactosidase and did not interfere with the assay. Low-molecular-weight phosphomannosyl receptor was readily quantitated in the range of 4 to 100 ng of receptor protein. Binding of beta-galactosidase to the receptor was specifically inhibited by 5 mM mannose 6-phosphate. The receptor exhibited optimum binding of beta-galactosidase at pH 5.7 and was saturated with beta-galactosidase at 320 munits/ml in the presence of 20 mM MnCl2. The requirement for MnCl2 was greatly diminished at higher concentrations of beta-galactosidase. Application of the assay procedure to the quantitation of the low-molecular-weight phosphomannosyl receptor in mammalian tissues is discussed.

Bovine testicular beta-galactosidase: purification of enzyme fractions that exhibit high affinity for phosphomannosyl receptors.

An improved method is described for the preparation of bovine testicular beta-galactosidase that allows the isolation of enzyme fractions that bind avidly to phosphomannosyl receptors. The procedure permits removal of a contaminating beta-hexosaminidase and yields nearly homogeneous beta-galactosidase. Enzyme eluted from DEAE-Sephacel was arbitrarily divided into pools that exhibited differing ability to bind phosphomannosyl receptors. A high binding fraction was rapidly assimilated by cultured cells and bound to both low and high molecular weight phosphomannosyl receptors. Carbohydrate analysis of the high binding fraction indicates an average content of one complex and one high mannose oligosaccharide chain per molecule and an average mannose 6-phosphate content of two residues per molecule. However, electrofocusing studies indicated that all the fractions were heterogeneous with respect to sialic acid and phosphate content. The purification procedure also provides highly purified beta-galactosidase suitable for removing beta-galactosidase residues from a variety of complex carbohydrates.

The binding specificity of high and low molecular weight phosphomannosyl receptors from bovine testes. Inhibition studies with chemically synthesized 6-O-phosphorylated oligomannosides.

A series of chemically synthesized oligomannosides that contain mannose 6-phosphate residues were utilized as inhibitors of the binding of beta-galactosidase to high (CI-MPR, 215 kDa) and low (CD-MPR, 41-46 kDa) molecular mass mannose 6-phosphate receptor from bovine testes in order to probe the specificity of each receptor. Mannobioside phosphorylated in the terminal position and linked alpha(1,2) was a 6-fold better inhibitor than the corresponding alpha(1,3)- and alpha (1,6)-linked isomers. Inhibition observed with a monophosphorylated alpha(1,2)-linked mannotrioside was approximately 6-fold greater than that with the corresponding mannobioside. Penultimate glycosidic linkages of the oligomannosides played little or no role in the inhibition of binding of ligand to the receptors. Monophosphorylated oligomannosides containing phosphomonoester groups on penultimate mannose residues were not inhibitors. Binding inhibition observed for biantennary oligomannosides with phosphate on terminal mannose residues of either alpha(1,3) or alpha(1,6) chains closely approximated the values obtained with analogous trimannosides. A biantennary oligomannoside on which each antennary chain contained a terminal phosphate exhibited approximately an 8-fold greater inhibition than monophosphorylated compounds. Although the receptors exhibited similar relative specificities for phosphomonoesters, phosphodiesters did not inhibit binding of ligand to CD-MPR and only weakly inhibited binding to CI-MPR.