Mannose 6-phosphate-independent endocytosis of beta-glucuronidase by human fibroblasts. I. Evidence for the existence of a membrane-binding activity.

Prior work has shown that endocytosis of bovine beta-glucuronidase by human fibroblasts can be mediated by the existence of a Man6P-independent receptor for the recapture and targeting to lysosomes. In this study, we have isolated a peptide (IIIb2) from pronase digested bovine beta-glucuronidase that behaved as competitive inhibitor of the endocytosis of bovine beta-glucuronidase by human fibroblasts. This peptide contained a Ser-X-Ser sequence, where X is probably a posttranslational modified Trp. Antibodies raised against this peptide impaired the endocytosis of the bovine but not the human beta-glucuronidase, implying that the new recognition marker for the endocytosis of acid hydrolases might reside in a single discrete stretch of amino acid sequence. On the other hand, bovine beta-glucuronidase has been shown to bind specifically to receptors of human fibroblast membranes. The binding was saturable, divalent cation-dependent and was competitively inhibited by the IIIb2 peptide, but not by mannose 6-phosphate. Results presented suggested an interplay between manganese concentrations, temperature and pH on the dissociation of the beta-glucuronidase-receptor complexes. All together, these data reinforce the presence of two endocytic systems for the recapture and targeting of beta-glucuronidase in human fibroblasts.

Mannose 6-phosphate-independent endocytosis of beta-glucuronidase. II. Purification of a cation-dependent receptor from bovine liver.

A new binding protein, which recognizes a specific peptide sequence from pronase digested bovine beta-glucuronidase, has been isolated from bovine liver membranes. Prior work has shown that this peptide (IIIb2) contains a Ser-X-Ser sequence, where X might be a posttranslational modified Trp. This receptor was detergent-extracted from total bovine liver membranes and purified by affinity chromatography on a bovine beta-glucuronidase-Sepharose and a IIIb2 peptide-Sepharose column. Binding of bovine beta-glucuronidase to the isolated receptor requires divalent cations, and their presence was necessary to maintain the receptor-ligand complex. Only the peptide sequence containing the fraction IIIb2 was able to impair the binding of the bovine enzyme to the receptor, no other peptide from bovine beta-glucuronidase had an effect on binding. When analyzed by SDS-PAGE under reducing conditions, two bands were observed, a major band of 78 kDa and a faint band of 72 kDa. Rabbit antibodies against this binding protein revealed the presence of the 78 kDa protein in membranes from bovine liver, human and bovine fibroblasts. These antibodies impaired human fibroblasts endocytosis of the bovine but not of the human beta-glucuronidase, which is taken up by a 300 kDa receptor that recognizes phosphomannosyl moieties in the enzyme.

Urinary glycosaminoglycan excretion in healthy subjects and in patients with mucopolysaccharidoses.

BACKGROUND: The mucopolysaccharidoses (MPS) are a group of lysosomal storage disorders caused by deficiency of enzymes catalyzing the stepwise degradation of glycosaminoglycans (GAGs), and are transmitted in an autosomal recessive manner, except for Hunter syndrome. METHODS: The levels of GAGs in 150 healthy subjects and 33 patients with MPS were determined, and results were expressed as milligrams of GAGs per grams of creatinine. RESULTS: We found that this ratio decreased with age during the first 15 years of life, but had a constant low rate between the ages of 17-40 years in healthy individuals. A different tendency was present in patients with MPS, because levels of GAG excretion in this group were higher (by four standard deviations up) compared with healthy individuals. The electrophoretic patterns of urinary GAGs in healthy subjects showed that the higher levels detected in urine were chondroitin sulfate (4 and 6) and a smaller quantity of dermatan sulfate, but in each MPS type its characteristic pattern was identified. CONCLUSIONS: This is a simple, reproducible method suitable for routine laboratory separation, identification, and quantity of urinary GAGs and for diagnosing MPS syndromes.

Mucopolysaccharidoses type II: enzymatic activity and quantitative and qualitative studies of urinary glycosaminoglycans in five patients.

Five patients presenting Hunter's syndrome were biochemically studied. Quantification of urinary glycosaminoglycans (GAGs), electrophoretic characterization and correlation with enzymatic activity in leucocytes were carried out. In all cases, urinary GAGs/creatinine ratio was increased. Electrophoresis revealed the presence of heparan sulfate (HS) and dermatan sulfate (DS) in four cases (80%), but in the remaining patient, only DS was present. In all patients, deficient enzymatic activity was demonstrated. These results show evidences of biochemical differences in this syndrome.

The residues Leu(Ile)475-Ile(Leu, Val, Ala)476, contained in the extended carboxyl cytoplasmic tail, are critical for targeting of the resident lysosomal membrane protein LIMP II to lysosomes.

LIMP II, a type II lysosomal integral membrane protein, and the CD36/LIMP II construct are targeted to lysosomes by means of a signal expressed in the tyrosine-lacking carboxyl cytoplasmic tail of LIMP II (Vega, M. A., Rodriguez, F., Seguí, B., Calés, C., Alcalde, J., and Sandoval, I. V. (1991) J. Biol. Chem. 266, 16269-16272; Vega, M. A., Seguí-Real, B., Garcia, J. A., Calés, C., Rodriguez, F., Vandekerckhove, J., and Sandoval, I. V. (1991) J. Biol. Chem. 266, 16818-16824). Substitution of Leu475 with Ile resulted in a decreased efficiency of targeting. Mutant forms produced by substituting Leu475 by hydrophobic residues with either large (Val) or small (Ala, Gly) side chains, or by a charged residue (Asp), showed inhibited targeting. In contrast, the contiguous Ile476 residue could be replaced by either Leu, without loss in the efficiency of targeting, or by Val or Ala, with some impediment. Substitution of Ile476 by either Gly or Asp inhibited completely the targeting. The addition of the sequence Ser-Trp-Asp to the carboxyl end of the construct did not interfere with targeting. Data from 1H NMR analysis of the icosapeptide corresponding to the carboxyl cytoplasmic tail of LIMP II indicated the predominance of structures with extended random coil conformations, suggesting that the targeting signal is contained in a domain with an extended configuration.

Isolation of an inhibitor for the independent mannose 6-phosphate receptor endocytosis of bovine beta-glucuronidase by human fibroblasts.

Adsorptive endocytosis of bovine beta-glucuronidase by human fibroblasts is mediated by two different membrane receptors: one recognizes phosphomannosyl residues on the enzyme, the other is yet a undefined recognition marker (A. González-Noriega, R. Coutiño, V. M. Saavedra, and R. Barrera (1989) Arch. Biochem. Biophys. 268, 649-658). We have purified a bovine liver inhibitor for the endocytosis of the bovine beta-glucuronidase mediated by the recently proposed recognition marker. The inhibitor is partially susceptible to periodate oxidation, can be released from a peptide backbone by mild alkali treatment, can be reduced by sodium borohydride, and can be adsorbed to anionic but not to cationic resins. Although the chemical structure of the isolated marker has not been determined, results indicate a 122-Da molecule which may contain amino alcohol groups and may be found in a 1800-Da glycosidic chain.

[Hereditary lysosomal diseases in Mexico. III. Laboratory diagnosis of sphingolipidosis]. / Enfermedades hereditarias lisosomales en México. III. Diagnóstico de laboratorio para esfingolipidosis.

Enzymatic determinations of the levels of lysosomal enzymes in serum or leukocytes samples have been carried out for the diagnosis of 7 sphingolipidosis. This methodology has allowed us to study 49 homozygotes and 33 close relatives at risk for the carrier state of a particular sphingolipidosis. So far we have diagnosed: 21 Gaucher's disease patients, 17 metachromatic leukodistrophy, 4 Niemann-Pick, 4 GM2 gangliosidosis, 2 Fabry and one GM1 gangliosidosis. Limitations in the performance and interpretation of the levels of the defective enzyme in heterozygotes, homozygotes and those variants not detected with the assays described are discussed.

[Hereditary lysosomal diseases in Mexico. II. Laboratory diagnosis of mucopolysaccharidosis and mucolipidosis]. / Enfermedades hereditarias lisosomales en México. II. Diagnóstico de laboratorio de mucopolisacaridosis y mucolipidosis.

Methods suitable for the diagnosis of the mucopolysaccharidoses and mucolipidosis using urine, serum and leucocytes are presented. The methodology includes a screening technique for the mucopolysaccharidoses, a determination of glycosaminoglycans excreted in urine, serum and leucocyte enzymatic determinations for deficient lysosomal enzymes in lysosomal storage diseases. Their use is validated in the diagnosis of 19 patient with mucopolysaccharidoses and 5 with mucolipidoses, and to establish a carrier state in 10 close relatives.

Association of pancreatic biotinidase activity and intestinal uptake of biotin and biocytin in hamster and rat.

Pancreatic biotinidase activity was higher in hamster than in rat; these results were reversed in plasma. Uptake was studied in everted intestinal rings. Saturation kinetics at 37 degrees C were observed for biotin in hamster and for biocytin in rat, with a Vmax of 1.83 and 1.05 nmol min-1 ml-1 and an apparent Kt of 25.14 and 40.7 microM, respectively. Biotin uptake by hamster intestine was reduced at 4 degrees C and when choline or potassium replaced sodium; it was inhibited by biocytin only at very high concentrations. Biocytin uptake in the rat was small compared to passive diffusion and was not influenced by sodium or temperature; it was not inhibited by biotin. We observed only passive diffusion of biotin in rat and of biocytin in hamster. Our results suggest that protein-bound biotin may be absorbed mainly in its free form in the hamster. In the rat, on the other hand, at least part of the dietary biotin may be absorbed lysine-bound, as biocytin.

[Hereditary lysosomal diseases. I. Initial results of a diagnostic program in Mexico]. / Enfermedades hereditarias lisosomales. I. Resultados iniciales del programa para su diagnóstico en México.

Results obtained by the Lysosome Storage Disease Diagnostic Program are described as well as the criteria used for the selection of patients to be studied. This program was started in 1983 and is sponsored by the National Reference Center for the Detection and Diagnosis of Inborn Errors of Metabolism in Mexico City. Laboratory tests include chemical determinations of urinary glycosaminoglycans and enzymatic assays of 15 lysosomal enzymes that allow the identification of 25 of the 35 lysosomal storage diseases known. A total of 259 patients with clinical phenotypes suggesting a lysosomal storage disease, and 47 individuals at risk for the carrier state were studied. The disease diagnosed were 35 patients with mucopolysaccharidoses, 27 with sphingolipidoses, 5 with mucolipidoses and 7 with glycogenoses; the most common lysosomal storage disease was Gaucher followed in decreasing frequency by Morquio, Hunter and glycogenoses Ia. The carrier state was confirmed in 29 of close relatives, one of them confirmed prenatally.

Adsorptive endocytosis of lysosomal enzymes by human fibroblasts: presence of two different functional systems that deliver an acid hydrolase to lysosomes.

Endocytosis of human spleen beta-glucuronidase by human fibroblasts can be completely impaired by the competitive inhibitor mannose 6-phosphate or by pretreatment with acid phosphatase or endoglycosidases H or F. However, endocytosis of bovine spleen and liver beta-glucuronidase is partially impaired by the same treatments, suggesting that the bovine enzyme contains two endocytosis recognition markers located in separate enzyme domains. The mannose 6-phosphate recognition marker seems to be responsible for approximately 23% of the bovine enzyme endocytosis. The existence of two lysosomal endocytosis systems in human fibroblasts is supported by the following facts: (a) the rate of endocytosis of mannose 6-phosphate-containing human beta-glucuronidase was not affected by the presence of high levels of the bovine enzyme (which has only the other marker). (b) Anti-215K mannose 6-phosphate receptor antibodies selectively impair the endocytosis of the beta-glucuronidase containing mannose 6-phosphate. (c) Weak bases exert a differential effect on human and bovine endocytosis. beta-Glucuronidase internalized by either system is targeted to secondary lysosomes of human beta-glucuronidase-deficient fibroblasts, where it is able to degrade accumulated glycosaminoglycans. These results suggest that human fibroblasts have two different and independent endocytic systems for targeting of acid hydrolases to lysosomes.

Subcellular distribution of the enzymes degrading thyrotropin releasing hormone and metabolites in rat brain.

In order to further understand the role of enzymes degrading Thyrotropin Releasing Hormone (TRH, pglu-his-proNH(2)) and metabolites, we studied their subcellular distribution in rat brain. Brain tissue was homogenized in 0.32 M sucrose, tris-HCl 0.01 M pH 7.4 and fractionated by differential and discontinuous gradient centrifugation; [(3)H]pro-TRH was incubated with the various subcellular fractions and the extent of degradation of each metabolite was measured after separation by thin layer chromatography. Several markers were simultaneously measured (lactate dehydrogenase, 5?-nucleotidase and hexosaminidase) to determine the pattern of distribution of the subcellular organelles. The post-proline cleaving enzyme responsible for pglu-his-pro formation and pyroglutamate amino-peptidase (which requires sulphydryl compounds for maximal activity) were found in cytosol but were barely detectable in the soluble component of synaptosomes; pyroglutamate aminopeptidase (dependent on metals) and post-proline dipeptidyl amino peptidase were found on the membranes of synaptosomes; imido peptidase was not enriched in any particular fraction. These data are consistent with the hypothesis that membrane-bound pyroglutamate aminopeptidase is responsible for TRH degradation once released into the synaptic cleft and that the post-proline dipeptidylaminopeptidase may participate in the extracellular catabolism of his-proNH(2) before it cyclizes to his-pro-DKP. They also suggest that post-proline cleaving enzyme and soluble pyroglutamate aminopeptidase may not play an important role in the regulation of TRH levels in nerve endings.

Phosphomannosyl-enzyme receptors in rat liver. Subcellular distribution and role in intracellular transport of lysosomal enzymes.

beta-Hexosaminidase B purified from human fibroblast secretions was used as a ligand to study phosphomannosyl-enzyme receptors in membranes from rat tissues. Enzyme binding to rat liver membranes was saturable, competitively inhibited by mannose 6-phosphate, not dependent on calcium, and destroyed by prior treatment of the hexosaminidase with either alkaline phosphatase or endoglycosidase H. Most (90%) of the phosphomannosyl-enzyme receptors were found in endoplasmic reticulum, Golgi apparatus, and lysosomes; 9.5% in the plasma membrane, and less than 1% in nuclei and mitochondria. Receptors were vesicle-enclosed in all fractions except plasma membrane. Receptors in the endoplasmic reticulum apparently were occupied by endogenous ligands, but most receptors in lysosomes and plasma membrane were unoccupied. Most of the endogenous beta-hexosaminidase was in lysosomes and was released from vesicles by detergent treatment. Displacement of the residual receptor-bound endogenous beta-hexosaminidase (mostly in endoplasmic reticulum and Golgi apparatus) from detergent-treated membranes by mannose 6-phosphate released high uptake enzyme with properties expected for phosphomannosyl-enzymes. Mannose 6-phosphate-inhibitable enzyme receptor activity was found in nine rat organs and correlated roughly with their lysosomal enzyme content. These data support a general model for lysosomal enzyme transport in which the phosphomannosyl-enzyme receptor acts as a vehicle for delivery of newly synthesized acid hydrolases from the endoplasmic reticulum to lysosomes.

Chloroquine inhibits lysosomal enzyme pinocytosis and enhances lysosomal enzyme secretion by impairing receptor recycling.

Adsorptive pinocytosis of acid hydrolases by fibroblasts depends on phosphomannosyl recognition markers on the enzymes and high-affinity pinocytosis receptors on the cell surface. In this study, beta-glucuronidase binding to the cell surface of attached fibroblasts was found to be saturable and inhibitable by mannose-6-phosphate (Man-6-P). Dissociation of cell-bound beta-glucuronidase occurred very slowly at neutral pH, but was greatly accelerated by lowering the pH below 6.0, or by exposure to Man-6-P. Comparison of the maximal cell surface binding and the observed rate of enzyme pinocytosis suggests that the pinocytosis receptors are replaced or reused about every 5 min. Enzyme pinocytosis was not affected by inhibition of new protein synthesis for several hours, suggesting a large pool of internal receptors and/or reuse of internalized receptors. Chloroquine treatment of normal human fibroblasts had three effects: (a) greatly enhanced secretion of newly synthesized acid hydrolases bearing the recognition marker for uptake, (b) depletion of enzyme-binding sites from the cell surface, and (c) inhibition of pinocytosis of exogenous enzyme. Only the third effect was seen in I-cell disease fibroblasts, which were also less sensitive than control cells to this effect. These observations are consistent with a model for transport of acid hydrolases that proposes that delivery of newly synthesized acid hydrolases to lysosomes requires the phosphomannosyl recognition marker on the enzymes, and intracellular receptors that segregate receptor-bound enzymes into vesicles for transport to lysosomes. This model explains how chloroquine, which raises intralysosomal pH, can disrupt both the intracellular pathway for newly synthesized acid hydrolases, and the one for uptake of exogenous enzyme by cell surface pinocytosis receptors.

Argininosuccinic acid synthetase deficiency in a hamster cell line and its complementation of argininosuccinic aciduria human fibroblasts.

Unlike normal human cells, cultured fibroblasts from patients with argininosuccinic aciduria cannot synthesize arginine from citrulline because they have a deficiency of argininosuccinic acid lyase (ASL). We have found that V79, a Chinese hamster cell line, cannot grow on citrulline. Although these cells show a normal uptake of citrulline and have levels of ASL comparable to a human cell line (HeLa) which can grow in citrulline-containing medium, V79 cells have less than 5% of the argininosuccinic acid synthetase (ASS) activity of HeLa and cannot convert citrulline to argininosuccinate and thence to arginine. When heterokaryocytes are formed between V79 and a human cell line derived from a patient with ASL deficiency, complementation takes place and citrulline is incorporated into cell protein, presumably after having been converted to arginine. This is the first time that a genetic defect of the urea cycle has been corrected in human cells.