Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The lysosomal storage disorder galactosialidosis results from a primary deficiency of the protective protein/cathepsin A (PPCA), which in turn affects the activities of beta-galactosidase and neuraminidase. Mice homozygous for a null mutation at the PPCA locus present with signs of the disease shortly after birth and develop a phenotype closely resembling human patients with galactosialidosis. Most of their tissues show characteristic vacuolation of specific cells, attributable to lysosomal storage. Excessive excretion of sialyloligosaccharides in urine is diagnostic of the disease. Affected mice progressively deteriorate as a consequence of severe organ dysfunction, especially of the kidney. The deficient phenotype can be corrected by transplanting null mutants with bone marrow from a transgenic line overexpressing human PPCA in erythroid precursor cells. The transgenic bone marrow gives a more efficient and complete correction of the visceral organs than normal bone marrow. Our data demonstrate the usefulness of this animal model, very similar to the human disease, for experimenting therapeutic strategies aimed to deliver the functional protein or gene to affected organs. Furthermore, they suggest the feasibility of gene therapy for galactosialidosis and other disorders, using bone marrow cells engineered to overexpress and secrete the correcting lysosomal protein.
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PMID:Mouse model for the lysosomal disorder galactosialidosis and correction of the phenotype with overexpressing erythroid precursor cells. 759 Feb 40

Lysosomal protective protein/cathepsin A is a serine carboxypeptidase that forms a complex with beta-galactosidase and neuraminidase. The enzyme is synthesized as a 54-kDa precursor/zymogen and processed into a catalytically active 32- and 20-kDa two-chain form. We have expressed in baculovirus-infected insect cells the human one-chain precursor as well as the two separate subunits in order to establish the mode of catalytic activation of the zymogen and the assembly and activation of the two subunits. Infected insect cells synthesize large quantities of the exogenous proteins, which are glycosylated and secreted but not processed. Co-expression of the two subunits results in their assembly into a two-chain form of 34- and 20-kDa with negligible enzymatic activity. Limited proteolysis with trypsin of the 54-kDa precursor and the reconstituted 34- and 20-kDa form gives rise to a fully active 32- and 20-kDa product. These results enabled us to map the sites of proteolytic cleavage needed for full activation of the cathepsin A zymogen. They further indicate that the 34- and 20-kDa form is a transient processing intermediate that is converted into a mature and active enzyme by removal of a 2-kDa "linker" peptide from the COOH terminus of the 34-kDa subunit.
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PMID:Lysosomal protective protein/cathepsin A. Role of the "linker" domain in catalytic activation. 759 59

An enzyme hydrolyzing the carboxyl terminus of endothelin-1 was detected in control human tissues but was deficient in tissues from a patient with galactosialidosis, a metabolic disease caused by the protective protein gene mutation. It was proportional to the amount of immunologically estimated mature protective protein. An antibody against the lysosomal protective protein/beta-galactosidase complex precipitated the enzyme activity almost completely. Transfection of the human cDNA for protective protein resulted in high expression of the enzyme activity in transformed fibroblasts from a galactosialidosis patient. These results indicated that the mature protective protein is a major soluble endogenous endothelin degradation enzyme in human tissues.
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PMID:Protective protein as an endogenous endothelin degradation enzyme in human tissues. 782 72

Human lysosomal beta-galactosidase is organized as a 680-kDa complex with cathepsin A (also named carboxypeptidase L and protective protein), which is necessary to protect beta-galactosidase from intralysosomal proteolysis. To understand the molecular mechanism of beta-galactosidase protection by cathepsin A, we defined the structural organization of their complex including the beta-galactosidase-binding interface on cathepsin A. Radiation inactivation analysis suggested the existence of a 168-kDa structural subunit of the complex containing both beta-galactosidase and cathepsin A. Chemical cross-linking of the complex confirmed the existence of this subunit and showed that it is composed of one cathepsin A dimer and one beta-galactosidase monomer. The modeling of the cathepsin A dimer tertiary structure based on atomic coordinates of a wheat carboxypeptidase suggested a putative beta-galactosidase-binding cavity formed by the association of two cathepsin A monomers. According to this model two exposed loops of cathepsin A bordering the cavity were chosen as part of a putative beta-galactosidase-binding interface. Synthetic peptides corresponding to these loops were found both to dissociate the complex and to inhibit its in vitro reconstitution from purified cathepsin A and beta-galactosidase. The defined location of the GAL monomer in the complex with 35% of its surface covered by the CathA dimer may explain the stabilizing effect of CathA on GAL in lysosome.
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PMID:Human lysosomal beta-galactosidase-cathepsin A complex: definition of the beta-galactosidase-binding interface on cathepsin A. 787 22

Cathepsin A (also named "protective protein" and carboxypeptidase L) stabilizes beta-galactosidase and activates neuraminidase by forming with them a high-molecular-weight lysosomal complex. We determined the main forms of the supramolecular organization of human placental cathepsin A and the quantitative relationship between them, using an affinity chromatography on agarose-Phe-Leu for direct purification of cathepsin A. We found that cathepsin A in human placenta exists as the following three forms: a 1270-kDa complex with beta-galactosidase and neuraminidase (about 1% of total cathepsin A), a 680-kDa complex with beta-galactosidase (30-40% of total), and a free 98-kDa cathepsin A dimer (60-70% of total). All forms are in dynamic equilibrium with each other, but almost all placental beta-galactosidase is associated with cathepsin A in the 680-kDa complex. The main properties of free cathepsin A (including the capacity to associate with beta-galactosidase) were found to be identical to those of cathepsin A obtained by dissociation of the 680-kDa complex. The presence of a free cathepsin A pool in the lysosome is connected with its sixfold overproduction in the cell compared to beta-galactosidase and may be necessary to ensure cathepsin A proteolytic function in addition to its protective role for beta-galactosidase and neuraminidase in the lysosomal multienzymatic complex. Such a dual function of cathepsin A is also confirmed by our finding that it is the only carboxypeptidase of placenta extract able to catalyze the hydrolysis of both carbobenzoxy (CBZ)-Glu-Tyr and CBZ-Phe-Leu dipeptide substrates.
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PMID:Direct affinity purification and supramolecular organization of human lysosomal cathepsin A. 805 88

We propose a method to study multienzyme complex formation in vitro based on nondenaturing agarose gel electrophoresis. The enzymes with different isoelectric points (pI) were loaded at the opposite ends of the same lane of agarose gel and electrophoresis was performed at a pH value intermediate between their pI's. In cases where a complex of the enzymes was formed, an additional protein band of low electrophoretic mobility was found corresponding to the point where they crossed on the gel. This band contained both enzyme activities. The method was used to demonstrate association between two enzymes of the mitochondrial citric acid cycle, malate dehydrogenase and citrate synthase, and between the lysosomal hydrolases, beta-galactosidase and cathepsin A. Relative proportions of free and bound enzymes after electrophoresis suggest that interaction between the mitochondrial enzymes is relatively weak compared to that of lysosomal hydrolases. Microdensitometric scanning of countermigration electrophoresis gels was used to determine the stoichiometry of components in the complex.
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PMID:Demonstration of enzyme associations by countermigration electrophoresis in agarose gel. 808 91

The deficiency of the lysosomal protective protein/carboxypeptidase L (CARB L) causes the lysosomal storage disorder, galactosialidosis, characterized by neuraminidase and beta-galactosidase deficiencies in patients' cells. The three enzymes form a complex inside the lysosome, and the neuraminidase and beta-galactosidase deficiencies are secondary to CARB L deficiency. Sequence similarity and common enzymological properties suggest that the protomeric tertiary structure of CARB L is conserved within a family of serine carboxypeptidases which includes the yeast carboxypeptidase Y, killer expression I gene product and several plant carboxypeptidases. We used this homology to build a model of the CARB L structure based on the recently published X-ray atomic coordinates of the wheat carboxypeptidase II (CPDW-II) which shares 32% primary structure identity with CARB L. Small insertions and deletions were accommodated into the model structure by energy minimization using the DREIDING II force field. The C alpha atomic coordinates of the final CARB L model have a RMS shift of 1.01 A compared to the corresponding conserved residues in the CPDW-II template structure. The correct orientation of the homologous catalytic triad residues Ser150, His429 and Asp392, the potential energy calculations and the distribution of hydrophobic and hydrophillic residues in the structure all support the validity of the CARB L model. Most missense mutations identified in galactosialidosis patients were located in secondary structural elements except for the Tyr211-->Asn mutation which is in a loop. The other mutant residues have their side chains deeply buried in the central beta-sheet of the model structure except for the Phe412-->Val mutation which is located in the dimer interface. The predicted effects of specific mutations on CARB L structural stability correlates well with recently published transient expression studies of mutant CARB L (Shimmoto, M. et al., J. Clin. Invest., 91:2393-2399, 1993).
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PMID:Homologous modeling of the lysosomal protective protein/carboxypeptidase L: structural and functional implications of mutations identified in galactosialidosis patients. 814 24

Galactosialidosis is a heterogeneous disorder that is manifested in infantile, late infantile, juvenile/adult, and atypical forms. In every instance the primary defect is in the ability of protective protein to associate with beta-galactosidase and neuraminidase to protect them from intralysosomal proteolysis. The protective protein is in reality a serine protease that displays both cathepsin A and C-terminal deamidase activity. We summarize the major clinical features of each form, and the range of storage products accumulated. The concept of an intralysosomal complex containing beta-galactosidase and neuraminidase in addition to protective protein seems irrefutable but major gaps exist in our understanding of how the complex is formed and in what subcellular organelles, how it is sustained, and the protein domains contributed by the constituent enzymes that play a pivotal role in this process.
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PMID:The biochemistry and clinical features of galactosialidosis. 831 69

Galactosialidosis is an autosomal recessive inherited metabolic disorder induced by the deficiency of beta-galactosidase and neuraminidase. It can be classified into the early infantile form, the late infantile form, and the juvenile/adult form, by clinical characteristics. This disease has been known to be caused by the lack of protective protein. The human protective protein is synthesized as a 54 kD precursor and then processed to the mature form, a heterodimer of 32 and 20 kD polypeptides. The mature protective protein forms a complex with beta-galactosidase and neuraminidase, stabilizing beta-galactosidase and activating neuraminidase. Recently, this protective protein was found to have other multiple functions including activities of carboxypeptidase, esterase and deamidase. The nature of abnormality of the protective protein in the three subtypes of galactosialidosis however has not yet been well elucidated. On the other hand, a cDNA of the protective protein was cloned, and point mutations in the protective protein gene were found in a Japanese family with the adult form, and in Canadian and Italian patients with the late infantile form. We also detected the same point mutation in two Japanese patients with the adult form. Discovery of the genetic defect in different subtypes of galactosialidosis will contribute to the study on the nature of abnormality in the protective protein itself.
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PMID:[Genetic advances in galactosialidosis]. 841 8

Chinese hamster ovary cells were transfected with a recombinant DNA containing the entire coding sequence of human lysosomal protective protein cDNA under the control of mouse metallothionein I promoter. Neomycin and methotrexate-resistant stably transformed cell lines expressing this protein were isolated. Immunoprecipitation of the product with antiserum against human placental protective protein-beta-galactosidase complex revealed a 52-kDa protective protein precursor, which was then processed to mature form, a heterodimer of 32- and 20-kDa polypeptides. The precursor secreted in the culture medium was taken up by the mannose 6-phosphate receptor system and restored acid carboxypeptidase, beta-galactosidase, and neuraminidase activities in galactosialidosis fibroblasts. The expressed protein showed a granular pattern in intracellular distribution, was fractionated at the density of lysosomes, and had serine esterase activities; acid carboxypeptidase at pH 5.6, esterase at pH 7.0, and carboxyl-terminal deamidase at pH 7.0. They were inhibited simultaneously by phenylmethylsulfonyl fluoride, N-benzyloxycarbonyl-L-phenylalanine chloromethyl ketone, or iodoacetamide. The acid carboxypeptidase activity of the purified monomeric mature protective protein was labile in vitro under the acidic condition. Saposins (sphingolipid activator proteins) stabilized the activity at micromolar level concentrations.
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PMID:Purification and characterization of human lysosomal protective protein expressed in stably transformed Chinese hamster ovary cells. 841 22


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