Desialylation of surface receptors as a new dimension in cell signaling.

Terminal sialic acid residues are found in abundance in glycan chains of glycoproteins and glycolipids on the surface of all live cells forming an outer layer of the cell originally known as glycocalyx. Their presence affects the molecular properties and structure of glycoconjugates, modifying their function and interactions with other molecules. Consequently, the sialylation state of glycoproteins and glycolipids has been recognized as a critical factor modulating molecular recognitions inside the cell, between the cells, between the cells and the extracellular matrix, and between the cells and certain exogenous pathogens. Until recently sialyltransferases that catalyze transfer of sialic acid residues to the glycan chains in the process of their biosynthesis were thought to be mainly responsible for the creation and maintenance of a temporal and spatial diversity of sialylated moieties. However, the growing evidence suggests that in mammalian cells, at least equally important roles belong to sialidases/neuraminidases, which are located on the cell surface and in intracellular compartments, and may either initiate the catabolism of sialoglycoconjugates or just cleave their sialic acid residues, and thereby contribute to temporal changes in their structure and functions. The current review summarizes emerging data demonstrating that mammalian neuraminidase 1, well known for its lysosomal catabolic function, is also targeted to the cell surface and assumes the previously unrecognized role as a structural and functional modulator of cellular receptors.

Cloning, expression and chromosomal mapping of human lysosomal sialidase and characterization of mutations in sialidosis.

Sialidase (neuraminidase, EC 3.2.1.18) catalyses the hydrolysis of terminal sialic acid residues of glyconjugates. Sialidase has been well studied in viruses and bacteria where it destroys the sialic acid-containing receptors at the surface of host cells, and mobilizes bacterial nutrients. In mammals, three types of sialidases, lysosomal, plasma membrane and cytosolic, have been described. For lysosomal sialidase in humans, the primary genetic deficiency results in an autosomal recessive disease, sialidosis, associated with tissue accumulation and urinary excretion of sialylated oligosaccharides and glycolipids. Sialidosis includes two main clinical variants: late-onset, sialidosis type I, characterized by bilateral macular cherry-red spots and myoclonus, and infantile-onset, sialidosis type II, characterized by skeletal dysplasia, mental retardation and hepatosplenomegaly. We report the identification of human lysosomal sialidase cDNA, its cloning, sequencing and expression. Examination of six sialidosis patients revealed three mutations, one frameshift insertion and two missense. We mapped the lysosomal sialidase gene to human chromosome 6 (6p21.3), which is consistent with the previous chromosomal assignment of this gene in proximity to the HLA locus.

Altered gene expression in cells from patients with lysosomal storage disorders suggests impairment of the ubiquitin pathway.

By comparing mRNA profiles in cultured fibroblasts from patients affected with lysosomal storage diseases, we identified differentially expressed genes common to these conditions. These studies, confirmed by biochemical experiments, demonstrated that lysosomal storage is associated with downregulation of ubiquitin C-terminal hydrolase, UCH-L1 in the cells of eight different lysosomal disorders, as well as in the brain of a mouse model of Sandhoff disease. Induction of lysosomal storage by the cysteine protease inhibitor E-64 also reduced UCH-L1 mRNA, protein level and activity. All cells exhibiting lysosomal storage contained ubiquitinated protein aggregates and showed reduced levels of free ubiquitin and decreased proteasome activity. The caspase-mediated apoptosis in E-64-treated fibroblasts was reversed by transfection with a UCH-L1 plasmid, and increased after downregulation of UCH-L1 by siRNA, suggesting that UCH-L1 deficiency and impairment of the ubiquitin-dependent protein degradation pathway can contribute to the increased cell death observed in many lysosomal storage disorders.

Clinical and genetic spectrum of Sanfilippo type C (MPS IIIC) disease in The Netherlands.

Mucopolysaccharidosis IIIC (MPS IIIC, Sanfilippo C syndrome) is a lysosomal storage disorder caused by deficiency of the lysosomal enzyme acetyl-CoA:alpha-glucosaminide N-acetyltransferase (HGSNAT). We performed a clinical study on 29 Dutch MPS IIIC patients and determined causative mutations in the recently identified HGSNAT gene. Psychomotor development was reported to be normal in all patients during the first year of life. First clinical signs were usually noted between 1 and 6 years (mean 3.5 years), and consisted of delayed psychomotor development and behavioral problems. Other symptoms included sleeping and hearing problems, recurrent infections, diarrhoea and epilepsy. Two sisters had attenuated disease and did not have symptoms until the third decade. Mean age of death was 34 years (range 25-48). Molecular analysis revealed mutations in both alleles for all patients except one. Altogether 14 different mutations were found: two splice site mutations, one frame shift mutation due to an insertion, three nonsense mutations and eight missense mutations. Two mutations, p.R344C and p.S518F, were frequent among probands of Dutch origin representing 22.0% and 29.3%, respectively, of the mutant alleles. This study demonstrates that MPS IIIC has a milder course than previously reported and that both severity and clinical course are highly variable even between sibs, complicating prediction of the clinical phenotype for individual patients. A clear phenotype-genotype correlation could not be established, except that the mutations p.G262R and p.S539C were only found in two sisters with late-onset disease and presumably convey a mild phenotype.

Lysosomal multienzyme complex: biochemistry, genetics, and molecular pathophysiology.

Lysosomal enzymes sialidase (alpha-neuraminidase), beta-galactosidase, and N-acetylaminogalacto-6-sulfate sulfatase are involved in the catabolism of glycolipids, glycoproteins, and oligosaccharides. Their functional activity in the cell depends on their association in a multienzyme complex with lysosomal carboxypeptidase, cathepsin A. We review the data suggesting that the integrity of the complex plays a crucial role at different stages of biogenesis of lysosomal enzymes, including intracellular sorting and proteolytic processing of their precursors. The complex plays a protective role for all components, extending their half-life in the lysosome from several hours to several days; and for sialidase, the association with cathepsin A is also necessary for the expression of enzymatic activity. The disintegration of the complex due to genetic mutations in its components results in their functional deficiency and causes severe metabolic disorders: sialidosis (mutations in sialidase), GM1-gangliosidosis and Morquio disease type B (mutations in beta-galactosidase), galactosialidosis (mutations in cathepsin A), and Morquio disease type A (mutations in N-acetylaminogalacto-6-sulfate sulfatase). The genetic, biochemical, and direct structural studies described here clarify the molecular pathogenic mechanisms of these disorders and suggest new diagnostic tools.

Regulation of the GM1-galactosidase supramolecular structure and catalytic activity in vitro.

Regulation of the supramolecular organization and the catalytic activity of GM1-galactosidase (EC 3.2.1.23) and neuraminidase (EC 3.2.1.18) from human kidney was studied in a system of hydrated reversed micelles of Aerosol OT in octane. It was shown that both the catalytic activity and the oligomeric structure of the GM1-galactosidase in reversed micelles depend on the [H2O]/[Aerosol OT] molar ratio (w(o)). GM1-galactosidase 64-67 kDa monomers, 260 kDa tetramers, and 660 kDa octamers were obtained in systems with w(o) = 0-20, 25-30 and 30-40, respectively. The association of GM1-galactosidase monomers into an octamer results in the cooperative increase in enzymatic activity. 'Protective protein', a component of the GM1-galactosidase-neuraminidase native complex, was found to improve this association significantly.

Localisation of a gene for mucopolysaccharidosis IIIC to the pericentromeric region of chromosome 8.

Mucopolysaccharidosis type IIIC (MPS IIIC, or Sanfilippo syndrome C) is a rare lysosomal storage disorder caused by a deficiency of acetyl-coenzyme A:alpha-glucosaminide-N-acetyltransferase. Patients develop progressive neuropsychiatric problems, mental retardation, hearing loss, and relatively minor visceral manifestations. The pattern of transmission is consistent with an autosomal recessive mode of inheritance. The aim of this study was to find a locus for MPS IIIC using a homozygosity mapping approach. A genomewide scan was performed on DNA from 27 affected individuals and 17 of their unaffected relatives. Additional patients were recruited, and DNA was obtained from a total of 44 affected individuals and 18 unaffected family members from 31 families from 10 countries. A working candidate interval was defined by looking for excess homozygosity in patients compared with their relatives. Additional markers were genotyped in regions of interest. Linkage analysis was performed to support the informal analysis. Inspection of the genomewide scan data showed apparent excess homozygosity in patients compared with their relatives for markers on chromosome 8. Additional genotyping identified 15 consecutive markers (from D8S1051 to D8S2332) in an 8.3 cM interval for which the genotypes of affected siblings were identical in state. A maximum multipoint lod score of 10.61 was found at marker D8S519. A locus for MPS IIIC maps to an 8.3 cM (16 Mbp) interval in the pericentromeric region of chromosome 8.

Solubilization of rat kidney lysosomes in reversed micelles of aerosol OT in octane.

Intact lysosomes from rat kidneys were solubilized in the ternary system: surfactant (Aerosol OT)-buffer-organic solvent. According to data of laser light-scattering analysis and kinetic experiments with the lysosomal marker enzyme, N-acetyl-beta-D-hexosaminidase (EC 3.2.1.30), the solubilization of lysosomes in this system resulted in the destruction of the lysosomes and the entrapping of their components in reversed micelles.

Alcohol dehydrogenase (ADH). Influence of homo- and heterologous ADH administration on albino rats craving for alcohol and on ADH isozyme activity in the liver.

The effects of homo- and heterologous alcohol dehydrogenase (ADH) administration into albino rats were investigated. It was found that homologous ADH increases and heterologous ADH decreases the craving for ethanol. The latter effect was accompanied by the appearance of anti-ADH-3 antibodies and by a decrease in ADH-3 activity in the liver. Craving for alcohol decreased after both active and passive immunization against ADH.

Caspase-3-dependent export of TCTP: a novel pathway for antiapoptotic intercellular communication.

The apoptotic program incorporates a paracrine component of importance in fostering tissue repair at sites of apoptotic cell deletion. As this paracrine pathway likely bears special importance in maladaptive intercellular communication leading to vascular remodeling, we aimed at further defining the mediators produced by apoptotic endothelial cells (EC), using comparative and functional proteomics. Apoptotic EC were found to release nanovesicles displaying ultrastructural characteristics, protein markers and functional activity that differed from apoptotic blebs. Tumor susceptibility gene 101 and translationally controlled tumor protein (TCTP) were identified in nanovesicle fractions purified from medium conditioned by apoptotic EC and absent from purified apoptotic blebs. Immunogold labeling identified TCTP on the surface of nanovesicles purified from medium conditioned by apoptotic EC and within multivesicular blebs in apoptotic EC. These nanovesicles induced an extracellular signal-regulated kinases 1/2 (ERK 1/2)-dependent antiapoptotic phenotype in vascular smooth muscle cells (VSMC), whereas apoptotic blebs did not display antiapoptotic activity on VSMC. Caspase-3 biochemical inhibition and caspase-3 RNA interference in EC submitted to a proapoptotic stimulus inhibited the release of nanovesicles. Also, TCTP siRNAs in EC attenuated the antiapoptotic activity of purified nanovesicles on VSMC. Collectively, these results identify TCTP-bearing nanovesicles as a novel component of the paracrine apoptotic program of potential importance in vascular repair.

Caspase-3-mediated secretion of connective tissue growth factor by apoptotic endothelial cells promotes fibrosis.

Apoptosis of endothelial cells (ECs) is an early pathogenic event in various fibrotic diseases. In this study, we evaluated whether paracrine mediators produced by apoptotic ECs play direct roles in fibrogenesis. C3H mice injected subcutaneously with serum-free medium conditioned by apoptotic ECs (SSC) showed increased skin thickness and heightened protein levels of alpha-smooth-muscle actin (alphaSMA), vimentin and collagen I as compared with mice injected with medium conditioned by non-apoptotic ECs. Fibroblasts exposed to SSC in vitro showed cardinal features of myofibroblast differentiation with increased stress fiber formation and expression of alphaSMA. Caspase-3 silencing in ECs prevented the release of mediators favoring myofibroblast differentiation. To identify the fibrogenic factor(s) released by ECs, the protein contents of media conditioned by either apoptotic or non-apoptotic ECs were compared using SDS-PAGE-liquid chromatography (LC)-tandem mass spectrometry (MS/MS) and two-dimensional LC-MS/MS. Connective tissue growth factor (CTGF) was the only fibrogenic protein found increased in SSC. Pan-caspase inhibition with ZVAD-FMK or caspase-3 silencing in ECs confirmed that CTGF was released downstream of caspase-3 activation. The fibrogenic signaling signatures of SSC and CTGF on fibroblasts in vitro were similarly Pyk2-, Src-family kinases- and PI3K dependent, but TGF-beta-independent. CTGF-immunodepleted SSC failed to induce myofibroblast differentiation in vitro and skin fibrosis in vivo. These results identify caspase-3 activation in ECs as a novel inducer of CTGF release and fibrogenesis.

Direct affinity purification and supramolecular organization of human lysosomal cathepsin A.

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.

Stoichiometry of the human lysosomal carboxypeptidase-beta-galactosidase complex.

The understanding of the lysosomal beta-galactosidase-carboxypeptidase-neuraminidase multienzymatic complex structure and function requires an efficient system for dissociation and association of its isolated protein components under controlled conditions. In this paper such a system was used to determine the stoichiometry of the two main components of this complex--beta-galactosidase and carboxypeptidase. The complex, after affinity purification from human placenta, was dissociated at pH 7.5 and beta-galactosidase and carboxypeptidase were separated and purified to homogeneity by FPLC anion-exchange chromatography. The 680 kD complex of beta-galactosidase and carboxypeptidase was reconstituted in vitro by mixing the isolated enzymes in a 1:2 molar ratio at pH 7.5 and then progressively acidifying the medium towards the intralysosomal pH value of 4.75. Under the same conditions, beta-galactosidase and carboxypeptidase independently existed as 306 kDa tetramer and 98 kDa dimer, respectively. Reconstitution experiments with various ratios of purified beta-galactosidase and carboxypeptidase allowed us to conclude that the whole complex is made of 4 beta-galactosidase and 8 carboxypeptidase monomers. Cross-linking of the native and reconstituted complexes with dimethylsuberimidate or glutaric dialdehyde suggested that the native and the reconstituted complexes have the same supramolecular structure.

Association of N-acetylgalactosamine-6-sulfate sulfatase with the multienzyme lysosomal complex of beta-galactosidase, cathepsin A, and neuraminidase. Possible implication for intralysosomal catabolism of keratan sulfate.

N-Acetylgalactosamine-6-sulfate sulfatase (GALNS) catalyzes the first step of intralysosomal keratan sulfate (KS) catabolism. In Morquio type A syndrome GALNS deficiency causes the accumulation of KS in tissues and results in generalized skeletal dysplasia in affected patients. We show that in normal cells GALNS is in a 1.27-MDa complex with three other lysosomal hydrolases: beta-galactosidase, alpha-neuraminidase, and cathepsin A (protective protein). GALNS copurifies with the complex by different chromatography techniques: affinity chromatography on both cathepsin A-binding and beta-galactosidase-binding columns, gel filtration, and chromatofocusing. Anti-human cathepsin A rabbit antiserum coprecipitates GALNS together with cathepsin A, beta-galactosidase, and alpha-neuraminidase in both a purified preparation of the 1. 27-MDa complex and crude glycoprotein fraction from human placenta extract. Gel filtration analysis of fibroblast extracts of patients deficient in either beta-galactosidase (beta-galactosidosis) or cathepsin A (galactosialidosis), which accumulate KS, demonstrates that the 1.27-MDa complex is disrupted and that GALNS is present only in free homodimeric form. The GALNS activity and cross-reacting material are reduced in the fibroblasts of patients affected with galactosialidosis, indicating that the complex with cathepsin A may protect GALNS in the lysosome. We suggest that the 1.27-MDa complex of lysosomal hydrolases is essential for KS catabolism and that the disruption of this complex may be responsible for the KS accumulation in beta-galactosidosis and galactosialidosis patients.

Human kidney hexosaminidase A and hexosaminidase B form a complex.

The isolation and purification of human kidney hexosaminidases A and B was carried out. Regulation of the supramolecular organization and catalytic activity of hexosaminidases was investigated in the bis(2-ethylhexyl)sulphosuccinate reversed micellar system modeling the enzyme microenvironment in the lysosomes. It was shown that hexosaminidases A and B associate forming 280-300-kDa dimeric complexes under these conditions. At pH 4.75, the hexosaminidases A and B can be isolated from kidney tissue only in the form of this complex.

Human lysosomal beta-galactosidase-cathepsin A complex: definition of the beta-galactosidase-binding interface on cathepsin A.

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.

Demonstration of enzyme associations by countermigration electrophoresis in agarose gel.

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.

Comparative modeling of substrate binding in the S1' subsite of serine carboxypeptidases from yeast, wheat, and human.

Human cathepsin A ("lysosomal protective protein"; E.C.3.4.16.5) is a multifunctional lysosomal protein which forms a high-molecular-weight complex with beta-galactosidase and alpha-neuraminidase, protecting them against intralysosomal proteolysis. In addition to this protective function, cathepsin A is a serine carboxypeptidase and the understanding of its catalytic function requires a definition of its substrate specificity. For this purpose, we used a combined experimental [Pshezhetsky, A. V., Vinogradova, M. V., Elsliger, M.-A., El-Zein, F., Svedas, V.K., & Potier, M. (1995) Anal. Biochem. 230, 303-307] and theoretical approach comparing cathepsin A to two different homologous carboxypeptidases of the same family: yeast carboxypeptidase Y and wheat carboxypeptidase II. We computed the energies involved in substrate binding to the S1' subsite (C-terminal) of cathepsin A using a structural model based on the X-ray structure of the homologous wheat carboxypeptidase II. The binding energies of N-blocked Phe-Xaa dipeptide substrates to the active sites of cathepsin A, wheat carboxypeptidase II, and yeast carboxypeptidase Y were estimated using a molecular mechanics force field supplemented with a solvation energy term. This theoretical analysis showed a good correlation with the experimentally determined free energies of substrate binding. This result validates the use of this approach to analyze the energetics of substrate binding to the S1' subsite and provides a rational interpretation of serine carboxypeptidase-substrate interactions in molecular terms. We conclude that the three serine carboxypeptidases have similar affinities for substrates with hydrophobic P1' amino acid residues but that the wheat enzyme has an additional capacity for binding positively charged P1' residues. Finally, the substrate specificity of human cathepsin A is very similar to that of carboxypeptidase Y, with a high binding affinity for substrates with hydrophobic P1' residues, but the affinity of cathepsin A for P1; Phe residue is higher than for the Leu residue.

3-Hydroxy-3-methylglutaryl coenzyme A lyase: targeting and processing in peroxisomes and mitochondria.

3-Hydroxy-3-methylglutaryl coenzyme A lyase (HL, E.C. 4.1.3.4) has a unique dual localization in both mitochondria and peroxisomes. Mitochondrial HL ( approximately 31.0 kDa) catalyzes the last step of ketogenesis; the function of peroxisomal HL ( approximately 33.5 kDa) is unknown. On density gradient fractionation, normal human lymphoblasts contain both peroxisomal and mitochondrial HL whereas in lymphoblasts from a patient with Zellweger syndrome, in which functional peroxisomes are absent, only the mitochondrial HL isoform was present. To study the kinetics of the dual targeting of HL, we performed pulse-chase experiments in normal and Zellweger cells. Pulse-chase studies revealed a biphasic curve for processing of the HL precursor. The first phase, with a calculated half-life of approximately 3 h in both normal and Zellweger fibroblasts and lymphoblasts and in HepG2 cells, presumably reflects mitochondrial import and processing of the precursor; the second (t1/2, 12-19 h) is present only in normal cells and presumably represents the half-life of peroxisomal HL. The half-life of mature mitochondrial HL was 14 to 19 h in both normal and Zellweger cells. Studies of the HMG-CoA lyase precursor in isolated rat mitochondria showed a rate of processing approximately 2.6-fold lower than that of the ornithine transcarbamylase precursor.

Identification of UDP-N-acetylglucosamine-phosphotransferase-binding sites on the lysosomal proteases, cathepsins A, B, and D.

A key step in the targeting of soluble lysosomal enzymes is their recognition and phosphorylation by a 540 kDa multisubunit enzyme, UDP-N-acetylglucosamine-phosphotransferase (phosphotransferase). The molecular mechanism of recognition is still unknown, but previous experiments suggested that the phosphotransferase-binding sites on lysosomal proteins are represented by structurally conserved surface patches of amino acids. We identified four such regions on nonhomologous lysosomal enzymes, cathepsins A, B, and D, which were superimposed by rotating their structures around the Calpha atom of the glycosylated Asn residue. We proposed that these regions represent putative phosphotransferase-binding sites and tested synthetic peptides, derived from these regions on the basis of surface accessibility, for their ability to inhibit in vitro phosphorylation of purified cathepsins A, B, and D. Our results indicate that cathepsin A and cathepsin D have one closely related phosphotransferase recognition site represented by a structurally and topologically conserved beta-hairpin loop, similar to that previously identified in lysosomal beta-glucuronidase. The most potent inhibition of phosphorylation was demonstrated by homologous peptides derived from the regions located on cathepsin molecules opposite the oligosaccharide chains which are phosphorylated by the phosphotransferase. We propose that recognition and catalytic sites of the phosphotransferase are located on different subunits, therefore, providing an effective mechanism for binding and phosphorylation of lysosomal proteins of different molecular size.