Identification and in vitro reconstitution of lysosomal neuraminidase from human placenta

Lysosomal neuraminidase from human placenta has been obtained in its active form by association of an inactive neuraminidase polypeptide with beta-galactosidase and the protective protein. Using a specific antiserum, we have now identified a 66-kDa protein as the inactive neuraminidase polypeptide. It is specifically recognized on immunoblots only in its nonreduced state, and it coprecipitates with neuraminidase activity. The 66-kDa polypeptide is substantially glycosylated (38-kDa protein core with 7-14 N-linked oligosaccharide chains), a feature characteristic of lysosomal integral membrane proteins. Specific removal of the 66-kDa neuraminidase polypeptide from glycoprotein preparations prevents the generation of neuraminidase activity. Removal of beta-galactosidase or destruction of the protective protein also hinders the formation of active neuraminidase. Reconstitution of neuraminidase activity is observed after mixing glycoprotein preparations, depleted in different components of the beta-galactosidase-neuraminidase-protective protein complex, indicating that all three components of the complex are required for neuraminidase activity. Association of the neuraminidase polypeptide and the protective protein generates unstable neuraminidase activity, whereas association with beta-galactosidase is required for stability.     

Lysosomal neuraminidase. Catalytic activation in insect cells is controlled by the protective protein/cathepsin A

Lysosomal N-Acetyl-alpha-neuraminidase is active in complex with the protective protein/cathepsin A (PPCA) and beta-galactosidase. The interaction with PPCA is essential for the correct intracellular routing and lysosomal localization of neuraminidase, but the mechanism of its catalytic activation is unclear. To investigate this process, we have used the baculovirus expression system to co-express neuraminidase and PPCA precursors in insect cells, which resulted in high enzymatic activity of neuraminidase. Both the 34- and 20-kDa PPCA subunits were required for the activation. We further demonstrated that when expressed alone, the neuraminidase precursor remained dimeric (114 kDa) and had low enzymatic activity, but when co-expressed with PPCA and beta-galactosidase, it multimerized in a complex of approximately 1350 kDa, together with the other two proteins. The fully active neuraminidase co-precipitated with full-length PPCA and beta-galactosidase precursors. However, when co-expressed with the individual PPCA subunits, neuraminidase co-precipitated only with the small 20-kDa polypeptide, which therefore must contain a neuraminidase-binding site. Our finding suggests a model of activation of neuraminidase dependent on its oligomerization at acidic pH that is mediated by interaction with PPCA.     

The relation between human lysosomal beta-galactosidase and its protective protein

Cultured skin fibroblasts from patients with the lysosomal storage disease galactosialidosis lack a 54-kDa protein which is a precursor of 32-kDa and 20-kDa proteins, which immunoprecipitate with human anti-beta-galactosidase antiserum. The lack of a 32-kDa "protective protein" results in a combined deficiency of beta-galactosidase and sialidase. The mechanism of protection of lysosomal beta-galactosidase against proteolytic degradation is elucidated by sucrose density gradient centrifugation and immunoprecipitation studies. In normal fibroblasts at the low intralysosomal pH, more than 85% of beta-galactosidase exists as a high molecular weight (600-700 kDa) multimer and about 10% as a monomer of 64-kDa. In mutant cells from galactosialidosis patients, the residual enzyme activity, about 10%, is present as a monomer and no multimer exists. After addition of the 54-kDa precursor form of the protective protein, the density pattern of beta-galactosidase in galactosialidosis cells is normalized. Immunoprecipitation studies after sucrose density gradient centrifugation on homogenate and on purified beta-galactosidase from normal fibroblasts show that the protective protein is associated only with the multimeric form of beta-galactosidase. We propose that intralysosomal protection against proteolysis of beta-galactosidase and sialidase is accomplished by aggregation into a high molecular weight complex consisting of multimeric beta-galactosidase, sialidase, and protective protein. The genetic deficiency of the latter, as in galactosialidosis, results in a rapid degradation of monomeric beta-galactosidase and a loss of sialidase activity.     

Galactosialidosis: molecular heterogeneity among distinct clinical phenotypes.

The lysosomal storage disorder galactosialidosis has been recognized as a distinct genetic and biochemical entity, associated with a combined beta-galactosidase and neuraminidase deficiency that is due to the lack of a 32-kilodalton (kDa) glycoprotein. The molecular basis of different clinical variants of galactosialidosis has been investigated. In the early-infantile form, the synthesis of the 52-kDa precursor of the 32-kDa "protective protein" is markedly reduced and the absence of the latter protein explains the severe neuraminidase deficiency. In the juvenile-adult form, there is relatively more 52-kDa precursor but no 32-kDa protein can be detected. Cells from the late-infantile form have in comparison with controls, besides a small amount of the 32-kDa glycoprotein, an accumulation of the 52-kDa precursor. Apparently, this protein is genetically altered in such a way that its further processing is impaired. Furthermore, in this mutant, the residual neuraminidase activity is stimulated four- to sixfold upon leupeptin treatment together with an increase of the 32-kDa glycoprotein.     

Deficient lysosomal carboxypeptidase activity in galactosialidosis

In the lysosome, the glycosidases neuraminidase (EC 3.2.1.18) and beta-galactosidase (EC 3.2.1.23) are associated to a 52 kDa "protective protein" to form a large multi-enzymatic complex. Deficient synthesis or inactivation of this protective protein causes galactosialidosis, a lysosomal storage disorder in man in which both neuraminidase and beta-galactosidase activities are deficient. Since the protective protein possesses extensive sequence homology with carboxypeptidase Y (carb Y) and the KEX 1 gene product from yeast, we have used the artificial substrate N-CBZ-Phe-Leu to detect and characterize the peptidase activity of the lysosomal carboxypeptidase (carb L). Using both a purified preparation of the lysosomal multi-enzymatic complex and cultured skin fibroblasts of patients affected with galactosialidosis, we demonstrate that the 52 kDa protective protein is responsible for carb L activity. The fibroblasts of three patients affected with late infantile and juvenile galactosialidosis were found to be deficient in carb L activity (1.4% of normal mean value).     

Human lysosomal protective protein. Glycosylation, intracellular transport, and association with beta-galactosidase in the endoplasmic reticulum.

In lysosomes beta-galactosidase and neuraminidase acquire a stable and active conformation through their association with the protective protein. The latter is homologous to serine carboxypeptidases and has cathepsin A-like activity which is distinct from its protective function towards the two glycosidases. To define signals in the human protective protein important for its intracellular transport, and to determine the site of its association with beta-galactosidase, we have generated a set of mutated protective protein cDNAs carrying targeted base substitutions. These mutants were either singly transfected into COS-1 cells or cotransfected together with wild type human beta-galactosidase. We show that all point mutations cause either a complete or partial retention of the protective protein precursor in the endoplasmic reticulum. This abnormal accumulation leads to degradation of the mutant proteins probably in this compartment. Only the oligosaccharide chain on the 32-kDa subunit acquires the mannose 6-phosphate recognition marker, the one on the 20-kDa subunit seems to be merely essential for the stability of the mature protein. In cotransfection experiments, wild type beta-galactosidase and protective protein appear to assemble already as precursors, soon after synthesis, in the endoplasmic reticulum. Mutated protective protein precursors that are retained in the endoplasmic reticulum or pre-Golgi complex interact with and withhold normal beta-galactosidase molecules in the same compartments, thereby preventing their normal routing.     

A sialidase complex from chicken liver: Characterization of a multienzyme complex with β-galactosidase and carboxypeptidase

Mammalian lysosomal sialidase exists as an enzyme complex with β-galactosidase and carboxypeptidase, so-called “protective protein.” In this article, we report that chicken sialidase also occurs as a complex with β-galactosidase and protective protein. The purified sialidase complex had a molecular weight > 700 kDa on gel filtration and showed four protein components of 76, 65, 54 and 48 kDa on SDS-PAGE under nonreducing conditions. N-Terminal sequences of the 65- and 48-kDa proteins were homologous to human lysosomal β-galactosidase and protective protein precursor, respectively. The purified sialidase complex also had carboxypeptidase activity. Both sialidase and carboxypeptidase activities were precipitated together by an antibody against chicken β-galactosidase. The complex reversibly dissociated into 120-kDa β-galactosidase dimer and 100-kDa carboxypeptidase dimer at pH 7.5, but the sialidase irreversibly inactivated during the depolymerization. These findings indicate that chicken sialidase exists as a multienzyme complex, by which the sialidase activity appears to be stabilized.     

Mouse "protective protein". cDNA cloning, sequence comparison, and expression

The "protective protein" is the glycoprotein that forms a complex with the lysosomal enzymes beta-galactosidase and neuraminidase. Its deficiency in man leads to the metabolic storage disorder galactosialidosis. The primary structure of human protective protein, deduced from its cloned cDNA, shows homology to yeast serine carboxypeptidases. We have isolated a full-length cDNA encoding murine protective protein. The nucleotide sequences as well as the predicted amino acid sequences are highly conserved between man and mouse. Domains important for the protease function are completely identical in the two proteins. Both human and mouse mature protective proteins covalently bind radiolabeled diisopropyl fluorophosphate. Transient expression of the murine cDNA in COS-1 cells yields a protective protein precursor of 54 kDa, a size characteristic of the glycosylated form. This cDNA-encoded precursor, endocytosed by human galactosialidosis fibroblasts, is processed into a 32- and a 20-kDa heterodimer and corrects beta-galactosidase and neuraminidase activities. A tissue-specific expression of protective protein mRNA is observed when total RNA from different mouse organs is analyzed on Northern blots.     

Molecular heterogeneity in human beta-galactosidase and neuraminidase deficiency

Human lysosomal beta-galactosidase and neuraminidase exist in a complex together with a 32-kilodalton (kd) glycoprotein. The latter protein was found to have a dual function: it is required for the aggregation of monomeric 64-kd beta-galactosidase into high molecular weight (600-700 kd) multimers and it is an essential subunit of neuraminidase together with a 76-kd polypeptide. The severe neurological disorder galactosialidosis, characterized by a coexistent deficiency of beta-galactosidase and neuraminidase, was found to be due to a genetic defect of the 32-kd protective protein. The molecular background of the clinical heterogeneity within this syndrome is described and will undoubtedly be further elucidated since we have recently isolated the gene coding for the protective protein. The sequence of normal and mutant (enzyme) proteins will also provide better insight into the characteristics of the beta-galactosidase-neuraminidase-protective protein complex. Another interesting model for the study of posttranslational processing is the defective phosphorylation of beta-galactosidase in cells from patients with GM1-gangliosidosis.     

Photoaffinity labeling of the lysosomal neuraminidase from bovine testis

ASA-NeuAc2en, a photoreactive arylazide derivative of sialic acid, is shown to be a powerful competitive inhibitor of lysosomal neuraminidase from bovine testis (Ki approximately 21 microM). Photoaffinity labeling and partial purification of preparations containing this lysosomal neuraminidase activity result in specifically and non-specifically labeled polypeptides. Only labeling in a 55 kDa polypeptide is found to be specific, since it could be prevented by the competitive neuraminidase inhibitor NeuAc2en. We conclude that the 55 kDa polypeptide in the bovine testis beta-galactosidase/neuraminidase/protective protein complex contains the catalytic site of neuraminidase.     

Structure of the lysosomal neuraminidase-beta-galactosidase-carboxypeptidase multienzymic complex.

Lysosomal neuraminidase (sialidase; EC 3.2.1.18) and beta-galactosidase (EC 3.2.1.23), together with a carboxypeptidase, the so-called 'protective protein', were co-purified from the human placenta by affinity chromatography on a concanavalin A-Sepharose column followed by a thiogalactoside-agarose affinity column for beta-galactosidase. Analysis of the purified material by gel-filtration h.p.l.c. revealed three distinct molecular forms, all with high beta-galactosidase specific activity, but only the largest one expressed neuraminidase activity. Rechromatography of each individual species separately indicated that all three are in fact part of an equilibrium system (the neuraminidase-beta-galactosidase-carboxypeptidase complex or NGC-complex) and that these species undergo slow conversion into one another through dissociation and association of protomeric components. Each species was sufficiently stable for the determination of their hydrodynamic properties by gel-filtration h.p.l.c. and sedimentation velocity. The largest species had an apparent sedimentation coefficient S20.w, of 18.8 S and a Stokes' radius of 8.5 nm, giving a molecular mass of 679 kDa and a fractional ratio, f/f min, of 1.47. The latter value indicates that the macromolecule is asymmetric or highly hydrated. This large species is composed of four types of polypeptide chains of molecular mass 66 kDa (neuraminidase), 63 kDa (beta-galactosidase), 32 kDa and 20 kDa (carboxypeptidase heterodimer). The 32 kDa and 20 kDa protomers are linked together by a disulphide bridge. Glycopeptidase F digestion of the NGC-complex transformed the diffuse 66-63 kDa band on the SDS gel into two close but sharp bands at 58 and 56 kDa. The two smaller species which were separated on the h.p.l.c. column correspond to tetrameric and dimeric forms of the 66-63 kDa protomers and express exclusively beta-galactosidase activity. Treatment of the NGC-complex with increasing concentrations of guanidinium hydrochloride up to 1.5 M also resulted in dissociation of the complex into the same smaller species mentioned above plus two protomers of molecular mass around 60 and 50 kDa. A model of the largest molecular species as a hexamer of the 66-63 kDa protomers associated to five carboxypeptidase heterodimers (32 kDa and 20 kDa) is proposed     

Purification and characterization of maturation-promoting factor in fish.

Maturation-promoting factor (MPF) activity has been demonstrated for the first time in fish oocytes. We purified MPF from a 100,000g supernatant of crushed, naturally spawned carp oocytes using four chromatography columns: Q-Sepharose Fast-Flow, p13suc1-affinity Sepharose, Mono S, and Superose 12. The final preparation was purified over 1000-fold with a recovery of about 1%. On Superose 12, MPF eluted as a single peak with an apparent molecular weight of 100 kDa. SDS-PAGE analysis of the active fractions after Superose 12 revealed the presence of four proteins of 33, 34, 46, and 48 kDa. A monoclonal antibody against the PSTAIR sequence of cdc2 kinase recognized the 33- and 34-kDa proteins for which the 46- and 48-kDa proteins are endogenous substrates. The 46- and 48-kDa proteins were recognized by a monoclonal antibody against Escherichia coli-produced goldfish cyclin B, but not by an anti-cyclin A antibody. When oocytes were matured in the presence of 32P, the labeling was seen with the 34-kDa protein, but not with the 33-kDa protein. The 34-kDa protein corresponded to the MPF activity, but the 33-kDa protein did not. These findings indicate that carp MPF is a complex of cdc2 kinase and cyclin B, and further that active MPF contains the phosphorylated form of cdc2 kinase.     

Properties of Leuconostoc mesenteroides B-512FMC constitutive dextransucrase

Leuconostoc mesenteroides B-512FMC, a constitutive mutant for dextransucrase, was grown on glucose, fructose, or sucrose. The amount of cell-associated dextransucrase was about the same for the three sugars at different concentrations (0.6% and 3%). Enzyme produced in glucose medium was adsorbed on Sephadex G-100 and G-200, but much less enzyme was adsorbed when it was produced in sucrose medium. Sephadex adsorption decreased when the glucose-produced enzyme was preincubated with dextrans of molecular size greater than 10 kDa. The release of dextransucrase activity from Sephadex by buffer (20 mM acetate, pH 5.2) was the highest at 28°–30°C. The addition of dextran to the enzyme stimulated dextran synthesis but had very little effect on the temperature or pH stability. Dextransucrase purified by ammonium sulfate precipitation, hydroxyapatite chromatography, and Sephadex G-200 adsorption did not contain any carbohydrate, and it synthesized dextran, showing that primers are not necessary to initiate dextran synthesis. The purified enzyme had a molecular size of 184 kDa on SDS-PAGE. On standing at 4°C for 30 days, the native enzyme was dissociated into three inactive proteins of 65, 62, and 57 kDa. However, two protein bands of 63 and 59 kDa were obtained on SDS-PAGE after heat denaturation of the 184-kDa active enzyme at 100°C. The amount of 63-kDa protein was about twice that of 59-kDa protein. The native enzyme is believed to be a trimer of two 63-kDa and one 59-kDa monomers.     

Immunological studies on the settlement-inducing protein complex (SIPC) of the barnacle Balanus amphitrite and its possible involvement in larva-larva interactions.

Immunological investigation has revealed that a settlement-inducing protein complex (SIPC), which induces cypris settlement of the barnacle Balanus amphitrite, is synthesized during larval development and accumulates in the cypris larva. We previously purified the SIPC from adult B. amphitrite, which was active when bound to a substratum. The SIPC is a glycoprotein of high molecular mass, consisting of three major subunits of 76, 88 and 98 kDa with lentil lectin (LCA)-binding sugar chains. In the present study, we prepared antiserum against each LCA-binding subunit of SIPC, and performed immunoblot analyses. Immunoblotting of adult extracts showed that anti-76-kDa antibody reacted only with the 76-kDa protein, whereas anti-88-kDa and anti-98-kDa antibodies reacted with both the 88-kDa and the 98-kDa proteins. Immunoblotting of larval extracts indicated that reactivity of the 76-kDa protein to anti-76-kDa antiserum increased during larval development and cyprid extracts reacted strongly. Moreover, by using immunostaining we found that the SIPC was contained in ''footprints'' of cyprids, which have been shown to act as a settlement-inducing pheromone, and is secreted onto the antennular attachment discs. The results suggest that the SIPC (or SIPC-like proteins) is involved in both adult-larva and larva-larva interactions during settlement of the barnacle B. amphitrite.     

Expression of cDNA encoding the human "protective protein" associated with lysosomal beta-galactosidase and neuraminidase: homology to yeast proteases

The "protective protein" is a glycoprotein that associates with lysosomal beta-galactosidase and neuraminidase and is deficient in the autosomal recessive disorder galactosialidosis. We have isolated the cDNA encoding human "protective protein". The clone recognizes a 2 kb mRNA in normal cells that is not evident in fibroblasts of an early infantile galactosialidosis patient. The cDNA directs the synthesis of a 452 amino acid precursor molecule that is processed in vivo to yield mature "protective protein," a heterodimer of 32 kd and 20 kd polypeptides held together by disulfide bridges. This mature form is also biologically functional since it restores beta-galactosidase and neuraminidase activities in galactosialidosis cells. The predicted amino acid sequence of the "protective protein" bears homology to yeast carboxypeptidase Y and the KEX1 gene product. This suggests a protease activity for the "protective protein."     

Emulsifying activities of purified Alasan proteins from Acinetobacter radioresistens KA53

The bioemulsifier of Acinetobacter radioresistens KA53, referred to as alasan, is a high-molecular-weight complex of polysaccharide and protein. The emulsifying activity of the purified polysaccharide (apo-alasan) is very low. Three of the alasan proteins were purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had apparent molecular masses of 16, 31, and 45 kDa. Emulsification assays using the isolated alasan proteins demonstrated that the active components of the alasan complex are the proteins. The 45-kDa protein had the highest specific emulsifying activity, 11% higher than the intact alasan complex. The 16- and 31-kDa proteins gave relatively low emulsifying activities, but they were significantly higher than that of apo-alasan. The addition of the purified 16- and 31-kDa proteins to the 45-kDa protein resulted in a 1.8-fold increase in the specific emulsifying activity and increased stability of the oil-in-water emulsion. Fast-performance liquid chromatography analysis indicated that the 45-kDa protein forms a dimer in nondenaturing conditions and interacts with the 16- and 31-kDa proteins to form a high-molecular-mass complex. The 45-kDa protein and the three-protein complex had substrate specificities for emulsification and a range of pH activities similar to that of alasan. The fact that the purified proteins are active emulsifiers should simplify structure-function studies and advance our understanding of their biological roles.     

Biochemical and immunological studies of purified mouse beta-galactosidase.

Beta-Galactosidase (EC 3.2.1.23) has been purified from the livers of C57BL/6J mice. The enzyme migrated as a single band of protein on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular weight of the denatured and reduced enzyme was 63,000. The native form of beta-galactosidase appeared to be a tetramer of 240,000 at pH 5.0, which was reversibly dissociated at alkaline pH to a dimer with apparent molecular weight of 113,000. Multiple charge isomers of beta-galactosidase were resolved by polyacrylamide gel electrophoresis and ion exchange chromatography. Treatment of beta-galactosidase with neuraminidase markedly reduced its electrophoretic mobility. Purified enzyme as well as crude liver extract hydrolyzed p-nitrophenyl-beta-D-fucoside at one-tenth the rate of hydrolysis of the beta-galactoside. Antiserum to the purified enzyme precipitated the major portion of beta-galactosidase activity of mouse liver, brain, and kidney. This antiserum cross-reacts with beta-galactosidases from rat and Chinese hamster, but not with human, porcine, or bovine beta-galactosidase.     

Ovigerous-hair stripping substance (OHSS) in an estuarine crab: purification, preliminary characterization, and appearance of the activity in the developing embryos

Ovigerous-hair stripping substance (OHSS) is an active factor in crab hatch water (i.e., filtered medium into which zoea larvae have been released). This factor participates in stripping off the egg attachment structures (i.e., egg case, funiculus, and the coat investing ovigerous hairs) that remain attached to the female's ovigerous hairs after larval release. Thus this activity prepares the hairs for the next clutch of embryos. OHSS activity of an estuarine crab, Sesarma haematocheir, eluted as a single peak on molecular-sieve chromatography, but this peak still showed two protein bands at 32 kDa and 30 kDa on SDS-PAGE. The two protein bands stained with a polyclonal antiserum raised to the active fractions from molecular-sieve chromatography. Moreover, antibodies purified from this polyclonal OHSS antiserum also recognized both the 32-kDa and 30-kDa bands. OHSS immunoreactivity and biological activity were associated with the attachment structures that remained connected to the ovigerous hairs after hatching. In developing embryos, both protein bands could be stained immunochemically at least 10 days before hatching. But OHSS biological activity appeared only 3 days before hatching. The immunoreactive protein bands were not observed in the zoea, but OHSS bioreactivity was present, though greatly reduced. The 32-kDa protein, at least, is probably an active OHSS, and the 30-kDa protein band may also be OHSS-related. The OHSS appears to be produced and stored by the developing embryo. Upon hatching, most of the material may be trapped by the remnant structures, and the remainder is released into the ambient water.     

Purification and biochemical analysis of catalytically active human cdc25C dual specificity phosphatase

We describe a reliable and efficient method for the purification of catalytically active and mutant inactive full-length forms of the human dual specificity phosphatase cdc25C from bacteria. The protocol involves isolating insoluble cdc25C protein in inclusion bodies, solubilization in guanidine HCL, and renaturation through rapid dilution into low salt buffer. After binding renatured proteins to an ion exchange resin, cdc25C elutes in two peaks at 350 and 450 mM NaCl. Analysis by gel exclusion chromatography and enzymatic assays reveals the highest phosphatase activity is associated with the 350 mM NaCl with little or no activity present in the 450 mM peak. Furthermore, active cdc25C has a native molecular mass of 220 kDa consistent with a potential tetrameric complex of the 55-kDa cdc25C protein. Assaying phosphatase activity against artificial substrates pNPP and 3-OMFP reveals a 220 kDa form of the phosphatase is active in a non-phosphorylated state. The protein effectively activates cdk1/cyclin B prokinase complexes in vitro in the absence of cdk1 kinase activity in an orthovanadate sensitive manner but is inactivated by A-kinase phosphorylation. In vitro phosphorylation of purified cdc25C by cdk1/cyclin B1, cdk2/cyclin A2 and cdk2/cyclin E shows that distinct TP/SP mitotic phosphorylation sites on cdc25C are differentially phosphorylated by these 3 cdk/cyclin complexes associated with different levels of cdc25C activation. Finally, we show that endogenous native cdc25C from human cells is present in high molecular weight complexes with other proteins and resolves mostly above 200-kDa. These data show that untagged cdc25C can be purified with a simple protocol as an active dual specificity phosphatase with a native molecular mass consistent with a homo-tetrameric configuration.