Immunochemical characterization of two activator proteins stimulating enzymic sphingomyelin degradation in vitro. Absence of one of them in a human Gaucher disease variant

Two nonenzymic activator proteins shown previously to strongly stimulate enzymic sphingomyelin degradation in vitro were purified from human Gaucher type 1 and control spleen. Activator A1 (molecular mass 6,500 Da) had affinity for ConA-Sepharose, while activator A2 (molecular mass 3,500 Da) did not. Monospecific antibodies to each activator protein were prepared in rabbits by immunization with protein purified from type 1 Gaucher spleen. A1 and A2 activators from Gaucher type 1 spleen were shown to be immunochemically identical to A1 and A2 activators from control spleen. However, A1 and A2 activators, whether isolated from Gaucher type 1 or control spleen, were shown to be distinct proteins. Immunochemical examination of all collected fractions during the purification revealed the existence of a third activator (molecular mass 6,000 Da), which was antigenically identical to A1 activator but had no affinity for ConA-Sepharose. The two forms of A1 activator showed similar mobility on immunoelectrophoresis differing from that of A2 activator. Fibroblast extracts from controls and patients with different variants of Gaucher disease were investigated using immunodiffusion against antisera to A1 or A2 activator. In contrast to normal and Gaucher (types 1, 2 and 3) cell extracts, those of a Gaucher patient with normal glucosylceramidase activity had no visible precipitin line towards the antiserum against the two forms of A1 activator. The lack of crossreacting material to antibodies against A1 activator was confirmed by radial immunodiffusion and rocket immunoelectrophoresis. A1 activator stimulated the basal glucosylceramidase activity 5-6 fold in fibroblasts from this patient, whereas the normal effect was only a 1.2-1.5-fold stimulation. The immunological results together with the biochemical data provide evidence for the lack of an activator protein in a variant form of human Gaucher disease for the first time.     

Analysis of the multiple forms of Gaucher spleen sphingolipid activator protein 2.

Gaucher spleen sphingolipid activator protein 2 was fractionated into concanavalin A binding- and non-binding fractions. These fractions each contained several bands on non-denaturing polyacrylamide gel electrophoresis (PAGE). The two fractions were further fractionated by electroblotting the proteins from preparative gels onto nitrocellulose, staining with Ponceau S to locate the bands of protein and then eluting the protein components from the nitrocellulose. A total of ten fractions, each containing only one or two major components, was collected. All of these subfractions activated beta-glucocerebrosidase and sphingomyelinase and most subfractions also activated beta-galactocerebrosidase. The structural relationship of the bands was investigated using endoglycosidase digestions. The results indicated that the two bands with the fastest mobility on non-denaturing PAGE did not contain any carbohydrate. The remaining bands showed only limited or partial digestion with endoglycosidase H and endoglycosidase D, but were readily hydrolysed with endoglycosidase F. The products of these digestions included bands with similar mobilities to the non-carbohydrate containing bands.     

Complete amino-acid sequence of the naturally occurring A2 activator protein for enzymic sphingomyelin degradation: identity to the sulfatide activator protein (SAP-1)

The naturally occurring A2 activator protein for enzymic sphingolipid degradation is characterized by complete amino-acid sequence and carbohydrate content. It consists of 79 amino-acid residues and has a molecular mass of 8.875 kDa. The polypeptide chain contains 2 mol of N-acetylglucosamine, bound to asparagine in position 21, as well as 2 mol of galactose and mannose per mol protein. The primary structure of the A2 activator protein is identical to that of the sulfatide activator protein (SAP-1). Possible differences in the carbohydrate content are discussed.     

High-resolution proton nuclear magnetic resonance studies of the glucocerebrosidase activator protein from Gaucher spleen

A heat-stable protein factor (HSF) obtained from the spleen of a patient with Gaucher's disease that activates glucocerebrosidase was studied by 600-MHz proton NMR spectroscopy. Assignments for a number of aromatic and aliphatic resonances were made on the basis of spin-decoupling, pH-titration, and resolution-enhancement experiments. The upfield ring current shifted aliphatic region and the downfield aromatic region were examined by nuclear Overhauser effect (NOE) methods using both pulsed Fourier-transform spectroscopy and correlation spectroscopy. It was found that a number of upfield-shifted methyl groups and certain methylene groups of specific aliphatic amino acid residues are in proximity relationships with several aromatic residues, forming a compact hydrophobic clustering site. Of special interest, tyrosine A, phenylalanine A, tryptophan B1, and tryptophan B2 were found to be located close to a cluster of aliphatic residues, indicating that the hydrophobic site of the HSF is conformationally rigid and its tertiary structure very compact. A two-dimensional structural model of the hydrophobic site of HSF is proposed.     

Isolation of a melibiose-binding protein from human spleen

A melibiose-binding protein was isolated from human spleen by serial affinity chromatography on lactose-, mannose-, and melibiose-Sepharose. The purified protein agglutinated rabbit erythrocytes and re-bound to melibiose, but did not bind to murine nor human laminin. The protein was composed of 58 kDA, 32 kDa and 26 kDa polypeptides. The polypeptides were detected in buffy coat cell extracts and they were synthesizedin vitro by B lymphoblastoid cells. The polypeptides did not react with anti-galaptin, anti-C-reactive protein, anti-amyloid P, anti-keratin, and anti-rat lung lectin 29 sera. The 58 kDa polypeptide reacted very weakly with anti-core-specific lectin serum and reacted with anti-IgG serum. The data suggest that the major protein isolated is an anti-Gall 6 immunoglobulin.Abbreviations ME mercaptoethanol - PMSF phenylmethylsufonyl fluoride - HEPES 4-(2-hydroxyethyl)-1-piperazine ethanosulfonate - PBS 0.01m PO₄, 0.12m NaCl, pH 7.3 - TBS 0.1m NaCl, 0.05m Tris, 0.05% NaN₃, 0.01m CaCl₂, 0.001m MgCl₂, pH 7.3 - BSA bovine serum albumin - GSI Griffonia simplicifolia I - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis     

Subcellular localization of the heat-stable glucocerebrosidase activator substance in Gaucher spleen

The spleen in Gaucher's disease contains relatively large quantities of a heat-stable activator of the glucocerebrosidase of normal human tissues (Ho, M. W., and O'Brien, J. S. (1971) Proc. Nat. Acad. Sci. USA68, 2810–2813) that has been shown to be an 11,000 molecular weight acidic glycoprotein (Peters, S. P., et al. (1977) J. Biol. Chem.252, 563–573). In an effort to determine the subcellular location of the activator, a mannitol-sucrose homogenate of fresh, unfrozen spleen obtained from a 26-year-old patient with adult, nonneuropathic (Type 1) form of Gaucher's disease was subjected to subcellular fractionation. The tissue used in these experiments exhibited a β-glucocerebrosidase deficiency (11% of control tissue characteristic of Gaucher's disease. Mitochondrial and lysosomal fractions obtained by centrifugation of the spleen homogenate at 6900 and and 20,000g, respectively, contained greater than 80% of the recovered acid phosphatase and heat-stable glucocerebrosidase activator activities. In addition, 60% of the residual glucocerebrosidase activity was recovered in the mitochondrial and lysosomal fractions. The lysosomal and mitochondrial fractions were subjected to equilibrium sucrose density gradient centrifugation. Analysis of the sucrose gradient of the crude mitochondrial fraction demonstrated the mitochondrial marker enzyme (cytochrome oxidase) banding with a specific gravity of 1.19 g/ml, whereas the heat-stable activating factor banded in an acid phosphatase-rich fraction having a specific gravity of 1.12 g/ml. Sucrose gradient analysis of the crude lysosomal fraction obtained from differential centrifugation indicated the activating factor banding with a specific gravity of 1.12 g/ml. Coincident with the activating factor was glucocerebrosidase and acid phosphatase activity. Electron microscopic examination of fractions from each of the sucrose density gradients demonstrated that the glucocerebrosidase activating factor was located in the same acid phosphatase-rich fractions that contained the characteristic Gaucher deposits. Furthermore, when Gaucher deposits were isolated and purified independently by a sucrose gradient procedure, they were found to contain high concentrations of the heat-stable glucocerebrosidase activator. The specific activity of the glucocerebrosidase activating factor was approximately 15-fold greater in the extensively purified Gaucher deposits than in the crude extract of Gaucher spleen from which the deposits were isolated. These observations indicate that the heat-stable activator is associated with the storage deposits contained in lysosomes of the Gaucher cell.     

Stimulation of lysosomal sphingomyelin degradation by sphingolipid activator proteins

Lysosomal breakdown of glycosphingolipids with short hydrophilic carbohydrate headgroups is achieved by the simultaneous action of specific hydrolases and sphingolipid activator proteins (SAPs). Activator proteins are considered to facilitate the enzyme/substrate interaction between water-soluble enzymes and membrane-bound substrates. Sphingomyelin, containing the small hydrophilic phosphorylcholine moiety, is hydrolysed by acid sphingomyelinase (acid SMase). Recent experimental data on the in vivo and in vitro role of activator proteins in sphingomyelin breakdown by acid SMase are reviewed. These data combined with the results using homogenous protein preparations as well as a liposomal assay system mimicking the physiological conditions suggest that lysosomal sphingomyelin degradation is not critically dependent on any of the known activator proteins. Moreover, evidence is provided that the assumed intramolecular activator domain of acid SMase and especially the presence of negatively charged lipids in the lysosomes are sufficient for sphingomyelin turnover.     

Isolation and characteristics of hexosaminidase A and activator protein from human kidney

Hexosaminidase A (HA) was isolated from human kidney and purified to an electrophoretically homogeneous state. The purification procedure included ion-exchange chromatography on DEAE-cellulose, gel filtration on Toyopearl HW-55 and chromatofocusing on PBE 94 (enzyme yield 26.6%, 1133.6-fold purification). The physico-chemical and kinetic properties of HA are as follows: Mr of the purified enzyme is approximately 100,000; Km for 4-methylumbelliferyl-2-acetamido-2-deoxy-beta-D-glucopyranoside is 0.6 mM; pH optimum is at pH 4.4-4.6; pI is 5.0. The amino acid composition of the purified enzyme was determined. A specific anti-HA antiserum was raised, which did not immunoprecipitate with fibroblast extracts characterized by a mutational blockade of HA synthesis. GM2 was isolated and purified from murine liver as well as from the brain of a female patient who died of Tay-Sachs disease. The label was introduced by way of treatment of GM2 with tritiated acetic anhydride. The specific radioactivity of [3H]GM2 was 521 and 2065 Ci/M, respectively. The label was introduced into the N-acetylneuraminic acid and GalNAc residues of these GM2 preparations. An activator protein capable of solubilizing the natural substrate of HA was isolated from human kidney and partially purified (with a 19.9% yield and 480-fold purification). The Mr of the purified activator protein was approximately 21,000. Purified HA hydrolyzed [3H]GM2 only in the presence of the activator protein. An addition of the activator to the incubation medium containing normal fibroblast culture extracts and [3H]GM2 caused an increase in the rate of substrate hydrolysis, tenfold, on the average.     

Isolation and characterization of two forms of malate dehydrogenase from human placenta

• 1.1. The purification and characterization of the cytoplasmic and mitochondrial forms of malate dehydrogenase from human placenta are described.
• 2.2. Both enzymes are composed of two subunits and have similar molecular weights and similar pH optima.
• 3.3. However, they differ with respect to thermal stability, excess substrate inhibition and electrophoretic mobility.

     

beta-Glucosidase activator protein from bovine spleen ("coglucosidase")

β-Glucosidase-stimulating proteins (“co-β-glucosidase”) have been isolated from bovine spleen by acidification of homogenized spleen, heat denaturation, and chromatography with DEAE-Sephacel, Sephadex G-75, hydroxyapatite, and decyl agarose columns. Gel electrophoresis of the product revealed a trace of inert protein and two fast-moving bands, a major diffuse band and a minor, faster-moving band. The latter two bands could be eluted from the gel and shown to stimulate a glucosidase preparation from bovine spleen. They both stained with Stains All and fast green, but poorly with Coomassie blue. The bands could also be visualized by ultraviolet scanning. Periodate-Schiff stain was positive for the major band. The M_r of the coglucosidase was about 20,400 as measured with the gel permeation column, but 4900 as measured with a Sephacryl S-200 column containing guanidine hydrochloride and roughly 6200 as measured by gel electrophoresis with Na dodecyl sulfate. A pI of 4.3–4.4 was indicated by isoelectric focusing. Neutral sugar was found to be present, but no sialic acid. It was destroyed by Pronase, but not by lyophilization, N-ethylmaleimide, or alkaline phosphatase. Stimulation of the basal activity (1 nmol/h assayed with methylumbelliferyl glucoside) was 50% when 0.15 μg/ml of coglucosidase was included in the incubation. The activating protein raised the V values and lowered the K_m values when both glucosyl ceramide and the artificial substrate were used. In contrast, phosphatidyl serine raised both the V, and the K_m for cerebroside hydrolysis. The activator protein was found to occur in the soluble part of spleen as well as in the mitochondrial and lysosomal fractions.     

An endogenous activator protein in human placenta for enzymatic degradation of glucosylceramide

An endogenous, heat-stable and pronase-sensitive activator for enzymatic hydrolysis of glucosylceramide was detected in the crude lysosome-mitochondria fraction of human placenta. Its properties differ distinctly in several important respects from those of the previously described glucosylceramidase activator. The activator reported here had no effect on crude glucosylceramidase with either glucosylceramide or 4-methylumbelliferyl-beta-D-glucopyranoside as the substrate in the presence of either sodium taurocholate or phosphatidylserine. On the contrary, glucosylceramide hydrolysis by the enzyme partially purified through Octyl-Sepharose 4B chromatography was stimulated by this activator 6-9-fold in the presence of either sodium taurocholate or phosphatidylserine. The Km for glucosylceramide in the presence of the activator was 1/3 of that without the activator. In the crude enzyme fraction, the activator was present in a 16-fold excess over the minimum amount necessary for full activation of the enzyme. Hydrolysis of the fluorogenic substrate by the post-Octyl-Sepharose enzyme, however, was not stimulated by the activator. Similarly, hydrolysis of galactosylceramide by galactosylceramidase obtained from the same Octyl-Sepharose chromatography was not stimulated. Our observations are consistent with the idea that glucosylceramidase is saturated by, or perhaps tightly associated with, this activator in the placenta and that they are dissociated by the Octyl-Sepharose chromatography. In fact, the properties of the combined post-Octyl-Sepharose enzyme and activator closely mimic those of the crude enzyme without added activator.     

Isolation of a cDNA encoding the human GM2 activator protein

The GM2 activator protein is a glycolipid-binding protein required for the lysosomal degradation of ganglioside GM2. A human fibroblast cDNA library was screened with mixtures of oligonucleotide probes corresponding to four different areas of the amino acid sequence. A putative clone (821 bp) which gave positive signals to all four probe mixtures was purified and sequenced. The sequence was colinear with the sequence of 160 amino acids of the mature GM2 activator protein. Availability of the cDNA clone should facilitate investigation into function of the GM2 activator protein and also into genetic abnormalities underlying GM2 gangliosidosis AB variant.     

Isolation of two forms of decay-accelerating factor (DAF) from human urine.

Decay-accelerating factor (DAF) was purified from human pooled urine by conventional techniques. The urine DAF was separated into two peaks, pool I and pool II, by gel chromatography. DAF-U1 was isolated from pool I by hydrophobic chromatography, and DAF-U2 from pool II by anti-DAF IgG column. The specific activities of DAF-U1 and DAF-U2 to decay membrane-phase C5 convertase were about 3% and 70% of membrane form DAF, respectively. However, both urine DAFs revealed a similar activity to each other and slightly higher activity than that of membrane form DAF in decay-accelerating fluid-phase C3 convertase of the alternative pathway.     

Isolation of two distinct activator proteins for lipoprotein lipase from ovine plasma

Two distinct activator proteins for lipoprotein lipase (LPL) have been isolated in approximately equal amounts from ovine plasma. These low molecular weight proteins were readily separated from each other on the basis of size and charge. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated proteins of Mr about 8000 and 5000, with pI in urea-containing gels of about 5.1 and 4.8 respectively. Each of the ovine activator proteins was as effective as human apolipoprotein C-II (apo C-II) in activating LPL, with 1 microgram/ml giving near to maximum activation, and in lowering the apparent Km of LPL for triolein substrate. As the ratio of activator to triolein increased from 0.16 to 5.2 (micrograms/mg) the apparent Km fell from about 0.5 to 0.18 mM. Whereas human apo C-II and the two ovine activators were equally effective in stimulating the hydrolysis of triolein, differences were observed between the human and ovine activators when p-nitrophenylbutyrate was used as substrate. Unlike human apo C-II, which produced significant inhibition of p-nitrophenylbutyrate hydrolysis, the ovine activators were without effect. This suggests that the interaction between the ovine activators and LPL is different from that of human apo C-II.     

Isolation and characterization of two distinct forms of protein kinase C

Protein kinase C (Ca2+- and phospholipid-dependent protein kinase) has been purified from rat brain by a three-step, 18-h procedure resulting in the isolation of milligram quantities of enzyme. Unlike previous preparations from published protocols, which yield a single polypeptide, this procedure yields a protein which consists of a 78/80-kDa doublet upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The two polypeptides have been characterized with respect to structure and function and are very similar in both regards. However, the two forms can be distinguished immunologically by polyclonal antisera generated against purified protein kinase C. The 78- and 80-kDa proteins do not appear to be related to one another by proteolytic cleavage or by differential phosphorylation, although the two purified proteins do contain stoichiometric amounts of phosphate. The 78- and 80-kDa polypeptides therefore appear to represent two distinct forms of protein kinase C, thus providing evidence for the existence of multiple isozymes of this key regulatory protein.     

Complete amino-acid sequence and carbohydrate content of the naturally occurring glucosylceramide activator protein (A1 activator) absent from a new human Gaucher disease variant

Two naturally occurring non-enzymic glucosylceramide activator proteins (A1a and A1b activator) shown previously to be immunochemically not detectable in a new variant of human Gaucher disease (glucosylceramide lipidosis) without glucosylceramidase deficiency, were characterized by amino-acid sequence and carbohydrate content. The complete amino-acid sequence of the A1a activator was determined. The protein consists of 80 amino-acid residues including three disulfide bridges lacking arginine and tryptophan. The molecular mass is 8.95 kDa. About 20% of the polypeptide chain are shorter by two amino-acid residues at the N-terminal end. The A1b activator was characterized by the amino-acid compositions of all tryptic peptides and of the entire protein; sequencing was performed of the regions 1-34 and 42-56. Identical results were obtained for the polypeptide chains of both A1 activators. This suggests that they do not differ in their primary structures which is in agreement with the immunochemical results. The difference between A1a and A1b activator is due to the carbohydrate part. The total amount of 49% carbohydrate in A1a and 76.7% in A1b consists mainly of hexoses. Both chains contain two moles of N-acetylglucosamine per mole protein bound to asparagine in position 22. A comparison of the primary structure of the A1 activator with the sulfatide activator sequence revealed an interesting similarity, especially of the cysteine residues and the carbohydrate-binding asparagine. Sequence homology was also found between a part of the A1 activator sequence and the hemagglutinin neuraminidase of influenza virus as well as to a hypothetical glycoprotein of the Epstein-Barr virus. The comparison with human lysosomal glucosylcerebrosidase showed no sequence similarity.     

Isolation and characterization of an anticoagulant protein from human placenta

An anticoagulant protein was purified from the EDTA extract of human placental tissue. The purified protein had a molecular weight of 73,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis under both reducing and non-reducing conditions. Because this protein had the ability to bind phospholipids such as phosphatidylserine, phosphatidylinositol, and cardiolipin in the presence of Ca2+, this protein was designated as calphobindin II (CPB-II). CPB-II prolonged the clotting time of normal plasma when coagulation was induced by tissue factor, cephalin and ellagic acid or recalcification, but did not affect thrombin-initiated fibrin formation. CPB-II also inhibited the activation of prothrombin by the complete prothrombinase complex or factor Xa-phospholipid-Ca2+ but not that by phospholipid-free factor Xa. In addition, CPB-II had an inhibitory activity against phospholipase A2.     

N-terminal amino-acid sequence of a sphingolipid activator protein missing in a new human Gaucher disease variant

A naturally occurring non-enzymic sphingolipid activator protein (A1a activator) shown previously to be immunochemically not detectable in a new variant of human Gaucher disease (glucosylceramide-lipidosis) without glucosylceramidase deficiency was characterized by partial sequence analysis. The N-terminal amino-acid sequence of the A1a activator--a glycoprotein with high carbohydrate content--could be determined up to position 38. About 20% of the polypeptide chain are shorter by two amino-acid residues at the N-terminal end. Position 22 seems to be occupied by a carbohydrate-binding asparagine. The N-terminus of the A1a activator does not show any homology with the activator for the enzymic sulfatide degradation.