The cerebroside sulfate activator from pig kidney: Derivitization,cerebroside sulfate binding,and metabolic correction

Highly purified cerebroside sulfate activator from pig kidneys was characterized by a number of chemical and biological procedures. Methods for chemical modifications were evaluated in an attempt to obtain biologically active derivatives. Iodination, dabsylation, and to a lesser degree reductive methylation provided useful products with good retention of cerebroside sulfate activator activity. Other procedures resulted in largely inactive derivatives or losses in both protein and biological activities. Attempts at renaturation of cerebroside sulfate activator subjected to various denaturing conditions appeared to be successful in many instances, but it was uncertain if the protein structure had actually been disrupted. The binding of cerebroside sulfate by activator was estimated by gel filtration under conditions similar to those of its assay. The formation of a relatively stable 1:1 complex was observed, collaborating results with the human protein. The complex was stable enough to be isolated and shown to be an efficient substrate for arylsulfatase A. The effectiveness of the pig kidney cerebroside sulfate activator for correcting the metabolic defect in activator-deficient human fibroblasts was compared with human materials. The pig kidney protein was taken up more efficiently by the cells and resulted in a better metabolic correction than material from human liver, but was somewhat less effective than a preparation from human urine.     

Genetic complementation in somatic cell hybrids of cerebroside sulfatase activator deficiency and metachromatic leukodystrophy fibroblasts

Summary Several cases of metachromatic leukodystrophy (MLD) have been described with normal or near normal activities of arylsulfatase A (cerebroside sulfatase). However, the ability of intact cultured fibroblasts to hydrolyze cerebroside sulfate was impaired. Since the impairment was corrected by cerebroside sulfatase activator, a deficiency of activator was implied. In the absence of direct demonstration of deficiency, other types of evidence were needed to support the premise that the genetic defect was not associated with the arylsulfatase A locus as in classical MLD. Therefore, somatic cell hybrids of activator deficiency and MLD fibroblasts were analyzed. Complementation was indicated by enhanced hydrolysis of cerebroside sulfate, supporting the view that cerebroside sulfatase activator deficiency and MLD are nonallelic.     

Activator protein required for the enzymatic hydrolysis of cerebroside sulfate. Deficiency in urine of patients affected with cerebroside sulfatase activator deficiency and identity with activators for the enzymatic hydrolysis of GM1 ganglioside and globotriaosylceramide

Urine specimens from two sibs affected with cerebroside sulfatase activator deficiency were examined to ascertain whether the deficiency of the supplementary activator protein required for the enzymatic hydrolysis of cerebroside sulfate was also evident in urine. Material from chromatographic fractionations was examined for the activator activity to avoid ambiguities resulting from protein inhibition. There were substantial deficits in all chromatographic fractions corresponding to activator-containing fractions of control urines. Since patient urines contained elevated amounts of lactosylceramide, digalactosylceramide, and globotriaosylceramide and since similarities between activators for cerebroside sulfate and GM1 ganglioside hydrolyses had been noted previously, the chromatographic fractions were also examined for activators in other glycosphingolipid hydrolase systems. There was coincidence of activators for the GM1 ganglioside/beta-galactosidase and the globotriaosylceramide/alpha-galactosidase A reactions with the cerebroside sulfatase activator in control urine fractions, and the patients' urines were deficient in activator activities for the three reactions. Identity of the three activators was suggested and antiserum to purified GM1 ganglioside activator was used to test this possibility. There were depressed levels of cross-reacting material in fractions of patient urines by Ouchterlony double diffusion and in unfractionated urine by enzyme-linked immunosorbent assay. Purified activators for the cerebroside sulfate and GM1 ganglioside systems showed lines of identity with no spurring on Ouchterlony double diffusion, identical mobility on immunoelectrophoresis, and similar stimulatory activities toward hydrolysis of the three glycosphingolipid species by their respective enzymes. Finally, the three activator activities were retained by anti-GM1-activator IgG coupled to Sepharose 4B. The results suggest strongly that the same protein entity serves as activator for the enzymatic hydrolysis of cerebroside sulfate, GM1 ganglioside, and globotriaosylceramide.     

Cerebroside sulfatase activator deficiency induced metachromatic leukodystrophy

Two siblings of consanguineous parents had presented with a variety of findings indicative of juvenile metachromatic leukodystrophy (MLD). However, instead of the expected profound deficiency of arylsulfatase A (ARS A), their enzyme levels were about half-normal, and enzyme from fibroblasts had properties identical with the properties of enzyme from normal fibroblasts. Nevertheless, the hydrolysis of cerebroside sulfate by growing fibroblasts was markedly attenuated. Supplementation of the fibroblasts with cerebroside sulfatase activator normalized the response in the loading test. These results imply that the fibroblasts, and by extension the patients, are deficient in activator. Although the defective catabolism of cerebroside sulfate and the clinical manifestations in these patients mimic MLD, the molecular basis is distinct from the classical forms of the disorder.     

The cerebroside sulfate activator from pig kidney: purification and molecular structure.

The activator protein for hydrolysis of cerebroside sulfate by arylsulfatase A was purified from pig kidney in high yield. This protein, also known as sphingolipid activator protein-1 and saposin-B, was particularly rich in pig kidney. Purification was achieved by a simple procedure involving homogenation and heat treatment followed by affinity, ion exchange, and gel filtration chromatographies. The final product was better than 90% pure by gel electrophoresis and HPLC. It was possible to sequence more than 60 amino acids from the N-terminus with only a few uncertain residues. The sequence differed from that predicted for the human protein by about 10%, with most amino acid variations being conservative. There appeared to be a residual glycosyl substituent on asparagine 21, but the sugar content was low and the protein failed to bind to concanavalin A. The cerebroside sulfate activator proved to be exceptionally resistant to denaturation or protease digestion. The apparent molecular mass was approximately 20,000 Da on preparative gel-filtration columns, but was variable when estimated by HPLC gel filtration. Values ranging from 30,000 to over 100,000 Da were observed in neutral buffers, while values around 15,000-16,000 Da were seen in acidic buffers such as those used for assay of the biological activity. This was further decreased to a putative subunit of 7000-8000 Da under severe denaturing conditions. Pig kidney is a convenient source for the large-scale preparation of this interesting protein which has heretofore been obtained from human sources.     

The activator of cerebroside sulphatase. Purification from human liver and identification as a protein.

1) A heat-stable activator of human sulphatase A (cerebroside sulphatase) was purified from human liver. It is required for the enzymatic degradation of cerebroside sulphates (sulphatides) in buffers (ionic strength greater than or equal 0.2) with osmolarity in the physiological range. 2) The purification steps involve extraction, acetone precipitation, heat treatment, isoelectric focusing and gel filtration. 3) Based on the definition of a specific activator unit, the purification of the final preparation was approximately 2000-fold over the acetone precipitation and several thousand-fold in the overall procedure. 4) The purified activator migrated as a single protein band when subjected to gel electrophoresis. Its effect was abolished after treatement with pronase E. The apparent molecular weight as determined by gel filtration was 21 500 +/- 1500; the isoelectric point was 4.3. 5) The activating effect of this protein factor and of taurodeoxycholate on cerebroside sulphatase activity was compared on a weight and molar basis.     

The activator of cerebroside sulphatase. Lysosomal localization.

1) An activator protein necessary for the enzymic hydrolysis of cerebroside sulphate could be partially purified from unfractionated rat liver. This activator, which is similar to that of human origin, proved to be a heat-stable, non-dialyzable, low molecular weight protein with an isoelectric point of 4.1. Its activity could be destroyed by pronase. 2) For elucidation of the subcellular localization of the activator, rat liver was fractionated by differential centrifugation. The intracellular distribution of the cerebroside sulphatase activator was compared to the distribution patterns of marker enzymes for different cell organelles and found to coincide with the lysosomal arylsulphatase, thus indicating a lysosomal localization. 3) This was confirmed using highly purified secondary, i.e. iron-loaded, lysosomes. After disruption by osmotic shock, these organelles hydrolyzed cerebroside sulphate when incubations were performed under physiological conditions with endogenous as well as exogenous sulphatase A as enzyme. 4) After subfractionation of the disrupted secondary lysosomes into membrane and lysosol fractions by high speed centrifugation, it was found that the activator protein was exclusively associated with the lysosol, whereas the acid hydrolases were distributed differently between the two fractions. 5) The lysosol was further fractionated by semi-preparative electrophoresis on polyacrylamide gels. Two protein fractions were obtained: a high molecular weight fraction, containing the activator-free acid hydrolases, and a low molecular weight fraction, containing the enzyme-free activator of cerebroside sulphatase. 6) The significance of these findings for the hydrolysis of sphingolipids in the lysosomes is discussed.     

Expression,purification, crystallization,and preliminary X-ray analysis of recombinant human saposin B

Saposin B (also known as cerebroside sulfate activator or CSAct) is a small non-enzymatic glycoprotein required for the breakdown of cerebroside sulfates (sulfatides) in lysosomes. Saposin B contains three intramolecular disulfide bridges, exists as a dimer and is remarkably heat, protease, and pH stable. We have expressed the protein in a thioredoxin reductase deficient strain of Escherichia coli and purified the protein by heat treatment, followed by ion-exchange, gel filtration, and hydrophobic interaction chromatographies. The protein is properly folded as judged by the observed disulfide bond topology, the hydrogen-deuterium exchange rate, and the level of stimulation of sulfatide hydrolysis by arylsulfatase A. Crystals of human saposin B were grown by vapor diffusion and diffract to a resolution of 2.2A. Despite obtaining only merohedrally twinned P3(1) native crystals, an untwined seleomethionine-substituted crystal belonging to space group P3(1)21 was also grown. The three-dimensional structure of saposin B protein will provide insights into how this 79 amino acid protein is able to solubilize relatively large membrane-bound lipid ligands.     

Synthesis, spectral properties and enzymatic hydrolysis of fluorescent derivatives of cerebroside sulfate containing long-wavelength-emission probes

Fluorescent derivatives of cerebroside sulfate (sulfogalactosyl ceramide, sulfatide) containing long-wavelength-emission fluorophores were synthesized. For this purpose a procedure was developed for preparing a cerebroside 3-sulfate derivative with an amino group on the terminal carbon atom of its fatty acyl residue. The latter compound has been used to prepare cerebroside 3-sulfate, coupled to lissamine-rhodamine, fluoresceine, eosine and NBD. The spectroscopic properties of these compounds, in different solvent systems and when incorporated into micelles of a non-ionic detergent or liposomes of a phospholipid, are reported. Incubation of these respective sulfatides with a human leukocyte preparation, resulted in the formation of the corresponding fluorescent cerebrosides.     

Synthesis of pyrene derivatives of cerebroside sulfate and their use for determining arylsulfatase A activity

Two fluorescent derivatives of cerebroside sulfate ('sulfatide') have been synthesized and used as substrates for determining arylsulfatase A activity. These were 12-(1-pyrene)dodecanoyl cerebroside sulfate (P12-sulfatide) and 12(1-pyrenesulfonylamido)dodecanoyl cerebroside sulfate (PSA12-sulfatide). When incubated at pH 5.0 in the presence of 5 mM MnCl2 and 5.5 mM of taurodeoxycholate, either substrate was hydrolyzed by arylsulfatase A of human leukocytes. The rate of hydrolysis was proportional to the incubation time and concentration of enzyme; Michaelis-Menten type kinetics were observed with increasing concentrations of substrate. For determining the rate of hydrolysis, each of the two products (i.e., P12- and PSA12-cerebrosides) were separated from the bulk of respective unreacted sulfatide on small columns of DEAE-Sephadex A-25 and their fluorescence intensities read at 343-378 and 350-380 nm for the excitation and emission wavelengths for P12- and PSA12-cerebrosides, respectively. When extracts of skin fibroblasts derived from normal individuals and patients with Maroteaux-Lamy (lacking arylsulfatase B) or metachromatic leukodystrophy (lacking arylsulfatase A) were used as source of enzyme, P12-sulfatide was hydrolyzed by the former two but not by the latter cell extract. Several derivatives of cerebroside sulfate were also synthesized and found to inhibit the hydrolysis of pyrenesulfatide by leukocyte arylsulfatase A. The results demonstrate that these two pyrene containing sulfatides can be effectively used as specific substrates for the determination of arylsulfatase A activity in extract of cells and most probably also of tissues.     

Metabolic responses of sulfatide and related glycolipids in Madin-Darby canine kidney (MDCK) cells under osmotic stresses

Incorporation of (35)S-sulfate into the polar molecular species of sulfoglycolipids (SM4s) in Madin-Darby canine kidney cells increased in a hypertonic medium (500 mOsm/L) supplemented with sodium chloride. The unknown sulfoglycolipid (SX) was identified as GlcCer sulfate based on the results of TLC, GLC, and mass spectra. The synthesis of SX increased in the hypotonic medium unlike that of SM4s and SM3. TLC showed that hypertonic stress induced the accumulation of GalCer as a precursor of SM4s, whereas hypotonic stress increased GlcCer as a precursor of GlcCer sulfate. The level of ceramide as a precursor of both GalCer and GlcCer increased under hypertonic stress and decreased under hypotonic stress. Cerebroside sulfotransferase mRNA was shown to be elevated in the hyperosmotic condition but not in the hypotonic condition. The increase in SM4s under hypertonic stress was induced by the activation of both the ceramide galactosyltransferase and the cerebroside sulfotransferase genes, whereas the increase in GlcCer sulfate under hypotonic stress was caused by the accumulation of GlcCer as the result of activation of ceramide glucosyltransferase.     

Disulfide connectivity in cerebroside sulfate activator is not necessary for biological activity or alpha-helical content but is necessary for trypsin resistance and strong ligand binding

Cerebroside sulfate activator (CSAct) protein is exceptionally resistant to heat denaturation and proteolytic digestion. Although water soluble the protein binds membrane-associated lipids. Its biological role is thought to be to transfer certain lipids between membranes and to facilitate their catabolism in the lysosomes. An example of the latter is the removal of the sulfate group from cerebroside sulfate by arylsulfatase A. The mechanism of lipid sequestration from membranes and presentation of the lipid-protein complex to catabolic enzymes is a crucial aspect of the function of this protein. The widespread occurrence of the protein class of which CSAct is one of the best known members underscores the significance of this protein. The preparation, purification and chemical and biological properties of a stable disulfide blocked derivative of CSAct is described. The pyridoethylated protein was susceptible to tryptic attack and devoid of a significant population of solvent-protected exchange resistant protons. It apparantly formed a CS complex. However, unlike the complex with the native protein, this was not sufficiently stable to remain intact during size exclusion chromatography. The disulfide-blocked protein had a similar CD spectrum as native protein, indicating similar alpha-helical content. Unexpectedly, the activities of disulfide-blocked protein in the arylsulfatse A catalyzed sulfate hydrolysis from cerebroside sulfate were substantial. Hitherto, it had been assumed that the disulfide connectivities were essential for the protein to maintain a correctly folded configuration to bind lipid ligands and potentiate their hydrolysis. Some revision of our thoughts on the importance of the disulfide connectivities in the structure and function of the protein are necessary.     

A novel mass spectrometric assay for the cerebroside sulfate activator protein (saposin B) and arylsulfatase A

A mass spectrometric method is described for monitoring cerebrosides in the presence of excess concentrations of alkali metal salts. This method has been adapted for use in the assay of arylsulfatase A (ASA) and the cerebroside sulfate activator protein (CSAct or saposin B). Detection of the neutral glycosphingolipid cerebroside product was achieved via enhancement of ionization efficiency in the presence of lithium ions. Assay samples were extracted into the chloroform phase as for the existing assays, dried, and diluted in methanol-chloroform-containing lithium chloride. Samples were analyzed by electrospray ionization mass spectrometry with a triple quadrupole mass spectrometer in the multiple reaction monitoring tandem mass spectrometric mode. The assay has been used to demonstrate several previously unknown or ambiguous aspects of the coupled ASA/CSAct reaction, including an absolute in vitro preference for CSAct over the other saposins (A, C, and D) and a preference for the non-hydroxylated species of the sulfatide substrate over the corresponding hydroxylated species. The modified assay for the coupled ASA/CSAct reaction could find applicability in settings in which the assay could not be performed previously because of the need for radiolabeled substrate, which is now not required.     

The activator of human cerebroside sulphatase. Activating effect on the acidic forms of the sulphatases from invertebrates.

1) Acidic forms of the sulphatase were partially purified from the following invertebrate species: Tethya aurantium (Porifera), Patella vulgata (mollusca), Maja squinado (Arthropoda), Marthasterias glacialis (Echinodermata) and Microcosmus sulcatus (Tunicata). Enzyme preparations thus obtained cleaved cerebroside sulphates (sulphatides) only in the presence of either specific detergents (e.g. taurodeoxycholate) or an activator protein isolated from human liver. This corresponds to the findings on purified sulphatase A of human origin. 2) At low concentrations, the activating effect was proportional to the amount of activator protein applied; at higher concentrations, proportionality was obtained only in some cases. On a molar basis, less of the activator protein was required to achieve the same activation as taurodeoxycholate. At optimum concentrations of the detergent however, the activation was much higher. 3) The enzyme specificity of the activator and some evolutionary implications are discussed.     

Origin of structural domains of the serum-albumin gene family and a predicted structure of the gene for vitamin D-binding protein

We have recently determined complete DNA sequences for the human albumin and alpha-fetoprotein [AFP] genes and thus have identified their detailed structures. Each is composed of three domains of four exons, three of which are internal and one of which is a domain-linking exon. Equivalent exons in each domain show sufficient sequence and structural similarity to be considered homologous; additional unique exons at each end of the gene show no similarity to the internal triplicated structures. Since earlier, conflicting evolutionary models were based on analysis of single gene structures, we derived from five genes a series of consensus sequences representing the three internal exons as well as the domain-linking exon. The five genes were human and rat albumin and human, mouse, and rat AFP genes. Structurally equivalent exons of the different domains are shown to have arisen from a single exon in a one-domain precursor. Exons that bridge the domains arose from an unequal crossover that fused two exons of the precursor. Our model suggests that part of the coding sequence of the one-domain precursor may have been derived from an intron, by way of loss of a splice site. The consensus sequences were used to propose an intron-exon structure for the related gene encoding the serum vitamin D-binding protein (DBP). DBP is truncated relative to albumin and AFP, and we submit that this results from deletion of two internal exons in the third domain of the gene rather than from premature termination of the coding sequence.     

Evidence of neuropeptide bivalency in the interaction of substance P with cerebroside sulfate.

A precipitin effect has been observed with mixtures of cerebroside sulfate and the neuropeptide substnace P. This phenomenon is attributed to multivalency of the lipid due to its existence in micellar form, and to bivalency of substance P. One of those neuropeptide sites is almost certainly the basic residue(s) located at the N-terminal of substance P, whereas the hydrophobic residues at the C-terminus are suggested as candidates for the other site on the basis of turbidimetric, circular dichroic, and fluorometric studies. An intrinsic association constant of 3.6 x 10(4)M-1 has been obtained from the cerebroside sulfate concentration associated with maximal turbidity of mixtures containing a fixed concentration of the neuropeptide.     

Opiate binding to cerebroside sulfate: a model system for opiate-receptor interaction.

Cerebroside sulfate was shown to bind etorphine and levorphanol with high affinity. The relative potency of narcotic analgesics in preventing the binding of levorphanol to cerebroside sulfate correlated well with their reported analgetic activity. The data indicate similarities between cerebroside sulfate and a purified opiate receptor from mouse brain which has been reported to be a proteolipid. Some preliminary animal data also imply the involvement of CS in opiate action We, therefore, propose that CS may serve as a useful “receptor” model for the study of opiate-receptor interaction $\text{in vitro}$.     

Kinetics of entry of galactolipids and phospholipids into myelin.

Abstract— Brain slices from 17 day rats were incubated with [³H]galactose and [³⁵S]sulphate to label cerebroside and sulphatide. Myelin was isolated by centrifugation on a sucrose density gradient. Following lipid extraction and alkaline methanolysis, cerebroside and sulphatide were isolated by tic, and radioactivity was measured. Appearance of [³H]cerebroside and [³H]sulphatide in myelin showed a lag of less than 15min, while appearance of [³⁵S]sulphatide in myelin showed a longer lag of about 30min. In chase experiments, the rate of appearance of [³H]cerebroside and [^3SS]sulphatide in the non-myelin fraction and of [³H]cerebroside in the myelin fraction slowed markedly after the chase. In contrast, [³⁵S]sulphatide continued to increase in myelin at a normal rate for 30min after the chase, then stopped, while ³H from galactose continued to accumulate in myelin sulphatides for 60 min. These data are interpreted to demonstrate an interval of 30 min between synthesis of cerebroside and its sulphation in the non-myelin fraction, and another delay of 30 min between sulphation and appearance in myelin. The distribution of newly synthesized cerebroside and sulphatide between myelin and non-myelin fractions also supported the concept that a complex metabolic pool of cerebroside in the non-myelin fraction is precursor to sulphatide of myelin. For comparison, entry of phosphatidyl choline and phosphatidyl ethanolamine into myelin was followed with [2-³H]glycerol as precursor. Like cerebroside, both phospholipids showed little delay in their initial appearance in myelin, and prompt cessation of their addition after a chase with unlabeled precursor. These results are consonant with either rapid entry of these three lipids into myelin after synthesis at an extra-myelin site, or synthesis of the lipids within myelin itself.     

The activator of cerebroside sulphatase. Binding studies with enzyme and substrate demonstrating the detergent function of the activator protein.

1. Sulphatase A (cerebroside sulphatase) (EC 3.1.6.1.) and a 12-fold excess of its physiological activator protein were chromatographed together on Sephadex G-75. The elution buffer was the same as that used in the enzymic degradation of sulphatides. The two proteins were eluted in different peaks indicating that no stable complex formed. 2. Activator protein was incubated with sulphatides under conditions used favouring the sulphatase activity. Incubation solutions were then examined by electrophoresis on a polyacrylamide gel gradient. An one-to-one complex between activator and sulphatides was observed. Half maximal binding occurred with 2.5 nmol of sulphatides together with 1 or 2 nmol of activator in 100 micronl. 3. Cerebrosides as the enzymic degradation products of sulphatides, bind also to the activator protein. A ratio of one-to-one could possibly be obtained at high cerebroside concentrations. The binding to cerebrosides is less specific than that to sulphatides. A 7-fold excess of cerebrosides was necessary for half maximal binding. 4. In a mixture of sulphatides and cerebrosides the formation of the complex with the activator protein is partly inhibited. The total amount of bound lipids changed as the composition of the lipid mixture was varied. In a one-to-one mixture of the two lipids 60% of the total bound lipids are sulphatides and 40% are cerebrosides.