Characterization of a recombinant molecule covalently indistinguishable from human cerebroside-sulfate activator protein (CSAct or Saposin B).

Humans deficient in the cerebroside-sulfate activator protein (CSAct or Saposin B) are unable to catabolize sulfatide and other glycosphingolipids leading to their accumulation and neurodegenerative disease. Clinically this usually manifests as a form of metachromatic leukodystrophy (MLD). CSAct is a small water-soluble glycoprotein that apparently functions in the lysosome to solubilize sulfatide and other lipids enabling their interaction with soluble lysosomal hydrolases. CSAct activity can be measured in vitro by assay of its ability to activate sulfatide-sulfate hydrolysis by arylsulfatase A or ex vivo by its ability to functionally complement CSAct deficient fibroblast cell lines derived from MLD patients. A recombinant form of CSAct has been expressed in E. coli and processed in vitro to a form covalently indistinguishable from deglycosylated human CSAct isolated from human urine. Size-exclusion chromatography in combination with multi-angle laser-light scattering (SEC-MALLS) measurements demonstrate that both native and recombinant forms of the molecule behave as a dimer in the pH range 7.0-4.5. The CSAct activity assay showed that both recombinant and deglycosylated human urine CSAct efficiently activated sulfatide sulfate hydrolysis and provided functional complementation of CSAct-deficient cells. However, a D21N mutant form of recombinant CSAct could not functionally complement these cells despite full activity in the in vitro assay. It is concluded that while glycosylation is unnecessary for in vitro and ex vivo activity of CSAct, modification of the native N21 is necessary to prevent loss of ex vivo activity, possibly via protection from degradation.

Comparative lipid binding study on the cerebroside sulfate activator (saposin B).

Cerebroside sulfate activator (saposin B) is a small protein involved in glycosphingolipid metabolism. It binds certain membrane lipids, making them available to water-soluble enzymes. Defects in this protein are responsible for a form of metachromatic leukodystropy, a progressive neurodegenerative condition. The protein participates in the catabolism of a number of lipids but does show lipid binding selectivity. However, the basis of this selectivity is unclear. Here we assess the relative binding of a number of lipids compared to cerebroside sulfate (sulfatide). We utilize a competitive binding paradigm, in which the lipids compete for protein under favorable conditions and are then switched to a condition in which the complex is stable. This study is unique in that a single molecular species of the activator is employed, and an expanded selection of natural and semisynthetic membrane lipids is surveyed. No simple "binding rule" can be ascertained from these data, but ligands with longer and/or more complex lipoidal and polar adducts appear to be favored.

Methionine oxidation within the cerebroside-sulfate activator protein (CSAct or Saposin B).

The cerebroside-sulfate activator protein (CSAct or Saposin B) is a small water-soluble glycoprotein that plays an essential role in the metabolism of certain glycosphingolipids, especially sulfatide. Deficiency of CSAct in humans leads to sulfatide accumulation and neurodegenerative disease. CSAct activity can be measured in vitro by assay of its ability to activate sulfatide-sulfate hydrolysis by arylsulfatase A. CSAct has seven methionine residues and a mass of 8,845 Da when deglycosylated. Mildly oxidized, deglycosylated CSAct (+16 Da), separated from nonoxidized CSAct by reversed-phase high-performance liquid chromatography (RP-HPLC), showed significant modulation of the in vitro activity. Because oxidation partially protected against CNBr cleavage and could largely be reversed by treatment with dithiothreitol, it was concluded that the major modification was conversion of a single methionine to its sulfoxide. High-resolution RP-HPLC separated mildly oxidized CSAct into seven or more different components with shorter retention times than nonoxidized CSAct. Mass spectrometry showed these components to have identical mass (+16 Da). The shorter retention times are consistent with increased polarity accompanying oxidation of surface-exposed methionyl side chains, in general accordance with the existing molecular model. A mass-spectrometric CNBr mapping protocol allowed identification of five of the seven possible methionine-sulfoxide CSAct oxoforms. The most dramatic suppression of activity occurred upon oxidation of Met61 (26% of control) with other residues in the Q60MMMHMQ66 motif falling in the 30-50% activity range. Under conditions of oxidative stress, accumulation of minimally oxidized CSAct protein in vivo could perturb metabolism of sulfatide and other glycosphingolipids. This, in turn, could contribute to the onset and progression of neurodegenerative disease, especially in situations where the catabolism of these materials is marginal.

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.

Hydrogen-deuterium exchange signature of porcine cerebroside sulfate activator protein.

Hydrogen-deuterium exchange can be a sensitive indicator of protein structural integrity. Comparisons were made between cerebroside sulfate activator protein (CSAct) in the native state and after treatment with guanidine hydrochloride plus dithiothreitol. Native protein has three internal disulfide bonds and treated protein has no internal disulfide bonds. The comparisons were made using hydrogen-deuterium exchange measured by electrospray ionization mass spectrometry, percentage alpha-helical content measured by circular dichroism and biological activity measured by the ability to support arylsulfatase A-catalyzed sulfate hydrolysis from cerebroside sulfate. In acidic solvent native protein has 59 exchange refractory protons and treated protein has 20 exchange refractory protons (44 and 14% of the exchangeable proton populations, respectively). In native protein the size of the exchange refractory proton population is sensitive to changes in pH, temperature and the presence of a ligand. It is uninfluenced by the presence or absence of glycosyl groups attached to Asn21. Helical content is virtually identical in native and treated protein. Biological activity is significantly reduced but not obliterated in treated protein. The hydrogen-deuterium exchange profile appears to be a sensitive signature of the correctly folded protein, and reflects a dimension of the protein structure that is not apparent in circular dichroic spectra or in the ability of the protein to support arylsulfatase A-catalyzed sulfate hydrolysis from sulfatide. The hydrogen-deuterium exchange profile will be a valuable criterion for characterizing mutant forms of CSAct produced by recombinant and synthetic paradigms and also the native and mutant forms of related proteins.

Structure of the asparagine-linked sugar chains of porcine kidney and human urine cerebroside sulfate activator protein.

The specific sugar residues and their linkages in the oligosaccharides from pig kidney and human urine cerebroside sulfate activator proteins (saposin B), although previously hypothesized, have been unambiguously characterized. Exhaustive sequential exoglycosidase digestion of the trimethyl-p-aminophenyl derivatives, followed by either matrix-assisted laser desorption/ionization and/or mass spectrometry, was used to define the residues and their linkages. The oligosaccharides were enzymatically released from the proteins by treatment with peptidyl-N-glycosidase F and separated from the proteins by reversed-phase high-performance liquid chromatography (HPLC). Reducing termini were converted to the trimethyl-p-aminophenyl derivative and the samples were further purified by normal-phase HPLC. The derivatized carbohydrates were then treated sequentially with a series of exoglycosidases of defined specificity, and the products of each digestion were examined by mass spectrometry. The pentasaccharides from pig kidney and human urine protein were shown to be of the asparagine-linked complex type composed of mannose-alpha 1-6-mannose-beta 1-4-N-acetylglucosamine-N-acetylglucosamine(alpha 1-6-fucose). This highly degraded structure probably represents the final product of intra-lysosomal exoglycosidase digestion. Oligosaccharide sequencing by specific exoglycosidase degradation coupled with mass spectrometry is more rapid than conventional oligosaccharide sequencing. The procedures developed will be useful for sequencing other oligosaccharides including those from other members of the lipid-binding protein class to which cerebroside sulfate activator belongs. (c) 2000 John Wiley & Sons, Ltd.

Preparation of the cerebroside sulfate activator (CSAct or saposin B) from human urine.

The purification of cerebroside sulfate activator (CSAct) or saposin B from pooled human urine is described. Urinary proteins are concentrated by ammonium sulfate precipitation. A suspension of the precipitate is heat-treated and the heat-stable proteins are fractionated through a series of chromatographic steps. An initial concanavalin A column retains little of the CSAct activity, but is important for subsequent purification. Passing the Con A effluent directly onto an octyl Sepharose column removes the protein of interest which is recovered by affinity elution with octyl glucoside. Subsequent ion-exchange and gel filtration chromatographies yield a protein of 80-90% purity, although it is sometimes necessary to repeat one or more steps. A small amount of CSAct can sometimes be recovered from the initial Con A Sepharose column by methyl mannoside elution and purified by a parallel chromatographic protocol. Mass spectral analysis suggests that the final material is a mixture of two major and several minor glycoforms of a 79 amino acid protein with the structure predicted from the human prosaposin cDNA by truncation of both N- and C-terminal regions. Sugar analysis revealed the presence of glucosamine, mannose, and fucose, consistent with the major isoforms bearing a five-sugar Man(2)GluNac(2)Fuc or a single GluNac substituent. The human urinary material is similar to the previously characterized pig kidney protein in most respects, but varies in some details.

Cerebroside sulfate activator protein (Saposin B): chromatographic and electrospray mass spectrometric properties.

Cerebroside sulfate activator protein is a small, heat-stable protein that is exceptionally resistant to proteolytic attack. This protein is essential for the catabolism of cerebroside sulfate and several other glycosphingolipids. Protein purified from pig kidney and human urine was extensively characterized by reversed-phase liquid chromatography and electrospray mass spectrometry. These two sources revealed 20 and 18 different molecular isoforms of the protein, respectively. Plausible explanations of the structures of the majority of these isoforms can be made on the basis of accurate molecular mass assignments. The reversed-phase chromatographic and electrospray mass spectrometric properties of enzymatically deglycosylated and disulfide-reduced protein were also compared. In addition to a demonstration of the power of electrospray ionization mass spectrometry for revealing a wealth of information on protein microheterogeneity and structural detail, the results also demonstrate the utility of this technique for monitoring spontaneous chemical and enzymatically mediated changes that occur as a result of metabolic processing and protein purification.

Two new arylsulfatase A (ARSA) mutations in a juvenile metachromatic leukodystrophy (MLD) patient.

Fragments of the arylsulfatase A (ARSA) gene from a patient with juvenile-onset metachromatic leukodystrophy (MLD) were amplified by PCR and ligated into MP13 cloning vectors. Clones hybridizing with cDNA for human ARSA were selected, examined for appropriate size inserts, and used to prepare single-stranded phage DNA. Examination of the entire coding and most of the intronic sequence revealed two putative disease-related mutations. One, a point mutation in exon 3, resulted in the substitution of isoleucine by serine. Introduction of this alteration into the normal ARSA cDNA sequence resulted in a substantial decrease in ARSA activity on transient expression in cultured baby hamster kidney cells. About 5% of the control expression was observed, suggesting a small residual activity in the mutated ARSA. The second mutation, a G-to-A transition, occurred in the other allele and resulted in an altered splice-recognition sequence between exon 7 and the following intron. The mutation also resulted in the loss of a restriction site. Apparently normal levels of mRNA were generated from this allele, but no ARSA activity or immuno-cross-reactive material could be detected. A collection of DNA samples from known or suspected MLD patients, members of their families, and normal controls was screened for these mutations. Four additional individuals carrying each of the mutations were found among the nearly 100 MLD patients in the sample. Gene segregation in the original patient's family was consistent with available clinical and biochemical data. No individuals homozygous for either of these two mutations were identified. However, combinations with other MLD mutations suggest that the point mutation in exon 3 does result in some residual enzyme activity and is associated with late-onset forms of the disease. The splice-site mutation following exon 7 produces late-infantile MLD when combined with other enzyme-null mutations, implying that it is completely silent enzymatically.

Molecular basis of different forms of metachromatic leukodystrophy.

BACKGROUND: Metachromatic leukodystrophy is an autosomal recessive inherited lysosomal storage disorder caused by a deficiency of arylsulfatase A. Three forms of the disease can be distinguished according to severity and the age at onset: late infantile (1 to 2 years), juvenile (3 to 16), and adult (greater than 16). METHODS AND RESULTS: To understand the molecular basis of the different forms of the disease, we analyzed arylsulfatase A alleles associated with metachromatic leukodystrophy. Two alleles (termed I and A) were identified and accounted for about half of all arylsulfatase A alleles among 68 patients with metachromatic leukodystrophy whom we examined. Sufficient information was available for 66 of the patients to allow classification of their disease. Of the six instances of homozygosity for allele I, all were associated with the late-infantile form of the disease; of the eight instances of homozygosity for allele A, five were associated with the adult form and three with the juvenile form. When both alleles were present, the juvenile form resulted (seven of seven instances). Heterozygosity for allele I (with the other allele unknown) is usually associated with late-infantile disease, and heterozygosity for allele A with a later onset of the disease. The clinical variability can be explained by the different levels of residual arylsulfatase A activity associated with these genotypes. CONCLUSIONS: Like many lysosomal storage disorders, metachromatic leukodystrophy shows clinical heterogeneity that seems to reflect genetic heterogeneity. One of the known alleles (allele I) is associated with earlier and more severe disease than the other (allele A).