The preparation of antibodies reactive against citrulline-containing charge isomers of myelin basic protein but not against the arginine-containing charge isomers.

Human myelin basic protein (MBP) is composed of several charge isomers, the result of various post-translational modifications. One of the charge isomers C-8, has been shown in our laboratory to contain six citrullinyl residues which replace arginyl residues at selected sites in the MBP. In order to determine the disposition of the citrulline-containing charge isomers in the myelin stack, we prepared specific antisera against the citrullinyl group. Since 9-fluorenylmethoxycarbonyl (Fmoc)-citrulline, required for the preparation of the synthetic peptides to be used for antibody production, was not commercially available, synthesis of the Fmoc-citrulline was a necessary prerequisite. The synthesis and purification of the N-9-fluorenylmethyloxycarbonyl derivative of citrulline is described. It was characterized by thin layer chromatography, 1H and 13C NMR spectroscopy, fast-atom bombardment mass spectroscopy, and thermal analyses. It was used in the automated peptide synthesis of a peptide Ala-Cit-His-Gly-Phe-Leu-Pro-Cit-His-Arg corresponding to residues 24-33 and Gly-Cit-Asp-Ser-Arg-Ser-Gly-Ser-Pro-Met-Ala-Cit-Arg, corresponding to residues 158-170 of the C-8 sequence, a naturally occurring charge isomer of human myelin basic protein, and a tetracitrulline peptide, Cit-Cit-Cit-Cit-Gly. The tetracitrulline peptide was used for the production of an antibody shown to react only with synthetic peptides and proteins containing citrulline. This antibody was used to distinguish between a citrulline-containing protein, C-8, a naturally occurring charge isomer of MBP, and a non-citrulline-containing charge isomer of MBP, C-1.     

Multiple charge isomers of human recombinant interleukin-1 beta

Human interleukin-1 beta (rhuIL-1 beta), obtained by DNA recombinant technology, was radiolabelled. Its isoelectric properties were determined by various analytical techniques such as high-voltage ultrathin layer isoelectric focusing (IEF) and chromatofocusing. The rhuIL-1 beta molecule had a molecular mass of 18 kDa, as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. When examined by IEF on a polyacrylamide gel of 1 mm thickness in the pH range of 3.5-9.5, it was resolved into two broad bands appearing in the pH range of 6.2-5.8 and 5.5-5.2. Each of the two bands was further resolved into multiple bands when electrofocused on (i) a thinner gel of 0.5 mm thickness and (ii) a narrower pH range of 5-8. Upon chromatofocusing in a liquid column, it was possible to isolate various charged components of rhuIL-1 beta. However, all these components reacted to the antiserum to rhuIL-1 beta and displayed a molecular mass of 18 kDa suggesting the charge heterogeneity of rhuIL-1 beta.     

A new electrophoretic variant of arylsulfatase A

Previous work in this laboratory has identified electrophoretic variant forms of arylsulfatase A in leucocyte plus platelets. During a study to replicate and extend these findings, a new seven-band variant of arylsulfatase A has been identified. Purified platelets gave a clearer, more distinct electrophoretic banding pattern than the leucocyte and platelet preparations.     

A new polymorphism of arylsulfatase A within the coding region

A 10-year-old boy with juvenile metachromatic leukodystrophy (MLD) presented with the 459 + 1G→A arylsulfatase A (ASA) mutation on one allele. To detect his complete genotype, the other ASA allele was sequenced and a T-to-C transition at nucleotide 376 in exon 2 was identified. This missense mutation results in a substitution of leucine 76 by proline. Of 20 MLD unrelated controls, 18 carried the L/P76 mutation either in the homozygous (n = 6) or heterozygous (n = 12) state. The presence or absence of L/P76 did not influence leukocyte ASA activity or urinary sulfatide excretion. Apparently, the substitution of leucine 76 by proline is a common ASA polymorphism, neither being related to MLD nor creating ASA pseudodeficiency. However, because of its frequency and location, L/P76 may be of particular importance in genetic studies requiring the differentiation of the ASA alleles within a kindred. Further studies are directed to the as yet unresolved genotype of the index case with juvenile MLD. Received: 5 March 1996 / Revised: 16 April 1996     

Attenuated activities and structural alterations of arylsulfatase A in tissues from subjects with pseudo arylsulfatase A deficiency

Summary It had been shown previously that arylsulfatase A activity was attenuated in pseudo arylsulfatase A deficiency fibroblasts and that subunits of the enzyme were smaller than subunits of the enzyme in normal fibroblasts. Attenuated enzyme activity has now been affirmed in other tissues. Subunits of the enzyme from these sources were also found to be smaller with apparent molecular size 59 and 56 kdaltons. Subunits of enzyme in corresponding control tissues were larger and there was heterogeneity in apparent molecular size as follows: fibroblast, 63 and 59 kdaltons; liver, 63 and 59 kdaltons; kidney, 62 and 58 kdaltons; and urine, 61 and 57 kdaltons. Attenuated enzyme activity and structurally altered enzyme in pseudo arylsulfatase A deficiency appears to be systemic. However, the reason for reduced amounts of structurally altered enzyme with normal catalytic activity is unresolved.     

Sulfhydryl groups of rabbit liver arylsulfatase A

Rabbit liver arylsulfatase A (aryl-sulfate sulfhydrolase, EC 3.1.6.1) monomers of 130 kDa contain two free sulfhydryl groups as determined by spectrophotometric titration using 5,5'-dithiobis(2-nitrobenzoate) and by labeling with the fluorescent probe 5-(iodoacetamidoethyl)aminonaphthalene-1-sulfonic acid. Fluorescence quenching data indicate that the reactive sulfhydryl is present in proximity to one or more tryptophan residues. Chemical modification of the sulfhydryl groups does not alter the distinctive pH-dependent aggregation property of the arylsulfatase A. The free sulfhydryls of the enzyme react with numerous sulfhydryl reagents. Based on the reactions of iodoacetic acid, methyl methanethiosulfonate, 5,5'-dithiobis(2-nitrobenzoate) and 5-(iodoacetamidoethyl)aminonaphthalene-1-sulfonic acid with the sulfhydryl groups of arylsulfatase A, it is concluded that free sulfhydryls are not essential for the enzyme activity. In contrast, the observed inactivation of the enzyme by p-hydroxymercuribenzoate or p-hydroxymercuriphenylsulfonate is probably due to a modification of a histidine residue, consistent with previous reports that histidine is near the active site of arylsulfatase A. p-Hydroxymercuribenzoate and p-hydroxymercuriphenylsulfonate are able to react both with cysteine and with histidine residues of the protein molecule.     

Cloning and expression of human arylsulfatase A

A full length cDNA for human arylsulfatase A was cloned and sequenced. The predicted amino acid sequence comprises 507 residues. A putative signal peptide of 18 residues is followed by the NH2-terminal sequence of placental arylsulfatase A. One of the arylsulfatase A peptides ends 3 residues ahead of the predicted COOH terminus. This indicates that proteolytic processing of arylsulfatase A is confined to the cleavage of the signal peptide. The predicted sequence contains three potential N-glycosylation sites, two of which are likely to be utilized. The sequence shows no homology to any of the known sequences of lysosomal enzymes but a 35% identity to human steroid sulfatase. Transfection of monkey and baby hamster kidney cells resulted in an up to 200-fold increase of the arylsulfatase A activity. The arylsulfatase A was located in lysosome-like structures and transported to dense lysosomes in a mannose 6-phosphate receptor-dependent manner. The arylsulfatase A cDNA hybridizes to 2.0- and 3.9-kilobase species in RNA from human fibroblasts and human liver. RNA species of similar size were detected in metachromatic leukodystrophy fibroblasts of two patients, in which synthesis of arylsulfatase A polypeptides was either detectable or absent.     

Antigen-antibody interactions and the anomalous kinetics of arylsulfatase A.

Antibodies against homogeneous rabbit liver arylsulfatase A (aryl-sulfatase sulfohydrolase, EC 3.1.6.1) were produced in a goat and the effects of these antibodies on the kinetic parameters of the enzyme have been studied. The results indicate that the binding of antibody to the enzyme does not alter the enzyme active site, since Km and -ki values are unaffected. However, a small reduction in the enzyme activity was observed as the result of a reduction of V in the enzyme-antibody complex. The binding of antibodies led to a change in the pH-rate profile, giving one broad pH optimum shifted toward higher pH value. The enzyme-antibody complex still showed the characteristic arylsulfatase A anomalous kinetics at pH 5.5, but the inactivation was significantly slower than for the native enzyme. As calculated from quantitative immuno-precipitation data, the native enzyme bound 5--7 molecules of IgG. The number of IgG molecules which bound to the turnover-modified enzyme was reduced to 3--4. The loss of antigenic determinants from the turnover-modified enzyme indicates that significant conformational changes occur during the turnover-induced modification, or that a covalent modification of residues present at the antigenic sites has occurred, or both.     

Studies in metachromatic leukodystrophy: XV. Purification of normal and mutant arylsulfatase A from human liver

In this report we describe a method to purify both normal and abnormal (inactive) arylsulfatase A. The abnormal enzyme protein was isolated both from cases of late infantile and early juvenile forms of metachromatic leukodystrophy. Conventional protein isolation methods reported earlier were followed by size exclusion high-performance liquid chromatography in the final purification stages. Both the mutant enzyme and the normal enzyme had the same HPLC elution behavior. They thus appeared to self-associate in a similar pH-dependent fashion. Both could be followed by their reaction to a rabbit antibody to normal human arylsulfatase A. The amount of homogenous protein obtained from about 500 grams of liver was 300-400 micrograms.     

Differentiation between arylsulfatase A deficiency and pseudo-deficiency

Metachromatic leukodystrophy (MLD)--lysosomal storage disease caused arylsulfatase A (ARSA) deficiency. Biochemical diagnostic of MLD is complicated by arylsulfatase A pseudodeficiency. There is possibility of mistake in MLD diagnoses in case of pseudodeficiency ARSA and non-MLD neurological disease combination. We suggest the new modification of arylsulfatase A activity detection method which allows to identify the arylsulfatase A pseudodeficiency without molecular genetic methods.     

Transport of arylsulfatase A across the blood-brain barrier in vitro

Enzyme replacement therapy is an option to treat lysosomal storage diseases caused by functional deficiencies of lysosomal hydrolases as intravenous injection of therapeutic enzymes can correct the catabolic defect within many organ systems. However, beneficial effects on central nervous system manifestations are very limited because the blood-brain barrier (BBB) prevents the transfer of enzyme from the circulation to the brain parenchyma. Preclinical studies in mouse models of metachromatic leukodystrophy, however, showed that arylsulfatase A (ASA) is able to cross the BBB to some extent, thus reducing lysosomal storage in brain microglial cells. The present study aims to investigate the routing of ASA across the BBB and to improve the transfer in vitro using a well established cell culture model consisting of primary porcine brain capillary endothelial cells cultured on Transwell filter inserts. Passive apical-to-basolateral ASA transfer was observed, which was not saturable up to high ASA concentrations. No active transport could be determined. The passive transendothelial transfer was, however, charge-dependent as reduced concentrations of negatively charged monosaccharides in the N-glycans of ASA or the addition of polycations increased basolateral ASA levels. Adsorptive transcytosis is therefore considered to be the major transport pathway. Partial inhibition of the transcellular ASA transfer by mannose 6-phosphate indicated a second route depending on the insulin-like growth factor II/mannose 6-phosphate receptor, MPR300. We conclude that cationization of ASA and an increase of the mannose 6-phosphate content of the enzyme may promote blood-to-brain transfer of ASA, thus leading to an improved therapeutic efficacy of enzyme replacement therapy behind the BBB.     

A kinetic method for determination of arylsulfatase activity

A new assay for arylsulfatase activity is described, which consists of direct kinetic measurements of pseudo-first-order rate constants by means of a spectrophotometric procedure. The assay is applicable for reactions occurring at different pH conditions and it can be used for a wide range of activities.