Identification of three amino acid residues in the B-chain of platelet-derived growth factor with different importance for binding to PDGF alpha- and beta-receptors.

The B-chain homodimer isoform of platelet-derived growth factor (PDGF) binds with high affinity both to alpha- and to beta-receptors. In order to localize amino acid residues in PDGF-BB of differential importance for the binding to the two receptors, PDGF-BB mutants were analyzed in which single amino acid residues were changed to alanine residues. We found that Phe-118 in loop 1 of the PDGF B-chain is crucial for binding to both receptors, and that the surrounding amino acids, Asn-117 and Leu-119, appear to be important primarily for binding to the beta-receptor. In contrast, Lys-161 in loop 3 was found to be more important for binding to alpha-receptors than beta-receptors. Previous studies have shown that the receptor binding epitope of PDGF-BB is composed mainly of loops 1 and 3; the findings of the present study show that the alpha- and beta-receptors interact with different amino acid residues in these regions.

Involvement of loop 2 of platelet-derived growth factor-AA and -BB in receptor binding.

Platelet-derived growth factor (PDGF) is a disulfide-bonded antiparallel dimer of A- and B-polypeptide chains. Each subunit contains two loops (loops 1 and 3) which point in the same direction, and which are located close to a region (loop 2) from the other subunit of the dimer. Previous studies have shown that epitopes in loops 1 and 3 are important for binding to PDGF alpha- and beta-receptors. The aim of the present investigation was to determine the importance of loop 2 for receptor interactions. PDGF A- and B-chain cDNA:s were mutated in the loop 2 regions and transfected into COS cells. Analyses of conditioned media of such cell cultures revealed that PDGF B-chain mutated in the loop 2 region lost its ability to compete with 125I-PDGF for binding to PDGF beta-receptors, but retained 2-5% of its binding of alpha-receptors. The A-chain binds only to alpha-receptors; 2-5% of this binding was also retained after mutation of the loop 2 region. In conclusion, the loop 2 region of PDGF is important for receptor binding, but appears to be more important for binding to the PDGF beta-receptors than to the alpha-receptors.

Translational control of arylsulfatase A expression in mouse testis.

Arylsulfatase A is a lysosomal enzyme that is involved in the degradation of sulfated glycolipids. High levels of arylsulfatase A mRNA are found in germ cells of mouse testis. In late pachytene and secondary spermatocytes the level of arylsulfatase A mRNA is increased 20-fold when compared with other tissues. These high levels of arylsulfatase A mRNA are maintained in round spermatids and decrease in late elongating spermatids. The increase of arylsulfatase A mRNA levels is not accompanied by a similar increase in enzyme activity or polypeptides. Subcellular fractionation revealed that the majority of arylsulfatase A mRNA is not associated with polysomes but is found in fractions of lower buoyancy. The failure to become translated is ascribed to the association of arylsulfatase A mRNA with nonpolysomal ribonucleoproteins. This translational repression may be due to proteins that bind to arylsulfatase A mRNA and prevent its translation. Within the 639-nucleotide 5'-untranslated region and the 700-nucleotide 3'-untranslated region of the arylsulfatase A mRNA, we identified two regions that specifically bind proteins present in extracts prepared from testicular cells. These RNA binding proteins were absent from extracts prepared from liver or brain.

Molecular genetics of metachromatic leukodystrophy.

Metachromatic leukodystrophy is a lysosomal storage disorder caused by the deficiency of arylsulphatase A. The disease is characterized by a progressive demyelination that causes a variety of neurological symptoms. Patients die within a few years after the age of onset. Clinically the disease is heterogeneous and according to the age of onset three different forms can be distinguished. The gene of arylsulphatase A has been cloned and several mutations causing metachromatic leukodystrophy have been characterized. The distribution of these alleles among patients with different clinical forms of the disease has revealed a genotype-phenotype correlation. A major determinant of the clinical phenotype is the residual enzyme activity that it associated with a particular genotype. Homozygosity for alleles that do not allow the synthesis of arylsulphatase A polypeptides causes the most severe form of disease, whereas homozygosity for alleles that encode arylsulphatase A with low residual enzyme activity is found in the mild late-onset forms of disease. A substantial arylsulphatase A deficiency can also be found in healthy individuals at high frequency. This phenomenon has been termed pseudodeficiency. It is often difficult to distinguish whether an arylsulphatase A deficiency is due to metachromatic leukodystrophy or harmless pseudodeficiency. The characterization of the mutations causing pseudodeficiency has allowed the detection of the pseudodeficiency allele in the DNA of probands and has thus improved the diagnosis and genetic counselling for metachromatic leukodystrophy.

Structure of the mouse arylsulfatase A gene and cDNA.

The murine arylsulfatase A (ARSA) gene and cDNA have been cloned and sequenced. The gene is 3.8 kb long and contains eight exons. All intron/exon splice junctions conform to the GT/AG consensus sequence. The genomic structure is similar to that of the human gene. One major RNA species of 3.2 kb is transcribed. This RNA species has a 5' untranslated region of 638 nucleotides and terminates in a region around nucleotide 700 downstream of the termination codon. In addition, a rare mRNA species terminating at a polyadenylation signal 135 nucleotides downstream of the termination codon has been found. A larger transcript of 4 kb can be detected in liver. The size difference is due to initiation of transcription 5' of the cap site of the 3.2-kb mRNA species. The entire ARSA cDNA has been cloned by PCR from reverse-transcribed RNA. The coding sequence has 1518 nucleotides and predicts a protein of 506 amino acids. The nucleotide as well as the amino acid sequence is highly conserved among humans and mice.

Genetics of metachromatic leukodystrophy.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease caused by the deficiency of arylsulfatase A (ASA). The mode of inheritance is autosomal recessive. The disease occurs panethnically and its frequency is 1 in 40000. The deficiency of the enzyme causes the accumulation of its substrate cerebroside sulfate. Since this sphingolipid is mainly found in the myelin membranes the disease primarily affects the oligodendrocytes. Patients suffer from a progressive demyelination and die due to a variety of neurologic symptoms. Clinically the disease is heterogeneous. Depending on the age of onset a late infantile, juvenile and adult form can be distinguished. We have cloned the cDNA and gene of arylsulfatase A. Several disease causing mutations have been identified and a simple genotype phenotype correlation has been revealed. Currently we try to develop a mouse model of MLD via homologous recombination in embryonic stem cells. The model will allow to elucidate the pathogenesis of the disease and to test possible therapeutic approaches.

High residual arylsulfatase A (ARSA) activity in a patient with late-infantile metachromatic leukodystrophy.

We identified a patient suffering from late-infantile metachromatic leukodystrophy (MLD) who has a residual arylsulfatase A (ARSA) activity of about 10%. Fibroblasts of the patient show significant sulfatide degradation activity exceeding that of adult MLD patients. Analysis of the ARSA gene in this patient revealed heterozygosity for two new mutant alleles: in one allele, deletion of C 447 in exon 2 leads to a frameshift and to a premature stop codon at amino acid position 105; in the second allele, a G-->A transition in exon 5 causes a Gly309-->Ser substitution. Transient expression of the mutant Ser309-ARSA resulted in only 13% enzyme activity of that observed in cells expressing normal ARSA. The mutant ARSA is correctly targeted to the lysosomes but is unstable. These findings are in contrast to previous results showing that the late-infantile type of MLD is always associated with the complete absence of ARSA activity. The expression of the mutant ARSA protein may be influenced by particular features of oligodendrocytes, such that the level of mutant enzyme is lower in these cells than in others.

Localization of lysosomal acid phosphatase mRNA in mouse tissues.

We studied the expression of lysosomal acid phosphatase (LAP) in mouse by hybridizing Northern blots and tissue sections with the mouse LAP cDNA. Three mRNA species of 2.3, 3.2 and 5.2 KB were identified, which differ in the length of their 3' untranslated region (UTR). The 3.2 KB mRNA is expressed in equal amounts in all tissues and represents the major species in most tissues, whereas the amounts of the 2.3 and 5.2 KB species differ. In situ hybridization of different tissues of adult mice showed a uniform expression of LAP, as expected for a housekeeping gene, except in testis and brain. In testis we found an increase in the LAP mRNA level in spermatocytes. By Northern blot analysis of young mouse testis, this increase could be attributed to late pachytene primary spermatocytes or secondary spermatocytes. In brain tissue the neurons were predominantly labeled, especially the Purkinje and pyramidal cells, whereas glial cells expressed only low amounts of LAP mRNA. Very high LAP expression was also found in the epithelial cells of the choroid plexus. Analysis of LAP expression during mouse embryonic development between Days 9.5 and 17.5 revealed a prominent expression relative to other tissues in the neural tube from Day 9.5 to Day 13.5.

Molecular genetics of metachromatic leukodystrophy.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease caused by the deficiency of arylsulfatase A (ASA). The ASA cDNA as well as the gene has been cloned. The gene is about 3 kb long and consists of 8 exons. The two most frequent alleles causing MLD have been characterized and the distribution of these alleles among patients with different clinical forms of MLD has revealed a simple genotype-phenotype correlation. Some individuals have low ASA activities but are healthy. This condition has been called ASA pseudodeficiency. These individuals are homozygous for the ASA pseudodeficiency allele which only encodes 5-10% of the ASA activity compared to the normal allele. The mutations in the PD allele have been characterized. Based on the knowledge of these mutations diagnostic assays have been developed to differentiate ASA deficiencies associated with PD or MLD.

Structure of the arylsulfatase A gene.

A 14-kb genomic clone containing the entire gene of human lysosomal arylsulfatase A was isolated. The arylsulfatase A gene is about 3.2 kb long and has eight exons (103-320 nucleotides in size). All intron-exon splice junctions conformed to the GT/AG consensus sequence. S1 nuclease mapping shows multiple transcription initiation sites between nucleotides -367 and -387. A fragment encompassing 360 nucleotides of the flanking sequence upstream of the transcription initiation site shows promoter activity when it was transiently expressed in COS cells using the gene for bacterial chloramphenicol acetyltransferase as a reporter gene. This putative promoter region shows four potential Sp1 binding sites but lacks typical TATA and CAAT box sequences. Three different mRNA species of 2.1, 3.7 and 4.8 kb are transcribed from the gene and arise probably from the use of different polyadenylation signals.

Arylsulfatase A pseudodeficiency: loss of a polyadenylylation signal and N-glycosylation site.

Metachromatic leukodystrophy is a metabolic disorder caused by the deficiency of arylsulfatase A. Deficiency of this enzyme is also found in apparently healthy individuals, a condition for which the term pseudodeficiency was introduced. The arylsulfatase A (cerebroside-3-sulfate 3-sulfohydrolase; EC 3.1.6.8) (ASA) encoding gene was isolated from an individual homozygous for the ASA pseudodeficiency allele. Sequence analysis revealed two A----G transitions. One changes Arg-350 to serine, which leads to the loss of a utilized N-glycosylation site. This loss explains the smaller size of ASA in ASA pseudodeficiency fibroblasts. The introduction of Ser-350 into normal ASA cDNA does not affect the rate of synthesis, the stability, or the catalytic properties of ASA in stably transfected baby hamster kidney cells. Therefore, the loss of the N-linked oligosaccharide does not contribute to the reduction of ASA activity in ASA pseudodeficiency. The other A----G transition changes the first polyadenylylation signal downstream of the stop codon from AATAAC to AGTAAC. The latter causes a severe deficiency of a 2.1-kilobase (kb) mRNA species. The deficiency of the 2.1-kb RNA species provides an explanation for the diminished synthesis of ASA seen in pseudodeficiency fibroblasts. Amplification of genomic DNA and hybridization with allele-specific oligonucleotides detected both mutations in four unrelated individuals with ASA pseudodeficiency.

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.