Brian M. Martin

A Mutation in the Human Glucocerebrosidase Gene in Neuronopathic Gaucher's Disease

To search for a genetic marker for type 2 Gauchers disease (acute neuronopathic form), we compared the nucleotide sequence of a cloned glucocerebrosidase gene from a patient with Gauchers disease with a normal gene. We found only a single base substitution (T----C) in exon X. This mutation results in the substitution of proline for leucine in position number 444 and produces a new cleavage site for the NciI restriction endonuclease. We analyzed NciI enzymatic digests of genomic DNA from 20 patients with type 1, 5 with type 2, and 11 with type 3 Gauchers disease, and 29 normal controls for a restriction-fragment-length polymorphism (RFLP). Four of 5 patients with type 2 disease and all 11 with type 3 disease had at least one allele with the mutation. Two of 5 patients with type 2 disease and 7 of 11 with type 3 were homozygous for this mutation. Only 4 of 20 patients with type 1 Gauchers disease had the mutant allele and were heterozygous for it. None of the 29 normal controls had the mutant allele. The high frequency of this mutation (444leucine----proline) in patients with neuronopathic Gauchers disease, detectable by the NciI RFLP, may be of value in the identification of patients who will have the neurologic sequelae of Gauchers disease.

The primary structure of noxiustoxin: A K+ channel blocking peptide, purified from the venom of the scorpion Centruroides noxius Hoffmann

Noxiustoxin, component II-11 from the venom of scorpion Centruroides noxiusHoffmann, was obtained in pure form after fractionation by Sephadex G-50 chromatography followed by ion exchange separation on carboxy-methylcellulose columns (17). The primary structure of Noxiustoxin, a polypeptide 39 amino acid residues long was determined by automaticEdman degradation and chemical cleavage with cyanogen bromide followed by amino acid analysis of the two resulting peptides. Its sequence is: Thr−Ile−Ile−Asn−Val−Lys−Cys−Thr−Ser−Pro−Lys−Gln−Cys−Ser−Lys−Pro−Cys−Lys−Glu−Leu−Tyr−Gly−Ser−Ser−Ala−Gly−Ala−Lys−Cys−Met−Asn−Gly−Lys−Cys−Lys−Cys−Tyr−Asx−Asn, with a molecular weight of 4,184±6. No histidine, arginine, tryptophan or phenylalanine was found. Noxiustoxin is the first short toxin directed against mammals and the first K+ channel blocking polypeptide-toxin (4) found in scorpion venoms.

Glucosylsphingosine accumulation in tissues from patients with Gaucher disease : correlation with phenotype and genotype

Gaucher disease, the inherited deficiency of lysosomal glucocerebrosidase, presents with a wide spectrum of clinical manifestations including neuronopathic and non-neuronopathic forms. While the lipid glucosylceramide is stored in both patients with Gaucher disease and in a null allele mouse model of Gaucher disease, elevated levels of a second potentially toxic substrate, glucosylsphingosine, are also found. Using high performance liquid chromatography, glucosylsphingosine levels were measured in tissues from patients with type 1, 2, and 3 Gaucher disease. Glucosylsphingosine was measured in 16 spleen samples (8 type 1; 4 type 2; and 4, type 3) and levels ranged from 54 to 728 ng/mg protein in the patients with type 1 disease, 133 to 1200 ng/mg protein in the patients with type 2, and 109 to 1298 ng/mg protein in the type 3 samples. The levels of splenic glucosylsphingosine bore no relation to the type of Gaucher disease, the age of the patient, the genotype, nor the clinical course. In the same patients, hepatic glucosylsphingosine levels were lower than in spleen. Glucosylsphingosine was also measured in brains from 13 patients (1 type 1; 8 type 2; and 4 type 3). While the glucosylsphingosine level in the brain from the type 1 patient, 1.0 ng/mg protein, was in the normal range, the levels in the type 3 samples ranged from 14 to 32 ng/mg protein, and in the type 2 samples from 24 to 437 ng/mg protein, with the highest values detected in two fetuses with hydrops fetalis. The elevated levels found in brains from patients with neuronopathic Gaucher disease support the hypothesis that glucosylsphingosine may contribute to the nervous system involvement in these patients.

CD4 receptor binding peptides that block HIV infectivity cause human monocyte chemotaxis. Relationship to vasoactive intestinal polypeptide.

The octapeptide Ala‐Ser‐Thr‐Thr‐Thr‐Asn‐Tyr‐Thr (peptide T) and two structural analogs are potent agonists of human monocyte chemotaxis, evincing identical rank potency orders as was previously shown for their inhibition of human immunodeficiency virus (HIV) envelope binding and T cell infectivity. Chemotactic activity could be inhibited by anti‐CD4 monoclonal antibodies (Mabs), but not other mononuclear cell Mabs. The core peptide required for chemotactic activity is a pentapeptide related to the sequence Thr‐Thr‐Asn‐Tyr‐Thr. Homologous pentapeptides, identified by computer search, were detected in several other non‐HIV‐related viruses as well as the neuropeptide vasoactive intestinal polypeptide (VIP). The CD4 molecule, therefore, appears to be a recognition molecule for a small signal peptide ligand whose active sequence is a homolog of peptide T [4–8] and which may be the neuropeptide VIP.

A metabolite of acetaminophen covalently binds to the 56 kDa selenium binding protein

Acetaminophen is metabolized by cytochrome P450 to a reactive metabolite that covalently binds to proteins and this binding correlates with the hepatotoxicity. The major protein adduct was previously reported to be a 55 kDa protein that was detected on Western blots using antisera specific for 3-(cystein-S-yl)acetaminophen. In this study, the 55 kDa protein was isolated using a combination of ion exchange fast flow chromatography, hydroxyapatite HPLC and anion exchange HPLC. Amino acid sequences of 8 internal peptides from a trypsin digestion of the 55 kDa protein were found to have 97% homology with the deduced amino acid sequence from a cDNA that corresponds to a 56 kDa selenium binding protein. This is the first report of a specific protein to which a metabolite of acetaminophen covalently binds.

Saposin D: A sphingomyelinase activator

Saposin D, a newly discovered heat-stable, 10 kDa glycoprotein, was isolated from Gaucher spleen and purified to homogeneity. Chemical sequencing from its amino terminus demonstrated colinearity between its amino acid sequence and the deduced amino acid sequence of the fourth domain of prosaposin, the precursor of saposin proteins. Saposin D specifically stimulates acid sphingomyelinase but has no significant effect on the other hydrolases tested.

A Novel Acidic Allergen, Hev b 5, in Latex PURIFICATION, CLONING AND CHARACTERIZATION

Latex allergy is recognized as a serious health problem among health care workers and children with spina bifida. A number of IgE-reactive proteins have been identified in natural and processed latex products. One of the most acidic proteins in the cytoplasm of lacticifer cells of rubber trees (Hevea brasiliensis) is demonstrated to be a potent allergen in eliciting allergic reactions in humans. This protein, with pI = 3.5, has a molecular mass of 16 kDa with a blocked N terminus and an unusual amino acid composition. This acidic protein was found in extracts prepared from latex gloves, which were shown to be allergenic. The purified protein elicits histamine release from human basophils passively sensitized with serum from latex-allergic individuals in a dose-dependent manner. From a latex cDNA library, the cDNA coding for this protein was isolated and sequenced. The deduced amino acid sequence shows a high degree of homology to another acidic protein identified in kiwifruit (Actinidia deliciosa var. deliciosa). The sequence homology (47% sequence identity) between these two acidic proteins suggests a molecular explanation for the high frequency of fruit hypersensitivity in latex-allergic patients.

Myoclonic Epilepsy in Gaucher Disease: Genotype-Phenotype Insights from a Rare Patient Subgroup

Gaucher disease, the inherited deficiency of lysosomal glucocerebrosidase, presents with a wide spectrum of manifestations. Although Gaucher disease has been divided into three clinical types, patients with atypical presentations continue to be recognized. A careful phenotypic and genotypic assessment of patients with unusual symptoms may help define factors that modify phenotype in this disorder. One such example is a rare subgroup of patients with type 3 Gaucher disease who develop progressive myoclonic epilepsy. We evaluated 16 patients with myoclonic epilepsy, nine of whom were diagnosed by age 4 y with severe visceral involvement and myoclonus, and seven with a more chronic course, who were studied between ages 22 and 40. All of the patients had abnormal horizontal saccadic eye movements. Fourteen different genotypes were encountered, yet there were several shared alleles, including V394L (seen on two alleles), G377S (seen on three alleles), and L444P, N188S, and recombinant alleles (each found on four alleles). V394L, G377S, and N188S are mutations that have previously been associated with non-neuronopathic Gaucher disease. The spectrum of genotypes differed significantly from other patients with type 3 Gaucher disease, where genotypes L444P/L444P and R463C/null allele predominated. Northern blot studies revealed a normal glucocerebrosidase transcript, whereas Western studies showed that the patients studied lacked the processed 56 kD isoform of the enzyme, consistent with neuronopathic Gaucher disease. Brain autopsy samples from two patients demonstrated elevated levels of glucosylsphingosine, a toxic glycolipid, which could contribute to the development of myoclonus. Thus, although there were certain shared mutant alleles found in these patients, both the lack of a shared genotype and the variability in clinical presentations suggest that other modifiers must contribute to this rare phenotype.

Glucosylsphingosine accumulation in mice and patients with type 2 Gaucher disease begins early in gestation.

Gaucher disease, the most common of the sphingolipidoses, results from the inherited deficiency of the enzyme glucocerebrosidase (EC 3.2.1.45). Although type 2 (acute neuronopathic) Gaucher disease is associated with rapidly progressive and fatal neurologic deterioration, the pathophysiologic mechanisms leading to the neurologic symptoms and early demise remain uncharacterized. While the pathology encountered in Gaucher disease has been attributed to glucocerebroside storage, glucosylsphingosine (Glc-sph), a cytotoxic compound, also accumulates in the tissues. Elevations of brain Glc-sph have been reported in patients with types 2 and 3 Gaucher disease. In this study, Glc-sph levels were measured using HPLC in tissues from mice with type 2 Gaucher disease created with a null glucocerebrosidase allele. Compared with unaffected littermates, homozygous mice with type 2 Gaucher disease had approximately a 100-fold elevation of Glc-sph in brain, as well as elevated levels in other tissues. This accumulation was detected in utero by E 13 and increased progressively throughout gestation. Similarly, elevated Glc-sph levels were seen in human fetuses with type 2 Gaucher disease, indicating that therapy initiated after birth may be too late to prevent the sequaelae of progressive neurologic damage that begins early in gestation. These findings suggest that the accumulation of Glc-sph may be responsible for the rapid demise of mice with type 2 Gaucher disease and the devastating clinical course seen in patients with type 2 Gaucher disease.

Purification and chemical characterization of the major toxins from the venom of the Brazilian scorpion Tityus serrulatus Lutz and Mello

The soluble venom from the Brazilian scorpion Tityus serrulatus Lutz and Mello was fractionated on Sephadex G-50 followed by ion exchange chromatography in carboxymethyl-cellulose, resolving at least 10 different toxic components. In this communication the purification and chemical characterization of four major components is reported. The toxins were found to be single polypeptide chains with molecular weights of approximately 7,000, 61–63 amino acid residues and four disulphide bridges. Amino terminal sequence determination of these toxins after reduction and alkylation revealed three unique sequences up until residue fourty-one. For toxin II-11 (14) and toxin III-10 the N-terminal sequence is: Lys-Glu-Gly-Tyr-Leu-Met-Asp-His-Glu-Gly-Cys-Lys-Leu-Ser-Cys-Phe-Ile-Arg-Pro-Ser-Gly-Tyr-Cys-Gly-Arg-Glu-Cys-Gly-Ile-Lys-Lys-Gly-Ser-Ser-Gly-Tyr-Cys-Ala-Trp-Pro. Toxin III-8 has the following N-terminal sequence: Lys-Glu-Gly-Tyr-Ala-Met-Asp-His-Glu-Gly-Cys-Lys-Phe-Ser-Cys-Phe-Ile-Arg-Pro-Ala-Gly-Phe-Cys-Asp-Gly-Tyr-Cys-Lys-Thr-His-Leu-Lys-Ala-Ser-Ser-Gly-Tyr-Cys-Ala-Trp-Pro. Toxin IV-5 has the N-terminal sequence: Lys-Lys-Asp-Gly-Tyr-Pro-Val-Glu-Tyr-Asp-Asn-Cys-Ala-Tyr-Ile- Cys-Trp-Asn-Tyr-Asp-Asn-Ala-Tyr-Cys-Asp-Lys-Leu-Cys-Lys-Asp-Lys-Lys-Ala-Asp-Ser-Gly-Tyr-Cys-Tyr-Trp-Val.