Human Acid Sphingomyelinase Research Articles

Isolation of cDNA clones encoding human acid sphingomyelinase: occurrence of alternatively processed transcripts.

Acid sphingomyelinase (sphingomyelin phosphodiesterase, EC 3.1.4.12) was purified from human urine and 12 tryptic peptides were microsequenced (128 residues). Based on regions of minimal codon redundancy, four oligonucleotide mixtures were synthesized and oligonucleotide mixture 1 (20mer; 256 mix) was used to screen 3 X 10(6) independent recombinants from a human fibroblast cDNA library. Putative positive clones (92) were purified and analyzed by Southern hybridization with oligonucleotide mixtures 2-4. These studies revealed two groups of clones; group 1 (80 clones; inserts ranging from approximately 1.2 to 1.6 kb) hybridized with oligonucleotides mixtures 1-4, while group II (12 clones; inserts ranging from approximately 1.2 to 1.4 kb) hybridized with oligonucleotide mixtures 1-3. Several group II clones had larger inserts than those in group I, but did not hybridize with oligonucleotide mixture 4. Screening of a human placental cDNA library with a 450 bp group I fragment, also resulted in the isolation of group I and II clones. Representative clones from group I (pASM-1) and group II (pASM-2) were sequenced. pASM-1 contained a 1879 bp insert which was colinear with 96 microsequenced amino acids, while the pASM-2 1382 bp insert was colinear with 78 microsequenced residues. Notably, pASM-2 did not have an internal 172 bp sequence encoding 57 amino acid residues, but had instead an in-frame 40 bp sequence encoding 13 amino acids which was not present in pASM-1. These findings demonstrate the presence of two distinct acid sphingomyelinase transcripts in human fibroblasts and placenta and suggest the occurrence of alternative processing of the mRNA encoding this lysosomal hydrolase.

The urine from patients with peritonitis as a rich source for purifying human acid sphingomyelinase and other lysosomal enzymes

Milligram amounts of human acid sphingomyelinase (EC 3.1.4.12) were purified to 95% homogeneity using urine from patients with acute peritonitis. The activity of this enzyme is elevated more than 200-times in the urine of these patients. To a lesser extent, levels of some other lysosomal hydrolases are also elevated.

Purification to homogeneity of human placental acid sphingomyelinase.

Acid sphingomyelinase was purified to homogeneity from human placenta in the presence of a dialyzable detergent, n-octyl-beta-D-glucopyranoside. The major steps in the procedure included column chromatographies with Con A-Sepharose, sphingosylphosphorylcholine-Sepharose 4B, hexyl-agarose, and Mono P. The purified enzyme with pI 7.4 had a specific activity of approx 170,000 units/mg protein with a yield of 3.6%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single protein band of Mr 62,000. Gel filtration with a Superose 12 column gave a single peak, and the enzyme in the presence 50 mM n-octyl-beta-D-glucopyranoside was of Mr 123,000, indicating that the native enzyme occurs in a dimeric form. The optimal pH was 5.5 with both sphingomyelin and an artificial substrate, 2-N-hexadecanoylamino-4-nitrophenylphosphorylcholine. The Km values were 55 microM with sphingomyelin and 340 microM with the artificial substrate. The enzyme activity was not affected by Mg2+ (1-5 mM), confirming that the enzyme is acid sphingomyelinase. The enzyme was stable at -80 degrees C for more than 4 months. In addition to the enzyme with pI 7.4, the Mono P chromatofocusing gave two peaks (pI 7.0 and 6.7) possessing the enzymatic activity.

An animal model of human acid sphingomyelinase deficiency (Niemannpick disease) and the study of its enzyme replacement

In December 1975, two 10-old-mice, one female and one mane, from a litter of the C57BL/KsJ inbred strain, which had been maintained in the Research Laboratories, Nippon Shinyaku Co., Ltd., since its transfer from the Jackson Laboratory in 1973 at F2~, were found to be suffering from behavioral abnormalities (Miyawaki et al., 1982). These abnormalities were characterized by motor incoordination with coarse tremor of the body and the extremities. During the following 2 weeks the tremor became more pronounced. Finally the animals were unable to walk and their body weight decreased markedly due to difficulty in obtaining food and water. Although these mice could not breed, mutant mice from the affected female have been bred by ovarian transplantation and maintained in the Research Laboratory. In 1982, we demonstrated a deficiency of sphingomyelinase activity and the accumulation of sphingomyelin in visceral organs of these mutant mice (Sakiyama et al., 1982). Hence, an animal model for Niemann-Pick disease has been developed.

804 cDNA CLONES FOR HUMAN SPHINGOMYELINASE ISOLATED USING THE λGT 11 SYSTEM

Human acid sphingomyelinase activity is deficient in two variants of Niemann-Pick disease, called Types A and B. Type C variants have near normal enzyme activity. The genetic expression of each of these diseases is poorly defined. Our approach to date has been to characterize the structural and kinetic properties of human sphingomyelinase and we have recently characterized a monoclonal antibody against the protein. Using the monoclonal antibody and a λgt 11 expression library, prepared by Drs.J.S.O'Brien and J.de Wet, with cDNA against human hepatoma mRNA, we have succeeded in isolating 6 immunopositive clones. The initial screening used 17×106 recombinants per dish and immunodetection used hybridoma culture fluid (10-30 μg of monoclonal antibody/ml) and peroxidase labeled goat antimouse antibody. One clone produced a fusion protein of about 122,000 daltons while a second produced one of 124,000 daltons corresponding to about 6000 and 8000 daltons of sphingomyelinase protein respectively. These were detected using a anti-E.coli β-galactosidase serum. Immunological identity with sphingomyelinase was shown by direct immunoprecipitation of the fusion protein by anti-sphingomyelinase antibody and by inhibition of immunoreaction with native enzyme. The corresponding cDNA inserts, released by EcoR1 digestion, are 150 bp and 250 bp respectively. These data demonstrate the application of monoclonal antibodies to cDNA cloning and the successful isolation of cDNA clones for human sphingomyelinase.

Monoclonal antibodies against human placental sphingomyelinase.

Monoclonal antibodies against human placental acid sphingomyelinase have been raised. The antibodies are all of the IgG1 type, and are quite specific for the enzyme. One of the antibodies has been used to demonstrate a common antigenic identity of three polypeptides (mol.wts. 90,000, 72,000, and 48,000) of a purified sphingomyelinase preparation.

Acid sphingomyelinase of human placenta: purification, properties, and 125iodine labeling.

Human placental acid sphingomyelinase was highly purified in the presence of Triton X-100. DEAE-Sephacel chromatography and chromatofocusing were the most effective steps in the purification procedure. Enzyme purification was 380,000 nmol/mg protein/h. Characterization and radioiodination were carried out with the chromatofocusing fraction containing highly purified enzyme. The purified enzyme contained no activity of eleven other lysosomal hydrolases but hydrolyzed bis-p-nitrophenyl phosphate slowly compared with [14C]sphingomyelin and chromogenic substrates. SDS-gel electrophoresis revealed two distinct protein bands with molecular weights of 70,500 and 39,800. This enzyme had a molecular weight of 200,000 as determined by analytical gel filtration. The pH optimum was 5.0 and Km was 52.6 x 10(-5) M for [14C]sphingomyelin. Highly purified sphingomyelinase was labeled with 125iodine by the use of Enzymobeads. Labeled sphingomyelinase preparation was rapidly cleared from blood with t1/2 of 1 min. It was absorbed mostly into the liver and presumably largely excreted from there. This labeled enzyme may be useful in metabolic studies in normal animals and animal models of genetic lysosomal storage disorders.

Toward enzyme therapy for lysosomal storage diseases.

articleToward enzyme therapy for lysosomal storage diseasesR. J. Desnick, S. R. Thorpe, and M. B. FiddlerR. J. Desnick, S. R. Thorpe, and M. B. FiddlerPublished Online:01 Jan 1976https://doi.org/10.1152/physrev.1976.56.1.57MoreSectionsPDF (6 MB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmailWeChat Previous Back to Top Next Download PDF FiguresReferencesRelatedInformationCited ByInborn Errors of Metabolism Involving Complex MoleculesPediatric Clinics of North America, Vol. 65, No. 2Inherited Metabolic Disease: Prospects for the Future in Both Basic and Clinical Research30 May 2008Enzyme replacement and enhancement therapies: lessons from lysosomal disordersNature Reviews Genetics, Vol. 3, No. 12Infusion of recombinant human acid sphingomyelinase into Niemann-Pick disease mice leads to visceral, but not neurological, correction of the pathophysiologyThe FASEB Journal, Vol. 14, No. 13Very early presentation of Pompe's disease and its cross-sectional echocardiographic featuresInternational Journal of Cardiology, Vol. 16, No. 3Immobilised enzymes as drugsAdvanced Drug Delivery Reviews, Vol. 1, No. 1Further studies on ectopic dendrite growth and other geometrical distortions of neurons in feline GMl gangliosidosisNeuroscience, Vol. 21, No. 2Ectopic axon hillock-associated neurite growth is maintained in metabolically reversed swainsonine-induced neuronal storage diseaseBrain Research, Vol. 410, No. 1Lysosomal enzymes in ocular tissues and diseasesSurvey of Ophthalmology, Vol. 27, No. 4Enzyme-protein conjugates: new possibilities for enzyme therapyPharmacology & Therapeutics, Vol. 21, No. 1Improved myelination in nerve grafts from the leucodystrophic twitcher into trembler mice: Evidence for enzyme replacementBrain Research, Vol. 237, No. 1Intracellular recording and HRP-staining of cortical neurons in feline ganglioside storage diseaseBrain Research, Vol. 181, No. 2Sulfatases, sulfate esters and their metabolic disordersClinica Chimica Acta, Vol. 100, No. 3Editorial Farewell26 June 2016 | Veterinary Pathology, Vol. 16, No. 6In vitro and in vivo activity of soluble cross-linked uricase-albumin polymers: A model for enzyme therapyLife Sciences, Vol. 24, No. 2IMMUNOGENICITY AND ANTIGENICITY OF SOLUBLE CROSS-LINKED ENZYME/ALBUMIN POLYMERS: ADVANTAGES FOR ENZYME THERAPYThe Lancet, Vol. 312, No. 8080Hypersialyloligosacchariduria in mucolipidoses: A method for diagnosisClinica Chimica Acta, Vol. 86, No. 2Properties of immobilized FIG α-galactosidase and effect of ceramide-3 content of plasma from patients with fabry's diseaseBiochimica et Biophysica Acta (BBA) - Enzymology, Vol. 527, No. 2Enzyme therapy—IX. stabilization of human splenic α-galactosidase a by zinc ionInternational Journal of Biochemistry, Vol. 9, No. 2Alpha-galactosidase a from human placentaBiochimica et Biophysica Acta (BBA) - Enzymology, Vol. 484, No. 2Enzyme therapy VI: Comparative in vivo fates and effects on lysosomal integrity of enzyme entrapped in negatively and positively charged liposomesBiochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 464, No. 3Differential distribution of liposome-entrapped [3H]methotrexate and labelled lipids after intravenous injection in a primateBiochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 448, No. 4Cardiac valvular anomalies in Fabry disease. Clinical, morphologic, and biochemical studies.Circulation, Vol. 54, No. 5Distortion of neuronal geometry and formation of aberrant synapses in neuronal storage diseaseBrain Research, Vol. 116, No. 1 More from this issue > Volume 56Issue 1January 1976Pages 57-99 Copyright & PermissionsCopyright © 1976 the American Physiological Societyhttps://doi.org/10.1152/physrev.1976.56.1.57PubMed813242History Published online 1 January 1976 Published in print 1 January 1976 Metrics