Correction of a defect in mammalian GPI anchor biosynthesis by a transfected yeast gene.

Glycosylphosphatidylinositol (GPI) serves as a membrane anchor for a large number of eukaryotic proteins. A genetic approach was used to investigate the biosynthesis of GPI anchor precursors in mammalian cells. T cell hybridoma mutants that cannot synthesize dolichol-phosphate-mannose (Dol-P-Man) also do not express on their surface GPI-anchored proteins such as Thy-1 and Ly-6A. These mutants cannot form mannose-containing GPI precursors. Transfection with the yeast Dol-P-Man synthase gene rescues the synthesis of both Dol-P-Man and mannose-containing GPI precursors, as well as the surface expression of Thy-1 and Ly-6A, suggesting that Dol-P-Man is the donor of at least one mannose residue in the GPI core.

Differential expression of glycosylphosphatidylinositol-anchored proteins in a murine T cell hybridoma mutant producing limiting amounts of the glycolipid core. Implications for paroxysmal nocturnal hemoglobinuria.

A T cell hybridoma mutant, which expressed a markedly reduced level of glycosylphosphatidylinositol (GPI)-anchored proteins on the cell surface, was characterized. The surface expression level of Thy-1 was approximately 17% of the wild-type level, whereas the surface expression of Ly-6A was approximately 2.4% of the wild-type level. We show here that these cells synthesized limiting amounts of the GPI core and that the underlying defect in these cells was an inability to synthesize dolichyl phosphate mannose (Dol-P-Man) at the normal level. The defect in Ly-6A expression could be partially corrected by tunicamycin, which blocked the biosynthesis of N-linked oligosaccharide precursors and shunted Dol-P-Man to the GPI pathway. Full restoration of Thy-1 and Ly-6A expression, however, required the stable transfection of a yeast Dol-P-Man synthase gene into the mutants. These results revealed that when the GPI core is limiting, there is a differential transfer of the available GPI core to proteins that contain GPI-anchor attachment sequences. Our findings also have implications for the elucidation of the defects in paroxysmal nocturnal hemoglobinuria.

Bone marrow-derived mesenchymal stem cells remain host-derived despite successful hematopoietic engraftment after allogeneic transplantation in patients with lysosomal and peroxisomal storage diseases.

Human bone marrow contains mesenchymal stem cells (MSCs) that can differentiate into various cells of mesenchymal origin. We developed an efficient method of isolating and culture expanding a homogenous population of MSCs from bone marrow and determined that MSCs express alpha-L-iduronidase, arylsulfatase-A and B, glucocerebrosidase, and adrenoleukodystrophy protein. These findings raised the possibility that MSCs may be useful in the treatment of storage disorders. To determine if donor derived MSCs are transferred to the recipients with lysosomal or peroxisomal storage diseases by allogeneic hematopoietic stem cell (HSC) transplantation, we investigated bone marrow derived MSCs of 13 patients 1-14 years after allogeneic transplantation. Highly purified MSCs were genotyped either by fluorescence in situ hybridization using probes for X and Y-chromosomes in gender mis-matched recipients or by radiolabeled PCR amplification of polymorphic simple sequence repeats. Phenotype was determined by the measurement of disease specific protein/enzyme activity in purified MSCs. We found that MSCs isolated from recipients of allogeneic HSC transplantation are not of donor genotype and have persistent phenotypic defects despite successful donor type hematopoietic engraftment. Whether culture expanded normal MSCs can be successfully transplanted into patients with storage diseases and provide therapeutic benefit needs to be determined.

Measurements from normal umbilical cord blood of four lysosomal enzymatic activities: alpha-L-iduronidase (Hurler), galactocerebrosidase (globoid cell leukodystrophy), arylsulfatase A (metachromatic leukodystrophy), arylsulfatase B (Maroteaux-Lamy).

Umbilical cord blood (UCB) has received increasing attention as a source of unrelated hematopoietic stem cells for transplantation. Lysosomal diseases have been effectively treated and normal enzymatic activity has occurred subsequent to engraftment using UCB. The use of donor cells with normal amounts of enzyme, rather than those from carriers whose level may be 50% or less, is an obvious goal. The frequency of such heterozygotes varies from 1:10 to 1:140 or lower depending upon the disease at issue. We assayed the levels of lysosomal enzymes in normal UCB in random samples as well as those used for transplantation. We measured the following enzymatic activities: alpha-l-iduronidase (Hurler), galactocerebrosidase (globoid cell leuko- dystrophy) and arylsulfatase A (metachromatic leukodystrophy). For the latter, levels of activity in UCB are comparable to those found in adult blood. In the case of arylsulfatase B (Maroteaux-Lamy) a level lower than adult level was found. An informed choice by the transplanting physician based on the activity of the relevant enzyme in the UCB donor will provide a better opportunity for an improved prognosis for more complete correction of the recipient's primary disease. Bone Marrow Transplantation (2000) 25, 541-544.

Urinary endo-beta-galactosidase capable of depolymerizing polylactosaminoglycans.

Human urine was found to contain an endo-beta-galactosidase capable of depolymerizing sulfated and non-sulfated polylactosaminoglycans. Using 0.05 M sodium phosphate buffer, pH 7.0, this enzyme was not retained by DEAE-Sephadex A-50 or concanavalin A-Sepharose. The urinary endo-beta-galactosidase liberated a disaccharide with chromatographic mobility identical to 6-O-sulfo-GlcNAc beta 1----3Gal as one of the major products from keratan sulfates isolated from whale nasal cartilage, bovine cornea, and human costal cartilage. It also liberated GlcNAc beta 1----3 Gal as one of the major oligosaccharides from erythroglycan. The oligosaccharide profiles produced from various keratan sulfates and erythroglycan by the action of urinary endo-beta-galactosidase are quite similar to those produced by Escherichia freundii endo-beta-galactosidase (Nakagawa, H., Yamada, T., Chien, J.-L., Gardas, A., Kitamikado, M., Li, S.-C., and Li, Y.-T. (1980) J. Biol. Chem. 255, 5955-5959). The presence of urinary endo-beta-galactosidase indicates the existence of a new catabolic pathway for polylactosaminoglycans. This pathway involves the cleavage of internal beta-galactosyl linkages of the glycan chain.

A GM2-specific beta-hexosaminidase from the roe of striped mullet (Mugil cephalus).

The roe of striped mullet (Mugil cephalus) was found to contain a beta-hexosaminidase different from the beta-hexosaminidases isolated from other sources. The enzyme from mullet roe is able to cleave GalNAc from GM2 without the assistance of either an activator protein or a detergent. It also cleaves the oligosaccharide derived from GM2 and other oligosaccharides containing the GM2 sequence GalNAc beta 4(NeuAc alpha 3)Gal-. However, it is not effective in hydrolyzing neutral glycosphingolipids containing terminal GalNAc or GlcNAc, such as GbOse4Cer, GgOse3Cer, or LcOse3Cer. These results indicate that mullet roe beta-hexosaminidase can specifically cleave GalNAc from the glycoconjugates containing the GM2 sequence. No beta-hexosaminidase with such specificity has been previously described. Thus, this unique enzyme should be very useful for the detection and analysis of glycoconjugates containing the oligosaccharide chains with GM2 sequence.

Isolation and characterization of an activator protein for the hydrolysis of ganglioside GM2 from the roe of striped mullet (Mugil cephalus).

After the revelation of the presence of ganglioside GM2 as the major ganglioside in the roe of striped mullet, Mugil cephalus [Li, Hirabayashi, DeGasperi, Yu, Ariga, Koerner & Li (1984) J. Biol. Chem. 259, 8980-8985], we have continued to investigate the catabolism of GM2 in this tissue. We have found that mullet roe contains a specific activator protein which stimulates the hydrolysis of GM2 carried out by the beta-hexosaminidase isolated from the same tissue. This activator has been purified by using conventional procedures including ammonium sulphate fractionation and chromatography on Sepharose 6B, DEAE-Sephadex A-50, octyl-Sepharose and Matrex Gel Blue A columns. This activator protein is also able to stimulate the hydrolysis of GM2 carried out by human beta-hexosaminidase A. Unlike human GM2-activator, the roe activator protein does not stimulate the hydrolysis of GgOse3Cer or GbOse4Cer. The molecular mass (18 kDa) of the roe activator protein was found to be similar to that of human GM2-activator; however, the pI (pH 4.1) was found to be lower than that of human GM2-activator. This is the first report on the presence of a GM2-activator protein in a source other than mammalian tissues.

Presence of two endo-beta-N-acetylglucosaminidases in human kidney.

We have isolated for the first time two kinds of endo-beta-N-acetylglucosaminidases (E-beta-GNases) simultaneously from human kidney. E-beta-GNase 1 was purified by water extraction, ammonium sulfate fractionation, and chromatography on Sephadex-G-200, DEAE-Sephadex, concanavalin A-Sepharose and Hypatite C columns. After the DEAE-Sephadex step, 107 units of E-beta-GNase 1 with a specific activity of 0.53 units/mg was obtained and after hydroxyapatite column, the enzyme recovery was 26 units with a specific activity of 10.4 units/mg. This enzyme hydrolyzed the high mannose-type asparaginylglycopeptide efficiently and had little activity toward the complex-type glycopeptide. This enzyme had an pH optimum at about 4.5 and was not inhibited by acetate ion. The Asn residue in a glycopeptide appeared not to be an important recognition site for E-beta-GNase 1 to express its activity because the acetylation or the dansylation of Asn residues as well as the elimination of Asn residue from the glycopeptide did not change the susceptibility of the oligosaccharide to E-beta-GNase 1. E-beta-GNase 2 was purified by water extraction, ammonium sulfate fractionation, and chromatography on Sephadex G-200, DEAE-Sephadex, concanavalin A-Sepharose, and Mono S columns. This enzyme was purified about 110-fold with 6.6% recovery. E-beta-GNase 2 was found to be a novel type of E-beta-GNase that hydrolyzed both the high mannose-type and the complex-type oligosaccharide with chitobiosyl group at the reducing end and without the Asn. E-beta-GNase 2 activity was found to be dependent on a L-aspartamido-beta-D-N-acetylglucosamine amidohydrolase (Asn-GNase) for the hydrolysis of asparaginylglycopeptide. Asn-GNase cleaved off the Asn residue from the glycopeptide, and the resulting oligosaccharide was hydrolyzed by E-beta-GNase 2. Because the acetylation or the dansylation of Asn residue in a glycopeptide rendered the glycopeptide resistant to Asn-GNase, the use of the modified asparaginylglycopeptide could not reveal the existence of E-beta-GNase 2 activity. The pH optimum of E-beta-GNase was found to be about 3.5. Like beta-hexosaminidases, this enzyme was inhibited by acetate ion, suggesting the recognition of GlcNAc moiety by this enzyme.

Accumulation of globotriaosylceramide in a case of leiomyosarcoma.

Analysis of the glycosphingolipid composition in one case of uterine leiomyosarcoma metastasized to the liver showed an accumulation of globotriaosylceramide as compared with normal liver and uterus from which the tumour originated. The structure and the amount of glycosphingolipids were established by using specific glycosidases, permethylation analysis and h.p.l.c. The reason for the accumulation of globotriaosylceramide in leiomyosarcoma remains to be answered.

A unique glycosphingolipid-splitting enzyme (ceramide-glycanase from leech) cleaves the linkage between the oligosaccharide and the ceramide.

A novel type of enzyme which hydrolyzes the linkage between the ceramide and the sugar chain in various glycosphingolipids has been found in the leech, Hirudo medicinalis. This enzyme releases the intact oligosaccharide from LacCer, GbOse3Cer, GbOse4Cer, GbOse5Cer, nLcOse4Cer, GM3, GM2, GM1, GD1a and GT1 with the concurrent release of ceramides. By using tritium-labeled GM1 as substrate we found the optimum pH of this enzyme to be between pH 4 and 5. Since the enzyme cleaves the linkage between the ceramide and the sugar chain in various glycosphingolipids with no apparent preference toward the sugar chain, we propose to call this enzyme ceramide-glycanase.

Human cell lines stably expressing HIV env and tat gene products.

A DNA fragment containing the tat, rev and env genes of the human immunodeficiency virus type 1 was inserted into the retroviral vector pZIPneoAU3. The resulting plasmid penvAU3 was transfected into HeLa and psi CRIP cells. Resulting recombinant retroviruses were used to infect HeLa and Jurkat cells. Immunoprecipitation analysis of stable transformants showed the expression of HIV env glycoproteins gp160, gp120 and gp41. Transactivation assays with a plasmid containing the gene for chloramphenicol acetyltransferase linked to HIV promoter-enhancer sequences demonstrated the expression of functional tat. These cells constitute virus-free tools for functional and structural studies of native env and tat.

Presence of activator proteins for the enzymic hydrolysis of GM1 and GM2 gangliosides in normal human urine.

Normal human urine has been found to contain activator proteins that stimulate the enzymic hydrolysis of GM1 and GM2 gangliosides. These two activators were partially purified by Sephadex G-200 filtration and DEAE-Sephadex A-50 chromatography. The presence of these two activators was assayed by demonstrating the stimulation of the in vitro hydrolysis of GM1 and GM2 gangliosides. As little as 50 ml of urine is sufficient to detect the presence of these two activators. The crude activator preparation from normal urine was also found to stimulate the hydrolysis of galactosylceramide sulfate by arylsulfatase A.

The substrate specificity of bovine and feline lysosomal alpha-D-mannosidases in relation to alpha-mannosidosis.

Lysosomal alpha-mannosidases were partially purified from bovine and feline liver and employed to digest a large number of oligosaccharides with structures corresponding to the oligomannosyl parts of complex, hybrid, and high-mannose glycans. The incubation products were identified by high pressure liquid chromatography with reference compounds of defined structure and by acetolysis. For all classes of substrates, the lysosomal alpha-mannosidases displayed a high degree of in vitro specificity with regard to the hydrolysis of mannose residues. Thus, in each case, 1 or at most 2 residues were always preferentially cleaved so that the degradative process proceeded down a well defined pathway. A comparison of the relative efficiency with which lysosomal alpha-mannosidases catalyzed the hydrolysis of particular oligosaccharides and of the structures of the resulting intermediates with those of the compounds accumulated in alpha-mannosidosis allows conclusions to be drawn regarding the nature of the enzymatic defect. In bovine alpha-mannosidosis, the oligosaccharides are those expected for a partial deficiency of normal lysosomal alpha-mannosidase, so that they correspond to intermediates in the normal catabolic pathway. In feline alpha-mannosidosis, in which the alpha-mannosidase deficiency is more severe than in cattle, the accumulated oligosaccharides primarily represent intact oligomannosyl moieties of N-linked glycans rather than the products of residual alpha-mannosidase activity.

Identification of a missing link in glycosylphosphatidylinositol anchor biosynthesis in mammalian cells.

A large number of mammalian proteins are anchored to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. Biosynthetic intermediates of the GPI anchor have been identified in mammalian cells. The early GPI precursors are sensitive to phosphatidylinositol (PI)-specific phospholipase C (PLC). However, all of the later GPI precursors, which contain 1 or more mannose residues, are PI-PLC-resistant, suggesting that there is another unidentified precursor. Here, we report the identification of this missing link. This GPI precursor can only be labeled with glucosamine and inositol, and is resistant to PI-PLC but sensitive to GPI-phospholipase D. It accumulates in large quantity only in mutants which are defective in the addition of the first mannose residue to the elongating GPI core. Thus, fatty acylation of glucosaminylphosphatidylinositol, to render it PI-PLC-resistant, is an obligatory step in the biosynthesis of mammalian GPI anchor precursors.

Negative regulation of the 5' long terminal repeat (LTR) by the 3' LTR in the murine proviral genome.

To assess the influence of the 3' long terminal repeat (LTR) on the promoter/enhancer activity of the 5' LTR, a set of isogenic retroviral vectors differing only in the U3 region of the 3' LTR was constructed. These U3 elements were derived from viruses with different tissue tropism. The 5' LTR originated from Moloney murine leukemia virus and directed the transcription of a reporter gene (chloramphenicol acetyltransferase [CAT] gene), giving rise to plasmids of the general configuration LTR-CAT-LTR'. Following transfection of these chimeric constructs into various cell types, the CAT activity in a given cell line was inversely related to the activity of the downstream U3 region when used in a single-LTR construct in that cell type, indicating negative regulation of the 5' LTR by the chimeric 3' LTR'. Our data indicate that a highly active 3' LTR interferes with gene expression from the 5' LTR. Potential mechanisms for this down-regulation are discussed.

Glycoprotein storage in Gaucher disease: lectin histochemistry and biochemical studies.

Lectin histochemical studies were performed on formalin-fixed, frozen, and paraffin-embedded tissue sections from 19 patients with glucosylceramide lipidosis (i.e., Gaucher disease). Eleven different lectins were used to identify the specific carbohydrate residues in the undegraded stored compounds in the cytoplasm of Gaucher cells. In all cases studied, Gaucher cells stained with Concanavalia ensiformis agglutinin, Datura stramonium agglutinin, Lens culinaris, Ricinus communis agglutinin-I, and wheat germ agglutinin. These results demonstrated common carbohydrate residues in the undegraded material stored within Gaucher cells and indicated the presence of fucosylated N-linked complex oligosaccharides, and glycans containing N-acetyllactosamine repeating sequences, as well as nonreducing terminal beta-galactosyl and sialyl residues. In order to confirm these findings using biochemical methods, livers and spleens from Gaucher patients and controls, and from a patient with Niemann-Pick disease type C (included for comparison) were digested with Pronase and the resulting glycopeptides separated by gel filtration into fractions with high and low molecular weight. In the high-molecular-weight fractions from livers of Gaucher patients, the levels of sugars corresponding to N-linked glycans, as measured by gas-liquid chromatography, were elevated over those in controls. In the high-molecular-weight fractions from spleens, the levels of the same sugars were elevated in both Gaucher and Niemann-Pick type C patients. Digestion of the glycopeptides with endo-beta-galactosidase, which specifically cleaves polylactosaminoglycans, showed the presence of material containing N-acetyllactosamine repeating units in Gaucher liver glycopeptide fractions, but not in control and Niemann-Pick type C derived glycopeptide fractions. Our histochemical and biochemical studies demonstrated that in addition to glucosylceramide, affected tissues of patients with Gaucher disease accumulate glycoproteins. This accumulation could not have been predicted on the basis of the primary enzymatic defect.

Storage of glycoprotein in NCTR-Balb/C mouse. Lectin histochemistry, and biochemical studies.

A strain of Balb/C mice carrying a lysosomal storage disorder exhibits metabolic and phenotypic abnormalities similar to patients with sphingomyelin-cholesterol lipidoses type II (i.e., Niemann-Pick C and D). Their foamy cells, which belong to the reticuloendothelial system, stained intensely by periodate-Schiff (PAS) reagent and were resistant to predigestion with diastase. To identify the chemical nature of the PAS-positive storage material, we applied lectin histochemistry and biochemical methods. Paraffin embedded sections, and delipidated frozen tissue sections, were treated with biotinylated lectins and localized with avidin-biotin-peroxidase complex. Araldite-embedded semithin sections were incubated with biotinylated lectins followed by avidin-gold and were enhanced with silver. By both histochemical methods the affected foamy cells stained positively as follows: Concanavalia ensiformis agglutinin, Datura stramonium agglutinin, Griffonia simplicifolia-I, Lens culinaris agglutinin, peanut agglutinin, Ricinus communis agglutinin-I, wheat germ agglutinin (WGA), and succinylated-WGA. Biochemical analysis of liver extracts complemented the histochemical data and demonstrated accumulation of glycoproteins containing polylactosaminoglycans in affected mice. Our findings indicate that the storage material in NCTR-Balb/C mice is heterogeneous. The lipids that are extracted by organic solvents during the histologic preparations mask the occurrence of polylactosaminoglycan containing glycoproteins in native frozen sections.

Characterization of human liver 3-O-beta-D-glucopyranuronosyl-cholesterol by mass spectrometry and nuclear magnetic resonance spectroscopy.

We have isolated an unusual acidic glycolipid which was detected in the lower phase of the Folch partition of the total lipid extract of human liver during a routine isolation of glycosphingolipids. With the solvent systems commonly used for thin-layer chromatography of glycosphingolipids, this glycolipid has a mobility similar to GbOse3Cer, one of the major glycosphingolipids in human liver. Free cholesterol was released from this glycolipid upon treatment with beta-glucuronidase. The electron impact mass spectrum of the permethylated derivative of this glycolipid showed an intense peak at m/e 369 which is consistent with the cholesterol part of the molecule. It also showed m/e 233 and 201 which are derived from the permethylated glucopyranuronosyl residue. The final proof of the structure was accomplished by high resolution NMR spectroscopy which revealed the presence of beta-linked glucopyranuronosyl residue and cholesterol. Thus, the structure of this acidic glycolipid was conclusively established to be 3-O-beta-D-glucopyranuronosyl-cholesterol.

Isolation and characterization of a novel phytosphingosine-containing GM2 ganglioside from mullet roe (Mugil cephalus).

The major ganglioside from the roe of striped mullet (Mugil cephalus) has been isolated and purified. Compositional analysis of this ganglioside revealed that it contained an equimolar ratio of the following residues: N-acetylneuraminic acid, N-acetylgalactosamine, galactose, glucose, and the long-chain base. Further structural studies by sequential enzymatic hydrolysis, permethylation analysis, and proton NMR spectroscopy indicated that the structure of the oligosaccharide moiety was identical to that of GM2 ganglioside from human brain: GalNAc beta 1----4Gal beta 1----4(3----2 alpha NeuAc)-Glc----ceramide. This ganglioside, however, differed from brain GM2 in its ceramide portion. The most striking differences are the presence of large amounts of C18 and C20 phytosphingosine (over 80% of the total long-chain bases) and the preponderance of monounsaturated alpha-hydroxy fatty acids (over 80%). Such a phytosphingosine-containing GM2 ganglioside has never been reported.