A distinct beta-hexosaminidase isoenzyme separated from human leukemic lymphocytes and myelocytes

The beta-hexosaminidase (EC 3.2.1.30) isoenzymes were separated on the basis of their carbohydrate moieties by an affinity chromatography using immobilized phenylboronate. Normal lymphocytes and granulocytes contain two major forms of beta-hexosaminidase, acute lymphoblastic, acute myeloblastic, chronic lymphocytic and chronic myelocytic leukemic cells contain an extra, distinct isoenzyme of beta-hexosaminidase. This extra isoenzyme may be a marker for the leukemic conversion of hematopoietic tissue.     

Metabolism of the human serum beta-hexosaminidase isoenzymes in the rat: an in vivo and in vitro study

Plasma clearance of purified human serum beta-hexosaminidase isoenzymes was studied in the rat. The serum beta-hexosaminidase isoenzymes (A, B and P) showed a slow clearance from circulation compared to their tissue counterparts. After desialylation, the clearance rate of all serum isoenzymes was markedly enhanced. The uptake of native as well as desialylated serum beta-hexosaminidase isoenzymes was studied in rat liver nonparenchymal cells and hepatocytes. No detectable uptake of any native serum isoenzyme was noticed in either cell type. However, when these isoenzymes were desialylated by neuraminidase treatment, isoenzymes A and B were taken up by the nonparenchymal cells. No uptake was observed for the P form. None of the desialylated serum forms was taken up by hepatocytes.     

Two mutations produce intron insertion in mRNA and elongated beta-subunit of human beta-hexosaminidase

An elongated beta-subunit of the lysosomal enzyme beta-hexosaminidase was found in fibroblast strains derived from two patients with juvenile Sandhoff disease and two asymptomatic individuals sharing an unusual isoenzyme pattern: a low level of residual A (alpha beta) isoenzyme activity (3-6% of normal for the juvenile Sandhoff and 9-10% for the asymptomatic strains) without B (beta beta) isoenzyme activity. The elongated beta-subunit was abnormal in other ways: It reacted with antiserum against the unfolded polypeptide, it was not phosphorylated on mannose residues, it was not processed to the mature form, and it was degraded rapidly. The increased length of the beta-subunit was caused by two different mutations. Cells from two juvenile Sandhoff and one asymptomatic individuals had the previously described G----A transition in intron 12 that creates a splice site, causing an in-frame insertion of 24 intronic nucleotides into mRNA (Nakano, T., and Suzuki, K. (1989) J. Biol. Chem. 264, 5155-5158). The second mutation was found in cells from the asymptomatic girl whose A+B- isoenzyme pattern had been designated "Hexosaminidase Paris" (Dreyfus, J. C., Poenaru, L., Vibert, M., Ravise, N., and Boue, J. (1977) Am. J. Hum. Genet. 29, 287-293); duplication of a region straddling the junction of intron 13 and exon 14 generates an alternate splice site, causing an in-frame insertion of 18 nucleotides into mRNA. Although the two new splice sites are used preferentially, the normal sites may be used to some extent, accounting for the residual A isoenzyme activity.     

A novel N-acetylglucosaminidase from neonatal rat enterocytes

The luminal surface of ileal enterocytes in fetal and neonatal mammals is covered by beta-hexosaminidase, which is attached to a fibrillar array. In this study, we have isolated this enzyme and subjected it to kinetic and structural analyses. The enzyme is identified as N-acetyl-beta-glucosaminidase (NA beta G) on the basis of substrate specificity and susceptibility to inhibition by N-acetylgalactosamine. Its catalytic properties and thermal stability are characteristics of the acidic, thermally labile human isoenzyme, but it differs from the human glycosidase in size. The neonatal NA beta G is a species of 225,000 relative mass (Mr), composed of two subunits of 125,000 and 115,000 Mr. Both its cellular location and differences in biophysical properties from the adult rat lysosomal forms and human glycosidases suggest that the neonatal rat NA beta G is a novel isoenzyme.     

Synthesis of a human lysosomal enzyme, beta-hexosaminidase B, using the baculovirus expression system

Human lysosomal beta-hexosaminidase exists in two major forms: the A isoform is composed of both alpha and beta chains, while the B form is a homopolymer of beta chains. Deficiency of beta-hexosaminidase underlies the GM2 gangliosidoses. We have produced active beta-hexosaminidase B in cultured insect (Sf9) cells by isolation of a recombinant insect virus (baculovirus) containing the cDNA for the beta chain within the viral polyhedron gene and infection of Sf9 cells with this construct. That portion of the enzyme secreted into the medium, 50%, was purified with concanavalin A Sepharose and subsequent affinity chromatography to yield beta-hexosaminidase B that is 75% pure. The product has an N-terminal amino acid sequence, specific activity, and size (M(r) 62,000) similar to that of the enzyme present in cultured human fibroblasts. However, endo H sensitivity studies revealed that the oligosaccharide structures present on recombinant beta-hexosaminidase B differ from those found on the enzyme synthesized in the human system. In addition, these structures lack the mannose 6-phosphate recognition marker that targets degradative hydrolases to lysosomes. Despite these differences, recombinant beta-hexosaminidase B does serve as a specific substrate for the mannose phosphorylating enzyme, N-acetylglucosaminyl phosphotransferase. Furthermore, the oligosaccharide moieties phosphorylated in vitro match those phosphorylated in vivo, pointing to the conformational integrity of the recombinant enzyme. Generous amounts of easily obtained, easily purified, and properly folded beta-hexosaminidase B will facilitate physical structural analysis of the enzyme.     

Molecular forms of beta-hexosaminidase and cathepsin D in serum and urine of healthy subjects and patients with elevated activity of lysosomal enzymes.

A procedure is described that allows the characterization of the molecular forms of beta-hexosaminidase and cathepsin D in controls and pathological specimens of human serum and human urine. The following observations were made. (1) In human serum, beta-hexosaminidase (alpha- and beta-chain) and cathepsin D are present predominantly in their high-molecular-weight precursor forms. In human urine, these enzymes exist as both precursor and mature forms. (2) Cathepsin D precursor from serum and urine differs in the number of oligosaccharides that are sensitive to endo-beta-N-acetylglucosaminidase H. Therefore the urine enzyme is not likely to originate from the serum. (3) The presence exclusively of precursors of beta-hexosaminidase and of cathepsin D in the sera of patients with hepatitis suggests that in hepatitis secretion of lysosomal enzymes is elevated, rather than the enzymes leaking from damaged cells. (4) In the urine of patients with nephrotic syndrome, beta-hexosaminidase and cathepsin D are present in grossly elevated amounts, but do not differ in the polypeptide patterns from controls. (5) In urine from a patient with mucolipidosis II, the elevated activity of beta-hexosaminidase is accounted for mainly by the precursor forms. Mature beta-chain of beta-hexosaminidase is lacking, and incompletely processed beta-hexosaminidase polypeptides are present. Both the precursor and the mature forms of cathepsin D are increased. They contain only complex oligosaccharides.     

Sequencing, expression, and enzymatic characterization of beta-hexosaminidase in rabbit lacrimal gland and primary cultured acinar cells

The lysosomal enzyme, beta-hexosaminidase, exists as two major isoforms; HexA and HexB. HexA is an alpha beta-subunit heterodimer and HexB a beta-subunit homodimer. Both isoforms can remove nonreducing beta-N-acetyl-D-glucosamine residues, whereas HexA hydrolyzes charged substrates as G(M2) gangliosides as well. beta-Hexosaminidase is present in both human and rabbit tear fluid and is secreted from rabbit lacrimal gland acinar cells in primary culture on stimulation with secretagogs. To further characterize the enzyme, the alpha- and beta-subunit mRNA expression was explored in rabbit lacrimal gland tissue as well as in cultured cells. Possible correlation between mRNA expression and HexA specific enzymatic activity was also investigated. Because existing beta-hexosaminidase antibodies are unable to recognize the rabbit enzyme, cloning and sequencing of the alpha- and beta-subunits were performed. Sequencing of the these subunits indicate that both are highly conserved between human, mouse, and rabbit. In contrast to the beta-subunit, showing an even mRNA expression between tissue and cultured cells, the level of alpha-subunit expression was higher in cultured acinar cells compared to tissue, with no alteration after cell stimulation. A minor but significant increase in total beta-hexosaminidase as well as HexA activity was observed in cultured cells compared to tissue. Enzymatic activity assays also revealed that HexA is the dominating isoform of beta-hexosaminidase in lacrimal gland and cultured acinar cells.     

Identification of plasma membrane associated mature beta-hexosaminidase A, active towards GM2 ganglioside, in human fibroblasts

Mature beta-hexosaminidase A has been found associated to the external leaflet of plasma membrane of cultured fibroblasts. The plasma membrane association of beta-hexosaminidase A has been directly determined by cell surface biotinylation followed by affinity chromatography purification of the biotinylated proteins, and by immunocytochemistry. The immunological and biochemical characterization of biotinylated beta-hexosaminidase A revealed that the plasma membrane associated enzyme is fully processed, suggesting its lysosomal origin.     

Properties of lysosomal beta-hexosaminidase accumulated in Niemann-Pick mouse liver

Various lysosomal acid hydrolases from tissues of Niemann-Pick mice, a mutant strain of C57BL/KsJ mice (spm/spm), were examined and compared to those from control mice. Activities of beta-hexosaminidase, beta-galactosidase, acid phosphatase, and cathepsin L were elevated in the liver and spleen of the affected mice, whereas no significant changes in beta-glucosidase and acid alpha-glucosidase were observed. Alpha-Mannosidase and neutral alpha-glucosidase activities were rather decreased in the affected mouse liver. The level of beta-hexosaminidase in the Niemann-Pick mice was raised sixfold in the liver and two- to threefold in the spleen and brain, whereas its total activity was decreased in the kidney. Sixty to ninety percent of total activity of lysosomal hydrolases was solubilized with 0.1% Triton X-100 in control mice, but most of the beta-hexosaminidase activity of the Niemann-Pick mice remained associated with the membrane fraction of liver lysosomes. The beta-hexosaminidase of the Niemann-Pick mice was appreciably stable when heated at 55 degrees C, while hydrolases of the affected mice and all of the enzymes tested in control mice were heat labile. The relative content of two beta-hexosaminidase fractions separated by DEAE-cellulose column chromatography was 8% for beta-hexosaminidase I and 92% for beta-hexosaminidase II in the case of the control mouse liver. The isozyme pattern of hexosaminidases in Niemann-Pick mice was similar to that of control enzymes. However, the beta-hexosaminidase II accumulated in Niemann-Pick mouse liver was different from that of the control in optimum pH, Km values and thermostability.     

Molecular cloning and sequence analysis of an ascidian egg beta-N-acetylhexosaminidase with a potential role in fertilization

Beta-N-acetylhexosaminidase, which is found almost ubiquitously in sperm of invertebrates and vertebrates, supposedly mediates a carbohydrate-based transient sperm-egg coat binding. In ascidians and mammals, beta-hexosaminidase released at fertilization from eggs has been proposed to modify sperm receptor glycoproteins of the egg envelope, thus setting up a block to polyspermy. Previously, it was shown that in potential sperm receptor glycoproteins of the ascidian Phallusia mammillata, N-acetylglucosamine is the prevailing glycoside residue and that the egg harbors three active molecular forms of beta-hexosaminidase. In the present study, P. mammillata beta-hexosaminidase cDNA was isolated from an ovarian cDNA library and characterized. The deduced amino acid sequence showed a high similarity with other known beta-hexosaminidases; however, P. mammillata beta-hexosaminidase had a unique potential N-glycosylation site. A phylogenetic analysis suggested that P. mammillata beta-hexosaminidase developed independently after having branched off from the common ancestor gene of the chordate enzyme before two isoforms of the mammalian enzyme appeared. In situ hybridization revealed stage-specific expression of beta-hexosaminidase mRNA during oogenesis in the oocyte and in the accessory test and follicle cells. This suggests that the three egg beta-hexosaminidase forms are specific for the oocyte, test cells and follicle cells.     

Comparative study of alkaline phosphatase isoenzymes,bone histology,and skeletal radiography in dialysis bone disease.

Liver, intestinal, and bone alkaline phosphatase isoenzymes were measured using heat stability and L-phenylalanine inhibition techniques in 78 patients on intermittent haemodialysis. Fifty-five patients had abnormalities in one or more of the isoenzymes. Changes in bone and intestinal alkaline phosphatase activities seemed to be related and raised liver isoenzyme activity was associated with the development of liver disease. Abnormal histological and radiological findings were better correlated with bone alkaline phosphatase levels than with total alkaline phosphatase, and serial estimations of bone isoenzyme activity were useful in assessing the response of renal osteodystrophy to treatment with a vitamin D analogue. Serum alkaline phosphatase isoenzyme measurement provides another useful and non-invasive index for monitoring metabolic bone disease in patients with chronic renal failure.     

Analysis of isoenzyme patterns of acid phosphatase in acute leukemias

The status of acid phosphatase isoenzymes was evaluated in cells of patients with acute lymphocytic leukemias or lymphomas by analytical isoelectric focusing in polyacrylamide gels (IEF) on horizontal thin-layer slabs. The isoenzyme patterns were correlated with routine immunological cell surface markers and the relationship of enzyme activity to specific immunological subclasses of ALL is discussed. By isoelectric focusing up to five isoenzyme groups (I-V) containing several isoenzyme were observed. No leukemia specific or additional isoenzyme could be demonstrated. This biochemical characterization showed a marked heterogeneity within two major immunologic subgroups indicating that various differentiation stages of cell maturation could be involved in cALL and T-ALL. According to their degree of maturation along T-cell differentiation axis the leukemic cells displayed no enzyme activity, weak isoenzyme bands or the incomplete or complete isoenzyme pattern seen with normal lymphocytes from human tonsils which were used as controls. The investigation of specific enzymatic patterns can lead to a further definition of subsets of acute leukemias and give insight into lymphopoietic differentiation.     

Cell disease: desialylation of beta-hexosaminidase and its effect on uptake by fibroblasts

The pinocytosis by fibroblasts of beta-hexosaminidase (EC 3.2.1.30) excreted by cultured skin fibroblasts from a patient with I-cell disease was not enhanced by neuraminidase treatment of the enzyme. The uptake of sialic acid-rich normal plasma beta-hexosaminidase was minimal and neuraminidase treatment did not appreciably enhance uptake. In contrast, sialic acid-rich normal seminal fluid beta-hexosaminidase was readily pinocytosed regardless of neuraminidase treatment. Thus the presence of sialic acid on beta-hexosaminidase does not influence uptake and a neuraminidase deficiency in I-cell disease may not be directly responsible for excessive extracellular enzyme.     

Lending a helping hand, screening chemical libraries for compounds that enhance beta-hexosaminidase A activity in GM2 gangliosidosis cells

Enzyme enhancement therapy is an emerging therapeutic approach that has the potential to treat many genetic diseases. Candidate diseases are those associated with a mutant protein that has difficulty folding and/or assembling into active oligomers in the endoplasmic reticulum. Many lysosomal storage diseases are candidates for enzyme enhancement therapy and have the additional advantage of requiring only 5-10% of normal enzyme levels to reduce and/or prevent substrate accumulation. Our long experience in working with the beta-hexosaminidase (EC 3.2.1.52) isozymes system and its associated deficiencies (Tay-Sachs and Sandhoff disease) lead us to search for possible enzyme enhancement therapy-agents that could treat the chronic forms of these diseases which express 2-5% residual activity. Pharmacological chaperones are enzyme enhancement therapy-agents that are competitive inhibitors of the target enzyme. Each of the known beta-hexosaminidase inhibitors (low microm IC50) increased mutant enzyme levels to >or= 10% in chronic Tay-Sachs fibroblasts and also attenuated the thermo-denaturation of beta-hexosaminidase. To expand the repertoire of pharmacological chaperones to more 'drug-like' compounds, we screened the Maybridge library of 50,000 compounds using a real-time assay for noncarbohydrate-based beta-hexosaminidase inhibitors and identified several that functioned as pharmacological chaperones in patient cells. Two of these inhibitors had derivatives that had been tested in humans for other purposes. These observations lead us to screen the NINDS library of 1040 Food and Drug Administration approved compounds for pharmacological chaperones. Pyrimethamine, an antimalarial drug with well documented pharmacokinetics, was confirmed as a beta-hexosaminidase pharmacological chaperone and compared favorably with our best carbohydrate-based pharmacological chaperone in patient cells with various mutant genotypes.     

Analysis of the glycosylation and phosphorylation of the lysosomal enzyme, beta-hexosaminidase B, by site-directed mutagenesis

Lysosomal enzymes require a mannose 6-phosphate recognition marker, constructed on asparagine-linked oligosaccharide chains, for targeting to lysosomes. We have identified the glycosylation sites of human beta-hexosaminidase B and have determined the influence of individual oligosaccharides on the phosphorylation, lysosomal targeting, and catalytic activity of the enzyme. The five potential glycosylation sites of the hexosaminidase beta-chain were modified individually by site-directed mutagenesis, and the constructs were expressed in COS 1 cells. By this analysis, we determined that four of the five potential sites were glycosylated. Two of the four oligosaccharides were preferentially phosphorylated. The absence of these two preferentially phosphorylated oligosaccharides resulted in greatly reduced amounts of the lysosomal form of the enzyme with increased secretion into the medium. The catalytic activity of beta-hexosaminidase B was not significantly altered by the absence of individual oligosaccharides suggesting the folding and assembly of the enzyme was not disrupted.     

Uptake of beta-hexosaminidase by nonparenchymal liver cells and peritoneal macrophages

The uptake of beta-hexosaminidase (EC 3.2.1.30) in nonparenchymal liver cells (i.e. endothelial and Kupffer's cells) and peritoneal macrophages has been determined by an enzymatic assay. A considerable uptake was noted in nonparenchymal liver cells, whereas no measurable uptake was seen in peritoneal macrophages. The endothelial cells were more active in the uptake of beta-hexosaminidase than were the Kupffer's cells. The uptake of beta-hexosaminidase by nonparenchymal liver cells showed saturation kinetics and was competitively inhibited by mannan. These findings support the concept that a cell surface receptor on nonparenchymal liver cells mediates uptake of beta-hexosaminidase and suggests a difference in the receptor mechanisms on liver and peritoneal macrophages.     

Release of beta-hexosaminidase after administration of different agents affecting the reticuloendothelial system. An experimental study in rats

Plasma levels of a lysosomal enzyme, beta-hexosaminidase (beta-N-acetylglucosaminidase, EC 3.2.1.30) were studied in Wistar rats after administration of 99mTc -sulfur colloid, 198Au colloid, gelatine (Haemaccel), alcohol, methylpalmitate and zymosan. The activity of beta-hexosaminidase was increased 10, 30 and 60 min after the zymosan injection. After 24 and 48 h, enzyme levels had returned to those at outset. The transient release of beta-hexosaminidase probably occurred only during the phagocytosis of zymosan which was evaluated by histological examination of lung, liver and spleen. After the injection of all other agents tested, no significant aberration of beta-hexosaminidase levels was seen. Activity distribution of the radio-labeled colloids revealed differences in organ uptake which were attributed to a difference in colloid particle size. Although the colloids tested have been used extensively for determination of reticuloendothelial function and histological studies suggest phagocytosis of the particles, their administration did not affect plasma beta-hexosaminidase levels. Since lysosomal enzymes are cleared from the blood predominantly by liver macrophages, the primary location of particle phagocytosis may explain the present findings.     

Intracellular transport of acid alpha-glucosidase in human fibroblasts: evidence for involvement of phosphomannosyl receptor-independent system

Intracellular transport of two lysosomal enzymes, acid alpha-glucosidase and beta-hexosaminidase, was analyzed in human fibroblasts. The precursors of beta-hexosaminidase in normal fibroblasts were released from the membrane fraction by treatment with mannose 6-phosphate, but the precursor of alpha-glucosidase was not. Percoll density gradient centrifugation revealed a normal amount of acid alpha-glucosidase activity in heavy lysosomes in I-cell disease fibroblasts despite impaired maturation and defective phosphorylation, and beta-hexosaminidase activity was markedly reduced in lysosomes. It was concluded that the membrane-bound precursor of acid alpha-glucosidase is transported to lysosomes by a phosphomannosyl receptor-independent system although the enzyme lacks the recognition marker for the phosphomannosyl receptor and processing of an intermediate form to mature forms does not occur in this disease.     

Isolation and characterization of boar spermatozoa with and without a cytoplasmic droplet

1. Boar semen was separated on a Percoll density gradient into three populations; a low-density band of immature sperm cells containing a cytoplasmic droplet and a high-density doublet band formed by spermatozoa without a cytoplasmic droplet. 2. In these three cell populations four acid hydrolases were determined, viz. (1) alpha-glucosidase; (2) alpha-mannosidase; (3) beta-galactosidase; (4) beta-hexosaminidase. 3. The release of the hydrolases (1), (2) and (3) from cytoplasmic droplet containing spermatozoa was stimulated whereas the release of beta-hexosaminidase was inhibited by calcium ions. 4. The results suggest that acid alpha-glucosidase, alpha-mannosidase and beta-galactosidase are situated in the acrosome whereas acid beta-hexosaminidase is localized predominantly in the cytoplasmic droplet of boar spermatozoa. 5. We conclude that beta-hexosaminidase should prove useful as a biochemical marker for cytoplasmic droplet containing spermatozoa and hence for the number of immature sperm cells in boar semen.