Variant of GM2-gangliosidosis with hexosaminidase A having a severely changed substrate specificity

The levels of hexosaminidase A activity in cultivated fibroblasts of two patients with GM2-gangliosidosis were close to the normal range with 4-methylumbelliferyl-beta-D-2-acetamido-2-deoxyglucopyranoside and 4-methylumbelliferyl-beta-D-2-acetamido-2-deoxygalactopyranoside as substrates, and the enzymes were normal in most parameters analyzed. However, the enzymes of both patients were almost completely inactive against two specific substrates for hexosaminidase A, rho-nitrophenyl-6-sulfo-2-acetamido-2-deoxy-beta-D-glucopyranoside, and ganglioside GM2 in the presence of GM2-activator. Fibroblast extracts of both patients showed normal hexosaminidase B and GM2-activator activity, the latter was strongly decreased in two cases with variant AB. It is suggested that human hexosaminidase A may contain two different active sites which might be inactivated separately by different mutations.     

Diagnosis of infantile and juvenile forms of GM2 gangliosidosis variant 0. residual activities toward natural and different synthetic substrates

Summary p-Nitrophenyl-6-sulfo-2-acetamido-2-deoxy--d-glucopyranoside, which is known to be a specific substrate for human hexosaminidase A, has recently been used successfully for diagnosis of variants B and B¹ of G_M2-gangliosidosis (Fuchs et al. 1983; Kytzia et al. 1983; Li et al. 1983). However, it is hydrolyzed by hexosaminidase S as well and is therefore not suitable for detection of patients with variant 0, who reach the normal range of activity toward this substrats. Assay of ganglioside G_M2 cleaving activity in fibroblast extracts in the presence of the natural G_M2 activator protein reveals residual hexosaminidase A activities of less than 2% of normal controls in two infantile and up to 7.5% in two juvenile patients with variant 0.     

Juvenile GM2 gangliosidosis (AMB variant): inability to activate hexosaminidase A by activator protein.

Two sibling from a consanguineous Puerto Rican marriage were found to have a juvenile-onset type of lipidosis first noted at age 2 1/2 by expressing difficulties with motor function and developmental delay. They continued to deteriorate, showing muscle atrophy, spasticity, and loss of speech, and death occurred at ages 7 and 8. Examination of the brains from these patients revealed that the concentration of GM2 ganglioside was about 56% of the total gangliosides. Hexosaminidase and percent hexosaminidase A (HEX A) and other lysosomal enzymes were normal in cultured skin fibroblasts, liver, and brain. The concentration of the activator protein required for the enzymatic hydrolysis of GM2 ganglioside was in high normal levels in the brain of the patient available. However, the HEX A from the patient's brain and liver as well as from skin fibroblast lysates could not be activated to hydrolyze GM2 ganglioside by the activator protein from a control or himself. The HEX A from a control could be activated by the activator protein from controls or this patient. These patients appear to have a defect in HEX A, which does not affect it heat stability, electrophoretic migration, and activity toward fluorogenic substrates, but may affect the binding of the activator protein required for GM2 ganglioside hydrolysis. We propose to call these patients the AMB variant of GM2 gangliosidosis to denote the mutation in HEX A but with normal levels of HEX A and B with synthetic substrates. This is to distinguish these patients from those missing the activator protein and normal HEX A and B levels.     

In Cellulo Examination of a Beta-Alpha Hybrid Construct of Beta-Hexosaminidase A Subunits,Reported to Interact with the GM2 Activator Protein and Hydrolyze GM2 Ganglioside

The hydrolysis in lysosomes of GM2 ganglioside to GM3 ganglioside requires the correct synthesis, intracellular assembly and transport of three separate gene products; i.e., the alpha and beta subunits of heterodimeric beta-hexosaminidase A, E.C. # 3.2.1.52 (encoded by the HEXA and HEXB genes, respectively), and the GM2-activator protein (GM2AP, encoded by the GM2A gene). Mutations in any one of these genes can result in one of three neurodegenerative diseases collectively known as GM2 gangliosidosis (HEXA, Tay-Sachs disease, MIM # 272800; HEXB, Sandhoff disease, MIM # 268800; and GM2A, AB-variant form, MIM # 272750). Elements of both of the hexosaminidase A subunits are needed to productively interact with the GM2 ganglioside-GM2AP complex in the lysosome. Some of these elements have been predicted from the crystal structures of hexosaminidase and the activator. Recently a hybrid of the two subunits has been constructed and reported to be capable of forming homodimers that can perform this reaction in vivo, which could greatly simplify vector-mediated gene transfer approaches for Tay-Sachs or Sandhoff diseases. A cDNA encoding a hybrid hexosaminidase subunit capable of dimerizing and hydrolyzing GM2 ganglioside could be incorporated into a single vector, whereas packaging both subunits of hexosaminidase A into vectors, such as adeno-associated virus, would be impractical due to size constraints. In this report we examine the previously published hybrid construct (H1) and a new more extensive hybrid (H2), with our documented in cellulo (live cell- based) assay utilizing a fluorescent GM2 ganglioside derivative. Unfortunately when Tay-Sachs cells were transfected with either the H1 or H2 hybrid construct and then were fed the GM2 derivative, no significant increase in its turnover was detected. In vitro assays with the isolated H1 or H2 homodimers confirmed that neither was capable of human GM2AP-dependent hydrolysis of GM2 ganglioside.     

A Cys138-to-Arg substitution in the GM2 activator protein is associated with the AB variant form of GM2 gangliosidosis.

The AB-variant form of GM2 gangliosidosis is an inherited lysosomal storage disease. Biochemical data have linked its cause to the lack of a functional GM2 activator protein (activator). In the present study we identify a mutation in the gene encoding the activator protein of an AB-variant patient. These data represent direct evidence that the disease in the patient described here is a result of mutations at the Activator gene locus. A T412----C transition was found in the homozygous form in cDNA and genomic DNA from the patient. This nucleotide change would result in the substitution of Cys138 by an Arg residue in the activator protein. Whereas the patient's fibroblasts produce apparently normal levels of activator mRNA, they lack a functional activator protein. Transfection of either a construct containing the normal activator cDNA, pAct1, or a cDNA construct containing the T----C transition caused COS-1 cells to transcribe high levels of activator mRNA. Lysates from cells transfected with pAct1 produced an elevated level of both pro- and mature forms of the activator protein, with an accompanying 11-fold enhancement in the ability of purified hexosaminidase A to hydrolyze GM2 ganglioside. However, lysates from cells transfected with the mutant cDNA construct contained only low levels of the pro-activator protein, which failed to enhance hexosaminidase A activity significantly above the endogenous level of mock transfected COS cells. We conclude that the T412----C transition in the GM2 Activator gene of the patient is responsible for the disease phenotype.     

Ganglioside GM3 sialidase activity in fibroblasts of normal individuals and of patients with sialidosis and mucolipidosis IV. Subcellular distribution and and some properties.

Sensitive assays for the determination of the ganglioside sialidase activity of fibroblast homogenates were established using ganglioside GM3, 3H-labelled in the sphingosine moiety, as a substrate. Ganglioside GM3 sialidase activity was greatly stimulated by the presence of the non-ionic detergent Triton X-100 and was further enhanced by salts such as NaCl; the optimal pH was 4.5. The subcellular localization of this activity was determined by fractionation using free-flow electrophoresis and found to be exclusively associated with the marker for the plasma membrane, but not with that for lysosomes. This Triton-stimulated ganglioside sialidase activity was selectively inhibited by preincubating intact cells in the presence of millimolar concentrations of Cu2+, suggesting that the activity resides on the external surface of the plasma membrane. In normal fibroblasts homogenates, ganglioside GM3 sialidase was also greatly stimulated by sodium cholate. In contrast to the Triton X-100-activated reaction, however, it was not diminished by prior incubation of intact cells in the presence of Cu2+. Only after cell lysis was Cu2+ inhibitory. the cholate-stimulated ganglioside sialidase activity thus paralleled the behaviour of the lysosomal 4-methylumbelliferyl-alpha-D-N-acetylneuraminic acid (4-MU-NeuAc) sialidase. In fibroblasts from sialidosis patients, the cholate-stimulated ganglioside GM3 sialidase activity, but not that of the Triton-activated enzyme, was profoundly diminished. In fibroblasts from patients with mucolipidosis IV (ML IV), both the Triton X-100- and the cholate-stimulated ganglioside GM3 sialidase activities were in the range of normal controls. The Triton-activated enzyme was associated with the plasma membrane in the same manner as in normal cells. Our findings suggest that, in human fibroblasts, there exist two sialidases that degrade ganglioside GM3: one on the external surface of the plasma membrane, and another that is localized in lysosomes and seems identical with the activity that acts on sialyloligosaccharides and 4-MU-NeuAc. As neither activity was found to be deficient in ML IV fibroblasts, our results argue against the hypothesis of a primary involvement of a ganglioside GM3 sialidase in the pathogenesis of ML IV.     

Accumulation of Lysosphingolipids in Tissues from Patients with GM1 and GM2 Gangliosidoses

By using a sensitive method, we assayed lysocompounds of gangliosides and asialogangliosides in tissues from four patients with GM2 gangliosidosis (one with Sandhoff disease and three with Tay-Sachs disease) and from three patients with GM1 gangliosidosis [one with infantile type (fetus), one with late-infantile, and one with adult type]. In the brain and spinal cord of all the patients except for an adult GM1 gangliosidosis patient, abnormal accumulation of the lipids was observed, though the concentration in the fetal tissue was low. In GM2 gangliosidosis, the amounts of lyso GM2 ganglioside accumulated in the brain were similar among the patient with Sandhoff disease and the patients with Tay-Sachs disease, whereas the concentration of asialo lyso GM2 ganglioside in the brain was higher in the former patient than in the latter patients. By comparing the sphingoid bases of neutral sphingolipids, gangliosides, and lysosphingolipids, it was suggested that lysosphingolipids in the diseased tissue are synthesized by sequential glycosylation from free sphingoid bases, but not by deacylation of the sphingolipids. Because lysosphingolipids are known to be cytotoxic, the abnormally accumulated lysophingolipids may well be the pathogenetic agent for the neuronal degeneration in gangliosidoses.     

Molecular forms of GM2-activator protein. A study on its biosynthesis in human skin fibroblasts

The biosynthesis and secretion of lysosomal GM2-activator was studied in fibroblasts from controls and patients of GM2 gangliosidosis metabolically labelled with [3H]-leucine. Immunoprecipitation was performed with affinity-purified antibodies to human kidney GM2-activator protein. Normal fibroblasts and fibroblasts of variant B and O of GM2 gangliosidosis secrete GM2-activator protein as a 24-kDa polypeptide, which is able to stimulate degradation of ganglioside GM2 by beta-hexosaminidase A in the in vitro assay. In the presence of 10mM NH4Cl the rate of secretion is twice as high as in normal fibroblasts. Intracellularly, GM2-activator protein is represented in these cell lines by polypeptides with apparent molecular masses ranging from 21 kDa-22.5 kDa. Under the same labelling conditions, in two cell lines of patients with variant AB of infantile GM2 gangliosidosis intracellularly only traces of GM2-activator were detectable, whereas significant amounts of polypeptides with molecular masses between 25 and 26.5 kDa could be precipitated from the media of these fibroblasts.     

Incorporation and metabolism of ganglioside GM2 in skin fibroblasts from normal and GM2 gangliosidosis subjects

Ganglioside GM2, 3H-labeled in the sphingoid base, was added to the culture medium of normal and GM2 gangliosidosis fibroblasts. Ganglioside was found to adsorb rapidly to the cell surface, most of it could however be removed by trypsination. The trypsin-resistant incorporation was about 10 nmol/mg cell protein, after 48 h. The rates of adsorption and incorporation depended strongly on the concentration of fetal calf serum in the medium, higher serum concentrations being inhibitory. After various incubation times, the lipids were extracted, separated by thin-layer chromatography and visualized by fluorography. In normal cells a variety of degradation products as well as sphingomyelin was found whereas in GM2 gangliosidosis cells, only trace amounts of such products (mainly GA2) were found. In contrast, the higher gangliosides GM1 and GD1a were formed in comparable amounts (2.2-3.6% of total radioactivity after 92 h) in normal and pathologic cell lines. Supplementation of cells from GM2 gangliosidosis, variant AB, with purified GM2-activator protein restored ganglioside GM2 degradation to almost normal rates but had no effect on its glycosylation to gangliosides GM1 and GD1a. From these results we conclude that the synthesis of higher gangliosides from incorporated GM2 can occur by direct glycosylation and not only via lysosomal degradation and resynthesis from [3H]sphinganine-containing degradation products. Preliminary studies with subcellular fractionation after various times of [3H]ganglioside incorporation indicated biphasic kinetics for the net transport of membrane-inserted ganglioside to lysosomes, compatible with the notion that a portion of the glycolipids can also escape from secondary lysosomes and migrate to Golgi compartment or cell surface.     

Complexing of glycolipids and their transfer between membranes by the activator protein for degradation of lysosomal ganglioside GM2

The lysosomal degradation of ganglioside GM2 by hexosaminidase A depends on the presence of the specific activator protein which mediates the interaction between micellar or membrane-bound ganglioside and water-soluble hydrolase. The mechanism and the glycolipid specificity of this activator were studied in more detail. 1. It could be shown with three different techniques (isoelectric focusing, centrifugation and electrophoresis) that the activator protein extracts glycolipid monomers from micelles or liposomes to give water-soluble complexes with a stoichiometry of 1 mol of glycolipid/mol of activator protein. Liposome-bound ganglioside GM2 is considerably more stable against extraction and degradation than micellar ganglioside. 2. In the absence of enzyme the activator acts in vitro as glycolipid transfer protein, transporting glycolipids from donor to acceptor membranes. 3. The activator protein is rather specific for ganglioside GM2. Other glycolipids (GM3 GM1, GD1a and GA2) form less stable complexes with the activator and are transferred at a slower rate (except for ganglioside GM1) than ganglioside GM2.     

Activating proteins for ganglioside GM2 degradation by beta-hexosaminidase isoenzymes in tissue extracts from different species

The existence of activator proteins that stimulate hydrolysis of ganglioside GM2 by beta-hexosaminidase was demonstrated in kidney extracts from four species (rat, mouse, cattle and pig). The extent to which these preparations, as well as their human counterpart, promote ganglioside GM2 catabolism by autologous and heterologous hexosaminidase isoenzymes was compared. It was found that these activators can replace each other functionally, although the animal activator proteins do not cross-react immunochemically with an antiserum against the human protein. All preparations examined catalysed the transfer of ganglioside GM2 between liposomal membranes, indicating that the animal activator proteins act by a mechanism similar to the human GM2 activator.     

Ganglioside depletion and EGF responses of human GM3 synthase-deficient fibroblasts

Recognition of important roles of gangliosides in normal and abnormal cell function has motivated pharmacological modification of cellular ganglioside content. However, constitutive depletion of gangliosides in untransformed human cells has not been reported. In this context, the recent identification of a kindred carrying a point mutation in the GM3 synthase [ST3Gal5, Siat9] gene (Simpson MA, Cross H, Proukakis C, Priestman DA, Neville DC, Reinkensmeier G, Wang H, Wiznitzer M, Gurtz K, Verganelaki A, Pryde A, Patton MA, Dwek RA, Butters TD, Platt FM, Crosby AH. 2004. Infantile-onset symptomatic epilepsy syndrome caused by a homozygous loss-of-function mutation of GM3 synthase. Nat Genet. 36:1225-1229) provided an opportunity to explore this possibility. We established primary cultures of skin fibroblasts of three patients homozygous for this autosomal recessive defect. They exhibited a 93% reduction in ganglioside content (0.8 +/- 0.2 nmol lipid-bound sialic acid per 10(7) cells versus 12.7 +/- 1.3 nmol per 10(7) normal fibroblasts). Importantly, this marked reduction was not compensated by the activation of an alternate pathway of ganglioside synthesis, as occurs in murine GM3 synthase knockout fibroblasts. Cell morphology appeared unaffected, but under stringent conditions EGF-induced proliferation and migration of the mutant fibroblasts were reduced by 80% and 60%, respectively. Probing potential explanations, we found that EGF binding (effective membrane EGF receptor (EGFR) number) was reduced by 52% (to 6.2 +/- 1.9 from 12.8 +/- 2.0 pmol/10(8) normal fibroblasts, P < 0.01), despite normal total EGFR protein. EGFR activation was likewise reduced as was EGF-induced Rho/Rac1 phosphorylation, which is associated with cell migration. We conclude that this GM3 synthase point mutation almost completely depletes human fibroblast cellular gangliosides, dampens membrane EGFR activation, and modulates related critical cell functions such as proliferation and migration. These cells offer a valuable model for the study of ganglioside modulation of cell function.     

GM2 ganglioside and pyramidal neuron dendritogenesis

GM2 ganglioside, although scarce in normal adult brain, is the predominant ganglioside accumulating in several types of lysosomal disorders, most notably Tay-Sachs disease. Pyramidal neurons of cerebral cortex in Tay-Sachs, as well as many other types of neuronal storage disorders, are known to exhibit a phenomenon believed unique to storage disorders: growth of ectopic dendrites. Recent studies have shown that a common metabolic abnormality shared by storage diseases with ectopic dendrite growth is the abnormal accumulation of GM2 ganglioside. The correlation between increased levels of GM2 and the presence of ectopic dendrites has been found in both ganglioside and nonganglioside storage disorders, the latter including sphingomyelin-cholesterol lipidosis, mucopolysaccharidosis, and -mannosidosis. Quantitative HPTLC analysis has shown that increases in GM2 occur in proportion to the incidence of ectopic dendrite growth, whereas, other gangliosides, including GM1, lack similar increases. Immunocytochemical studies of all nonganglioside storage diseases which exhibit ectopic dendritogenesis have revealed heightened GM2 ganglioside-immunoreactivity in the cortical pyramidal cell population, whereas neurons in normal adult brain exhibit little or no staining for this ganglioside. Further, studies examining disease development have consistently shown that accumulation of GM2 gangliosideprecedes growth of ectopic dendrites, indicating that it is not simply occurring secondary to new membrane production. These findings have prompted an examination for a similar relationship between GM2 ganglioside and dendritogenesis in cortical neurons of normal developing brain. Results show that GM2 ganglioside-immunoreactivity is consistently elevated in immature neurons during the period when they are undergoing active dendritic initiation, but this staining diminishes dramatically as the dendritic tress of these cells mature. Collectively, these studies on diseased and normal brain offer compelling evidence that GM2 ganglioside plays a pivotal role in the regulation of dendritogenesis in cortical pyramidal neurons.Special issue dedicated to Dr. Leon S. Wolfe.     

An alpha-subunit loop structure is required for GM2 activator protein binding by beta-hexosaminidase A

The alpha- and/or beta-subunits of human beta-hexosaminidase A (alphabeta) and B (betabeta) are approximately 60% identical. In vivo only beta-hexosaminidase A can utilize GM2 ganglioside as a substrate, but requires the GM2 activator protein to bind GM2 ganglioside and then interact with the enzyme, placing the terminal GalNAc residue in the active site of the alpha-subunit. A model for this interaction suggests that two loop structures, present only in the alpha-subunit, may be critical to this binding. Three amino acids in one of these loops are not encoded in the HEXB gene, while four from the other are removed posttranslationally from the pro-beta-subunit. Natural substrate assays with forms of hexosaminidase A containing mutant alpha-subunits demonstrate that only the site that is removed from the beta-subunit during its maturation is critical for the interaction. Our data suggest an unexpected biological role for such proteolytic processing events.     

Purification, biochemical and immunological characterisation of hexosaminidase A from variant AB of infantile GM2 gangliosidosis.

Variant AB of infantile GM2 gangliosidosis is a fatal disease leading invariably to death within the first few years of life, due to the excessive storage of the glycolipids GM2 and GA2 which occurs in the nervous tissue of the patient. Unlike other variants of this hereditary disease, where a deficiency of hexosaminidase A, the ganglioside-GM2-degrading enzyme, could be demonstrated, the variant AB is characterized by a normal or even elevated level of this enzyme. To examine the possibility of a mutant hexosaminidase A, well capable of hydrolyzing the fluorogenic synthetic substrates but unable to attack the ganglioside, the enzyme was isolated from a patients tissue and characterized biochemically and immunologically in comparison with an enzyme preparation from normal control tissue. No differences between hexosaminidase A from normal and variant AB tissue could be detected indicating that the defect involved in this disease is not at the genetic level of production of either alpha or beta chains of hexosaminidase A.     

Alteration of ganglioside synthesis by GM3 synthase knockout in murine embryonic fibroblasts

To probe the functions of membrane gangliosides, the availability of ganglioside-depleted cells would be a valuable resource. To attempt to identify a useful genetic model of ganglioside depletion, we assessed ganglioside metabolism in murine GM3 synthase (GM3S)-/- knockout primary embryonic fibroblasts (MEF), because normal fibroblast gangliosides (GM3, GM2, GM1, and GD1a), all downstream products of GM3S, should be absent. We found that heterozygote MEF (GM3S+/-) did have a 36% reduced content of qualitatively normal gangliosides (7.0+/-0.8 nmol LBSA/mg cell protein; control: 11+/-1.6 nmol). However, two unexpected findings characterized the homozygous (GM3-/-) MEF. Despite complete knockout of GM3S, (i) GM3-/- MEF retained substantial ganglioside content (21% of normal or 2.3+/-1.1 nmol) and (ii) these gangliosides were entirely different from those of wild type MEF by HPTLC. Mass spectrometry identified them as GM1b, GalNAc-GM1b, and GD1alpha, containing both N-acetyl and N-glycolylneuraminic acid and diverse ceramide structures. All are products of the 0 pathway of ganglioside synthesis, not normally expressed in fibroblasts. The results suggest that complete, but not partial, inhibition of GM3 synthesis results in robust activation of an alternate pathway that may compensate for the complete absence of the products of GM3S.     

Characterization of unusual hexosaminidase A (HEX A) deficient human mutants.

Two families with unusual hexosaminidase A (HEX A) mutations are described. In one, the proband had the Tay-Sachs disease phenotype with considerable HEX A activity. In the second, the proband was phenotypically normal with absent HEX A activity. Activities using ganglioside GM2 as substrate demonstrate markedly reduced activities in the first case and half-normal activities in the second. Pedigree analyses indicate the presence of two different mutations. In the first, the proband appears to be an allelic compound HEX A 2-4 where mutation HEX A 4 leads to a diminution of HEX A activity against GM2 but not for the synthetic substrate, 4MU-beta-D-N-acetyl-glucosaminide, with HEX A 2 being the Tay-Sachs disease (or similar) mutation. In the second family, the proband is an allelic compound HEX A 2-5 where mutation HEX A 5 leads to a diminution of HEX A activity against the synthetic substrate, 4MU-beta-D-N-acetyl-glucosaminide, but not for GM2. The presence of either mutation will lead to false-negative (HEX A 4) or false-positive (HEX A 5) assignments of heterozygosity or homozygosity for GM2 gangliosidosis when synthetic substrates are employed. In both families, DM2 N-acetyl-beta-D-galactosaminidase activity in fibroblasts was an accurate determinant of phenotype.     

The human GM2 activator protein. A substrate specific cofactor of beta-hexosaminidase A.

Ganglioside GD1a-GalNAc was isolated from Tay-Sachs brain, tritium-labeled in its sphingosine moiety, and its enzymic degradation studied in vitro and in cultured fibroblasts. When offered as micelles, GD1a-GalNAc was almost not hydrolyzed by Hex A or Hex B, while after incorporation of the ganglioside into the outer leaflet of liposomes, the terminal GalNAc residue was rapidly split off by Hex a. In striking contrast to ganglioside GM2, the major glycolipid substrate of Hex A, the enzymic hydrolysis of GD1a-GalNAc was not promoted by the GM2 activator protein, although the activator protein did bind GD1a-GalNAc to form a water-soluble complex. Pathobiochemical studies corroborate these results. After incorporation of [3H]GD1a-GalNAc into cultured skin fibroblasts from healthy subjects and from patients with different variants of GM2 gangliosidosis, its degradation was found to be strongly attenuated in mutant cells with Hex A deficiencies such as variant B (Tay-Sachs disease), variant B1 and variant 0 (Sandhoff disease), while in cells with variant AB (GM2 activator deficiency), its catabolism was blocked only at the level of GM2. In line with these metabolic studies, a normal content of GD1a-GalNAc was found in brains of patients who had succumbed to variant AB of GM2 gangliosidosis whereas in brains from variants B, B1, and 0, its concentration was considerably elevated (up to 19-fold). Together with studies on the enzymic degradation of GM2 derivatives with modifications in the ceramide portion, these results indicate that mainly steric hindrance by adjacent lipid molecules impedes the access of Hex A to membrane-bound GM2 (whose degradation therefore depends on solubilization by the GM2 activator) and in addition that the interaction between the GM2. GM2 activator complex and the enzyme must be highly specific.     

Sialidase activities of cultured human fibroblasts and the metabolism of GM3 ganglioside

Free sialic acid has been found in the cell-conditioned medium of human foreskin fibroblasts. It is proposed that the accumulation of extracellular sialic acid may result from the hydrolysis of GM3 ganglioside on the cell surface of these fibroblasts. Sialidase activities with GM3 ganglioside and sialyllactitol as substrates were demonstrated in cell-conditioned medium, and the levels of their activities correlated positively with cell density. The GM3 sialidase activity at pH 4.5 was 4.1 and 38 pmol/h/ml of medium at sparse and confluent densities, respectively; the corresponding activities with sialyllactitol as the substrate were 12 and 75 pmol/h/ml of medium (pH 4.5). The pH versus activity profiles with GM3 as the substrate suggested the presence of a second sialidase with an optimal activity at pH 6.5 in the conditioned medium of preconfluent cells. This activity was virtually absent in the medium of contact-inhibited cells and could not be assayed with sialyllactitol as the substrate. The turnover of cell surface GM3 was assessed by pulse labeling human foreskin fibroblasts with a radioactive precursor of sialic acid ([1-14C]N-acetylmannosamine) and a radioactive precursor of ceramide ([3,3-3H2]serine). During a chase period of 24 h turnover of the doubly labeled cellular GM3 was observed; there was a loss of about 35% of the 14C-labeled sialic acid without any measureable loss of 3H-labeled ceramide from GM3. We have speculated that the enzyme-catalyzed removal of sialic acid from the GM3 ganglioside on the extracellular aspect of the plasma membrane may be a necessary event involved in the modulation of cell growth.