Interactions of pig brain cytosolic sialidase with gangliosides. Formation of catalytically inactive enzyme-ganglioside complexes

82

Biochimica et Bioph_wicaActu 833 (1985) 82-92 Elsevier

BBA 51818

Interactions of pig brain cytosolic sialidase with gangliosides. Formation of cat~yticalIy inactive enzyme-ganglioside

complexes

Bruno Venerando, Amelia Fiorilli, Massimo Masserini, Attilia Giuliani and Guido Tettamanti * Study Center for the Funciional Biochemistry oj Brain Lipids, Department of Biological Chemistry, The Medical School, Universi[y of Milan, Milan (Italy) (Received

Key words:

Sialidase;

Ganglioside;

July 5th, 1984)

Enzyme kinetics;

Lipid-protein

interaction;

(Pig brain cytosol)

Cytosolic siaiidase A was extracted from pig brain and purified about 2000-fold with respect to the starting homogenate (about SO-fold relative to the cytosolic fraction). The enzyme preparation provided a single peak on Uhrogel AcA-34 column chromatography and had an apparent molecular weight of 4 * 104. On incubation with micellar ganglioside GTlb, (molecular weight of the micelle, 3.5 105) under the conditions used for the enzyme assay, brain cytosoiic siahdase A formed two ganglioside-enzyme complexes, I and II, which were isolated and characterized. Complex II had a molecular weight of 4.2 lo’, and a gangIioside/ protein ratio (w/w) of 4 : 1. This is consistent with a stoichiometric combination of one ganglioside miceile and two enzyme molecules. Complex I was probably a dimer of complex II. In both complexes I and II cytosolic sialidase was completely inactive. Inactivation of cytosolic sialidase by formation of the corresponding complexes was also obtained with gangliosides Gnu and GDn,, which, like GTu,, are potential substrates for the enzyme and GM,, which is resistant to the enzyme action. Therefore, the enzyme becomes inactive after ~teracting with g~glioside micelles. G rn,-sialidase complexes acted as excellent substrates for free cytosolic sialidase, as did the complexes with Gol, and Go,,. l

l

Introduction Soluble extracts from the brain of different animals display sialidase activity [1,2]. The source of brain soluble sialidase, from the cytosol or from intracellular organelles and membranous structures which can release intraterminal or loosely * To whom correspondence should be addressed at Dipartimento di Chimica e Biochimica Medica. via Saldini 50, 20133, Milano, Italy. Abbreviations: the paper follows the ganglioside nomenclature of Svennerholm [31j and the IUPAC-IUB recommendations I13ruNeuAc-GgOse,-Cer;GD1=. II’aNeuAc, 1321. G,,, IV3aNeuAc-GgOsed-Cer; Go,,, 113(aNeuAc)2-GgOse,-Cer; G T,h, I13(aNeuAc),,IV”aNeuAc-GgOse,-Cer. 0005-2760/85/$03.30

0 1985 Elsevier Science Publishers

B.V.

bound proteins upon tissue homogenization, has been investigated. Experiments carried out in the brain of adult pig and developing rat [3-51 ruled out any release of the enzyme from the lysosomes or from the membranes which contain a tightly bound sialidase [6,7] and indicated a behaviour for the soluble enzyme parallel to that of authentic cytosolic components. The soluble, possibly ‘cytosolic’, sialidase from pig brain was also shown to occur in two forms, A and B, which were purified and characterized in their action toward a soluble substrate of low molecular weight, namely 3’sialosyllactose [2f. The functional relationships between brain cytosolic sialidase and the sialosylglycoconjugates occurring in brain - gangliosides

83

and sialosylglycoproteins - are still to be defined. The present work was undertaken with the aim of studying the interactions of brain cytosolic sialidase(s) with gangliosides and the mode of the enzyme(s) action on the same substances. Preliminary experiments showed the extreme complexity of the enzyme(s)-ganglioside(s) interactions. On this basis, and in order to simplify the approach, the cytosolic sialidase A (which could be obtained from pig brain with the highest yield and purity) and trisialogangliosde G-r,,,, toward which the highest enzyme activity was recorded, were employed in the study. In order to better appreciate the importance of the amphiphilic properties of ganglioside in the interactions with the enzyme, the lipid-free carbohydrate component of GTlb (which is freely soluble in water solution) was also used as enzyme substrate. Materials and Methods Chemicals and other products Commercial chemicals were of the highest available grade. Solvents were redistilled before use. N-Acetylneuraminic acid (NeuAc) and crystalline bovine serum albumin were purchased from Sigma Chem. Co. (St. Louis, MO, U.S.A.); Affi-Gel Blue (SO-100 mesh), Bio-Gel A, Bio-Gel HTP (hydroxylapatite), Cellex N-l (highly purified cellulose powder) and Dowex 2-X8 resin (200-400 mesh), prepared in acetate form according to Svennerholm [8], were from Bio-Rad Laboratories (Richmond, VA, U.S.A.); 4-methylumbelliferyl-cu-D_Nacetylneuraminic acid (MUB-NeuAc) and 4-methylumbelliferone (MUB) from Koch-Light (Colnbrook, U.K.); ultrafiltration apparatus, Mod. 52, and Diaflo membranes UM 10 from Amicon GmbH (Witten, F.R.G.); Ultrogel AcA-34 and AcA-44 from LKB (Stockholm, Sweden); dialysis tubing from A. Thomas (Philadelphia, PA, U.S.A.); preblended liquid scintillation solution, Instagel. from Packard (Downers Grove, IL, U.S.A.). Water was doubly distilled in a glass apparatus and used to prepare the different solutions. Brains of adult pigs were obtained at the slaughter-house immediately after death and kept on ice until processed (generally 30-40 min). Meninges were removed and the gray matter, grossly dissected, was washed in ice-cold homogenizing

solution

and homogenized

as previously

described

]21. Preparation of substrates Sialosyllactose (isomer C-3, or 3’-sialosyllactose) was isolated from l-day-postpartum cow colostrum according to the method of Ohman and Hygstedt [9]. Gangliosides G,,,, G,,,,, G,,, and G , extracted and purified from pig brain by the mzhod of Tettamanti et al. [lo], were structurally analyzed and identified as described by Ghidoni et al. [ll]. Their purity was over 99%. The long-chain base composition (as mol’%) of GTlb ganglioside was as follows: C,, sphingosine, 30.5%; C,, sphinganine, 3.1%; C,, sphingosine, 62.5%; C,, sphinganine, 3.9%. The predominant fatty acid (95%) was stearic acid. The average molecular weight of GTlh micelle, determined by laser light scattering technique [12], was 350000 _t 35000, Ganglioside G nla was ‘H-labelled at the C-3 position of the long-chain base using the method of Ghidoni et al. [13]. The radiochemical purity was over 99% and the specific radioactivity 0.96 Ci/mmol. The radioactive compound was stored at 0-4°C in n-propanol/water, 1 : 1, by vol. The lipid-free carbohydrate component of ganglioside Gr,,, (G.,,-carbohydrate) was obtained after ozonolysis and alkaline treatment of the ganglioside [14]. The product was purified from the reaction mixture and chemically characterized as described by Venerando et al. [15]. Its purity was over 95%. Preparation of brain cytosolic sialidase A All work was done at 4°C unless otherwise stated. Cytosolic sialidase A was extracted and purified from pig brain gray matter, following an improved version of the procedure described by Venerando et al. [2]. After the first three steps of the original procedure (ammonium sulphate fractionation; chromatography on Bio-Gel A-5m column; chromatography on hydroxylapatite/ celh$ose gel column), the material containing the enzyme was dialyzed overnight against 100 vol. of 10 mM potassium phosphate buffer (pH 6.8) changed twice. After addition of solid CaCl, till 0.5% (final concentration), the mixture was rapidly heated (using a boiling-water bath) till 52°C kept at this temperature for 3 min, and then cooled in

84

an ice bath. The suspension was centrifuged at 105 000 X g for 30 min and the supernatant submitted to dialysis for 8 h against 50 vol. of 10 mM potassium phosphate buffer (pH 6.8) changed three times. The dialyzed mixture was poured on the top of an Affi-Gel Blue column (3 ml of sedimented gel/l0 mg protein) previously equilibrated with the above buffer. After washing with 5 column volumes of the same buffer, protein elution was accomplished with a continuous gradient of NaCl in 10 mM potassium phosphate buffer (pH 6.8) 4-ml fractions were automatically collected and protein elution was monitored by flow-through recording of ultraviolet absorbance at 280 nm. Sialidase activity was present only on the protein peak eluted with 0.7 M NaCl. The fractions carrying sialidase activity were pooled, dialyzed for 12-14 h against 100 vol. of the above buffer, changed three times, and concentrated to a small volume (10 mg protein/ml) by an Amicon ultrafiltration apparatus (Diablo membranes, UM-IO). The concentrated solution was stored at -2O’C until use. The specific activity of the final preparation of sialidase, assayed on 3’-sialosyllactose, was 7.4 nmol liberated NeuAc/min per mg protein, corresponding to about 2000-fold purification with respect to the starting homogenate (about 550-fold relative to the cytosolic fraction). The specific activity of the enzyme was 34-fold higher than that obtained with the original purification procedure [2]. The enzyme displayed a single symmetrical peak on Ultrogel AcA-34 column, and the apparent molecular weight, determined as described below, was 4. 104. Formation and separation of non-catalytic GTlbsialidase complexes G Tlb was dissolved in chloroform/meth~ol,

2: 1 by vol. A known portion of the mixture, containing the required amount of ganglioside, was transferred into a glass tube, added with 0,15 PCi of [3H]GDla as tracer, and the solvent was completely removed by a gentle flow on N,. The residue, kept under vacuum over P,O, until processed, was dissolved with 0.6 ml of redistilled water and the solution was allowed to stand overnight at room temperature. The ganglioside was thus present in the form of stable micelles [16]. Then sodium acetate/acetic acid buffer (pH 4.8)

(the same buffer used for sialidase assay), final concentration 0.1 M, and known amounts of purified sialidase were added; the volume was brought to 1.2 ml, and the mixture incubated at 37°C for an established time. After stopping the reaction by immersion in an ice bath, a 0.2 ml aliquot was analyzed for released sialic acid (see sialidase assay), 0.1-0.15 ml used for measurement of radioactivity, and 0.85 ml submitted to chromato~aphy on an Ultrogel AcA-34 column (2 X 45 cm) previously equilibrated with 0.1 M sodium acetate/acetic acid buffer (pH 4.8). The following conditions were employed: temperature, 4’C; eluting solvent, 0.1 M sodium acetate/acetic acid buffer (pH 4.8); flow rate, 0.9 ml/mm; 1.5~ml fractions automatically collected; elution monitoring by measurement of protein and radioactivity. The eluted fractions which constituted each of the two peaks containing both protein and radioactivity (G,,,-sialidase complexes I and II, see Fig. 1) were pooled, concentrated to a small volume (about 1 mg gan~ioside-bound Ne~c/ml) by ultrafiltration on an Amicon apparatus with Diaflo membrane UM-10, and stored at 4’C until further processing or analysis. Generally storage did not exceed 8 h. Sialidase assay (a) Calorimetric method. The incubation

mixtures contained 5-120 pg (as protein) of purified sialidase, given amounts of substrate (3’-sialyllactose; ganglioside Gri,,; Gary-carbohydrate; G,,,sialidase complex I or II, see below); 0.1 M sodium acetate/acetic acid buffer (pH 4.8) (which was optimal; see the Results section), in a final volume of 0.2 ml. When using Gri,, a known portion of a chlorofo~/methanol, (2 : 1, by vol.) solution of the ganglioside, containing the required amount of compound, was transferred into the incubation tube and evaporated to dryness. The residue was dissolved with 0.1 ml of redistilled water, allowed to stand overnight at room temperature (in order to have stable micelles) and then the incubation mixture was completed. All the incubation rnixtures, which were started by addition of the enzyme, were placed in an incubator shaker and incubated at 37°C for an established time (see Results). The enzyme reaction was stopped by

85

immersing the tubes in an acetone/solid CO, bath. The liberated NeuAc was determined by the chromatographic microprocedure of Caimi et al. [17]. The yield by this procedure, checked each time with pure standard NeuAc, was higher than 92% and reproducibility was good (+ 2%) in the range of 3-50 nmol of NeuAc. An absorbance of 0.100 (as corrected absorbance, see Warren [18]) corresponded to 14-14.5 nmol of free NeuAc. The control incubation mixtures (blanks) were complete incubation mixtures but lacked the enzyme. The optimum buffer and pH for the enzyme action was assessed using the following buffer systems at different molarity and pH values: sodium acetate/acetic acid, sodium citrate/citric sodium phosphate/ citric acid, sodium acid, maleate/Tris. (b) Fluorimetric method. Incubation mixtures containing, in a final volume of 0.1 ml, 5-80 pg (as protein) of purified sialidase, 0.1 M sodium acetate/acetic acid buffer (pH 4.8) (optimum value), 400 pg of bovine serum albumin, given amounts (see below) of MUB-NeuAc, were incubated at 37’C for an established time. The enzyme reaction was stopped by addition of 1.8 ml of 0.5 M glycine/NaOH buffer (pH 10.8) and fluorescence was immediately measured with a Perkin-Elmer Mod 2000 fluorimeter (excitation wavelength, 365 nm; emission wavelength, 445 nm). A fluorescence of 0.1 corresponded to 0.35 nmol liberated MUB. The control incubation mixtures (blanks) were complete incubation mixtures lacking the enzyme. (c) Expression of sialidase activity. The activity of sialidase was expressed in International Units (I.U.), pmol liberated NeuAc, or MUB, per min at 37°C. K, and V,,, values were determined by the method of Lineweaver and Burk [19]. Identification of the glycolipid products of G,, enzymatic hydrolysis Incubation mixtures containing G,,,-sialidase complexes prepared as specified above in the volume of 1 ml were incubated at 37°C for an established time, then cooled and dialyzed at 4°C for 12-15 h against 200 vol. of bidistilled water, changed three to four times. Lipids were extracted from the dialyzed mixture by the tetrahydrofuran method of Tettamanti et al. [lo] and gangliosides

were extracted by partitioning following the same procedure [lo]. The final aqueous phase was dialyzed at 4°C for 2 days against 200 vol. of redistilled water changed three to four times and then lyophilized. The gangliosides contained in the residue were separated by TLC or column chromatography and submitted to chemical analysis. TLC was accomplished under the following conditions: HPTLC plates, heated at 120°C for 2 h solvent system, chloroform/ prior to use; methanol/0.2% aqueous CaCl, 50 : 42 : 11, by vol.; temperature, 18-2O’C; run time, 1.5 h; detection of the spots by treating with an Ehrlich spray reagent and heating at 120°C for 15 min [ll]; identification of the spots by co-chromatography with pure standard gangliosides. Column chromatographic separation of gangliosides and chemical analysis of the separated gangliosides were performed according to Ghidoni et al. [ll]. Molecular weight determination The molecular weight of purified cytosolic sialidase and of the G,,,-sialidase complexes was estimated by the gel filtration method suggested by Andrews [20]. To this purpose a column of Ultrogel AcA-44 or Ultrogel AcA-34 (1.8 x 50 cm) [21] was equilibrated with 0.1 M sodium acetate/ acetic acid buffer (pH 6.8) and developed with the same buffer at a flow rate of 54 ml per h (V, and V,: 52.5 and 166.5 ml for AcA-44 column; 69.5 and 178 ml for AcA-34 column, respectively). The following standard proteins (from Sigma St. Louis, MO, U.S.A.) were used for calibration: cytochrome c, myoglobin, chymotrypsinogen A, ovalbumin, cu-amylase, bovine serum albumin (monomer and dimeric forms), transferrin, alcohol dehydrogenase, catalase, fibrinogen. The partition coefficient, K,,, was calculated using the equation of Laurent and Killander [22]. Other analytical methods Ganglioside-bound NeuAc was determined by the method of Svennerholm [8]. Protein content was determined by the method of Lowry et al [23] with bovine serum albumin as standard. Results The cytosolic sialidase A obtained from pig brain at the final stage of purification was able to

86

release NeuAc from 3’-sialosyflactose, confirming previous evidence [2], MUB-NeuAc, G.,.,,-carbohydrate and gangliosides GTlb, GDlh and GDla. G M1 was resistant to the enzyme action. Among the gangliosidic substrates the highest enzyme activity was recorded on GT,,,. In all cases the buffer providing the highest enzyme activity was 0.1 M sodium acetate/acetic acid (pH 4.8). The activity of cytosolic sialidase A on MUB-NeuAc was markedly enhanced by the presence of bovine serum albumin, this effect being optimal at a bovine serum albumin concentration of 4 mg/ml in the incubation mixture. Patterns of cytosofic sialidase activip on ~~~NeuAc, Gary-~arbohydrute and micellar G,, The main features of the activity of cytosolic sialidase on MUB-NeuAc and on GTlbcarbohydrate are shown in Fig. 1. In both cases the V/(substrate) relationships obeyed regular hyperbolic kinetics, and the enzyme activity was linear with the amount of enzyme present from 5 to at least 100 pg, as protein. The enzyme activity was linear with incubation time for 40-45 (GTlbcarbohydrate) and 60-70 min (MUB-NeuAc). The activity on these sialosyl compounds closely resembled that already described for the same enzyme, at a lower degree of purification, on 3’sialosyllactose [2]. As shown in Fig. 2 the kinetics of cytosolic sialidase A action on micellar GTlht studied by conventional experimental approaches, appeared quite anomalous. The V/(substrate) and the V/ enzyme protein relationships were biphasic and sigmoidal shaped in the first phase of the curve. In particular, the transition points between the first and second phase were strictly dependent upon the enzyme/ganglioside ratio. The enzyme activity toward GTlh was linear with time for 40-45 min. The profile of sialidase activity on GDla and G Dlh was quite similar. Formation and separation of non-catalytic G,,sialidase complexes A series of experiments, performed with the aim of clarifying the anomalous behaviour of cytosolic sialidase kinetics on micellar G,,,, suggested, as a possible explanation, the formation of non-catalytic GTlb- sialidase complexes during incubation.

INCUBATION

TIME

HOURS

Fig. 1. Kinetics of pig brain cytosolic sialidase action on low molecular weight substrates. See Materials and Methods for assay conditions. A, G T,b-carbohydrate as substrate; 0, MUB-NeuAc as substrate. Substrate molarity is expressed as bound NeuAc.

Should this be verified, the conventional approach to V/(substrate) or V/enzyme studies would be invalidated owing to either enzyme or substrate subtraction from catalysis, or to constitution of new forms of substrate. Therefore the formation of such complexes, under the conditions used for the enzyme assay, became the object of accurate investigations. As shown in Fig. 3, when a mixture of cytosolic sialidase and G,,, in 0.1 M sodium acetate/acetic acid buffer (pH 4.8) (the same buffer used for sialidase assay) at zero time of incubation was submitted to column chromatography on Ultrogel AcA-34, the enzyme protein was eluted as an individual, retarded peak, which

87

MICELLAR GANGLIOSIOE

A

FREE ENZYME

GTlb.mM

ELUTION

ENZYME

INCUBATION

PROTEIN,

TIME,

~9

VOLlJME.ml

Fig. 3. Formation of G,,,- sialidase complexes under the conditions used for enzyme assay. Column chromatography on Ultrogel AcA-34 was carried out as described in Materials and Methods. Enzyme, 0.25 mg/ml; GTlb (with the addition of 0.15 PCi of 13H]G,,,,), 0.1 mM: incubation time, 30 min. The elution of ganglioside was monitored by measurement of radioactivity, that of protein by flow through reading of ultraviolet absorbance at 280 nm. Sialidase activity was assayed by the fluorimetric method. Similar results were obtained with ganglioside G,,,. Go,, and G,,.

HOURS

Fig. 2. Apparent kinetics of pig brain cytosolic sialidase action on ganglioside G T1b. The enzyme was directly incubated in the presence of GTlb in micellar form. V/substrate relationships were studied with different enzyme amounts in the incubation mixture; V/enzyme-protein relationships were studied at different ganglioside concentrations. The time-course of enzymatic hydrolysis of GTlb was studied using 25 pg of enzyme and 0.1 mM ganglioside. GTlb molarity is expressed as bound NeuAc. For details see Materials and Methods. Similar results were obtained with gangliosides G,,, and GDlb. Top panel, cytosolic sialidase; 0. 25 pg, * , 50 pg; A, 80 pg. Centre panel, G,t,; 0, 0.1 mM; *, 0.2 mM; A, 0.5 mM. Bottom panel, enzyme protein (pg).

was nicely separated from that of gangliosidebound radioactivity, corresponding to mixed micelles of GTn, and tracer [3H]GDla [16]. The peak of enzyme activity, assayed on MUB-NeuAc, closely overlapped that of protein, indicating that the enzyme maintained full activity during chromatography. After 30 min of incubation at 37°C followed by column chromatography, three protein peaks appeared. The most retarded of them had the same elution volume as the enzyme protein peak at zero time, although much smaller than that. It also contained sialidase activity which was

lower than that recorded in the enzyme protein peak at zero time. Therefore, it likely constituted the ‘free’ form of the enzyme which remained after incubation. The other two protein peaks, designated I and II, were eluted closer to the void volume of the column, indicating the presence of material of increased molecular weight. Both peaks I and II contained ganglioside, as shown by the occurrence of bound radioactivity. On the other hand, the peak of micellar ganglioside almost disappeared, only a small shoulder of it being visible on the descending line of peak II. Therefore, peak I and II, which contained both protein and ganglioside, likely corresponded to two G ,,,-sialidase complexes, designated complex I and complex II, formed during incubation. It is noteworthy that both complexes did not contain sialidase activity on MUB-NeuAc. The observed behaviour was quite reproducible, provided that new columns were used for each experiment. This renewal was necessary, owing to the behaviour of radioactivity on the column. In fact, only 85-90% of loaded radioactivity could be recovered after elution and the remaining radioac-

88

tivity stuck to the column. This portion of radioactivity probably corresponded to monomeric gangliosides, which are known to firmly adhere to the gel materials [24]. The formation of G,,,-sialidase complexes was a process dependent upon incubation time (Fig. 4) and the starting ratio between enzyme protein and ganglioside (Fig. 5). Using a fixed enzyme/G,,, ratio (0.25 mg/ml of enzyme protein and 0.1 mM G rib in the experiments reported in Fig. 4), complex II tended to be formed first and complex I with prolonging incubation, suggesting possible conversion of complex II to complex I with time. The effect of the proportions -between enzyme protein and ganglioside on the formation of complexes I and II was studied at a fixed incubation time (30 mm), using enzyme protein ranging from from 0.05 to 1 mM. A 5 to 120 pg and G,, selection of the results obtained, which better illustrate the phenomenon, is shown in Fig. 5. In the condition characterized by the ratio most favorable to enzyme protein (0.80 mg/ml enzyme protein, 0.1 mM GTn,, Fig. 5c) the free form of both enzyme and GTlb was preponderant and the bound

COHPLEXES HICELLAR

FREE

-0.5 -0.3

62)

2-

- 0.5 -0.4

l-

COMPLEXES I II

!

-0.3 0.

MICELLAR GANGLIOSIOE

0 iREE ENZYME

I

-0.1 -0

a0

O5

ELUTION VOLUME,ml

c I 2-

4

,

I

~05 -04 03

1.

Fig. 5. Effect of the starting proportions between cytosoIic sialidase and G,,, on the formation of G,,,-sialidase complexes I and II. Same conditions as described in the legend to Fig. 3. Incubation time, 30 min. a. 0.1 mM GTlb and 0.25 mg /ml enzyme. b, 0.1 mM G,,, and 0.50 mg/ml enzyme. c, 0.1 mM GTI~ and 0.80 mg/mI enzyme. d, 0.5 mM G,,, and 0.25 mg/ml enzyme. e, 0.5 mM G,,,. 0.50 mg/ml enzyme. f, 0.5 mM GTlb and 0.80 mg/ml enzyme.

-01 10

so ELUTION

100

150

VOL~ME,m~

Fig. 4. Effect of storage at 37*C on the formation of GTILsialidase complexes I and II. Same conditions as described in the legend to Fig. 3. Storage time: A, 15 min; B, 45 min; C, 90 min.

form was present only as complex II. Under the same conditions, complex II became the preponderant form by increasing G,,, concentration from 0.1 to 0.5 mM (Fig. 5f). Conversely, the condition characterized by the lowest enzyme protein/Gri, ratio (0.25 mg/ml enzyme protein, 0.5

89

mM GTIbyFig. 5d) led to almost stoichiometric interaction of the enzyme protein with ganglioside in the form of complex I, with virtual disappearance of the free forms of both enzyme protein and ganglioside. Under this condition, only traces of complex II could be detected. At intermediate values of enzyme protien/ ganglioside ratio, intermediate patterns of free and complexed forms of enzyme protein and ganglioside were observed (Fig. 5a, b e). It is thus evident that both complex I and complex II could arise from direct interaction of the enzyme protein with ganglioside. The formation of one, or the other, complex mainly depended on the ratio between the two starting components. Similar ganglioside-sialidase complexes were formed using gangliosides G,,ia, GDlb and G,,. In the experiments performed for exploring the formation of Grit,-cytosolic sialidase complexes, the release of NeuAc from ganglioside was also measured. It was observed that in all the conditions yielding complete transformation of the enzyme protein into complexes no free NeuAc could be detected. Instead, in all the conditions where the free form of the enzyme was present, some release of NeuAc occurred. The extent of release depended on the amount of free enzyme and of the GTlb- sialidase complex present, especially of complex I. In no case did the enzymatic hydrolysis of Gr,, during incubation exceed 1.5% of releasable NeuAc. Isolation and partial characterization

of G,,-siali-

dase complexes

As shown in Fig. 5d, incubation (30 min at 37°C) of 0.25 mg/ml of cytosolic sialidase with 0.5 mM Grit, in 1.2 ml of 0.1 M sodium acetate/ acetic acid buffer (pH 4.8) led to almost complete transformation of the enzyme into complex I. Also, the condition employing 0.80 mg/ml of enzyme (Fig. 5f) was the most suitable for formation of complex II. These conditions were employed for preparative purposes. The eluted fractions corresponding to complexes I and II, respectively, were pooled and concentrated at 4°C to 1.5-2 ml by the ultrafiltration apparatus described in Materials and Methods. Each complex, when submitted to rechromatography under the same conditions, produced a peak which exactly corresponded to the

original one. Upon storage at 37°C complex II underwent partial transformation to complex I (8% after 1 h, 12% after 2 h, 14% after 3 h). At 4°C the transformation of complex II to complex I occurred at a much slower rate. Generation of free enzyme and free ganglioside from complexes I and II upon storage was never observed. Moreover, incubation of complex I and II with various amounts of G,,, (containing the same amounts of unlabelled GDla as [ 3H]GDla in the starting conditions) followed by chromatographic isolation of the complex did not cause any release of bound radioactivity. Addition of bovine serum albumin was not able to release radioactivity from the complexes. This indicates that both complexes are quite stable and that the reaction leading to formation of the complexes is practically irreversible. The content of ganglioside-bound NeuAc relative to that of protein, w/w, was 3.6 and 4.0 mg/mg protein for complex I and complex II, respectively. The apparent molecular weight of complex II, established by the gel filtration method, was 4.2. 105. That of complex I, which could not be accurately established, was in the range (g-10). 105. Complexes I and II, incubated at 37°C in 0.1 M sodium acetate/acetic acid buffer for up to 14 h, did not release NeuAc, indicating that the enzyme incorporated in the complex was not active on complex-bound G rib. Both complexes incubated under the same conditions in the presence of varying amounts of MUB-NeuAc, 3’-sialosyllactose, G r,,-carbohydrate and G,,, did not display any hydrolytic activity. All this is consistent with the statement that cytosolic sialidase, when involved in the formation of complex I and complex II, is completely inactive. Treatment of complex I and complex II with Triton X-100 (I’%, w/v, final concentration) for 60 min at 37°C was followed by substantial recovery of sialidase activity, measured on MUB-NeuAc. Identical results, concerning sialidase inactivation by complexation and recovery of activity by treatment with Triton X-100, were obtained also with Goi,, Gnu, and G,,. Action of cytosolic sialidase on complex plex II

I and com-

Incubation mixtures were set up containing purified cytosolic sialidase and the catalytically inactive G r,,,-sialidase complexes I and II. These

90

mixtures were submitted to incubation in order to assess whether the two complexes might serve as substrates for the added enzyme. As shown in Fig. 6, cytosolic sialidase affected the complexes and released sialic acid. The enzyme activity was linear with the enzyme protein up to 25 pg and 60 pg with complex II and complex I, respectively, and was linear with incubation time for up to 40 min in both cases. The V/(substrate) relationships obeyed regular hyperbolic kinetics with both complexes and displayed an early inhibition by excess substrate. The values of K, and V,,, of cytosolic sialidase with different substrates (complex I, complex II, G,,,-carbohydrate, 3’-sialosyllactose, and MUB-NeuAc) are reported in Table I. The highest value of V,,, was obtained with complex II, followed by complex I. A comparison of the enzyme kinetic parameters on the two complexes and on G,,,-carbohydrate shows that the affinity of the enzyme for Gri,, in the complexes is 25-30-fold higher than that recorded for the free oligosaccharide of G,,, and that the maximum velocity on complex-bound G,,, is 3-5-fold higher than that recorded on the free oligosaccharide. This means that complex-bound G,,, is a much better substrate for cytosolic sialidase than the low molecular weight, freely soluble, oligosaccharide portion of the same ganglioside.

TABLE

I

K, AND v,,, VALUES TION ON DIFFERENT Assay conditions

are as described

in Materials

Km 3’-Sialosyllactose MUB-NeuAc G,,,-carbohydrate G,,,-sialidase complex G r,,-sialidase complex

I II

AC-

and Methods.

(10m5 M)

Vma (mu per mg protein)

62.5 2.9 217 7.1 8.7

7.4 5.9 6.6 20 33.3

The data given in Fig. 7 show that upon incubation of complexes I and II in the presence of cytosolic sialidase, G,,, was transformed mainly into a ganglioside which co-chromatographed with G on,, and in much lower amounts in a ganglioside behaving as G,,. The main ganglioside enzymatitally produced from complex I and II was isolated from incubation mixtures, set up and processed for this purpose, and subjected to chemical analyses. The results obtained were consistent with the composition and structure of ganglioside GDlb.

GTlb

u

A

Fig. 6. Kinetics of pig brain cytosolic sialidase action on G,,,-sialidase complexes I and II. Grit,-sialidase complexes I and II were prepared and separated as described in the text and submitted to the action of free cytosolic sialidase. The V/ enzyme protein experiments were carried out at 25 gM GTlbsialidase complex I (A) and 50 /.tM Gr,,-sialidase complex II (0). Free enzyme, 25 pg. Substrate molarity is expressed as ganglioside-bound NeuAc.

OF CYTOSOLIC SIALIDASE SIALOSYL DERIVATES

B

am

C

D

E

F

G

Fig. 7. Thin-layer chromatography of the products of cytosoeic sialidase promoted hydrolysis of G,,,-sialidase complexes I and II. A, reference standard gangliosides; B, Grit,-sialidase complex I, blank incubation mixture after 1 h incubation; C, complex I, complete incubation mixture after 1 G r,,-sialidase h incubation (enzyme, 60 pg); D, same as C, co-chromatographed with standard authentic GDlb; E, Grit,-sialidase complex II, blank incubation mixture after 1 h incubation; F, complex II, complete incubation mixture after 1 G r,,-sialidase h incubation (enzyme, 30 pg); G, same as F, co-chromatographed with standard authentic Goit,. Temperature of incubation, 37°C.

91

Discussion Gangliosides are amp~p~lic substances which possess a high binding potential. This is based on the presence in their molecule of groups capable of sharing hydrogen bonds and of determining ionic, hydrophobic, dipole-dipole interactions. A peculiar feature of gangliosides is the ability to form micelles in water solution in a wide range of concentrations, from lo-‘* to lo-* M [l&25]. Owing to these properties the interactions of gangliosides with enzymes are very complex and the experimental approaches of ganglioside enzymology are different from the conventional ones [26]. Examples of the peculiarity of ganglioside enzymology are the requirement of protein activators for the enzymatic hydrolysis of some gangliosides 127,281 and the ability of some enzymes to recognize the supramolecular organization of the system into which substrate gangliosides are embedded [15,29]. The present investigation illustrates a new aspect of ganglioside-enzyme interactions, that is, the enzyme inactivation after binding to ganglioside micelles. The enzyme which was found to exhibit this feature is cytosolic sialidase A, purified from pig brain. We have observed that incubation of a purified preparation of cytosolic sialidase with G,t, (or other gangliosides) under the conditions used for enzyme assay was followed by formation of one or two ganglioside-protein complexes. These complexes, which were isolated and partially characterized, displayed no sialidase activity on any of the substrates used. Conditions were provided under which no protein of the enzyme preparation remained free after interaction with G,,, and, concurrently, all protein participated to form complexes with ganglioside. Therefore, the enzyme protein itself was involved in the formation of complexes, regardless of the degree of homogeneity of the enzyme preparation. Since aqueous solu-’ tions were used and ganghoside concentration was always over lo-’ M, GTlb was present and interacting as micelles. On the other hand, no such complex was formed with G,,,-carbohydrate, which does not micellize, nor was Grit,carbohydrate capable of inhibiting the enzyme. Moreover, cytosolic sialidase formed complexes

and underwent inactivation also with gangliosides G Dla and G DID,, which are potential substrates for the enzyme, and with G,,, which is resistant to the enzyme action. Thus, the complexes are a combination of the enzyme protein with ganglioside micelle and the combination results in complete enzyme inactivation. The average molecular weight of G,,, micelle was 3.5 . 105, that of the enzyme was 4 - lo4 and that of complex II, the smaller of the two GTn,sialidase complexes, about 4.2 *10’. The molecular weight of complex I was in the lo6 range. In addition, the ganglioside/protein ratios (w/w) were 4 : 1 and 3.6 : 1 for complex II and complex I, respectively. These data are consistent with the hypothesis that complex II resulted from association of one ganglioside micelle with two enzyme protein molecules, and complex I from association of two II complexes, in analogy with the complexes formed by bovine serum albumin and ganglioside G,, (241. The complexes were easily affected by free cytosolic sialidase with release of NeuAc, indicating that the carbohydrate portion of Grib was readly available to the enzyme and exposed on the surface. Also, isolated complexes did not originate or exchange the starting components upon prolonged storage or treatment with ganglioside micelles, indicating that the formation of the complexes was an apparently irreversible phenomenon and that the complexes were quite stable. However, treatment of complexes with the non-ionic detergent Triton X-100 resulted in reappearance of sialidase activity on MUB-NeuAc. These observations suggest that the enzyme protein penetrated into the ganglioside micelle and resided in the inner core of it, surrounded by ganglioside molecules. Thus, the ‘inactivity’ of complex-bound sialidase would be the consequence of non-accessibility of the enzyme to added substrate, in other words, of ‘crypticity’ of the enzyme. A quite similar behaviour has been observed for another soluble enzyme, cr-L-fucosidase from Octopus uulguris, acting on fucose-containing gangliosides (unpublished data), suggesting that micellar gangliosides might be capable of inactivating some enzymes in a potentially reversible way. Verification of these peculiar binding capacities of gangliosides with respect to enzymes, for which gangliosides are not substrates but that can

92

be connected with gangliosides in the physiological milieu, is in progress. A more general extension of the present evidence would imply that interaction with ganglioside micelles has dramatic effects on the structure-function properties of proteins. Very likely ganglioside micelles do not occur in cell fluids under physiological conditions. However, it cannot be excluded that, in particular sites of the plasma membrane, where gangliosides reside, they can occasionally arrange in micelle-like structures (clusters) [30] which interact with neighbouring proteins. The resulting modifications of the functional state of proteins (activation-inactivation, accessib~ty-c~ticity) might be of physiological importance. It is curious that GTlb- sialidase complexes acted as excellent substrates for cytosolic sialidase. In other words, pure micelles of Gri, were enzyme inactivators, while the complexes, which are lipoproteic micelles, were substrates. This explains why the kinetics of cytosolic sialidase action on micellar Grrb were anomalous. In fact, during incubation the enzyme gave origin to G,,,-enzyme complexes, which were inactive forms of the enzyme; concurrently, the remaining free form of enzyme could split NeuAc from the formed G,,,-enzyme complexes. Therefore, the release of NeuAc was not dependent on the amount of enzyme present nor on the starting ganglioside concentration but on the amount of ‘free’ enzyme and G,tb-enzyme complexes formed. Acknowledgements The present work was supported in part by (No. 83.0285.56) from the Consiglio Naz-

grants

ionale delle Ricercher, CNR, Rome, Italy. Pig brains were a generous gift of the E. Galbani S.p.A., Melzo, Milan, Italy. References 1 Carubelli, R. and Tulsiani, D.R.P. (1971) B&him. Biophys. Acta 231,78-87 2 Venerando, B., Tettamanti, G., Cestaro, B. and Zambotti, V. (1975) Biochim. Biophys. Acta 403,461-472 3 Venerando, B., Preti, A., Lombardo, A., Cestaro, B. and Tettamanti, G. (1978) Biochim. Biophys. Acta 527, 17-30 4 Venerando, B., Goi, G.C., Preti, A., Fiorilli, A., Lombardo, A. and Tettamanti, G. (1982) Neurochem. Int. 4, 313-320

5 Venerando, B., Fiorilli, A., Malesci, A., Goi, G.C., Lombardo, A., Preti, A. and Tettamanti, G. (1983) Neurothem. Int. 5, 619-624 6 Schengrund, CL. and Rosenberg, A. (1970) J. Biol. Chem. 545,6196-6200 7 Tettamanti, G., Morgan, LG., Gombos, G., Vincendon, G. and Mandel, P. (1972) Brain Res. 47, 515-518 8 Svennerhohn, L. (1957) Biochim. Biophys. Acta 24,604-611 9 Ohman, R. and Hygstedt, 0. (1968) Anal. Biochem. 23, 391-402 10 Tettamanti, G., Bonah, F., Marchesini, S. and Zambotti, V. (1973) Biochim. Biophys. Acta 296, 160-170 11 Ghidoni, R., Sonnino, S., Tettamanti, G., Baumann, N., Reuter, G. and Schauer, R. (1980) J. Biol. Chem. 255, 6990-6995 12 Corti, M., Degiorgio, V., Ghidoni, R., Sonnino, S. and Tettamanti, G. (1980) Chem. Phys. Lipids 26, 225-238 13 Ghidoni, R., Sonnino, S., Masserim, M., Orlando, P. and Tettamanti, G. (1981) J. Lipid Res. 22, 1286-1295 14 Wiegandt, H. and Baschang, G. (1965) Z. Naturforsch. Teil 8. 20, 164-166 15 Venerando, B., Cestaro, B., Fiorilli, A., Ghidoni, R., Preti, A. and Tettamanti, G. (1982) Biochem. J. 203,735-742 16 Formisano, S., Johnson, M.L., Lee, G., Aloj, SM. and Edelhoch, H. (1979) Biochemistry 18, 1119-1121 17 Caimi, L., Lombardo, A., Preti, A,, Wiesmann, U. and Tettamanti, G. (1979) B&him. Biophys. Acta 571,137-146 18 Warren, L. (1963) Methods Enzymol. 6,465 19 Lineweaver, H. and Burk, D. (1934) J. Am. Chem. Sot. 56, 659-665 20 Andrews, P. (1964) Biochem. J. 91,222-223 21 U&da, Y., Tsukada, Y. and Sugimori, T. (1979) J. Biothem. 86,1573-1585 22 Laurent, T.C. and Killander, J. (1964) J. Chromatogr. 14, 317-330 23 Lowry, O.H., Rosebrougb, N.J., Fan; A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275 24 Tomasi, M., Roda, L.G., Ausiello, C., D’Agnolo, G., Venerando, B., Ghidoni, R., Sonmno, S. and Tettamanti, G. (1980) Eur. J. B&hem. 111, 315-324 25 Curatolo, W., Small, D.M. and Shipley, G. (1977) Biochim. Biophys. Acta 468, 11-20 26 Gatt, S. and Bartfai, T. (1977) Biochim. Biophys. Acta 488, 1-12 27 Sandhoff, K. and Conzehnann, E. (1979) Trends B&hem. Sci. 4, 231-233 28 Li, Y.T. and Li, S.C. (1983) The Enzymes, Vol. XVI, pp. 427-445, Academic Press, New York 29 Masserini, M., Sonnino, S., Ghidoni, R., Chigomo, V. and Tettamanti, G. (1982) Biochim. Biophys. Acta 688, 333-340 30 Sharom, F.J. and Grant, C.W.M. (1978) Biochim. Biophys. Acta 507, 280-293 31 Svennerholm, L. (1970) in Handbook of Neurochemistry (Lajtha, A., ed.), Vol. III, pp. 425-452, Plenum Press, New York 32 IUPAC-IUB, Commission on Biochemical Nomenclature (1972) Lipids 12, 455-458; (1982) J. Biol. Chem. 257, 3347-3351