Int J. Biochem. Vol. 23, No. 9, pp. 969-972, 1991 Printed in Great Britain
0020-711X/91$3.00+ 0.00 Pergamon Press plc
fi-HEXOSAMINIDASE EXPRESSION IN CHICK FIBROBLASTS IN VK’-‘RO T.
BECCARI, M.
Dipartimento
BODO, E. BECCHETTI,F. PEzzET~I, G.
BELLACH~OMA
EMBRYO
and A.
ORLACCHIO*
di Medicina Sperimentale e Scienze Biochimiche, UniversitB di Perugia, Via de1 Giochetto, 06100 Perugia, Italy [Fax 075-585-34431 (Received 24 September 1990)
Abstract-l. Two forms of /I-hexosaminidase, similar to hexosaminidase A and hexosaminidase C, were separated by DEAE-cellulose chromatography in chick embryo skin fibroblasts in uitro. 2. /3-Hexosaminidase specific activity increases during development in cultured chick embryo skin fibroblasts in vitro. 3. Concanavalin-A treatment determines the increase of the neutral form, hexosaminidase C, during development. 4. Concanavalin-A reduces the specific activity of /?-hexosaminidase during development.
INTRODUCIION
The lysosomal enzyme /I-N-acetylhexosaminidase (E.C. 3.2.1.52) (Hex) is widely distributed in the animal kingdom and its general distribution in nature is a reflection of the vital metabolic role of the enzyme. Hexosaminidase has been found to exist in many mammalian tissues, in two major forms, A and B, which differ in thermal stability and electrophoretic mobility, A being more heat labile and having greater anodic mobility than B (Robinson and Stirling, 1968). In addition to these forms with acidic pH optimum, forms with neutral pH optimum, called Hex C or “neutral”, have been described in several mammalian tissues (Braidman et al., 1974; Overdijk et al., 1975; Izuni and Suzuki, 1983), including chicken brain (Bernal et nl., 1973), skin and lung (Beccari et al., 1989). Hex C shows a higher molecular weight than A and B, is not bound to concanavalinA-Sepharose and has little N-acetylgalactosaminidase activity (Braidman et af., 1974; Swallow et al., 1976). We are interested in the study of Hex C in chick embryo because changes in lysosomal exoglycosidases during organogenesis suggest a possible mechanism of regulation of glycosaminoglycans (GAG) turnover via developmental modulation of these enzymes (Becchetti et al., 1989). We have previously studied the expression of Hex in chick skin and lung during development in uivo (Beccari et al., 1989). In this paper, we have analyzed Hex expression in chick embryo skin fibroblasts during development in vitro. The biochemical properties of Hex isoenzymes have been compared with those obtained in viva The effect of concanavalin-A (con-A) on Hex expression has been studied in an attempt to show a possible relationship between GAG composition and Hex expression. In fact, lectins, which bind specifically saccharide moieties of membrane receptors, are able
*To whom all correspondence should be addressed.
to modulate mesenchymal GAG composition in vitro (Locci et al., 1990).
MATERIALS AND
METHODS
4-Methylumbelliferyl-2-acetamido-2-deoxy-~-D-glucopyranoside (4-MUGlcNAc), 4-methylumbelliferyl-2-a&amido-2-deoxy$-o-galactopyranoside (4-MUGalNAc), amethyl-o-mannoside and concanavalin-A lectin (con-A) were obtained from Sigma Chemical Co, St Louis, MO, U.S.A. DEAEcellulose DE-52 was from Whatman Biochemicals Ltd, Maidstone, Kent, U.K. ConcanavalinA-Sepharose, Sephacryl S-300 and molecular weight standards were from Pharmacia, Uppsala, Sweden. Bio-Rad protein assay reagent concentrate was from Bio-Rad Laboratories, Richmond, CA, U.S.A. Medium 199, fetal calf serum, was from GIBCO, Grand Island, NY, U.S.A. All other reagents were purchased from Baker Chemicals B.V., Deventer, The Netherlands. 4-Methylumbelliferyl-2-acetamido-2-deoxy-/I-o-glupyranoside-6-sulphate (4-MUGlcNAc-6-S04) was obtained from HSC, Research Development Corporation, Toronto, Canada. Cell cultures and preparation of cell extract Back skin fragments from 7-, 11- and 14-day-old chick embryos were dissociated in 0.25% trypsin at room temperature for 30 min. The dissociated cells were filtered and suspended in medium 199 plus 10% fetal calf serum. Serum was treated at 70°C for 30min to inactivate endogenous Hex. Fifteen ml of cell suspension (300,000 cells/ml) were plated in Falcon flasks in a humidity-saturated atmosphere (5% CO,, at 37°C) for 3-4 days to obtain confluent cultures. Nutrient was then exchanged either for medium 199 alone (controls), or for medium 199 plus con-A (20 pg/ml), and incubated for another 24 hr. Treated cultures exhibited no changes in viability, even if the fibroblasts were more globular. A parallel set of controls was performed, adding con-A in the presence of 0.1 M a-methylD-mannoside. At the end of the incubation period, the cells were recovered in 10 mM Na-phosphate buffer, pH 7.0, and homogenized in a Potter-Elvehjem homogenizer. The homogenates were centrifuged at 25,000 g for 30 min at 4°C in a Sorvall RC-5B refrigerated centrifuge. The resulting pellets were discarded and the supematants were the extracts to be loaded on DEAE-cellulose chromatography. 969
T.
970 Enzyme
BECCARI et ai.
assay
Enzyme activity was measured as previously described (Orla&hio et al., I984) using 4-MUGliNAc and 4-MUGalNAc as substrates in 0.1 M citrate/O.2 M phosphate. uH 4.0 and 6.0. The assay with 4-MU~l~NA~-6-S-~ ias performed as previously described (Beccari et al., 1987). Fluorescence of the liberated 4-methylumbelliferone was measured on a Perkin-Elmer LS-3 fluorimeter, with excitation at 360 nm and emission measured at 446 nm. The fluorimeter was calibrated with 4-methylumbelliferone solutions in 0.4 M glycine buffer, pH 10.6. One unit was defined as the amount of the enzyme which converts 1nmol of substratejhr into 4-methylumbelliferone at 37°C. Protein determination
Proteins were measured according to the method of Bradford (1976) using crystalline bovine gammaglobulin as standard. The specific activity was calculated as nmol of 4-methylumbelliferone released/hr per mg of protein. DEAE-cellulose
chromatography
Chromatography on DEAEcelIulose was performed on columns (1 cm x 5 cm) equilibrated with 10 mM sodium phosphate buffer, pH 7.0. Enzyme activity retained by the column was eluted using a linear gradient of NaCl (t&O.3M) in 60 ml of column buffer. Finally, the column was eluted with 1.0 M NaCl in the same buffer. Proteins in the eluates were monitored at 280nm. Hexosa~nidase activity was measured as indicated above, using 4-MUGlcNAc and 4-MUGlcNAc-6-SO4 as substrates. Determrnatlon of pH optimum
The optimum pH was determined under standard assay conditions m 0.2 M phosphat-itrate buffer for pH range 2.5-6.5 and 0.2 M sodium phosphate buffer for pH range 7.0-8.5, using 4-MUGl~NAc as substrate, Molecular
weight determination
Gel
filtration on columns of Sephacryl S-300 (1.6 cm x 78 cm) equilibrated in 10 mM sodium phosphate buffer. pH 6.8. containing 0.1 M NaCl, was used to estimate the molecular weight of hexosaminidase isoenzymes. Molecular weight standards for gel filtration were: cytochrome c (12,~0);-ovalbumin (45,OO~);aldolase (1 SS,OOt$;catalase (232,000) and ferritin (440.000). Fractions (l.Oml) were / collected and assayed for enzyme activity. Proteins were monitored at 280 nm. I
,
0
10
20
30
Fraction
number
40
50
1. DEAE-cellulose profile of j.?-N-acetylhexosaminidase from IQday-old chick embryo skin fibroFig.
blasts treated with con-A. The column was equilibrate with 10 mM phosphate buffer, pH 7.0, and eluted with a linear gradient of NaCl (O-O.3M) as described in the experimental section. Hex activity was determined using 4-MUGlcNAc, pH 4.0 (0) and pH 6.0 (O), as substrates. All the kinetic determinations were made at the optimal pH values. RESULTS AND
DISCUSSION
Extracts of 7-, ll- and lCday-old cultured chick embryo skin fibroblasts gave one peak of Hex activity on DEAE-cellulose chromatography using 4MUGlcNAc as substrate at pH 4.0 (Fig. 1). A second peak, eluted with the salt gradient later than the first peak, was detected when the assay was performed at pH 6.0 (Fig. 1). This “neutral” form of hexosaminidase was present in all the stages of the development of chick embryo cultured skin fibroblasts. The percentage of the “neutral” form increased with development, to reach 8% of total activity measured at pH 6.0 in 14-day-old chick skin cultured fibroblasts (Fig. 2). Con-A treatment affects the increasing the amount of isoenzyme pattern, “neutral” form, which reaches 30% of total activity
Thermal stability
Heat Inactivation of hexosaminidase was performed at 50°C. The samples were diluted to equivalent activity and suspended in the standard assay mixture minus the substrate. After incubation for specific intervals of times the samples were maintained at 4°C for 2-4 hr and then warmed to 37% The addition of substrate initiated the reaction.
30 r
~On~anal~alin~A-~epharose chromatography
Concanavalin A-Sepharose affinity chromato~aphy was performed using a column (0.5 cm x 4 cm) equilibrated with 20 mM Tris-HCl buffer, pH 7.4, containing 1mM CaCl,, 1 mM MgCl, and 1 mM MnCl,. Samples after DEAEcellulose chromatography were dialysed against the above buffer and then applied to the column. A linear gradient containing a-methyl-n-mannoside (o-0.5 M) and 1.0 M NaCl in the starting buffer was used to elute the glycoproteins. Determinations of K,,,
The Michaelis-Menten constants were determined from the reciprocal plots (Lineweaver and Burk, 1934). The substrate concentrations ranged from 0.4 to 2.1 mM for 4-MUGlcNAc and from 0.05 to 2.1 mM for 4-MUGalNAc
Days Fig. 2. Percentage of Hex C in control and con-A-treated chick embryo skin fibroblasts of 7, I1 and 14 days of incubation. The percentage of Hex C was calculated in enzyme units as percentage of total activity at pH 6.0 eluted from DEAE-cellulose chromatography in control (A) and con-A-treated (A) fibroblasts.
Chick fibroblast B-hexosaminidase
971
Table 1. Effect of con-A on hexosamimdase specific activity in 7-, I l- and 14-day-old cultured skin fibroblasts; specific activity is expressed as units/q of protein
Days of incubation 7
11
14
Control Con-A Con-A + a-CH3-D-mannoside
181 + 10’ 130+9 178k4
451+18 330 + 15 446f16
640?21 450 f 20 630 f 18
Control Con-A Con-A + a-CH3-o-mannoside
100*8 73 + 8 95*4
250 * 12 18Ok9 240*8
370 * 10 340*7 357 + 5
Substrate MUGlcNAc, pH 4.0 MUGlcNAc, pH 6.0 ‘Values
are the means of 6 measurements
k SD.
at pH 6.0 in lCday-old skin cultured fibroblasts (Fig. 2). Only the acidic form of hexosaminidase hydrolyses the 4-MUGlcNAc-6-SO4 substrate, which, in human tissues, is hydrolysed by the active a subunit (Kytzia and Sandhoff, 1985). Thus it appears to contain the a subunit, and because of this, together with its behaviour on DEAE-cellulose chromatography, it can be considered to be hexosaminidase A. Similar to the neutral form isolated from fibroblasts in viuo (Beccari et al., 1989), the neutral form does not contain the active CIsubunit. There is no peak of Hex activity eluted with the void volume, as observed in fibroblasts in vivo, and the percentage of the “neutral” form present during development is lower in vitro than in vivo (Beccari et al., 1989.) The specific activity of Hex, measured at pH 6.0 and 4.0, using 4-MUGlcNAc as substrate, increased in the fibroblasts from 7 to 14 days (Table 1). This finding suggests that there could be an increased synthesis of both Hex isoenzymes during development in cultured fibroblasts or a decrement of their degradation. Con-A reduces the Hex specific activity during development to a greater extent at pH 4.0 than at pH 6.0 because it increases the synthesis of the “neutral” form of hexosaminidase (Table 1). Thermal stability at 50°C of the acidic and neutral hexosaminidases after DEAE-cellulose chromatography showed that the first 70% of its activity was lost after 30 min of incubation and the remainder was partially thermostable. The acidic form was retained by a column of concanavalin A-Sepharose and all the activity was recovered in a single peak after elution with methylmannoside. In contrast, the neutral form was not retained by the concanavalin A-Sepharose column. Thus acidic form is a glycoprotein, as are all the acidic forms isolated from mammalian tissues (Mahuran et al., 1985) whereas the neutral form is not a glycoprotein, or at least has a different carbohydrate moiety, and in this resembles other neutral forms (Swallow et al., 1976). Table 2. Michaelis constants for cultured skm fibroblast /3-N-acetylhexosaminidase using 4-MUGlcNAc and 4-MUGalNAc as substrates K, (mW Substrate 4-MUGlcNAc 4-MUGalNAc
Audit
form
0.530 0.060
Values are the means of 5 detemxnations.
Neutral
form
0.150 5.400
The molecular weights of the acidic and neutral forms, determined by gel filtration chromatography, were 100,000 and 150,000, respectively. As observed in skin fibroblasts in uivo, the neutral form has a higher molecular weight than the acidic form, resembling hexosaminidase C from other sources (Braidman et al., 1974). The K,,, values of acidic and neutral forms separated by DEAE-cellulose chromatography, calculated with Lineweaver-Burk plots using the 4-MUGlcNAc and 4-MUGalNAc substrates, are shown in Table 2. They are similar to the values already found in skin fibroblasts in ho, with the neutral form showing a very low affinity for the 4MUGalNAc substrate and a greater affinity than the acidic form for the 4-MUGlcNAc substrate. These properties of the two forms are consistent with those expected for hexosaminidase A and C respectively. Exogenous lectin administration in vitro can be used as a suitable experimental model to study the influence of environmental factors on fibroblasts. Con-A, SBA, WGA and PNA lectins affect GAG secretion (Evangelisti et al., 1983) and the specific activity of glycosidases in cultured embryonic fibroblasts (Bodo et al., 1988). In particular, con-A, which binds specifically to a-D-mannoside moieties, seems to modulate hexosaminidase expression in cultured fibroblasts, decreasing the Hex specific activity and increasing the amount of neutral form during development. These effects are age dependent. The addition of c(-methyl-D-mannoside, which specifically binds to con-A, completely prevented the inhibition induced by the lectin at pH 4.0 and 6.0 (Table 1). We conclude that in 7-, 11- and lCday-old chick embryo skin fibroblasts in vitro there are two forms of Hex, the first of which is similar to Hex A and the second of which is similar to the neutral form Hex C. Con-A modulates the expression of Hex isoenzymes, reducing the amount of Hex A and increasing the amount of Hex C, and this effect is age dependent. It is possible to hypothesize that the binding of con-A to glycoside moieties of surface receptors could be responsible for the observed effect. GAG, the secretion of which is modified by con-A (Evangelisti et al., 1983; Locci et al., 1990), could also be involved in changing Hex expression. At present, it is difficult to explain the exact mechanism by which con-A modulates Hex expression or to determine the metabolic function of Hex C. Acknowledgements-This Italian CNR and MPI.
work
was
supported
by
the
T.
972
BECCARIet al.
REFERENCES
Beccari I., Emiliani C., Hosseini R., Orlacchio A. and Stirling J. L. (1987) Intermediate forms of human B-Nacetylhexosaminidase lack activity towards 4-methylumbelliferyl-b-acetylglucosamine-6-sulphate. Biochem. J. 244(3), 801-804. Beccari T., Pezzetti F., Belardinelli R., Bodo M., Becchetti E. and Orlacchio A. (1989) p-N-Acetylhexosaminidase isoenzymes during chick embryo development. Int. J. Biochem. 21, 769-776. Becchetti E., Evangelisti R., Bodo M., Pezzetti F., Orlacchio A. and Carinci P. (1989) /?-iV-Acetylglucosaminidase activity in embryonic chick skin and lung tissue and cultured fibroblasts. Cell. Mol. Biol. 35, 187-198. Bemal C. S., Barrientos J. M. and Cabezas J. A. (1983) Separation and properties of a “neutral” hexosaminidase from embryonic chicken brain. Znt. J. Biochem. 15, 703-708. Bodo M., Pezzetti R., Becchetti E., Orlacchio A., Evangelisti R. and Carinci P. (1988) Age related and lectin influenced changes of exoglycosidases activity m cultured chick embryonic skin fibroblasts. Ceil Bzol. Int. Rep. 12, 459464. Bradford M. M. (1976) A rapid and sensitive method for the quantitation of micrograms of protein utilizing the principles of proteindye binding. Analyt. Biochem. 12, 248-254. Braidman I., Carrol M., Dance N. and Robinson D. (1974) Separation and properties of human brain hexosaminid&e C. Bioche& .I. 143, 295-301. Evangelisti R., Bodo M., Caruso A. and Carinci P. (1983) Conca!tavalin-A affects glycosaminiglycans synthesis by cultured fibroblasts. IRC’S 11, 629.
Izuni T. and Suzuki K. (1983) Neutral b-N-acetylhexosaminidase of rat brain. J. biol. Chem. 258, 69916999. Kytzia H. J. and Sandhoff K. (1985) Evidence for two different active sites on human B-hexosaminidase A. J. biol. Chem. 260, 7568-7572. Lineweaver H. and Burk D. (1934) The determination of the enzyme dissociation constants. 1. Am. them. Sot. 56, 658-666. Locci P., Evangelisti R., Lilli C., Becchetti E. and Carinci P. (1990) Concanavalin-A affects glycosaminoglycans cellular and extracellular accumulation in cultured embryonic fibroblasts. Cell. Biol. Inf. Rep. 14, 641648. Mahuran D., Novak A. and Gravel J. A. (1985) The lysosomal hexosaminidase isozymes. Zsozymes Curr. Top. Biol. Med. Res. 12, 229-288. Orlacchio A., Maffei C., Emiliani C., Rambotti P. and Davis S. (1984). A distinct B-hexosaminidase isoenzyme separated from human leukemic lymphocytes and myelocytes. Biochem. biophys. Res. Commun. 122, 966-973. Overdijk E., Van Der Kroef W. M. J., Veltkamp W. A. and Hooghwinkel G. J. M. (1975) The separation of bovine brain b-N-acetyl-o-hexosaminidase. Biochem. J. 151, 257-26 1. Robinson D. and Stirling J. L. (1968) N-Acetylglucosaminidase in human spleen. Biochem. J. 107, 321-327. Swallow D. M., Evans L., Saha E. N. and Harris H. (1976) Characterization and tissue distribution of N-acctylhexosaminidase C: suggestive evidence for a separate hexosaminidase locus. Ann. Hum. Genef. 40, 5565.
0020-711X/91$3.00+ 0.00 Pergamon Press plc
fi-HEXOSAMINIDASE EXPRESSION IN CHICK FIBROBLASTS IN VK’-‘RO T.
BECCARI, M.
Dipartimento
BODO, E. BECCHETTI,F. PEzzET~I, G.
BELLACH~OMA
EMBRYO
and A.
ORLACCHIO*
di Medicina Sperimentale e Scienze Biochimiche, UniversitB di Perugia, Via de1 Giochetto, 06100 Perugia, Italy [Fax 075-585-34431 (Received 24 September 1990)
Abstract-l. Two forms of /I-hexosaminidase, similar to hexosaminidase A and hexosaminidase C, were separated by DEAE-cellulose chromatography in chick embryo skin fibroblasts in uitro. 2. /3-Hexosaminidase specific activity increases during development in cultured chick embryo skin fibroblasts in vitro. 3. Concanavalin-A treatment determines the increase of the neutral form, hexosaminidase C, during development. 4. Concanavalin-A reduces the specific activity of /?-hexosaminidase during development.
INTRODUCIION
The lysosomal enzyme /I-N-acetylhexosaminidase (E.C. 3.2.1.52) (Hex) is widely distributed in the animal kingdom and its general distribution in nature is a reflection of the vital metabolic role of the enzyme. Hexosaminidase has been found to exist in many mammalian tissues, in two major forms, A and B, which differ in thermal stability and electrophoretic mobility, A being more heat labile and having greater anodic mobility than B (Robinson and Stirling, 1968). In addition to these forms with acidic pH optimum, forms with neutral pH optimum, called Hex C or “neutral”, have been described in several mammalian tissues (Braidman et al., 1974; Overdijk et al., 1975; Izuni and Suzuki, 1983), including chicken brain (Bernal et nl., 1973), skin and lung (Beccari et al., 1989). Hex C shows a higher molecular weight than A and B, is not bound to concanavalinA-Sepharose and has little N-acetylgalactosaminidase activity (Braidman et af., 1974; Swallow et al., 1976). We are interested in the study of Hex C in chick embryo because changes in lysosomal exoglycosidases during organogenesis suggest a possible mechanism of regulation of glycosaminoglycans (GAG) turnover via developmental modulation of these enzymes (Becchetti et al., 1989). We have previously studied the expression of Hex in chick skin and lung during development in uivo (Beccari et al., 1989). In this paper, we have analyzed Hex expression in chick embryo skin fibroblasts during development in vitro. The biochemical properties of Hex isoenzymes have been compared with those obtained in viva The effect of concanavalin-A (con-A) on Hex expression has been studied in an attempt to show a possible relationship between GAG composition and Hex expression. In fact, lectins, which bind specifically saccharide moieties of membrane receptors, are able
*To whom all correspondence should be addressed.
to modulate mesenchymal GAG composition in vitro (Locci et al., 1990).
MATERIALS AND
METHODS
4-Methylumbelliferyl-2-acetamido-2-deoxy-~-D-glucopyranoside (4-MUGlcNAc), 4-methylumbelliferyl-2-a&amido-2-deoxy$-o-galactopyranoside (4-MUGalNAc), amethyl-o-mannoside and concanavalin-A lectin (con-A) were obtained from Sigma Chemical Co, St Louis, MO, U.S.A. DEAEcellulose DE-52 was from Whatman Biochemicals Ltd, Maidstone, Kent, U.K. ConcanavalinA-Sepharose, Sephacryl S-300 and molecular weight standards were from Pharmacia, Uppsala, Sweden. Bio-Rad protein assay reagent concentrate was from Bio-Rad Laboratories, Richmond, CA, U.S.A. Medium 199, fetal calf serum, was from GIBCO, Grand Island, NY, U.S.A. All other reagents were purchased from Baker Chemicals B.V., Deventer, The Netherlands. 4-Methylumbelliferyl-2-acetamido-2-deoxy-/I-o-glupyranoside-6-sulphate (4-MUGlcNAc-6-S04) was obtained from HSC, Research Development Corporation, Toronto, Canada. Cell cultures and preparation of cell extract Back skin fragments from 7-, 11- and 14-day-old chick embryos were dissociated in 0.25% trypsin at room temperature for 30 min. The dissociated cells were filtered and suspended in medium 199 plus 10% fetal calf serum. Serum was treated at 70°C for 30min to inactivate endogenous Hex. Fifteen ml of cell suspension (300,000 cells/ml) were plated in Falcon flasks in a humidity-saturated atmosphere (5% CO,, at 37°C) for 3-4 days to obtain confluent cultures. Nutrient was then exchanged either for medium 199 alone (controls), or for medium 199 plus con-A (20 pg/ml), and incubated for another 24 hr. Treated cultures exhibited no changes in viability, even if the fibroblasts were more globular. A parallel set of controls was performed, adding con-A in the presence of 0.1 M a-methylD-mannoside. At the end of the incubation period, the cells were recovered in 10 mM Na-phosphate buffer, pH 7.0, and homogenized in a Potter-Elvehjem homogenizer. The homogenates were centrifuged at 25,000 g for 30 min at 4°C in a Sorvall RC-5B refrigerated centrifuge. The resulting pellets were discarded and the supematants were the extracts to be loaded on DEAE-cellulose chromatography. 969
T.
970 Enzyme
BECCARI et ai.
assay
Enzyme activity was measured as previously described (Orla&hio et al., I984) using 4-MUGliNAc and 4-MUGalNAc as substrates in 0.1 M citrate/O.2 M phosphate. uH 4.0 and 6.0. The assay with 4-MU~l~NA~-6-S-~ ias performed as previously described (Beccari et al., 1987). Fluorescence of the liberated 4-methylumbelliferone was measured on a Perkin-Elmer LS-3 fluorimeter, with excitation at 360 nm and emission measured at 446 nm. The fluorimeter was calibrated with 4-methylumbelliferone solutions in 0.4 M glycine buffer, pH 10.6. One unit was defined as the amount of the enzyme which converts 1nmol of substratejhr into 4-methylumbelliferone at 37°C. Protein determination
Proteins were measured according to the method of Bradford (1976) using crystalline bovine gammaglobulin as standard. The specific activity was calculated as nmol of 4-methylumbelliferone released/hr per mg of protein. DEAE-cellulose
chromatography
Chromatography on DEAEcelIulose was performed on columns (1 cm x 5 cm) equilibrated with 10 mM sodium phosphate buffer, pH 7.0. Enzyme activity retained by the column was eluted using a linear gradient of NaCl (t&O.3M) in 60 ml of column buffer. Finally, the column was eluted with 1.0 M NaCl in the same buffer. Proteins in the eluates were monitored at 280nm. Hexosa~nidase activity was measured as indicated above, using 4-MUGlcNAc and 4-MUGlcNAc-6-SO4 as substrates. Determrnatlon of pH optimum
The optimum pH was determined under standard assay conditions m 0.2 M phosphat-itrate buffer for pH range 2.5-6.5 and 0.2 M sodium phosphate buffer for pH range 7.0-8.5, using 4-MUGl~NAc as substrate, Molecular
weight determination
Gel
filtration on columns of Sephacryl S-300 (1.6 cm x 78 cm) equilibrated in 10 mM sodium phosphate buffer. pH 6.8. containing 0.1 M NaCl, was used to estimate the molecular weight of hexosaminidase isoenzymes. Molecular weight standards for gel filtration were: cytochrome c (12,~0);-ovalbumin (45,OO~);aldolase (1 SS,OOt$;catalase (232,000) and ferritin (440.000). Fractions (l.Oml) were / collected and assayed for enzyme activity. Proteins were monitored at 280 nm. I
,
0
10
20
30
Fraction
number
40
50
1. DEAE-cellulose profile of j.?-N-acetylhexosaminidase from IQday-old chick embryo skin fibroFig.
blasts treated with con-A. The column was equilibrate with 10 mM phosphate buffer, pH 7.0, and eluted with a linear gradient of NaCl (O-O.3M) as described in the experimental section. Hex activity was determined using 4-MUGlcNAc, pH 4.0 (0) and pH 6.0 (O), as substrates. All the kinetic determinations were made at the optimal pH values. RESULTS AND
DISCUSSION
Extracts of 7-, ll- and lCday-old cultured chick embryo skin fibroblasts gave one peak of Hex activity on DEAE-cellulose chromatography using 4MUGlcNAc as substrate at pH 4.0 (Fig. 1). A second peak, eluted with the salt gradient later than the first peak, was detected when the assay was performed at pH 6.0 (Fig. 1). This “neutral” form of hexosaminidase was present in all the stages of the development of chick embryo cultured skin fibroblasts. The percentage of the “neutral” form increased with development, to reach 8% of total activity measured at pH 6.0 in 14-day-old chick skin cultured fibroblasts (Fig. 2). Con-A treatment affects the increasing the amount of isoenzyme pattern, “neutral” form, which reaches 30% of total activity
Thermal stability
Heat Inactivation of hexosaminidase was performed at 50°C. The samples were diluted to equivalent activity and suspended in the standard assay mixture minus the substrate. After incubation for specific intervals of times the samples were maintained at 4°C for 2-4 hr and then warmed to 37% The addition of substrate initiated the reaction.
30 r
~On~anal~alin~A-~epharose chromatography
Concanavalin A-Sepharose affinity chromato~aphy was performed using a column (0.5 cm x 4 cm) equilibrated with 20 mM Tris-HCl buffer, pH 7.4, containing 1mM CaCl,, 1 mM MgCl, and 1 mM MnCl,. Samples after DEAEcellulose chromatography were dialysed against the above buffer and then applied to the column. A linear gradient containing a-methyl-n-mannoside (o-0.5 M) and 1.0 M NaCl in the starting buffer was used to elute the glycoproteins. Determinations of K,,,
The Michaelis-Menten constants were determined from the reciprocal plots (Lineweaver and Burk, 1934). The substrate concentrations ranged from 0.4 to 2.1 mM for 4-MUGlcNAc and from 0.05 to 2.1 mM for 4-MUGalNAc
Days Fig. 2. Percentage of Hex C in control and con-A-treated chick embryo skin fibroblasts of 7, I1 and 14 days of incubation. The percentage of Hex C was calculated in enzyme units as percentage of total activity at pH 6.0 eluted from DEAE-cellulose chromatography in control (A) and con-A-treated (A) fibroblasts.
Chick fibroblast B-hexosaminidase
971
Table 1. Effect of con-A on hexosamimdase specific activity in 7-, I l- and 14-day-old cultured skin fibroblasts; specific activity is expressed as units/q of protein
Days of incubation 7
11
14
Control Con-A Con-A + a-CH3-D-mannoside
181 + 10’ 130+9 178k4
451+18 330 + 15 446f16
640?21 450 f 20 630 f 18
Control Con-A Con-A + a-CH3-o-mannoside
100*8 73 + 8 95*4
250 * 12 18Ok9 240*8
370 * 10 340*7 357 + 5
Substrate MUGlcNAc, pH 4.0 MUGlcNAc, pH 6.0 ‘Values
are the means of 6 measurements
k SD.
at pH 6.0 in lCday-old skin cultured fibroblasts (Fig. 2). Only the acidic form of hexosaminidase hydrolyses the 4-MUGlcNAc-6-SO4 substrate, which, in human tissues, is hydrolysed by the active a subunit (Kytzia and Sandhoff, 1985). Thus it appears to contain the a subunit, and because of this, together with its behaviour on DEAE-cellulose chromatography, it can be considered to be hexosaminidase A. Similar to the neutral form isolated from fibroblasts in viuo (Beccari et al., 1989), the neutral form does not contain the active CIsubunit. There is no peak of Hex activity eluted with the void volume, as observed in fibroblasts in vivo, and the percentage of the “neutral” form present during development is lower in vitro than in vivo (Beccari et al., 1989.) The specific activity of Hex, measured at pH 6.0 and 4.0, using 4-MUGlcNAc as substrate, increased in the fibroblasts from 7 to 14 days (Table 1). This finding suggests that there could be an increased synthesis of both Hex isoenzymes during development in cultured fibroblasts or a decrement of their degradation. Con-A reduces the Hex specific activity during development to a greater extent at pH 4.0 than at pH 6.0 because it increases the synthesis of the “neutral” form of hexosaminidase (Table 1). Thermal stability at 50°C of the acidic and neutral hexosaminidases after DEAE-cellulose chromatography showed that the first 70% of its activity was lost after 30 min of incubation and the remainder was partially thermostable. The acidic form was retained by a column of concanavalin A-Sepharose and all the activity was recovered in a single peak after elution with methylmannoside. In contrast, the neutral form was not retained by the concanavalin A-Sepharose column. Thus acidic form is a glycoprotein, as are all the acidic forms isolated from mammalian tissues (Mahuran et al., 1985) whereas the neutral form is not a glycoprotein, or at least has a different carbohydrate moiety, and in this resembles other neutral forms (Swallow et al., 1976). Table 2. Michaelis constants for cultured skm fibroblast /3-N-acetylhexosaminidase using 4-MUGlcNAc and 4-MUGalNAc as substrates K, (mW Substrate 4-MUGlcNAc 4-MUGalNAc
Audit
form
0.530 0.060
Values are the means of 5 detemxnations.
Neutral
form
0.150 5.400
The molecular weights of the acidic and neutral forms, determined by gel filtration chromatography, were 100,000 and 150,000, respectively. As observed in skin fibroblasts in uivo, the neutral form has a higher molecular weight than the acidic form, resembling hexosaminidase C from other sources (Braidman et al., 1974). The K,,, values of acidic and neutral forms separated by DEAE-cellulose chromatography, calculated with Lineweaver-Burk plots using the 4-MUGlcNAc and 4-MUGalNAc substrates, are shown in Table 2. They are similar to the values already found in skin fibroblasts in ho, with the neutral form showing a very low affinity for the 4MUGalNAc substrate and a greater affinity than the acidic form for the 4-MUGlcNAc substrate. These properties of the two forms are consistent with those expected for hexosaminidase A and C respectively. Exogenous lectin administration in vitro can be used as a suitable experimental model to study the influence of environmental factors on fibroblasts. Con-A, SBA, WGA and PNA lectins affect GAG secretion (Evangelisti et al., 1983) and the specific activity of glycosidases in cultured embryonic fibroblasts (Bodo et al., 1988). In particular, con-A, which binds specifically to a-D-mannoside moieties, seems to modulate hexosaminidase expression in cultured fibroblasts, decreasing the Hex specific activity and increasing the amount of neutral form during development. These effects are age dependent. The addition of c(-methyl-D-mannoside, which specifically binds to con-A, completely prevented the inhibition induced by the lectin at pH 4.0 and 6.0 (Table 1). We conclude that in 7-, 11- and lCday-old chick embryo skin fibroblasts in vitro there are two forms of Hex, the first of which is similar to Hex A and the second of which is similar to the neutral form Hex C. Con-A modulates the expression of Hex isoenzymes, reducing the amount of Hex A and increasing the amount of Hex C, and this effect is age dependent. It is possible to hypothesize that the binding of con-A to glycoside moieties of surface receptors could be responsible for the observed effect. GAG, the secretion of which is modified by con-A (Evangelisti et al., 1983; Locci et al., 1990), could also be involved in changing Hex expression. At present, it is difficult to explain the exact mechanism by which con-A modulates Hex expression or to determine the metabolic function of Hex C. Acknowledgements-This Italian CNR and MPI.
work
was
supported
by
the
T.
972
BECCARIet al.
REFERENCES
Beccari I., Emiliani C., Hosseini R., Orlacchio A. and Stirling J. L. (1987) Intermediate forms of human B-Nacetylhexosaminidase lack activity towards 4-methylumbelliferyl-b-acetylglucosamine-6-sulphate. Biochem. J. 244(3), 801-804. Beccari T., Pezzetti F., Belardinelli R., Bodo M., Becchetti E. and Orlacchio A. (1989) p-N-Acetylhexosaminidase isoenzymes during chick embryo development. Int. J. Biochem. 21, 769-776. Becchetti E., Evangelisti R., Bodo M., Pezzetti F., Orlacchio A. and Carinci P. (1989) /?-iV-Acetylglucosaminidase activity in embryonic chick skin and lung tissue and cultured fibroblasts. Cell. Mol. Biol. 35, 187-198. Bemal C. S., Barrientos J. M. and Cabezas J. A. (1983) Separation and properties of a “neutral” hexosaminidase from embryonic chicken brain. Znt. J. Biochem. 15, 703-708. Bodo M., Pezzetti R., Becchetti E., Orlacchio A., Evangelisti R. and Carinci P. (1988) Age related and lectin influenced changes of exoglycosidases activity m cultured chick embryonic skin fibroblasts. Ceil Bzol. Int. Rep. 12, 459464. Bradford M. M. (1976) A rapid and sensitive method for the quantitation of micrograms of protein utilizing the principles of proteindye binding. Analyt. Biochem. 12, 248-254. Braidman I., Carrol M., Dance N. and Robinson D. (1974) Separation and properties of human brain hexosaminid&e C. Bioche& .I. 143, 295-301. Evangelisti R., Bodo M., Caruso A. and Carinci P. (1983) Conca!tavalin-A affects glycosaminiglycans synthesis by cultured fibroblasts. IRC’S 11, 629.
Izuni T. and Suzuki K. (1983) Neutral b-N-acetylhexosaminidase of rat brain. J. biol. Chem. 258, 69916999. Kytzia H. J. and Sandhoff K. (1985) Evidence for two different active sites on human B-hexosaminidase A. J. biol. Chem. 260, 7568-7572. Lineweaver H. and Burk D. (1934) The determination of the enzyme dissociation constants. 1. Am. them. Sot. 56, 658-666. Locci P., Evangelisti R., Lilli C., Becchetti E. and Carinci P. (1990) Concanavalin-A affects glycosaminoglycans cellular and extracellular accumulation in cultured embryonic fibroblasts. Cell. Biol. Inf. Rep. 14, 641648. Mahuran D., Novak A. and Gravel J. A. (1985) The lysosomal hexosaminidase isozymes. Zsozymes Curr. Top. Biol. Med. Res. 12, 229-288. Orlacchio A., Maffei C., Emiliani C., Rambotti P. and Davis S. (1984). A distinct B-hexosaminidase isoenzyme separated from human leukemic lymphocytes and myelocytes. Biochem. biophys. Res. Commun. 122, 966-973. Overdijk E., Van Der Kroef W. M. J., Veltkamp W. A. and Hooghwinkel G. J. M. (1975) The separation of bovine brain b-N-acetyl-o-hexosaminidase. Biochem. J. 151, 257-26 1. Robinson D. and Stirling J. L. (1968) N-Acetylglucosaminidase in human spleen. Biochem. J. 107, 321-327. Swallow D. M., Evans L., Saha E. N. and Harris H. (1976) Characterization and tissue distribution of N-acctylhexosaminidase C: suggestive evidence for a separate hexosaminidase locus. Ann. Hum. Genef. 40, 5565.
