Eq.
Eye Res. (1977) 25, 39-45
Lens Glycosidases in Human and Bovine Species II. The Isozymes of fi-Hexosaminidases in Human and Bovine Lens L.
POESARU*,
H.
SCALA;*
Y.
COURTOIS~
AND
J.-C.
DREYPCS”
The isozymes of /? hexosaminiclase were studied in extracts from human lens and from various parts of bovine lens. (1) Kinetic modifications were found, involving varktions in the specificity and in the pH activity curve. (2) Electrophoretic modifications were found, resulting in the prevalence of one band in the C position. (3) Bovine cultured cells from lens epithelium a.nd lung fibroblasts showed no band B. (4) The interpretation of the above facts are discussed in regard to the aging of proteins in the lens, and to the genetics of hexosaminidsses A and B in human and bovine species. age dependence; bovine; human glycosidases; Key ~uwds : lens; nucleus; cortex; !lexosaminidases; isozymes.
1. Introduction The activity of several glycosidases has been measured in extracts from lenses of various species (Carlin and Cotlier, 1971; Dre>fus, Poenaru and Skala, 1976) showing that active glycosidases are present in this tissue, but at very Iow levels. There is a steady decrease from the epithelial layer to the cortex and a further decrease to the nucleus of the lens. In the nucleus, proteins can remain for a very long time, undergoing post-translational changes during aging of the tissue. Among the glycosidasev only ,&hexosaminidases retain enough activity to make a qualitative study possible. Hexosaminidases possess several isozymes, which have been well studied in human and bovine species. Upon electrophoresis on cellulose acetate at pH 6.5, three bands can be recognized in both species (Poenaru and Dreyfus, 1973; Hooghwinkel, Veltkamp, Overdijk and Lisman, 1972). Two major bands have been called B and B. One minor band has been called C. It seems that the latter band consists of two different enzymes that can be separated or recognized by various procedures. We have undertaken a study of hexosaminidases in human and bovine lens, compa,ring their properties to those of other tissues. In addition, cells cultured from bovine lens have been used in order to compare in vivo to in vitro evolution. Characteristic changes have been observed in lens extracts, which we have tried to correlate wit,11 the relative longevity of the various isozymes. 2. Materials and Methods Lemes
Human lenses were obtained from operated cataracts or within 24 hr after death. Bovine lenses were obtained from the slaughterhouse and separated into three pnrtb: epithelial membrane, cortex and nucleus. As control tissue, iris was sometimes used as well as other bovine tissues. $ U.129 de 1’Institut National la Recherche Scient,ifique.
de la Ssnt6 et de la Recherche Mkdicale, LA 85 du Centre National 39
de
L. POENARU
40
ET AL.
Extraction was carried out in distilled water by grinding in a Potter Elvehjem followed by centrifugation at 20 000 xg for 10 min.
apparatus,
Chenzicals
Methylumbelliferyl from Merck.
derivatives
were from Koch-Light
Laboratories,
buffer reagents
Assay of enzymes Assays were performed as described previously (Leaback and Walker, 1961; Dreyfus and Poenaru, 1975). For pH activity curves we used a 0.4 M-Citrate phosphate buffer which was diluted with equal parts of a 2 mM-4-methylumbelliferyl ,&glucosaminide solution in water. Final concentration of buffer in the incubation test was 0.17 M. This relatively high concentration was necessary because of the high protein concentration of lens extracts. Electrophoresis Electrophoresis was performed on Cellogel according to Fluharty, Lassila, Porter and Kihara (1971) with minor modifications (Poenaru and Dreyfus, 1973). Buffer was 0.06 n;r-potassium phosphate pH 6.5. Electrophoresis lasted for 2 hr at 200 V. The cellulose acetate strip was then incubated by contact with a Whatman 3 MM paper impregnated with the substrate, generally 10-3~r-4-methylun~belliferyl-/3-glucosan~inide pH 4.5. After 15-30 min this paper was replaced by another one, impregnated with 1 MM-glycine buffer pH IO. Hexosaminidase appeared as fluorescent bands which were photographed with a Polaroid film (3000 ASA). In some cases the extra,cts were incubated for 2 hr prior to electrophoresis in the presence of 5 mg per ml of Concanavalin A (Sigma). Concanavalin A precipitates glycoproteins without inactivating them. They do not migrate and appear at the origin. Hexosamiuidase C is not a glycoprot,ein and its migration remains unchanged (Swallow, Evans, Saha and Harris, 1976).
The lens capsule was removed from the eye of freshly killed cows. The epithelial cells which remained attached to the capsule were trypsinized and cultured in Falcon Petri dishes as described previously (Hughes, Laurent, Lonchampt and Courtois, 1975) using a minimum essential medium containing 6% calf serum, 100 U/ml penicillin and 100 pg/ml streptomycin. When the cells reached confluency, they were subcultured using a trypsinEDTA mixture (EDTA 020 g, trypsin 0.50 g, glucose 1 g, bicarbonate 0.58 g, KC1 0.40 g and NaC18 g per litre). For these experiments, cells were grown in Roller Bottles (area 980 cm2) at 0.6 rev/min. Cells from first and fifth passages were used.
3. Results ContparisorL of the nctivifiies of glucosaminidnse glucosccmilziclase~ylactosiclase)
and galactosawinidase
(Yatio
Activity was assayed at various pHs and the results are given at optimal pH. They are summarized in Table I. (a) in extracts from human lenses the mean ratio at pH 5.0 was found to be 8.5. This value is intermediate to that of hexosaminidase A and B found to be 5 and that of hexosaminidase C, close to 20 (Penton, Poenaru and Dreyfus, 1975).
HEXOXAMINIDBSES
IN
HUMAN TABLE
AND
BOVINE
41
LENS
I
Activity of l~,ezosnnainidasesin human ,whole lens ad;
in beef lens epithelium nanomoles per hour per mg protein at 37°C
Human lens Glucosaminidase
Mean
2.40 2.70 I.10 3.00 1.45 2-ll+-0.85
Glu c;ral
8,5
Beef epithelium
and cortex:
Beef cortex
Galactosaminidase
GIUCOSaminidase
Galactosaminidase
Glucosaminidase
0.27 0.29
200 97 13.5 148
59 22 31 59
8.00 6.00 3.00 1.20
4.4 3.8 2.0 1.0
145+43
43flS
4Y%G&3~0
243* 1.5
0.11 0.39 0.18 0.24S*O.l
Galactosaminidase
1.63
3.37
(b) in extracts of bovine lenses the mean ratio was 3.37 jn the epithelium and l-63 in the cortex. In t,his case the gaIactosaminidase activity was relatively higher than in the other tissues.
Figure l(a) shows the effect of pH on hexosaminidase activity in man. The curve with. a control organ had a maximum at pH 4.5 and decreased rapidly on the alkaline side. The maximum in lens was shifted to pH 5. The decrease at more alkaline pHs was slower. Only glucosaminidase was tested on account. of the very 10~ level of galactosaminidase in human lens. Figure I(b) shows the results in beef lens for 100 -
4.0
4.5
50
55
GO
4.0
6.5
45
5.0
5.5
6.0
6.:
PH
FIG. 1. Efkct of pH on hexosaminidase activity in (a) Human lens and (b) bovine lens. (a) Human white blood cells (u - - - n ), lens (0-a). (b) B ovine epithelium glu (O-e), gal (0-O); cortex glu (A - - - A)- gal (a - - - A).
epithelial and cortex hexosaminidases. In the epithelium the maximum of activity was at pH 5.0 for both glucosaminidase and galactosaminidase. A rather sharp decrease occurred towards alkaline pHs. In lens cortex a considerable shift towards alkaline pHs was observed, with a maximum at pH 5.5 (galactosaminidase) and 6.0 (glucosaminidase). Activities at acid pHs were very low. The shift seemed to be great.er for glucosaminidase than for galactosaminidase.
41
L. POENARU
ET AL.
A
8
2
1
FIG. 3. @ucosaminidase electrophoresis concanavalin A; (4) Tny-Sachs leucocytes;
3
4
5
7
6
-
of hun1a.n: (1) adult brain: (2) fetal brain; (8) fetal brain ; (5) Sandhoff leucocytes; (6) lens; (7) lens+concavanalin A.
Eleetrophoresis (a) Man. The results are shown in Fig. 2. In normal organs three bands are seen, reading from cathode to anode. They are B, B and C (lane 1). Isozyme C is much less visible than the others, except in fetal organs, especially in fetal brain (lane 2). In Tay-Sachs disease there is no band A (lane 4)$ and in Sandhoff’s disease no band A and B, while band C becomes accentuated (lane 5). Lens extracts showed a major band at the position of hexosaminidase C (lane 6). In some extracts a faint additional band could be seen at a position intermediary’ between bands B and 6. When tissue extracts were preincubated with
CO
I
2
3
4
5
6
7
8
9
IO
-
FIG. 3. ,B-glueosaminidase electrophoresis in beef: (1) brain ; (2) heart; (3) lung; (4) leucocytes; (5) Iens epithelium; (6) lens cortex: (7) lens cortex+conoanavalin A: (8) fibroblasts from lung; (9) cultured cells from lens first passage; (10) cultured cells from lens 5th passage.
HEXOSAMINIDASES
IK
HUMAN
AND
BOVINE
LEBS
43
Concanavalin, bands ,4 and B disa,ppeared and only C remained unchanged (lane 3). Band C was attenuated in lens extracts, but did not disappear (lane 5). (1)) Berf. The results are shown in Fig. 3. In most organs (lane 1 to 4) the picture was comparable to that of human tissues, with a t’hree-banded pattern, band C showing up as particularly intense in brain and heart extracts. In our bands, A was closer to B, and farther from C, in bovine as compared to human extracts. Epithelial membrane extracts showed only A and B with a preponderance of A and practically no C (lane 5). In contrast, cortex extracts $homed only one band in the C position (lane 6). Addition of concanavalin A suppressed bands A and B and decreased intensity of ba.nd C. In cortex extracts, the fast band ,&owed a decrease of intensity but did not disappear (lane 7). No picture could be taken of lens nucleus extracts, which showed only a very faint band in the position of band C. Cultured bovine cells showed a peculiar pattern: lanes 8: 9 and 10 respectively show the result of electrophoresis of fibroblasts (cultured from embryonic lung), lens cells at first passa,ge,and lens cells at fifth passage. In all cases one major band was observed, in the position of ba,nd 9, and one minor in the position of band C. No band B was seen. 4. Discussion The present work has demonstrated the following facts: (1) Modifications of hexosaminidase kinetics appeared in lens extracts. They involved variation in the specificity (activities towards glucosaminides and galactosaminides) and in the pH activity curve. (2) Major electrophoretic modifications were found. Both in human and bovine lens the two major bands more or less disappeared, and only one band remained, in the posit#ion of band C. The situation is even paradoxical in bovine lenses, since band C was not apparent in extracts of epithelial cells. (3) Bovine cultured cells originating from lung or from lens epithelium from short term and long term cultures, showed no band B. This contrasts with the findings for human species in which cultured cells (fibroblasts or amniotic cells) show the same hexosaminidase pattern as other tissues. These results can be discussed from two points of view: (1) effect of age on hexosarninida’se in the lens, (2) compared genetics of hexosaminidases in man and ox. The hexosaminidase system in man is relatively well known. The most probable interpretation of the data is that of Beutler and Kuhl (1975) : hexosaminidase B is a homopolpmer made of identical subunits ,L?;hexosaminidase A is a heteropolymer made of a combination of subunits p and c(. The cx homopolymer migrates to the position of band C. This may explain what happens in diseases : in Tay-Sachs disease subunit u is not synthesized and band 9 disappears; in Sandhoff’s disease subunit ,8 is lacking, and excess of a reinforces the fast band. This does not explain the persistence of band C in Tay-Sachs disease (Penton, Poenaru and Dreyfus, 1975). Hexosaminidase C indeed has a different, non lysosomal origin; its optimum pII activity is neutral, and it has nearly no galactosaminidase activity; so the homopolymer ‘J.(n) has been called hex S. Hex C and hex S isozymes migrate identically in most electrophoretic systems. They could be separat’ed in a complex discontinuous Suffer, and by the fact that form 8, but not C, binds to Concanavalin A (Swallow et al., 1976). The bovine hexosaminidase system is at least as complex as the human one; but is
44
L. POENARU
ET AL.
less well understood. Frohwein and Gatt (1967) were the first to separate two fomw of hexosaminidase from beef spleen. 9 third form was described by Hooghwinkel et al. (1972). Overdijk, Van der Kreef, Veltkamp and Hooghwinkel (1975) recently showed that it is possible to separate two forms, one acting on glucosaminide, called type C, the other acting on galactosaminide, called type D, which could be homologous to the human S enzyme. Both bovine and human enzymes could then be regarded as a set of three isozymes resulting from the combination of two types of subunits, 0: and p, while t.ype C would be genetically quite distinct. Lens extracts, both of human and bovine origin, retain essentially the fast band. In human lens extracts; the low activity towards galactosaminide, and the shift of optimum pH towards a more alkaline zone, point to a predominance of hexosaminidase C. After electrophoresis, human lens extracts could not be stained with galactosaminide as substrate. However Concanavalin A decreased the activity of this fast band. SO it must be concluded that either we are dealing with a mixture of forms C and S, or form C alone remains but has been somewhat modified. The persistence of hex C alone is somewhat paradoxical, since in vitro at least this isozyme is more labile than the others. Whether the prevalence of hex C indicates that this enzyme plays a biological role in the lens is still unknown. It should be remembered that the natural substrates of hexosaminidases do accumulate in the case of hex A and B deficiency (Sandhoff disease). The electrophoretic picture of bovine lens extracts is,quite similar to that obtained with human extracts, and only the fast band remains visible. High activity towards galactosaminide, partial disappearance in the presence of Concanavalin A; shift of pH activity curve towards alkaline zone, also point to a mixture of forms C and D. In addition several problems arise which complicate the int’erpretation. The first problem is that in extracts from the epithelial layer we did not see the fast bands, and only bands A and B are apparent. Unless synthesis of band C takes place in the cortex itself and not in the epithelium, either band A or B has been modified during aging, or a C or D form is present in a concentration too low to be visible and is concentrated later in the lens cortex. A more difficult and general problem arises from the observations made on cultured cells. Indeed, the same results were obtained with all cells which were assayed. It is known that bovine lens cells become transformed after a. few passages. We therefore examined both non-transformed cells, at first and fifth passages, and transformed cells after 15 passages, as well as lung fibroblasts in culture (Fig. 3). In all cases the picture was the same. Cultured cells showed no visible B band, but one major band in the A position, and a minor band in the C position. This observation leads us to question the genetic make up of hexosaminidases in the bovine species. If hexosaminidase B is the p,8 h omopolymer, it appears difficult to understand how this homopolymer can be absent in cultured cells, since subunit p is necessarily synthesized as a contribution to the G$ hybrid, hexosaminidase A. In conclusion, our work demonstrates that the in vivo pattern of lens hexosaminidase isozymes undergoes considerable change which may alter the physiological function of the enzyme. In vitro, the observed modifications in cells derived from lens cells or lung lead us to question the genetic relationships of human and bovine hexosaminidases. ACKNOWLEDGMENTS
This work was supported bp a grant INSERM No. 14.75.37. We tha’nk J. Tassin for skilful technical help.
HEXOSAMIKIDASES
IN
HUMAN
AKD
BOVIriE
LENS
45
REFERENCES verified: interBeutler. E. and Kuhl, W. (1975). Su b unit structure of human hexosaminidase convertibility of hexosaminidase isozymes. Nature, Lond. 258, 2624. Carlin, R. and Cotlier, E. (1971). Glycosidases of the crystalline lens. Inwst. Ophthalmol. 10 887-97. Dreyfus, J. C. and Poenaru, L. (1975). Le diagnostic enzymatique dans les maladies lysosomiales Arch. Franc. Pkdiat. 32, 503-14. Dreyfus, J. C., Poenaru, L. and Skala, H. (1977). Lens glycosidases in human and bovine lens. Exp. Eye Res. 25, 47-51. Pluhart,y, A. L., Lassila, E. L., Porter, M. T. and Kihara, H. (1971). The electrophoretic separation of human P-galactosidases on cellulose acetate. Biochem. Med. 5, 158-64. Frohwein. Y. Z. and Gatt, S. (1967). Evolution of /3-N-acetylhexosaminidase, P-N-acetylglucosaminidase and /3-N-acetylgalactosaminidase from calf brain. Biochemistry 6, 2775-82. Hooghwinkel, G. J. M., Veltkamp, W. A., Overdijk, B. and Lisman, J. J. W. (1972). Electrophoretic separation of P-N-acetylhexosaminidases of human and bovine brain and liver and of Tay-Sachs brain tissue. Hoppe-Seyler’s 2. Ph.ysiol. Chem. 353, 83941. Hughes, R. C., Laurent, M., Lonchampt. M. 0. and Courtois, Y. (1975). Lens glycoproteins: Biosynthesis in cultured epithelial cells of bovine lens. Eur. J. Biochem. 52, 143-55. Leaback, D. H. and Walker, P. G. (1961). Studies on glucosaminidase. 4: The fluorimetric assay of N-acetyl-P-glucosaminidase. Biochem. J. 78, 151-6. Overdijk, B., Van der Kroef, W. M. J., Veltkamp, W. A. and Hooghwinkel, G. J. M. (1975). The separation of bovine brain /?-N-acetyl-n-hexosaminidases. Bioch,em. J. 151, 257-61. Penton, E., Poenaru, L. and Dreyfus, J. C. (1975). Hexosaminidase C in Tay-Sachs and Sandhoff disease. Biochim. Biophys. Acta 391, 162-9. Poenaru. L. and Dreyfus, J. C. (1973). Electrophoretic study of hexosaminidases: Hexosaminidase C. Clin. Chim. Acta 43, 43942. Swallow, D. M.. Evans, L., Saha, N. and Harris, H. (1976). Characterization and tissue distribution of N-acetyl hexosaminidase 6: suggestive evidence for a separate hexosaminidase locus. Ann. Hum,. Genet., London 40, 55-66.
Eye Res. (1977) 25, 39-45
Lens Glycosidases in Human and Bovine Species II. The Isozymes of fi-Hexosaminidases in Human and Bovine Lens L.
POESARU*,
H.
SCALA;*
Y.
COURTOIS~
AND
J.-C.
DREYPCS”
The isozymes of /? hexosaminiclase were studied in extracts from human lens and from various parts of bovine lens. (1) Kinetic modifications were found, involving varktions in the specificity and in the pH activity curve. (2) Electrophoretic modifications were found, resulting in the prevalence of one band in the C position. (3) Bovine cultured cells from lens epithelium a.nd lung fibroblasts showed no band B. (4) The interpretation of the above facts are discussed in regard to the aging of proteins in the lens, and to the genetics of hexosaminidsses A and B in human and bovine species. age dependence; bovine; human glycosidases; Key ~uwds : lens; nucleus; cortex; !lexosaminidases; isozymes.
1. Introduction The activity of several glycosidases has been measured in extracts from lenses of various species (Carlin and Cotlier, 1971; Dre>fus, Poenaru and Skala, 1976) showing that active glycosidases are present in this tissue, but at very Iow levels. There is a steady decrease from the epithelial layer to the cortex and a further decrease to the nucleus of the lens. In the nucleus, proteins can remain for a very long time, undergoing post-translational changes during aging of the tissue. Among the glycosidasev only ,&hexosaminidases retain enough activity to make a qualitative study possible. Hexosaminidases possess several isozymes, which have been well studied in human and bovine species. Upon electrophoresis on cellulose acetate at pH 6.5, three bands can be recognized in both species (Poenaru and Dreyfus, 1973; Hooghwinkel, Veltkamp, Overdijk and Lisman, 1972). Two major bands have been called B and B. One minor band has been called C. It seems that the latter band consists of two different enzymes that can be separated or recognized by various procedures. We have undertaken a study of hexosaminidases in human and bovine lens, compa,ring their properties to those of other tissues. In addition, cells cultured from bovine lens have been used in order to compare in vivo to in vitro evolution. Characteristic changes have been observed in lens extracts, which we have tried to correlate wit,11 the relative longevity of the various isozymes. 2. Materials and Methods Lemes
Human lenses were obtained from operated cataracts or within 24 hr after death. Bovine lenses were obtained from the slaughterhouse and separated into three pnrtb: epithelial membrane, cortex and nucleus. As control tissue, iris was sometimes used as well as other bovine tissues. $ U.129 de 1’Institut National la Recherche Scient,ifique.
de la Ssnt6 et de la Recherche Mkdicale, LA 85 du Centre National 39
de
L. POENARU
40
ET AL.
Extraction was carried out in distilled water by grinding in a Potter Elvehjem followed by centrifugation at 20 000 xg for 10 min.
apparatus,
Chenzicals
Methylumbelliferyl from Merck.
derivatives
were from Koch-Light
Laboratories,
buffer reagents
Assay of enzymes Assays were performed as described previously (Leaback and Walker, 1961; Dreyfus and Poenaru, 1975). For pH activity curves we used a 0.4 M-Citrate phosphate buffer which was diluted with equal parts of a 2 mM-4-methylumbelliferyl ,&glucosaminide solution in water. Final concentration of buffer in the incubation test was 0.17 M. This relatively high concentration was necessary because of the high protein concentration of lens extracts. Electrophoresis Electrophoresis was performed on Cellogel according to Fluharty, Lassila, Porter and Kihara (1971) with minor modifications (Poenaru and Dreyfus, 1973). Buffer was 0.06 n;r-potassium phosphate pH 6.5. Electrophoresis lasted for 2 hr at 200 V. The cellulose acetate strip was then incubated by contact with a Whatman 3 MM paper impregnated with the substrate, generally 10-3~r-4-methylun~belliferyl-/3-glucosan~inide pH 4.5. After 15-30 min this paper was replaced by another one, impregnated with 1 MM-glycine buffer pH IO. Hexosaminidase appeared as fluorescent bands which were photographed with a Polaroid film (3000 ASA). In some cases the extra,cts were incubated for 2 hr prior to electrophoresis in the presence of 5 mg per ml of Concanavalin A (Sigma). Concanavalin A precipitates glycoproteins without inactivating them. They do not migrate and appear at the origin. Hexosamiuidase C is not a glycoprot,ein and its migration remains unchanged (Swallow, Evans, Saha and Harris, 1976).
The lens capsule was removed from the eye of freshly killed cows. The epithelial cells which remained attached to the capsule were trypsinized and cultured in Falcon Petri dishes as described previously (Hughes, Laurent, Lonchampt and Courtois, 1975) using a minimum essential medium containing 6% calf serum, 100 U/ml penicillin and 100 pg/ml streptomycin. When the cells reached confluency, they were subcultured using a trypsinEDTA mixture (EDTA 020 g, trypsin 0.50 g, glucose 1 g, bicarbonate 0.58 g, KC1 0.40 g and NaC18 g per litre). For these experiments, cells were grown in Roller Bottles (area 980 cm2) at 0.6 rev/min. Cells from first and fifth passages were used.
3. Results ContparisorL of the nctivifiies of glucosaminidnse glucosccmilziclase~ylactosiclase)
and galactosawinidase
(Yatio
Activity was assayed at various pHs and the results are given at optimal pH. They are summarized in Table I. (a) in extracts from human lenses the mean ratio at pH 5.0 was found to be 8.5. This value is intermediate to that of hexosaminidase A and B found to be 5 and that of hexosaminidase C, close to 20 (Penton, Poenaru and Dreyfus, 1975).
HEXOXAMINIDBSES
IN
HUMAN TABLE
AND
BOVINE
41
LENS
I
Activity of l~,ezosnnainidasesin human ,whole lens ad;
in beef lens epithelium nanomoles per hour per mg protein at 37°C
Human lens Glucosaminidase
Mean
2.40 2.70 I.10 3.00 1.45 2-ll+-0.85
Glu c;ral
8,5
Beef epithelium
and cortex:
Beef cortex
Galactosaminidase
GIUCOSaminidase
Galactosaminidase
Glucosaminidase
0.27 0.29
200 97 13.5 148
59 22 31 59
8.00 6.00 3.00 1.20
4.4 3.8 2.0 1.0
145+43
43flS
4Y%G&3~0
243* 1.5
0.11 0.39 0.18 0.24S*O.l
Galactosaminidase
1.63
3.37
(b) in extracts of bovine lenses the mean ratio was 3.37 jn the epithelium and l-63 in the cortex. In t,his case the gaIactosaminidase activity was relatively higher than in the other tissues.
Figure l(a) shows the effect of pH on hexosaminidase activity in man. The curve with. a control organ had a maximum at pH 4.5 and decreased rapidly on the alkaline side. The maximum in lens was shifted to pH 5. The decrease at more alkaline pHs was slower. Only glucosaminidase was tested on account. of the very 10~ level of galactosaminidase in human lens. Figure I(b) shows the results in beef lens for 100 -
4.0
4.5
50
55
GO
4.0
6.5
45
5.0
5.5
6.0
6.:
PH
FIG. 1. Efkct of pH on hexosaminidase activity in (a) Human lens and (b) bovine lens. (a) Human white blood cells (u - - - n ), lens (0-a). (b) B ovine epithelium glu (O-e), gal (0-O); cortex glu (A - - - A)- gal (a - - - A).
epithelial and cortex hexosaminidases. In the epithelium the maximum of activity was at pH 5.0 for both glucosaminidase and galactosaminidase. A rather sharp decrease occurred towards alkaline pHs. In lens cortex a considerable shift towards alkaline pHs was observed, with a maximum at pH 5.5 (galactosaminidase) and 6.0 (glucosaminidase). Activities at acid pHs were very low. The shift seemed to be great.er for glucosaminidase than for galactosaminidase.
41
L. POENARU
ET AL.
A
8
2
1
FIG. 3. @ucosaminidase electrophoresis concanavalin A; (4) Tny-Sachs leucocytes;
3
4
5
7
6
-
of hun1a.n: (1) adult brain: (2) fetal brain; (8) fetal brain ; (5) Sandhoff leucocytes; (6) lens; (7) lens+concavanalin A.
Eleetrophoresis (a) Man. The results are shown in Fig. 2. In normal organs three bands are seen, reading from cathode to anode. They are B, B and C (lane 1). Isozyme C is much less visible than the others, except in fetal organs, especially in fetal brain (lane 2). In Tay-Sachs disease there is no band A (lane 4)$ and in Sandhoff’s disease no band A and B, while band C becomes accentuated (lane 5). Lens extracts showed a major band at the position of hexosaminidase C (lane 6). In some extracts a faint additional band could be seen at a position intermediary’ between bands B and 6. When tissue extracts were preincubated with
CO
I
2
3
4
5
6
7
8
9
IO
-
FIG. 3. ,B-glueosaminidase electrophoresis in beef: (1) brain ; (2) heart; (3) lung; (4) leucocytes; (5) Iens epithelium; (6) lens cortex: (7) lens cortex+conoanavalin A: (8) fibroblasts from lung; (9) cultured cells from lens first passage; (10) cultured cells from lens 5th passage.
HEXOSAMINIDASES
IK
HUMAN
AND
BOVINE
LEBS
43
Concanavalin, bands ,4 and B disa,ppeared and only C remained unchanged (lane 3). Band C was attenuated in lens extracts, but did not disappear (lane 5). (1)) Berf. The results are shown in Fig. 3. In most organs (lane 1 to 4) the picture was comparable to that of human tissues, with a t’hree-banded pattern, band C showing up as particularly intense in brain and heart extracts. In our bands, A was closer to B, and farther from C, in bovine as compared to human extracts. Epithelial membrane extracts showed only A and B with a preponderance of A and practically no C (lane 5). In contrast, cortex extracts $homed only one band in the C position (lane 6). Addition of concanavalin A suppressed bands A and B and decreased intensity of ba.nd C. In cortex extracts, the fast band ,&owed a decrease of intensity but did not disappear (lane 7). No picture could be taken of lens nucleus extracts, which showed only a very faint band in the position of band C. Cultured bovine cells showed a peculiar pattern: lanes 8: 9 and 10 respectively show the result of electrophoresis of fibroblasts (cultured from embryonic lung), lens cells at first passa,ge,and lens cells at fifth passage. In all cases one major band was observed, in the position of ba,nd 9, and one minor in the position of band C. No band B was seen. 4. Discussion The present work has demonstrated the following facts: (1) Modifications of hexosaminidase kinetics appeared in lens extracts. They involved variation in the specificity (activities towards glucosaminides and galactosaminides) and in the pH activity curve. (2) Major electrophoretic modifications were found. Both in human and bovine lens the two major bands more or less disappeared, and only one band remained, in the posit#ion of band C. The situation is even paradoxical in bovine lenses, since band C was not apparent in extracts of epithelial cells. (3) Bovine cultured cells originating from lung or from lens epithelium from short term and long term cultures, showed no band B. This contrasts with the findings for human species in which cultured cells (fibroblasts or amniotic cells) show the same hexosaminidase pattern as other tissues. These results can be discussed from two points of view: (1) effect of age on hexosarninida’se in the lens, (2) compared genetics of hexosaminidases in man and ox. The hexosaminidase system in man is relatively well known. The most probable interpretation of the data is that of Beutler and Kuhl (1975) : hexosaminidase B is a homopolpmer made of identical subunits ,L?;hexosaminidase A is a heteropolymer made of a combination of subunits p and c(. The cx homopolymer migrates to the position of band C. This may explain what happens in diseases : in Tay-Sachs disease subunit u is not synthesized and band 9 disappears; in Sandhoff’s disease subunit ,8 is lacking, and excess of a reinforces the fast band. This does not explain the persistence of band C in Tay-Sachs disease (Penton, Poenaru and Dreyfus, 1975). Hexosaminidase C indeed has a different, non lysosomal origin; its optimum pII activity is neutral, and it has nearly no galactosaminidase activity; so the homopolymer ‘J.(n) has been called hex S. Hex C and hex S isozymes migrate identically in most electrophoretic systems. They could be separat’ed in a complex discontinuous Suffer, and by the fact that form 8, but not C, binds to Concanavalin A (Swallow et al., 1976). The bovine hexosaminidase system is at least as complex as the human one; but is
44
L. POENARU
ET AL.
less well understood. Frohwein and Gatt (1967) were the first to separate two fomw of hexosaminidase from beef spleen. 9 third form was described by Hooghwinkel et al. (1972). Overdijk, Van der Kreef, Veltkamp and Hooghwinkel (1975) recently showed that it is possible to separate two forms, one acting on glucosaminide, called type C, the other acting on galactosaminide, called type D, which could be homologous to the human S enzyme. Both bovine and human enzymes could then be regarded as a set of three isozymes resulting from the combination of two types of subunits, 0: and p, while t.ype C would be genetically quite distinct. Lens extracts, both of human and bovine origin, retain essentially the fast band. In human lens extracts; the low activity towards galactosaminide, and the shift of optimum pH towards a more alkaline zone, point to a predominance of hexosaminidase C. After electrophoresis, human lens extracts could not be stained with galactosaminide as substrate. However Concanavalin A decreased the activity of this fast band. SO it must be concluded that either we are dealing with a mixture of forms C and S, or form C alone remains but has been somewhat modified. The persistence of hex C alone is somewhat paradoxical, since in vitro at least this isozyme is more labile than the others. Whether the prevalence of hex C indicates that this enzyme plays a biological role in the lens is still unknown. It should be remembered that the natural substrates of hexosaminidases do accumulate in the case of hex A and B deficiency (Sandhoff disease). The electrophoretic picture of bovine lens extracts is,quite similar to that obtained with human extracts, and only the fast band remains visible. High activity towards galactosaminide, partial disappearance in the presence of Concanavalin A; shift of pH activity curve towards alkaline zone, also point to a mixture of forms C and D. In addition several problems arise which complicate the int’erpretation. The first problem is that in extracts from the epithelial layer we did not see the fast bands, and only bands A and B are apparent. Unless synthesis of band C takes place in the cortex itself and not in the epithelium, either band A or B has been modified during aging, or a C or D form is present in a concentration too low to be visible and is concentrated later in the lens cortex. A more difficult and general problem arises from the observations made on cultured cells. Indeed, the same results were obtained with all cells which were assayed. It is known that bovine lens cells become transformed after a. few passages. We therefore examined both non-transformed cells, at first and fifth passages, and transformed cells after 15 passages, as well as lung fibroblasts in culture (Fig. 3). In all cases the picture was the same. Cultured cells showed no visible B band, but one major band in the A position, and a minor band in the C position. This observation leads us to question the genetic make up of hexosaminidases in the bovine species. If hexosaminidase B is the p,8 h omopolymer, it appears difficult to understand how this homopolymer can be absent in cultured cells, since subunit p is necessarily synthesized as a contribution to the G$ hybrid, hexosaminidase A. In conclusion, our work demonstrates that the in vivo pattern of lens hexosaminidase isozymes undergoes considerable change which may alter the physiological function of the enzyme. In vitro, the observed modifications in cells derived from lens cells or lung lead us to question the genetic relationships of human and bovine hexosaminidases. ACKNOWLEDGMENTS
This work was supported bp a grant INSERM No. 14.75.37. We tha’nk J. Tassin for skilful technical help.
HEXOSAMIKIDASES
IN
HUMAN
AKD
BOVIriE
LENS
45
REFERENCES verified: interBeutler. E. and Kuhl, W. (1975). Su b unit structure of human hexosaminidase convertibility of hexosaminidase isozymes. Nature, Lond. 258, 2624. Carlin, R. and Cotlier, E. (1971). Glycosidases of the crystalline lens. Inwst. Ophthalmol. 10 887-97. Dreyfus, J. C. and Poenaru, L. (1975). Le diagnostic enzymatique dans les maladies lysosomiales Arch. Franc. Pkdiat. 32, 503-14. Dreyfus, J. C., Poenaru, L. and Skala, H. (1977). Lens glycosidases in human and bovine lens. Exp. Eye Res. 25, 47-51. Pluhart,y, A. L., Lassila, E. L., Porter, M. T. and Kihara, H. (1971). The electrophoretic separation of human P-galactosidases on cellulose acetate. Biochem. Med. 5, 158-64. Frohwein. Y. Z. and Gatt, S. (1967). Evolution of /3-N-acetylhexosaminidase, P-N-acetylglucosaminidase and /3-N-acetylgalactosaminidase from calf brain. Biochemistry 6, 2775-82. Hooghwinkel, G. J. M., Veltkamp, W. A., Overdijk, B. and Lisman, J. J. W. (1972). Electrophoretic separation of P-N-acetylhexosaminidases of human and bovine brain and liver and of Tay-Sachs brain tissue. Hoppe-Seyler’s 2. Ph.ysiol. Chem. 353, 83941. Hughes, R. C., Laurent, M., Lonchampt. M. 0. and Courtois, Y. (1975). Lens glycoproteins: Biosynthesis in cultured epithelial cells of bovine lens. Eur. J. Biochem. 52, 143-55. Leaback, D. H. and Walker, P. G. (1961). Studies on glucosaminidase. 4: The fluorimetric assay of N-acetyl-P-glucosaminidase. Biochem. J. 78, 151-6. Overdijk, B., Van der Kroef, W. M. J., Veltkamp, W. A. and Hooghwinkel, G. J. M. (1975). The separation of bovine brain /?-N-acetyl-n-hexosaminidases. Bioch,em. J. 151, 257-61. Penton, E., Poenaru, L. and Dreyfus, J. C. (1975). Hexosaminidase C in Tay-Sachs and Sandhoff disease. Biochim. Biophys. Acta 391, 162-9. Poenaru. L. and Dreyfus, J. C. (1973). Electrophoretic study of hexosaminidases: Hexosaminidase C. Clin. Chim. Acta 43, 43942. Swallow, D. M.. Evans, L., Saha, N. and Harris, H. (1976). Characterization and tissue distribution of N-acetyl hexosaminidase 6: suggestive evidence for a separate hexosaminidase locus. Ann. Hum,. Genet., London 40, 55-66.
