221
Clinica Chimica Acta, 93 (1979) 221-225 0 Elsevier~North-Holland Biomedical Press
CGA 10069
ISOELECTRIC FOCUSING OF ACID HYDROLASES FIBROBLASTS AND LEUCOCYTES
IN HUMAN
B. HULTBERG Department
of Clinical Chemistry,
University Hospital, S-221 8.5 Lund (Sweden)
(Received September 2&h, 1978)
Summary
Lysosomal hydrolases in fibrobl~~ and leucocytes exhibited the same isoelectric focusing pattern as found in liver. Treatment with neuraminidase had a great effect on the pattern of several glycosidases while treatment with alkaline phosphatase did not significantly change the pattern. These findings are discussed with special reference to the different “uptake” systems that exist for acid hydrolases.
Introduction Lysosomal hydrolases exist in several molecular forms. Recently I investigated acid hydrolases in serum and liver tissue and found that acid hydrolases in serum seemed to be more sialylated than those in liver tissue [I]. In this communication fibroblasts and leucocytes are investigated by isoelectric focusing, with special reference to the different “uptake” systems that exist for acid hydrolases [ 2-91, using alkaline phosphatase and neuraminidase treatment. The available information on normal isoelectric pattern in leucocytes and cultiva~d fibrobl~~ is rather insufficient [lo]. Another aim of this study therefore is to compare the activity profile of acid hydrolases obtained after isoelectric focusing of liver, serum, fibroblast and leucocyte preparations. Material and methods Isoelectric focusing, enzyme assay and neur~inidase treatment as described before [l] (see also legend to Fig. 1). Treatment of lysosomal enzymes with alkaline phosphatase was performed as described elsewhere [2]. Leucocyte preparation has been described earlier [ 111. The crude leucocyte suspension from 40 ml of blood in physiological saline (4 ml) was homogenized, frozen and thawed and thereafter dialysed
222
A 0.4
(5’. 10
20
, 30
40
Tube number
,I
,
50 Tube number
a2
10 Tube number
20
30
40
50 Tube number
223 against 10 mM sodium phosphate, pH 6.0 over night. The dialysate was ultracentrifuged (100 000 X g for 60 min). The supernatant was added to the isoelectric column. Fibroblast preparation has been described earlier [ 121. Fibroblasts from four confluent culture bottles (Falcon plastics, Los Angeles, U.S.A., volume 250 ml) were homogenized in 4 ml of aq. dest. and ultracentrifuged. The supernatant was added to the isoelectric column. Results The investigated hydrolases (Fig. 1) from fibroblasts and leucocytes preparations showed about the same molecular forms as seen in liver (Ref. 1 and unpublished findings). Also in these tissues the A-form of N-acetyl-fl-glucosaminidase was divided into two components (Fig. l), one form around pH 4.5 (serum form) and the other around pH 4.8-5.0 (tissue form). P-Glucuronidase seemed to have a predominance of the acidic forms (at pH 4.4) in fibroblasts, while in leucocytes and liver, forms around pH 4.8-5.0 dominated in the acidic peak of /3-glucuronidase activity (Fig. 1). In liver preparations the activity around pH 7.5 consisted about 70--80% of the total P-glucuronidase activity. The isofocusing pattern of N-acetyl-/3-glucosaminidase and ol-mannosidase showed no change after treatment with alkaline phosphatase. ,&Galactosidase activity was almost completely destroyed by the alkaline milieu itself. The pattern of @-glucuronidase seemed to have changed somewhat, at least in the fibroblast preparation. The activity around pH 4.4 had decreased and that of pH 4.8-5.0 and 7.5 showed some increase. However, even this discrepancy was very discrete, not more than 10% of the total &glucuronidase activity could have been affected. Neuraminidase treatment affected N-acetyl-@-glucosaminidase and CY-mannosidase as described earlier [ 11. Thus the serum form of iV-acetyl$-glucosaminidase A was transformed to about pH 5.5-5.7. The tissue form, was not affected. Acidic forms of a-mannosidase were transformed to pH 7.0-7.5. /3-Glucuronidase activity in the acidic peak was almost completely transformed to around pH 7.0-7.5. Acidic forms of fl-galactosidase were likewise changed to more basic forms. Fig. 1. Isoelectric focusing of acid hydrolases in fibroblasts (upper figure) and 1eucocYteS flower figure). Preparation of fibroblasts and leucocytes before applying to the column is described in methods. Column size was 110 ml (LKB products. Stockholm, Sweden). Ampholine used had a PH range of 43 (LKB products). Samples were electrofocused for 42 h. Voltage at the end of experiment 550 V and.the current 0.8 mA. 2-ml fractions were collected. For enzyme assay 100 ~1 of column fractions were incubated with 100 1.11of 1 mM solution of 4.methylumbelliferyl glycoside dissolved in citrate-phosphate buffer (100 mM citric acid/200 mM NaZHP04). Ac&ity is expressed as nmol substrate split per h under the conditions described above. Fig.
Enzyme
A B
N-Acetyl-fl-glucosaminidase wMannosidase (EC 3.2.1.24)
C D
&Galactosidase P-Glucuronidase
(EC 3.2.1.23) (EC 3.2.1.31)
(EC 3.2.1.30)
PH
Incubation
4.5 4.5 ( -) 5.5 (---) 4.5 4.5
120 180 180 180 180
time (min)
224
Discussion Recent evidence from several laboratories suggests that rapid in vivo clearance of glycoproteins (acid hydrolases) from serum is mediated by at least two distinct recognition systems. The work of Ashwell and colleagues [ 131 has revealed the hepatocyte-dependent receptor-mediated plasma clearance of galactose-terminal (i.e., asialo-) glycoproteins. Based on work from several laboratories [6-91 evidence for a second pathway has emerged. The latter accomodates many lysosomal hydrolases and glycoproteins having mannose and/or N-acetylglucosamine as their respective terminal sugars. It seems as if this receptor is connected to cells of the reticula endothelial system [9]. Thus, removal of sialic acid from glycoproteins (e.g. acid hydrolases) in sera, exposes the rest of their cabohydrate chain(s) to these receptors and the glycoproteins are immediately cleared from plasma. In this investigation and in the earlier report [l] it was also found that serum exhibited a predominance of acidic forms compared to those found in liver, fibroblasts and leucocytes (unpublished observation for P-galactosidase and P-glucuronidase). The dominance of acidic forms in serum probably reflected the rapid uptake of asialo-acid hydrolases. Fibroblasts, however, seem to internalize some of their secreted lysosomal hydrolases by another receptor system, namely a cell-surface receptor recognizing a phosphorylated carbohydrate residue, perhaps phosphomannose [ 2-51. The alkaline phosphatase treatment of the acid hydrolases in fresh fibroblasts and leucocyte preparations did not seem to significantly change the pattern of the different molecular forms on isofocusing. These findings were also found in serum and liver preparation (1). Thus the molecular forms were the same in liver and fibroblasts in spite of the different receptor systems in these two tissues. It seems that even in fibroblasts the phosphomannose groups are restricted to a very small fraction of the total population of enzyme molecules. The low level of enzymes with phosphomannose residues in the fibroblasts is expected, because once taken up the groups should be split off and the only enzymes with phosphomannose residues are those which just are being secreted. The charge heterogeneity of acid hydrolases in the tissues I have examined are thus mainly found because of sialic acid residues. The quantitative determination of acid hydrolases has been greatly simplified by the use of synthetic substrate. However, as a prerequisite for diagnosis of lysosomal storage diseases, the isoenzyme pattern of acid hydrolases in normal cells and body fluids, available for enzyme assay, should be known. One aim of this study has therefore been to study the activity profile of acid hydrolases obtained after isoelectric focusing of liver, serum, fibroblasts and leucocytes. References 1
Hultberg.
2
Ullrich,
K.,
B. (1978) Mersmann,
3
Kaplan,
A.,
Fischer,
D..
4
Kaplan,
A.,
Achord.
D. and
5
Sando,
G.N.
and
Clin.
Cbim.
G.,
Neufeld,
Acta
Wever.
Achord, Sly.
E.F.
88,
441-448
E. and D. and W.
(1977)
van Sly,
(1977) Cell
Figwa, W.
(1977)
Proc. 12,
Natl.
619427
K.
(1978)
Biochem.
J. Clin. Acad.
Invest.
Sci.
U.S.A.
60,
J. 170,
643-650
1088-1093 14.
2026-2030
225 6 Stahl,
P., Six, H., Rodman.
J.S.,
Schlesinger.
Acad. Sci. U.S.A. 73.4045-4049 7 Furbish, F.S., Steer, CJ.. Barranger,
J.A.,
P., Tulsiani. Jones.
E.A.
D.R.P. and
and Touter,
Brady,
R.O. (1978)
0.
(1976) Biochem.
Proc.
Natl.
Biophys.
Res. Commun. 81.1047-1053 8 Bearpark, T. and Stirling, J.L. (1977) Biochem. J. 168.435-439 9 Stahl. P.D., Rodman. J.S., Miller, M.J. and Schlesinger, P.H. (1978) Proc. Natl. Acad. Sci. U.S.A. 1399-1403 10 Christomanou, D.. Cap. C. and Sandhoff, K. (1977) Neuropiidiatrie 8.238-262 11 Hultberg. B.. Autio. S.. Berg, 8. and &kerman. P.A. (1973) Stand. J. Haematol. 10. 265-272 12 Hultberg. B., SjBblad. S. and &kerman. P.A. (1975) Acta Paediatr. 64, 123-131 13 Ashwell. G. and Morell, A. (1974) Adv. Enzymol. 41.99-128
75,
Clinica Chimica Acta, 93 (1979) 221-225 0 Elsevier~North-Holland Biomedical Press
CGA 10069
ISOELECTRIC FOCUSING OF ACID HYDROLASES FIBROBLASTS AND LEUCOCYTES
IN HUMAN
B. HULTBERG Department
of Clinical Chemistry,
University Hospital, S-221 8.5 Lund (Sweden)
(Received September 2&h, 1978)
Summary
Lysosomal hydrolases in fibrobl~~ and leucocytes exhibited the same isoelectric focusing pattern as found in liver. Treatment with neuraminidase had a great effect on the pattern of several glycosidases while treatment with alkaline phosphatase did not significantly change the pattern. These findings are discussed with special reference to the different “uptake” systems that exist for acid hydrolases.
Introduction Lysosomal hydrolases exist in several molecular forms. Recently I investigated acid hydrolases in serum and liver tissue and found that acid hydrolases in serum seemed to be more sialylated than those in liver tissue [I]. In this communication fibroblasts and leucocytes are investigated by isoelectric focusing, with special reference to the different “uptake” systems that exist for acid hydrolases [ 2-91, using alkaline phosphatase and neuraminidase treatment. The available information on normal isoelectric pattern in leucocytes and cultiva~d fibrobl~~ is rather insufficient [lo]. Another aim of this study therefore is to compare the activity profile of acid hydrolases obtained after isoelectric focusing of liver, serum, fibroblast and leucocyte preparations. Material and methods Isoelectric focusing, enzyme assay and neur~inidase treatment as described before [l] (see also legend to Fig. 1). Treatment of lysosomal enzymes with alkaline phosphatase was performed as described elsewhere [2]. Leucocyte preparation has been described earlier [ 111. The crude leucocyte suspension from 40 ml of blood in physiological saline (4 ml) was homogenized, frozen and thawed and thereafter dialysed
222
A 0.4
(5’. 10
20
, 30
40
Tube number
,I
,
50 Tube number
a2
10 Tube number
20
30
40
50 Tube number
223 against 10 mM sodium phosphate, pH 6.0 over night. The dialysate was ultracentrifuged (100 000 X g for 60 min). The supernatant was added to the isoelectric column. Fibroblast preparation has been described earlier [ 121. Fibroblasts from four confluent culture bottles (Falcon plastics, Los Angeles, U.S.A., volume 250 ml) were homogenized in 4 ml of aq. dest. and ultracentrifuged. The supernatant was added to the isoelectric column. Results The investigated hydrolases (Fig. 1) from fibroblasts and leucocytes preparations showed about the same molecular forms as seen in liver (Ref. 1 and unpublished findings). Also in these tissues the A-form of N-acetyl-fl-glucosaminidase was divided into two components (Fig. l), one form around pH 4.5 (serum form) and the other around pH 4.8-5.0 (tissue form). P-Glucuronidase seemed to have a predominance of the acidic forms (at pH 4.4) in fibroblasts, while in leucocytes and liver, forms around pH 4.8-5.0 dominated in the acidic peak of /3-glucuronidase activity (Fig. 1). In liver preparations the activity around pH 7.5 consisted about 70--80% of the total P-glucuronidase activity. The isofocusing pattern of N-acetyl-/3-glucosaminidase and ol-mannosidase showed no change after treatment with alkaline phosphatase. ,&Galactosidase activity was almost completely destroyed by the alkaline milieu itself. The pattern of @-glucuronidase seemed to have changed somewhat, at least in the fibroblast preparation. The activity around pH 4.4 had decreased and that of pH 4.8-5.0 and 7.5 showed some increase. However, even this discrepancy was very discrete, not more than 10% of the total &glucuronidase activity could have been affected. Neuraminidase treatment affected N-acetyl-@-glucosaminidase and CY-mannosidase as described earlier [ 11. Thus the serum form of iV-acetyl$-glucosaminidase A was transformed to about pH 5.5-5.7. The tissue form, was not affected. Acidic forms of a-mannosidase were transformed to pH 7.0-7.5. /3-Glucuronidase activity in the acidic peak was almost completely transformed to around pH 7.0-7.5. Acidic forms of fl-galactosidase were likewise changed to more basic forms. Fig. 1. Isoelectric focusing of acid hydrolases in fibroblasts (upper figure) and 1eucocYteS flower figure). Preparation of fibroblasts and leucocytes before applying to the column is described in methods. Column size was 110 ml (LKB products. Stockholm, Sweden). Ampholine used had a PH range of 43 (LKB products). Samples were electrofocused for 42 h. Voltage at the end of experiment 550 V and.the current 0.8 mA. 2-ml fractions were collected. For enzyme assay 100 ~1 of column fractions were incubated with 100 1.11of 1 mM solution of 4.methylumbelliferyl glycoside dissolved in citrate-phosphate buffer (100 mM citric acid/200 mM NaZHP04). Ac&ity is expressed as nmol substrate split per h under the conditions described above. Fig.
Enzyme
A B
N-Acetyl-fl-glucosaminidase wMannosidase (EC 3.2.1.24)
C D
&Galactosidase P-Glucuronidase
(EC 3.2.1.23) (EC 3.2.1.31)
(EC 3.2.1.30)
PH
Incubation
4.5 4.5 ( -) 5.5 (---) 4.5 4.5
120 180 180 180 180
time (min)
224
Discussion Recent evidence from several laboratories suggests that rapid in vivo clearance of glycoproteins (acid hydrolases) from serum is mediated by at least two distinct recognition systems. The work of Ashwell and colleagues [ 131 has revealed the hepatocyte-dependent receptor-mediated plasma clearance of galactose-terminal (i.e., asialo-) glycoproteins. Based on work from several laboratories [6-91 evidence for a second pathway has emerged. The latter accomodates many lysosomal hydrolases and glycoproteins having mannose and/or N-acetylglucosamine as their respective terminal sugars. It seems as if this receptor is connected to cells of the reticula endothelial system [9]. Thus, removal of sialic acid from glycoproteins (e.g. acid hydrolases) in sera, exposes the rest of their cabohydrate chain(s) to these receptors and the glycoproteins are immediately cleared from plasma. In this investigation and in the earlier report [l] it was also found that serum exhibited a predominance of acidic forms compared to those found in liver, fibroblasts and leucocytes (unpublished observation for P-galactosidase and P-glucuronidase). The dominance of acidic forms in serum probably reflected the rapid uptake of asialo-acid hydrolases. Fibroblasts, however, seem to internalize some of their secreted lysosomal hydrolases by another receptor system, namely a cell-surface receptor recognizing a phosphorylated carbohydrate residue, perhaps phosphomannose [ 2-51. The alkaline phosphatase treatment of the acid hydrolases in fresh fibroblasts and leucocyte preparations did not seem to significantly change the pattern of the different molecular forms on isofocusing. These findings were also found in serum and liver preparation (1). Thus the molecular forms were the same in liver and fibroblasts in spite of the different receptor systems in these two tissues. It seems that even in fibroblasts the phosphomannose groups are restricted to a very small fraction of the total population of enzyme molecules. The low level of enzymes with phosphomannose residues in the fibroblasts is expected, because once taken up the groups should be split off and the only enzymes with phosphomannose residues are those which just are being secreted. The charge heterogeneity of acid hydrolases in the tissues I have examined are thus mainly found because of sialic acid residues. The quantitative determination of acid hydrolases has been greatly simplified by the use of synthetic substrate. However, as a prerequisite for diagnosis of lysosomal storage diseases, the isoenzyme pattern of acid hydrolases in normal cells and body fluids, available for enzyme assay, should be known. One aim of this study has therefore been to study the activity profile of acid hydrolases obtained after isoelectric focusing of liver, serum, fibroblasts and leucocytes. References 1
Hultberg.
2
Ullrich,
K.,
B. (1978) Mersmann,
3
Kaplan,
A.,
Fischer,
D..
4
Kaplan,
A.,
Achord.
D. and
5
Sando,
G.N.
and
Clin.
Cbim.
G.,
Neufeld,
Acta
Wever.
Achord, Sly.
E.F.
88,
441-448
E. and D. and W.
(1977)
van Sly,
(1977) Cell
Figwa, W.
(1977)
Proc. 12,
Natl.
619427
K.
(1978)
Biochem.
J. Clin. Acad.
Invest.
Sci.
U.S.A.
60,
J. 170,
643-650
1088-1093 14.
2026-2030
225 6 Stahl,
P., Six, H., Rodman.
J.S.,
Schlesinger.
Acad. Sci. U.S.A. 73.4045-4049 7 Furbish, F.S., Steer, CJ.. Barranger,
J.A.,
P., Tulsiani. Jones.
E.A.
D.R.P. and
and Touter,
Brady,
R.O. (1978)
0.
(1976) Biochem.
Proc.
Natl.
Biophys.
Res. Commun. 81.1047-1053 8 Bearpark, T. and Stirling, J.L. (1977) Biochem. J. 168.435-439 9 Stahl. P.D., Rodman. J.S., Miller, M.J. and Schlesinger, P.H. (1978) Proc. Natl. Acad. Sci. U.S.A. 1399-1403 10 Christomanou, D.. Cap. C. and Sandhoff, K. (1977) Neuropiidiatrie 8.238-262 11 Hultberg. B.. Autio. S.. Berg, 8. and &kerman. P.A. (1973) Stand. J. Haematol. 10. 265-272 12 Hultberg. B., SjBblad. S. and &kerman. P.A. (1975) Acta Paediatr. 64, 123-131 13 Ashwell. G. and Morell, A. (1974) Adv. Enzymol. 41.99-128
75,
