Synthesis of lysosomal α-mannosidase in normal and mannosidosis fibroblasts

Vol. 115, No. 3, 1983

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September 30, 1983

Pages 1 083-1089

SYNTHESIS OF LYSOSOMAL e-MANNOSIDASE IN NORMAL AND MANNOSIDOSIS FIB~OBLASTS R. Pohlmann, A. Hasilik, S. Cheng +, S. Pemble +, B. Winchester + and K. von Figura Institute of Physiological C~emistry, University of Muenster, FRG, +Department of Biochemistry, Queen Elizabeth College, University of Lcndon, Campden Hill Road, Lcndon W8 7AH, England

Received August 22, 1983

SU~4ARY: The biosynthesis and secretion of lysoscmal e-nmnnosidase was studied in metabolically labelled fibrd01asts frcm ccntrols and two patients with mannosidosis. Nompal fibroblasts secrete ~-mannosidase as a 110kDa polypeptide. Intracellularly ~-mannosidase is represented by several polypeptides with apparent M s ranging frcm 40 to 67kDa. In two mannosidosis cell lines none of intra- a~rd extracellular polypeptides of ~-mannosidase were detectable. The mannosidosis fibroblasts secreted acid ~-mannosidase activity at one third of the normal rate. In contrast to normal cells the secretion was not enhanced by NH.CI and the secreted activity was not immunoprecipitable, indicating that th4 acid ~-mannosldase activity secreted by mannosidosis fibroblasts is not related to the lysescmal ~-mannosidase.

INTRODLL-~ION: Mannosidosis is an autosomal recessive tran~nitted storage disease, in which the deficiency in the acid hydrolase ~-D-mannosidase

(EC 3.2. I.

24) leads to intralysosomal acc~nulaticn and massive urinary excretion of mannose-rich oligosaccharides (I ,2). The ~-mannosidase activities with neutral or intermediate pH optima localized in Golgi membranes (3,4) are normal in mannosidosis (5-7, 15). Heterogeneity within htmmn mannosidosis is suggested both clinically (2) and biochemically. Patients with high residual activity (8, 9), With (10, 11) or without (12) crossreacting material have been described. In ccntrast to intracellular deficiency an apparently t~laffected acid ~-mannosidase activity is forbid in the culture medit~n of fibroblasts (I 3-15). These results have been interpreted to indicate normal synthesis and partial secreticn of acid ~-mannosidase and loss of enzyme activity after delivery to lysosc~es (15). In the present study we report on the biosynthesis and processing of acid ~-

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mannosidase in ccntrol and mannosidosis fibroblasts. Our findings indicate that the synthesis of acid e-mannosidase was deficient in the two mannosidosis cell lines studies and that the e-mannosidase activity secreted by these cells is immunologically not related to the lysosoaml a-mannosidase. METHODS: Cell culture: Hunan diploid fibroblasts were maintained at 37°C in 5 % CO 9 in Eagle's minimal essential medimn supplemented with antibiotics, non essentYal amino acids and 7.5 % fetal calf ser~n (Boehringer Mannheim) as described by Cantz, M. et al. (16). Skin fibroblasts of mannosidosis cell lines ware kindly provided by Dr. Farriaux, Lille, France (cell line G) and the Institut fUr Humangenetik, Marburg, W.-Germany, (cell line B). Normal fibroblasts were obtained from biopsies submitted to our laboratory for diagnosis. Secretion of lysoscnml enzymes: Confluent cultures were incubated for 6 to 24 h with culture median supplemented with 10 % pH inactivated fetal calf serun (I7). Activity of a-mannosidase and B-hexosaminidase in cells and medium was determined as described (18). In experiments where secreted a-mannosidase activity was inmunoprecipitated serum free Waymouth mediun (19), as formulated in the Gibco catalogue) supplemented with 2 % (w/v) of bovine serum alb~nine was used. Labelling of cells: Radioactive labelling of cells was performed as described by H a s i l ~ and Neufeld (20) with minor medif~ations (21). Confluent cultures in 75 Qn flasks were labelled with 50 DCi [ JS]methionine (1190 C½/nmol). Iiqdocytosis of labelled ~-mannosidase: Confluent cultures in 75 an flasks were incubated for 24-33 h w i ~ 5 ml of labelling mediun supplemented with 10 r@4 NH4CI and 0.05-0.15 mCi [ ~S]methionine. The secretions were precipitated with 2.~ g (NHz)gSO 4. ]]%e precipitate was dissolved in H20 and dialysed overnight against F/gle '~ minimal essential medium. After Dialysis the v o ~ n e was adjusted to 5 ml with culture median. Confluent cultures in 75 an- flasks were incubate~ for 6 to 24 h with the medium ccntaining the labelled secretions (30 x IO~ clan) followed by incubation for 3 d in the culture medium (24) . Inlnunoprecipitation: Antibodies to human liver e-mannosidase B were produced in rabbits by intradermal injections at multiple sites (0.2 mg/animal) of enzyme preparation emulsified with equal volune of Freund's incomplete adjuvant. The injections were repeated after 21 d and 42 d and serum was prepared 1 7 d later. Preparation of extracts of cells and media for inTnunoprecipitation (20) of a-mannosidase, polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate and fluorography (20) were performed as described. Acid s-mannosidase was adjusted in extracts of cells and media to 12 mU/ml with a partially purified ~-mannosidase frem human placenta. Antiserum was used at a final dilution of I : 10. For inmunoprecipitation of secreted ~-mam/losidase activity and detemnination of residual ~-mannosidase activity in the supernates immunoglobulins from antiserum and preinmm%e serun inn~bilized to Protein A-Sepharose CL-4B (Sigma, St. Louis) were used to avoid interference by the high endogenous a-mannosidase activities in the sera. RESULTS: Acid a-mannosidase activity secreted by normal and mannosidosis fibroblasts: Stirmllation by NH4CI of secretion of precursor forms is characteristic for lysoscmal enzymes in cultured fibrdolasts (20). We assayed therefore the effect of NH4CI on the secretion of acid ~-mannosidase activity in controls and mannosidosis fibroblasts. Normal fibroblasts secreted ~-mannosidase at a rate of 0.54 m units/d and ng of cell protein. In the presenoe of 10 nN NH4CI the rate of secretion was 2-6-fold elevated. In two mannosidosis fibroblasts lines

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the secretion of ~-mannosidase activity was only about cne third of ccntrols and was not stimulated by 10 n~4 NH4CI. Secreticn of B-hexosaminidase served as a control. The rate of secretion of this enzyme (30-35mt~its/d per mg of oell protein) and the stimulation by 10 mM NH4CI (2-4-fold) was similar in ccntrols and mannosidosis cell lines. About 80 % of the ~-mannosida~e activity secreted by controls was precipitable with antiserum against purified htmkln liver acid ~-mannosidase (as measured by enzyme activity recovered in the supernates above the immune precipitates). In contrast, only 20 % of the ~-mannosidase activity in secretions of mannosidosis fibrcblasts were immunoprecipitable. Biosynthesis and processing of acid ~-mannosidase: Fibroblasts were grcwn for 24 h in the presence of [35S]methionine. Polypeptides immunoprecipitable with antiserum against purified human liver acid a-mannosidase frcm extracts of cells and mediua were analyzed by polyacrylamide gel electrophoresis and fluorography. A single 110kDa polypeptide was inmunoprecipitated fran the median. In the cells rather diffuse appearing polypeptides with apparent MrS of 67kDa, 63kDa and 40-46kDa were immunoprecipitable. ~ e n

cells were labelled in the pre-

sence of 10 mM NH4CI the amount of the secreted 110kDa polypeptide was greatly enhanced (Fig. I, lane 6) and intracellular polypeptides greatly reduced (not shown). I-cell fibroblasts were likewise deficient in intracellular polypeptides (Fig. I, lane 4) and contained increased anounts of the 110kDa polypeptide in the medium (Fig. I, lane 10). In analogy to other lysosomal enzymes we ass~ne that ~-mannosidase is synthesized and partly secreted as a 110kDa precursor and processed intracellularly to a family of polypeptides with M s between 40 to r 67kDa. In accordance with other lysosomal enzymes (20), the secretion of the precursor is greatly enhanced in the presence of 10 KM NH4C1 and in I-cell fibroblasts, which lack the intracellular forms of lysosomal enzymes.

The pre-

sumed precursor-product relationship between the 110kDa polypeptide and the snaller intracellular polypeptides was s~pported by an endocytosis experiment. Radioactive secretions enriched in lysosomal enzyme precursors were prepared frQm fibroblasts labelled in the presence of 10 mM NH4CI. Fibrdolasts incubated for 6 or 24 h with the radioactive secretions contained the family of 40

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Cells

"1

2

3

Medium

4

5

6 7

8

9 IO

! ,11 -- Precursor (110 K)

u

Niature C67 Mature (63 ~/

46--

Mature (40-46 K)

30-

12.3-

Fig. I Synthesis of acid ~-mannosidase in hi,nan skin fibroblasts Fibroblasts of control and patient affected wi N mannosidosis and I-cell disease were grown for 24 h in the presenoe of [ S]methionine. ~-Mannosidase was immunoprecipitated from cells and media. The p o s ~ o n s of =-mannosidase polypeptides are indicated by arrow~. The following -~C-methylated standards were used: phosphorylase B, 92.5kDa; bovine serum albumin, 69kDa; ovalbumint 46kDa; carbonic anhydrase, 30kDa and cytochrcrn c, 12.3kDa. The numbers indicate imm~loprecipitates of ~-mannosidase from: I. normal cells treahed with preimaune ser~n as a control; 2. normal cells; 3. mannosidosis fibroblasts (cell line B); 4. I-cell disease fibreblasts; 5. the medium of a normal culture; 6. the medium of a normal culture treated with 10 ~4 NH4CI; 7. the medium of mannosidosis cell line B; 8. the medium of mannosidosis cell line B treated with 10 mM NHACI; 9. the medium of mannosidosis cell line G; 10. the medium of an I-cell ~sease culture. Contaminants are seen: lane 2 and 3 at 60 kDa (marked by asterisk); lane 3 and 4 at 48kDa (marked by asterisk) ; lanes 2-10 the polypeptides located between carbonic anhydrase and cytcehrcrn c. qhis judgment is based on their irregular occurence and intensity in this and other experiments.

to 67kDa polypeptides

(Fig. 2). After a chase for 3 d the polypeptides pattern

showed no apparent change

(Fig. 2). These observaticns indicate that a 110kDa

precursor of acid ~-mannosidase is intraoellularly processed to a family of snaller polypeptides, which are relatively stable. In fibroblasts from two patients with mannosidosis polypeptides were absent

the 110, 67, 63 and 40-46Da

(Fig. I, lanes 3, 7 and 8). In addition the 110kDa

polypeptide was not found in secretions of mannosidosis in the presence of NH4C1

fibroblasts incubated

(Fig. 2, lane 9). Finally, none of the above menticned

radioactive polypeptides was recovered frcm normal cells incubated with radioactive secretions obtained frGm mannosidosis

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fibroblasts

(not shown).

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Endacytosis(h) Chase (h) Precursor (110 K)

Mature (67 K) Mature (63 K)

6K1

~!

Mature 143 K I

142 K[

i~o Kj

Fig. 2 EndocTtosis of acid enmannosidase The secreticns containing labelled a-mannosidase precursor were added to confluent cultures of fibrcblasts. After incubation for 6 and 24 h and a chase for 3 d cells were harvested and ~-mannosidase immunoprecipitated frcrn the cell extracts. The m e d i a recovered after incubaticn with the cells for 6 h and 24 h (not shown)contained cnly the precursor and the cells contained only processed forms of ~-mannosidase. For standards see Fig. I.

DISCUSSION: Newly synthesized acid a-mannosidase is secreted as a 110kDa polypeptide. Intracellularly only smaller polypeptides with MrS ranging from 40 to 67kDa are detectable. We propose that the 1 IOkDa polypeptide (or a closely related molecule) is the precursor of the intracellular forms. By analogy to other lysosomal enzymes (16/17) it may be assuned that the precursor is transiently present in the cells.

The relation of the 110 kDa and the 40 to 67kDa polypeptides to acid ~-mannosidase was strongly stloported by the absence of these polypeptides in two cell lines from patients genetically deficient in acid e-mannosidase activity. Furthermore, the secretion of the 110kDa precursor was enhanced in the presence of NH4CI and in mucolipidosis II fibroblasts, whereas intracellularly prooessed forms of acid a-mannosidase were absent which is characteristic for lysosomal enzyme polypeptides. From cells incubated with secretions containing the radioactive 110kDa polypeptide, the processed forms of acid ~-mannosidose -were re-

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covered. The processed polypeptides were not detectable, when secretions deficient in the 11OkDa polypeptide were offered for endocytosis. This oberservaticn establishes a product-precursor relationship between the extracellular 110kDa polypeptide and the smaller intrcellular polypeptides. Endocytosis of lysosomal enzyme precursors and its processing to mature forms intracellularly has been established for several lysosomal enzymes (22-24). ~hdocytosis by fibroblasts is dependent on mannose 6-phosphate residues in the lysosomal enzymes. In accordance with that we found that the 11OkDa polypeptide is phosphorylated and that the phosphate resides in oligosaccharides cleavable by ende-BN-acetylglucosaminidase H (unpublished).

Previous reports had suggested that an apparently normal acid ~-mannosidase is synthesized and secreted in mannosidosis fibroblasts and that its intracellular deficiency results frGm a defect which manifests itself only after delivery of the enzyme to the lysosome (13-15). The two mannosidosis cell lines assayed secreted acid ~-mannosidase activity at a rate of about third of controls. In contrast to normal fibroblasts the secretion was not stimulated by 10 nM NH4CI and the secreted enzyme was not i~nunoprecipitable suggesting different properties of the acid a-mannosidase activity secreted by normal mannosidosis fibroblasts. Furthermore the synthesis of the lysosomal ~-mannosidase was deficient in the two mannosidosis cell lines tested indicating that the secreted acid ~mannosidase activity is due to a protein unrelated to lysosomal acid ~-mannosidase. In view of the apparent heterogeneity within hum%an mannosidosis T our observation does not preclude the synthesis of lysosc~al ~-mannosidase in other mannosidosis cell lines. As shown here, secretion of acid ~-mannosidase activity in mannosidosis cell lines, however, does not necessarily indicate the synthesis of lysesomal ~-mannosidase. The protein with which the acid e-mannosidase activity secreted by mannosidosis cells is associated remains to be identified.

ACKNOWLEDC~%Zf: This work was supported by the Deutsche Forschungsgemeinschaft and the "Fonds der chemischen Industrie".

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REFENENUES I. 0ckermann, P.A. (1967) Lancet 2, 239-241 2. Beaudet, A.L. (1983) in "The metabolic~basis of inherited disease" (Stanburg), J.B., Wyngaarden, J.B., Frederickson, D.S., Goldstein, J.L. and Brown, M.S. eds.) pp 788-802 Mc Graw Hill, New York 3. Tulsiani,D.R.P., Opheim,D.J.and Touster,O. (1977) J.Biol.Chem. 252,3227-3233 4. Tabas, J. and Kornfeld, S. (1979) J. Biol. C~em. 254, 11655-11663 5. Carrol, N., Dance, N., Masson, P.K., Robinson, D. and Winchester, B.G. (1972) Biechem. Biophys. Res. Cc~mun. 49, 479-583 6. Taylor, H.A., Thomas, G.H., Ayls~Drth, A., Stevenson, R.E. and Reynolds, L.W. (1975) Clin. Chim. Acta 59, 93-99 7. Andria, G. and Sly, W.S. (1981) Pediatr. Res. 15, 70-73 8. Kistler,J.P., Lott,J.T., Kolodny,E.H. ,Friedman, R.B., Nerasian,R. ,Schnur,J., Milun,M.C., Dvorak,A.M. and Dickersin, R. (1977) Arch. Neurol. 34, 45-51 9. Bach, G., Kohn, G., Lasch, E.E., E1 Massn, M., Ornoy, A., Sekeles, E., Legion, C. and Cohen, M.M. (1978) Ped. Res. 12, 1010-1015 10. Mermnann, G., Buddecke, E. (1977) FEBS Lett. 73, 123-126 11. Poenaru, L., Miranda, C., Dreyfus, J.C. (1980) Am.J.Hum.Genet. 32, 354-363 12. Burditt, L.J., Chotai,K.A. and Winchester,B.G. (1978) FEBS Lett.91, 186-189 13. Hultberg, B., Masson, P.K. (1977) Bioch~n. Biophys. Acta 481, 573-577 14. Halley, D. JJ., Winchester, B.G., Burditt, L.J., d'Azzo, A., Robinson, D. and Galjaard, H. (1980) Bioch~n. J. 187, 541-543 15. Ben-Yoseph, Y., De Franco, C.L., Charrow, J., Hahn, L.C. and Nadler, H.L. (1982) Am. J. Hum. Genet. 34, 100-111 16. Cantz, M., Kresse, H., Barton, R.W. and Neufeld, E.F. (1972) Methods Enzymolog. 28, 884-897 17. yon Figura, K. (1978) Exp. Cell. Res. 111, 15-21 18. von Figura, K. (1977) Eur. J. Biochem. 80, 525-528 19. Gorham, L.W. and Walanouth, C. (1965) Proc. Soc. Exp. Biol. Med.119, 287-291 20. Hasilik, A. and Neufeld, E.F. (1980) J. Biol. Chem. 255, 4937-4945 21. Waheed, A., Hasilik, A. and yon Figura, K. (1982)Eur.J.Biochem.123,317-321 22. Frisch,A. and Neufeld, E.F. (1981) J. Biol. Chem. 256, 8242-8246 23. Robbins, A.R. and Myerowitz, R. (1981) J. Biol. Chem. 256, 10623-10627 24. yon Figura,K., Steckel,F. and Hasilik,A. (1983) Proc. Natl. Acad. Sci, U.S.A., in press

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