NOTES
Dolichyl-pyrophosphoryloligosaccharideprotein oligosaccharide transferase in neuronal ceroid-lipofuscinosis
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'
GUIDOVANDESSEL,ALBERTLAGROU,HERWIGHILDERSON, AND WILFRIEDDIERICK Universitaire Instelling Antwerpen and Rijksuniversitaire Centrum Antwerpen Laboratories for Pathological and Human Biochemistry, University of Antwerp, Groenenborgerlaan 171, B2020 Antwerp, Belgium Received July 27. 1991 VAN DESSEL,G., LAGROU,A., HILDERSON, H., and DIERICK,W. 1992. Dolichyl-pyrophosphoryloligosaccharide protein oligosaccharide transferase in neuronal ceroid-lipofuscinosis. Biochem. Cell Biol. 70: 515-518. The kinetics of the oligosaccharidetransfer from oligosaccharylpyrophosphoryldolichol to endogeneous protein acceptors in human fibroblasts were studied. No alterations in the transferase activity and enzyme characteristics could be observed in fibroblasts from neuronal ceroid-lipofuscinosis (NCL) patients. Analysis of urinary dolichol of two NCL patients also did not reveal substantial differences with respect to controls. Key words: dolichol, fibroblast, neuronal ceroid-lipofuscinosis, oligosaccharide transfer, urine. VAN DESSEL,G., LAGROU,A., HILDERSON,H., et DIERICK,W. 1992. Dolichyl-pyrophosphoryloligosaccharide protein oligosaccharide transferase in neuronal ceroid-lipofuscinosis. Biochem. Cell Biol. 70 : 515-518. Nous avons CtudiC le transfert des oligosaccharides depuis l'oligosaccharyl pyrophosphoryldolichol aux accepteurs prottiques endogknes dans les fibroblastes humains. Nous n'avons observe aucune alttration de I'activitt transftrasique et des caracteristiques de I'enzyme dans les fibroblastes des patients atteints de ctroyde-lipofuscinose neuronale (NCL). L'analyse du dolichol urinaire de deux patients NCL ne rCvkle pas non plus de difftrences substantielles par rapport aux contrales. Mots clPs : dolichol, fibroblaste, ctroide-lipofuscinose neuronale, transfert des oligosaccharides, urine. [Traduit par la redaction]
Introduction Ceroid-lipofuscinoses (NCL) represent a group of progressive neurological disorders characterized by the lysomal accumulation of autofluorescent lipopigments in the brain and other tissues of human and animals. The exact nature of the underlying defect is still obscure (Zeman 1974; Svennerholm et al. 1975; Wolfe et al. 1983). Several lines of evidence suggest that dolichol and (or) derivatives might be implicated in the aetiology of the disease. (i) Wolfe et al. (1983) have reported a 10- to 30-fold increase of free dolichol in brain and urine of NCL patients. (ii) Keller et al. (1984) and Hall and Patrick (1985) both have found an accumulation of dolichyl phosphate in animal forms of NCL (iii) Recently Hall and Patrick (1985) and Pullarkat et al. (1988), as well as Wolfe et al. (1988), all refer to an elevated level of dolichyl-pyrophosphoryloligosaccharide in several tissues of NCL patients. (iv) In skin fibroblasts of late infantile NCL patients, Dawson (1982) has shown a small but consistent increase in the incorporation of labelled glucosamine in glycoproteins, while Pullarkat et al. (1988) have observed an accumulation of total phosphorylated dolichol in fibroblasts from three juvenile NCL patients. The latter findings prompted us to investigate the possible role of the oligosaccharide transferase activity in NCL. The enzyme, an integral membrane protein located in the rough endoplasmic reticulum, accelerates the cotranslational transfer of oligosaccharide from oligosaccharyl pyro-
Cell culture Fibroblasts, derived from skin biopsies at the Laboratory of Genetics, University of Antwerp, were cultured according to routine procedures in HAM'S F10 medium supplemented with 10% fetal bovine serum and antibiotics.
ABBREVIATIONS: NCL, neuronal ceroid-lipofuscinosis; HPLC, high performance liquid chromatography; C,,, dolichol molecule consisting of 85 carbons; BSA, bovine serum albumin; dpm, disintegrations per minute (60 dpm = 1 Bq). ' ~ u t h o rto whom all correspondence should be addressed.
Analysis of urine samples Dolichols were extracted according to Sakakihara and Kamoshita (1991; personal communication). Briefly, after addition of the internal standard, the urine (20 mL) was extracted with 20 mL of chloroform-methanol (2: 1, v/v). The lower phase was evaporated and dissolved in an appropriate HPLC solvent. HPLC analyses
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phosphoryldolichol to the nascent polypeptide acceptors (Welply et al. 1983). To check for a possible defect at the level of N-glycoprotein assembly in NCL, the oligosaccharide transferase activity, as well as a number of enzyme characteristics, have been compared in skin fibroblast cultures from NCL patients and healthy controls. In parallel, qualitative and quantitative HPLC analyses of dolichol in whole urine have been performed. Urinary dolichol analyses have been frequently used as a diagnostic tool for screening NCL patients (Bennett et al. 1985; Ohashi et al. 1986; Wolfe et al. 1986; Paton and Poulos 1987; Salaspuro and Korri 1987). Material and methods Patients Urine samples were obtained from two cases of juvenile ceroidlipofuscinosis (two male, age 26-44 years). Fibroblasts were obtained from four cases of juvenile ceroid-lipofuscinosis (two male and two female, age 14-19 years). Diagnosis of the patients was based on case histories supplied by their attending clinicians and from histological examinations of biopsies. Age-matched controls were healthy individuals.
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BIOCHEM. CELL BIOL. VOL. 70, 1992
Time (min)
Dolichololigosoccharide (prnol)
Protah ( p g )
PH
FIG. 1. Oligosaccharide transferase activity in human fibroblasts. Transferase activity is shown as a function of (a) time, ( b ) protein concentration, (c) substrate concentration, and (d) pH (buffers: A, imidazole; o,Tris; o , glycine). Incubations were performed at standard assay conditions. were performed on a HP 1090M apparatus equipped with a diode array detector using the method of Elmberger et al. (1988). Identification of the respective dolichol homologues rested on the chromatographic behaviour of pig liver and bovine thyroid dolichol (Van Dessel et al. 1979). Dolichol peaks corresponding to C,,, CgO,C,,, C,,, and C,,, were quantitated using C,,, dolichol as an internal standard. Biosynthesis of lipid linked oligosaccharide ~olichol-P-P- an-''~]oligosaccharide was prepared from bovine thyroid microsomes, purified, and characterized as described by Ronin and Bouchilloux (1978). Homogenization procedure Human fibroblasts were suspended in 2 mL of incubation buffer (50 mM Tris-HC1, pH 7.4) and sonicated (2 x 10 s with a 30-s interval cooling on ice) and homogenized with a Potter Elvehjem (five strokes), yielding the oligosaccharide transferase suspension. Protein concentration was measured according to Lowry et al. (1951) with BSA as a standard. Oligosaccharide transfer assay An aliquot of dolichol-P-P- an-''~]oligosaccharide (10 000 dpm; 30 m ~ i - m m o l of substrate; 150 pmol; 1 Ci = 37 GBq) was dispersed in 50 mM Tris-HC1 (pH 7.4) containing 60 mM P-mercaptoethanol, 8 mM MnCl,, and 0.09% Triton X-100. The reaction was started by the addition of the enzyme preparation ( * 100 pg protein) yielding a final volume of 0.130 mL. The incubation (room temperature, 30 min) was terminated by adding 1 mL of 10% trichloroacetic acid. After an extra boiling for 5 min, the mixture was filtered through Whatman GF/C and the residue was processed further for scintillation counting as described by Voets et al. (1979).
-'
Results In normal fibroblasts the transfer of the dolicho1-P-P[~an-'~~]oli~osaccharide t o endogeneous proteins in function of time proved t o be linear for about 30 min (Fig. la). The enzymic activity was proportional to the amount of protein up t o 600 pg protein (Fig. lb). With respect t o substrate concentration, an increase in oligosaccharide transfer was noted up to 0.900 pM (Fig. lc). The p H optimum was found to be around pH 7.5 (Fig. Id). The transfer into glycoproteins required divalent cations with the following order of effectiveness, Mn2+ > ca2+ > Mg2+, as sustained by the strong annihilating effect of EDTA (Table I). When performing similar experiments with fibroblasts from NCL patients, no significant differences with respect t o controls could be observed. Control and NCL fibroblasts transferred oligosaccharides at similar rates (Table 2). Both also showed equivalent curves for the oligosaccharide activity as a function of time, protein concentration, substrate concentration, and p H (data not shown). Finally no evidence could be established for an elevation in dolichol concentration in whole urine of two NCL patients; 7 1 and 76 pg .L - was found versus a control mean value of 64 pg L (n - 12). Further it must be stressed that a wide variation occurs in the urinary dolichol level between individuals (controls, 45-125 pg L - I , n = 12). Additionally, the same homologue HPLC profiles were obtained for both NCL patients and controls: dolichol-90, dolichol-95, and dolichol-100, together representing 85% of the sum of dolichol species, with a slight
- -'
'
.
517
NOTES
TABLE1. Influence of divalent cations on the oligosaccharide transfer activity in normal human skin fibroblasts Cation None ~ n
Concentration (mM)
Activity* (qo)
Subjects
0 10 10 10 10 5
100 560 50 150k 12 200+17 8 0 k 10 Ok6
Batten (n = 4) Controls (n = 5)
~
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M ~ ~ +
ca2+ zn2+ EDTA
2. Oligosaccharidetransfer activity to endogenous TABLE acceptors in fibroblasts of NCL patients and healthy controls
+
*
'The activities represent the mean of three assays.
(statistically not significant) shift towards shorter chain dolichol homologues in the NCL patients.
pmol oligosaccharide.mg protein transferred - ' .30 min - '
*The slight elevation is exclusively due to the elevated enzyme activity observed in one NCL patient who displayed an oligosaccharidetransferase activity twice the control value.
et al. 1986; Paton and Poulos 1987). Moreover, elevated levels in urine seem not to be NCL specific (Salaspuro and Korri 1987). Acknowledgments We thank Mrs. Sonia Plompen for her excellent technical assistance, and Mr. Paul Van Dyck and Mr. Jos Van Sonhoven for their care in layout. We also acknowledge the participation in this study of Drs. H. Carton, W. Robbrechts, and L. Vanderwegen of the Catholic University of Leuven and Drs. G. Franck and B. Rogister of Centre Hospitalier Universitaire de Likge for providing the urine samples, and Dr. A. Van Elsen for the preparation of the cell cultures. We are grateful for the generous financial support of Belgian Nationaal Fonds voor Wetenschappelijk Ondersoek (grant 3.0083.87) and Belgian Lotto (grant 9.0013.88).
Discussion Disturbance of dolichol metabolism as a possible cause for NCL still remains debatable (Wolfe et al. 1983; Paton and Poulos 1984; Pullarkat et al. 1988). From the results presented here, it is most unlikely that NCL does originate from a defect at the level of a gene locus coding for the oligosaccharide transferase polypeptide. Indeed, no deficiency nor alteration in the enzyme characteristics could be noted in patients with Batten's disease (Table 2; Fig. 1). Our data are in accordance with the observation made by Pullarkat et al. (1988), showing a normal transfer activity when incubating skin fibroblasts with [3~]glucosamine,a core Bennett, M., Mathers, N., Hemming, F., et al. 1985. Urinary sedisugar of the oligosaccharide chain (no data on the enzyme ment dolichol excretion in patients with Batten disease and other activity were given). However, it should be stressed that the neurodegenerative and storage disorders. Pediatr. Res. 19: 213-216. oligosaccharide transfer assay is only an indicator of the Daniel, P., Sauls, D., and Boustany, R.-M. 1989. Structure of transfer of sugar to a bulk of proteins. A small, specific, unusual lipid-linked oligosaccharides isolated from brains of regulatory protein fraction, in some way involved in the patients with neuronal ceroid lipofuscinosis. J. Cell. Biochem. disturbed metabolism of NCL patients, could not be Suppl. 13A: 141. detected by our experimental approach. Dawson, G . 1982. Approaches to the detection of neuronal ceroid Very recently Daniel et al. (1989) and Hall et al. (1989) lipofuscinosis in cultured skin fibroblasts. In Ceroidoreported the oligosaccharide composition of the stored lipidlipofuscinosis (Batten's disease). Edited by D. Armstrong, linked oligosaccharides, respectively, in human brain and N. Koppang, and J. Rider. Elsevier Biomedical Press, ovine brain and pancreas from NCL-affected individuals. Amsterdam. pp. 229-240. Both groups of investigators showed that the major accuElmberger, P., Engfeldt, P., and Dallner, G. 1988. Presence of dolichol and its derivatives in human blood. J. Lipid Res. 29: mulated oligosaccharide contained an incomplete sugar 1651-1662. sequence. Moreover, Daniel et al. (1989) demonstrated that Hall. N., and Patrick, A. 1985. Dolichol and phosphorylated this oligosaccharide was not an intermediate in the biosynthesis of the G-oligosaccharide (the ( G ~ c N A c ) ~ - dolichol content of tissues in ceroid-lipofuscinosis. J. Inherited Metab. Dis. 8: 178-183. Man9Glc3 oligosaccharide linked t o dolichol via a Hall, N., Jolly, R., Palmer, D., et al. 1989. Analysis of dolichyl pyrophosphate bridge) and that the storage of this lipidpyrophosphoryl oligosaccharides in purified storage cytosomes linked oligosaccharide was organ specific. Combining these from ovine ceroid-lipofuscinosis. Biochim. Biophys. Acta, 993: findings with our results, one could argue that the oligo245-25 1. saccharide transfer proceeds at normal rates on the condiKeller, R., Armstrong, D., Crum, F., and Koppang, N. 1984. tion that the right substrate is made available to the enzyme. Dolichol and dolichylphosphate levels in brain tissue from English setters with ceroid lipofuscinosis. J. Neurochem. 42: NCL could then be a result of a preoligosaccharide-transfer 1040- 1047. defect, unless one accepts that the transferase deficiency does Lowry, O., Rosebrough, N., Farr, A., and Randall, R. 1951. Pronot manifest itself in skin fibroblasts. However, such a tein measurement with the folin phenol reagent. J. Biol. Chem. defect at the level of the G-oligosaccharide assembly does 193: 265-275. not fit with Pullarkat's data already referred to (Pullarkat Ohashi, T., Kanamoto, Y., Yamaguchi, S., et al. 1986. Abnormal et al. 1988). excretion of autofluorescent lipids in urine from patients with Finally, with regard to the urinary dolichol content, no neuronal ceroid lipofuscinosis. Tohoku J. Exp. Med. 148: elevated level could be demonstrated in our two NCL 335-339. patients. Measurement of urinary dolichol cannot replace Paton, B., and Poulos, A. 1984. Dolichol metabolism in cultured the current diagnostic methods (Bennett et al. 1985; Ohashi skin fibroblasts from patients with "neuronal" ceroid
BIOCHEM. CELL BIOL. VOL. 70, 1992
518
lipofuscinosis (Batten's disease). J. Inherited Metab. Dis. 7: 112-1 16.
Paton, B., and Poulos, A. 1987. Normal dolichol concentration in urine sediments from four patients with neuronal ceroid lipofuscinosis (Batten's disease). J. Inherited Metab. Dis. 10:
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28-32.
Pullarkat, R., Kim, K., Sklower, S., and Patel, V. 1988. Oligosacchary1 diphosphodolichols in the ceroid-lipofuscinoses. Am. J. Med. Genet. Suppl. 5: 243-251. Ronin, C., and Bouchillow, S. 1978. Cell-free labeling in thyroid rough microsomes of lipid-linked and protein-linked oligosaccharides. Biochim. Biophys. Acta, 539: 470-480. Sakakihara, Y., and Kamoshita, S. 1991. Partition of free dolichol into urinary sediments and supernatant. In Dolichol and other lipids related to glycoconjugate metabolism. Satellite meeting to the 11th International Symposium on Glycoconjugates, Kimberley, Ont. Abstr. 25. Salaspuro, M., and Korri, U.M. 1987. Blood acetate and urinary dolichols-new markers of heavy drinking and alcoholism. In Genetics and Alcoholism. Alan R. Liss Inc., New York. pp. 231-240. Svennerholm, L., Hagberg, B., Haltia, M., et al. 1975. Poly-
unsaturated fatty acid lipidosis. 11. Lipid biochemical studies. Acta Paediatr. Scand. 64: 489-496. Van Dessel, G., Lagrou, A., Hilderson, H.J.J., et al. 1979. Isolation and identification of polyprenols from bovine thyroid gland. Biochim. Biophys. Acta, 573: 296-300. Voets, R., Lagrou, A., Hilderson, H.J.J., et al. 1979. RNA synthesis in isolated nuclei and nucleoli from bovine thyroid. HoppeSeyler's Z. Physiol. Chem. 360: 1271-1283. Welply, J., Shenbagamurthi, P., Lennarz, W., and Naider, F. 1983. Substrate recognition by oligosaccharyltransferase. J. Biol. Chem. 258: 11 856 - 11 863. Wolfe, L., Ng, Y., Palo, J., and Haltia, M. 1983. Dolichols in brain and urinary sediment in neuronal ceroid lipofuscinosis. Neurology, 33: 103-106. Wolfe, L., Palo, J., Santavuori, P., et al. 1986. Urinary sediment dolichols in the diagnosis of neuronal ceroid-lipofuscinosis. Ann. Neurol. 19: 270-274. Wolfe, L., Gauthier, S., Haltia, M., and Palo, J. 1988. Dolichol and dolichylphosphate in the neuronal ceroid-lipofuscinosis and other diseases. Am. J. Med. Genet. Suppl. 5: 233-242. Zeman, W. 1974. Studies in the neuronal ceroid-lipofuscinoses. J. Neuropathol. Exp. Neurol. 33: 1-12.
Partition of free dolichol in human urine YOICHI SAKAKIHARA AND SHIGEHIKO KAMOSHITA Department of Pediatrics, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-Ku Tokyo, Japan Received September 3, 1991 SAKAKIHARA, Y., and KAMOSHITA,S. 1992. Partition of free dolichol in human urine. Biochem. Cell Biol. 70: 518-521.
We have demonstrated that dolichol is present in the urinary supernatant. Most of the dolichol present in the supernatant seems to be associated with cellular debris or membrane fragments. The amount of sediment in healthy subjects correlate well with the volume of urine. Although it is illogical to express urinary dolichol relative to urine volume, a good correlation between the amount of sediment and urine volume has made its use justifiable. Because of the presence of a substantial amount of dolichol in the supernatant, it seems better to use uncentrifuged whole urine as the sample for measurement of dolichol. Key words: dolichol, urine, sediment, supernatant. SAKAKIHARA, Y., et KAMOSHITA,S. 1992. Partition of free dolichol in human urine. Biochem. Cell Biol. 70 : 518-521.
Nous avons dtmontrt la prtsence du dolichol dans le surnageant urinaire. La plus grande partie du dolichol prtsent dans le surnageant semble associt B des dtbris cellulaires ou des fragments membranaires. Chez les sujets en santt, la quantitt de ce stdiment correspond ttroitement au volume de l'urine. I1 est peut-&treillogique d'exprimer le dolichol urinaire par rapport au volume de I'urine, mais I'ttroite relation entre la quantitk de sediment et le volume d'urine justifie son emploi. fitant donnt la prhence d'une quantitt substantiellede dolichol dans le surnageant, il semble prtftrable d'utiliser I'urine totale non centrifugke comme tchantillon pour la mesure du dolichol. Mots clks : dolichol, urine, ddiment, surnageant. [Traduit par la redaction]
Introduction Dolicol and its derivatives are lipid intermediates participating in the synthesis of N-linked glycoprotein. Its clinical implication was not recognized until Wolfe reported elevated levels of free dolichol in the brains of patients with Alzheimer's disease (Wolfe et al. 1982). Since then, dolichol accumulation has been reported in the brains of patients with several neurodegenerative disorders (Ng Yink Kin and Wolfe 1982; Sakakihara et al. 1987), in the urine of chronic alcoholics and patients with ceroid-lipofuscinosis (Ng Ying Kin and Wolfe 1982; Wolfe et al. 1983, Pullarkat et al. 1984; Wolfe et al. 1986), and in the serum from patients with aspartylglucosarninuria (Salaspuro et al. 1990). ABBREVIATIONS: PBS, phosphate buffered saline; M,, relative mass. Printed in Canada / Im~rimbau Canada
Dolichyl-pyrophosphoryloligosaccharideprotein oligosaccharide transferase in neuronal ceroid-lipofuscinosis
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'
GUIDOVANDESSEL,ALBERTLAGROU,HERWIGHILDERSON, AND WILFRIEDDIERICK Universitaire Instelling Antwerpen and Rijksuniversitaire Centrum Antwerpen Laboratories for Pathological and Human Biochemistry, University of Antwerp, Groenenborgerlaan 171, B2020 Antwerp, Belgium Received July 27. 1991 VAN DESSEL,G., LAGROU,A., HILDERSON, H., and DIERICK,W. 1992. Dolichyl-pyrophosphoryloligosaccharide protein oligosaccharide transferase in neuronal ceroid-lipofuscinosis. Biochem. Cell Biol. 70: 515-518. The kinetics of the oligosaccharidetransfer from oligosaccharylpyrophosphoryldolichol to endogeneous protein acceptors in human fibroblasts were studied. No alterations in the transferase activity and enzyme characteristics could be observed in fibroblasts from neuronal ceroid-lipofuscinosis (NCL) patients. Analysis of urinary dolichol of two NCL patients also did not reveal substantial differences with respect to controls. Key words: dolichol, fibroblast, neuronal ceroid-lipofuscinosis, oligosaccharide transfer, urine. VAN DESSEL,G., LAGROU,A., HILDERSON,H., et DIERICK,W. 1992. Dolichyl-pyrophosphoryloligosaccharide protein oligosaccharide transferase in neuronal ceroid-lipofuscinosis. Biochem. Cell Biol. 70 : 515-518. Nous avons CtudiC le transfert des oligosaccharides depuis l'oligosaccharyl pyrophosphoryldolichol aux accepteurs prottiques endogknes dans les fibroblastes humains. Nous n'avons observe aucune alttration de I'activitt transftrasique et des caracteristiques de I'enzyme dans les fibroblastes des patients atteints de ctroyde-lipofuscinose neuronale (NCL). L'analyse du dolichol urinaire de deux patients NCL ne rCvkle pas non plus de difftrences substantielles par rapport aux contrales. Mots clPs : dolichol, fibroblaste, ctroide-lipofuscinose neuronale, transfert des oligosaccharides, urine. [Traduit par la redaction]
Introduction Ceroid-lipofuscinoses (NCL) represent a group of progressive neurological disorders characterized by the lysomal accumulation of autofluorescent lipopigments in the brain and other tissues of human and animals. The exact nature of the underlying defect is still obscure (Zeman 1974; Svennerholm et al. 1975; Wolfe et al. 1983). Several lines of evidence suggest that dolichol and (or) derivatives might be implicated in the aetiology of the disease. (i) Wolfe et al. (1983) have reported a 10- to 30-fold increase of free dolichol in brain and urine of NCL patients. (ii) Keller et al. (1984) and Hall and Patrick (1985) both have found an accumulation of dolichyl phosphate in animal forms of NCL (iii) Recently Hall and Patrick (1985) and Pullarkat et al. (1988), as well as Wolfe et al. (1988), all refer to an elevated level of dolichyl-pyrophosphoryloligosaccharide in several tissues of NCL patients. (iv) In skin fibroblasts of late infantile NCL patients, Dawson (1982) has shown a small but consistent increase in the incorporation of labelled glucosamine in glycoproteins, while Pullarkat et al. (1988) have observed an accumulation of total phosphorylated dolichol in fibroblasts from three juvenile NCL patients. The latter findings prompted us to investigate the possible role of the oligosaccharide transferase activity in NCL. The enzyme, an integral membrane protein located in the rough endoplasmic reticulum, accelerates the cotranslational transfer of oligosaccharide from oligosaccharyl pyro-
Cell culture Fibroblasts, derived from skin biopsies at the Laboratory of Genetics, University of Antwerp, were cultured according to routine procedures in HAM'S F10 medium supplemented with 10% fetal bovine serum and antibiotics.
ABBREVIATIONS: NCL, neuronal ceroid-lipofuscinosis; HPLC, high performance liquid chromatography; C,,, dolichol molecule consisting of 85 carbons; BSA, bovine serum albumin; dpm, disintegrations per minute (60 dpm = 1 Bq). ' ~ u t h o rto whom all correspondence should be addressed.
Analysis of urine samples Dolichols were extracted according to Sakakihara and Kamoshita (1991; personal communication). Briefly, after addition of the internal standard, the urine (20 mL) was extracted with 20 mL of chloroform-methanol (2: 1, v/v). The lower phase was evaporated and dissolved in an appropriate HPLC solvent. HPLC analyses
Printed in Canada / Imprime au Canada
phosphoryldolichol to the nascent polypeptide acceptors (Welply et al. 1983). To check for a possible defect at the level of N-glycoprotein assembly in NCL, the oligosaccharide transferase activity, as well as a number of enzyme characteristics, have been compared in skin fibroblast cultures from NCL patients and healthy controls. In parallel, qualitative and quantitative HPLC analyses of dolichol in whole urine have been performed. Urinary dolichol analyses have been frequently used as a diagnostic tool for screening NCL patients (Bennett et al. 1985; Ohashi et al. 1986; Wolfe et al. 1986; Paton and Poulos 1987; Salaspuro and Korri 1987). Material and methods Patients Urine samples were obtained from two cases of juvenile ceroidlipofuscinosis (two male, age 26-44 years). Fibroblasts were obtained from four cases of juvenile ceroid-lipofuscinosis (two male and two female, age 14-19 years). Diagnosis of the patients was based on case histories supplied by their attending clinicians and from histological examinations of biopsies. Age-matched controls were healthy individuals.
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BIOCHEM. CELL BIOL. VOL. 70, 1992
Time (min)
Dolichololigosoccharide (prnol)
Protah ( p g )
PH
FIG. 1. Oligosaccharide transferase activity in human fibroblasts. Transferase activity is shown as a function of (a) time, ( b ) protein concentration, (c) substrate concentration, and (d) pH (buffers: A, imidazole; o,Tris; o , glycine). Incubations were performed at standard assay conditions. were performed on a HP 1090M apparatus equipped with a diode array detector using the method of Elmberger et al. (1988). Identification of the respective dolichol homologues rested on the chromatographic behaviour of pig liver and bovine thyroid dolichol (Van Dessel et al. 1979). Dolichol peaks corresponding to C,,, CgO,C,,, C,,, and C,,, were quantitated using C,,, dolichol as an internal standard. Biosynthesis of lipid linked oligosaccharide ~olichol-P-P- an-''~]oligosaccharide was prepared from bovine thyroid microsomes, purified, and characterized as described by Ronin and Bouchilloux (1978). Homogenization procedure Human fibroblasts were suspended in 2 mL of incubation buffer (50 mM Tris-HC1, pH 7.4) and sonicated (2 x 10 s with a 30-s interval cooling on ice) and homogenized with a Potter Elvehjem (five strokes), yielding the oligosaccharide transferase suspension. Protein concentration was measured according to Lowry et al. (1951) with BSA as a standard. Oligosaccharide transfer assay An aliquot of dolichol-P-P- an-''~]oligosaccharide (10 000 dpm; 30 m ~ i - m m o l of substrate; 150 pmol; 1 Ci = 37 GBq) was dispersed in 50 mM Tris-HC1 (pH 7.4) containing 60 mM P-mercaptoethanol, 8 mM MnCl,, and 0.09% Triton X-100. The reaction was started by the addition of the enzyme preparation ( * 100 pg protein) yielding a final volume of 0.130 mL. The incubation (room temperature, 30 min) was terminated by adding 1 mL of 10% trichloroacetic acid. After an extra boiling for 5 min, the mixture was filtered through Whatman GF/C and the residue was processed further for scintillation counting as described by Voets et al. (1979).
-'
Results In normal fibroblasts the transfer of the dolicho1-P-P[~an-'~~]oli~osaccharide t o endogeneous proteins in function of time proved t o be linear for about 30 min (Fig. la). The enzymic activity was proportional to the amount of protein up t o 600 pg protein (Fig. lb). With respect t o substrate concentration, an increase in oligosaccharide transfer was noted up to 0.900 pM (Fig. lc). The p H optimum was found to be around pH 7.5 (Fig. Id). The transfer into glycoproteins required divalent cations with the following order of effectiveness, Mn2+ > ca2+ > Mg2+, as sustained by the strong annihilating effect of EDTA (Table I). When performing similar experiments with fibroblasts from NCL patients, no significant differences with respect t o controls could be observed. Control and NCL fibroblasts transferred oligosaccharides at similar rates (Table 2). Both also showed equivalent curves for the oligosaccharide activity as a function of time, protein concentration, substrate concentration, and p H (data not shown). Finally no evidence could be established for an elevation in dolichol concentration in whole urine of two NCL patients; 7 1 and 76 pg .L - was found versus a control mean value of 64 pg L (n - 12). Further it must be stressed that a wide variation occurs in the urinary dolichol level between individuals (controls, 45-125 pg L - I , n = 12). Additionally, the same homologue HPLC profiles were obtained for both NCL patients and controls: dolichol-90, dolichol-95, and dolichol-100, together representing 85% of the sum of dolichol species, with a slight
- -'
'
.
517
NOTES
TABLE1. Influence of divalent cations on the oligosaccharide transfer activity in normal human skin fibroblasts Cation None ~ n
Concentration (mM)
Activity* (qo)
Subjects
0 10 10 10 10 5
100 560 50 150k 12 200+17 8 0 k 10 Ok6
Batten (n = 4) Controls (n = 5)
~
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M ~ ~ +
ca2+ zn2+ EDTA
2. Oligosaccharidetransfer activity to endogenous TABLE acceptors in fibroblasts of NCL patients and healthy controls
+
*
'The activities represent the mean of three assays.
(statistically not significant) shift towards shorter chain dolichol homologues in the NCL patients.
pmol oligosaccharide.mg protein transferred - ' .30 min - '
*The slight elevation is exclusively due to the elevated enzyme activity observed in one NCL patient who displayed an oligosaccharidetransferase activity twice the control value.
et al. 1986; Paton and Poulos 1987). Moreover, elevated levels in urine seem not to be NCL specific (Salaspuro and Korri 1987). Acknowledgments We thank Mrs. Sonia Plompen for her excellent technical assistance, and Mr. Paul Van Dyck and Mr. Jos Van Sonhoven for their care in layout. We also acknowledge the participation in this study of Drs. H. Carton, W. Robbrechts, and L. Vanderwegen of the Catholic University of Leuven and Drs. G. Franck and B. Rogister of Centre Hospitalier Universitaire de Likge for providing the urine samples, and Dr. A. Van Elsen for the preparation of the cell cultures. We are grateful for the generous financial support of Belgian Nationaal Fonds voor Wetenschappelijk Ondersoek (grant 3.0083.87) and Belgian Lotto (grant 9.0013.88).
Discussion Disturbance of dolichol metabolism as a possible cause for NCL still remains debatable (Wolfe et al. 1983; Paton and Poulos 1984; Pullarkat et al. 1988). From the results presented here, it is most unlikely that NCL does originate from a defect at the level of a gene locus coding for the oligosaccharide transferase polypeptide. Indeed, no deficiency nor alteration in the enzyme characteristics could be noted in patients with Batten's disease (Table 2; Fig. 1). Our data are in accordance with the observation made by Pullarkat et al. (1988), showing a normal transfer activity when incubating skin fibroblasts with [3~]glucosamine,a core Bennett, M., Mathers, N., Hemming, F., et al. 1985. Urinary sedisugar of the oligosaccharide chain (no data on the enzyme ment dolichol excretion in patients with Batten disease and other activity were given). However, it should be stressed that the neurodegenerative and storage disorders. Pediatr. Res. 19: 213-216. oligosaccharide transfer assay is only an indicator of the Daniel, P., Sauls, D., and Boustany, R.-M. 1989. Structure of transfer of sugar to a bulk of proteins. A small, specific, unusual lipid-linked oligosaccharides isolated from brains of regulatory protein fraction, in some way involved in the patients with neuronal ceroid lipofuscinosis. J. Cell. Biochem. disturbed metabolism of NCL patients, could not be Suppl. 13A: 141. detected by our experimental approach. Dawson, G . 1982. Approaches to the detection of neuronal ceroid Very recently Daniel et al. (1989) and Hall et al. (1989) lipofuscinosis in cultured skin fibroblasts. In Ceroidoreported the oligosaccharide composition of the stored lipidlipofuscinosis (Batten's disease). Edited by D. Armstrong, linked oligosaccharides, respectively, in human brain and N. Koppang, and J. Rider. Elsevier Biomedical Press, ovine brain and pancreas from NCL-affected individuals. Amsterdam. pp. 229-240. Both groups of investigators showed that the major accuElmberger, P., Engfeldt, P., and Dallner, G. 1988. Presence of dolichol and its derivatives in human blood. J. Lipid Res. 29: mulated oligosaccharide contained an incomplete sugar 1651-1662. sequence. Moreover, Daniel et al. (1989) demonstrated that Hall. N., and Patrick, A. 1985. Dolichol and phosphorylated this oligosaccharide was not an intermediate in the biosynthesis of the G-oligosaccharide (the ( G ~ c N A c ) ~ - dolichol content of tissues in ceroid-lipofuscinosis. J. Inherited Metab. Dis. 8: 178-183. Man9Glc3 oligosaccharide linked t o dolichol via a Hall, N., Jolly, R., Palmer, D., et al. 1989. Analysis of dolichyl pyrophosphate bridge) and that the storage of this lipidpyrophosphoryl oligosaccharides in purified storage cytosomes linked oligosaccharide was organ specific. Combining these from ovine ceroid-lipofuscinosis. Biochim. Biophys. Acta, 993: findings with our results, one could argue that the oligo245-25 1. saccharide transfer proceeds at normal rates on the condiKeller, R., Armstrong, D., Crum, F., and Koppang, N. 1984. tion that the right substrate is made available to the enzyme. Dolichol and dolichylphosphate levels in brain tissue from English setters with ceroid lipofuscinosis. J. Neurochem. 42: NCL could then be a result of a preoligosaccharide-transfer 1040- 1047. defect, unless one accepts that the transferase deficiency does Lowry, O., Rosebrough, N., Farr, A., and Randall, R. 1951. Pronot manifest itself in skin fibroblasts. However, such a tein measurement with the folin phenol reagent. J. Biol. Chem. defect at the level of the G-oligosaccharide assembly does 193: 265-275. not fit with Pullarkat's data already referred to (Pullarkat Ohashi, T., Kanamoto, Y., Yamaguchi, S., et al. 1986. Abnormal et al. 1988). excretion of autofluorescent lipids in urine from patients with Finally, with regard to the urinary dolichol content, no neuronal ceroid lipofuscinosis. Tohoku J. Exp. Med. 148: elevated level could be demonstrated in our two NCL 335-339. patients. Measurement of urinary dolichol cannot replace Paton, B., and Poulos, A. 1984. Dolichol metabolism in cultured the current diagnostic methods (Bennett et al. 1985; Ohashi skin fibroblasts from patients with "neuronal" ceroid
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Paton, B., and Poulos, A. 1987. Normal dolichol concentration in urine sediments from four patients with neuronal ceroid lipofuscinosis (Batten's disease). J. Inherited Metab. Dis. 10:
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Partition of free dolichol in human urine YOICHI SAKAKIHARA AND SHIGEHIKO KAMOSHITA Department of Pediatrics, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-Ku Tokyo, Japan Received September 3, 1991 SAKAKIHARA, Y., and KAMOSHITA,S. 1992. Partition of free dolichol in human urine. Biochem. Cell Biol. 70: 518-521.
We have demonstrated that dolichol is present in the urinary supernatant. Most of the dolichol present in the supernatant seems to be associated with cellular debris or membrane fragments. The amount of sediment in healthy subjects correlate well with the volume of urine. Although it is illogical to express urinary dolichol relative to urine volume, a good correlation between the amount of sediment and urine volume has made its use justifiable. Because of the presence of a substantial amount of dolichol in the supernatant, it seems better to use uncentrifuged whole urine as the sample for measurement of dolichol. Key words: dolichol, urine, sediment, supernatant. SAKAKIHARA, Y., et KAMOSHITA,S. 1992. Partition of free dolichol in human urine. Biochem. Cell Biol. 70 : 518-521.
Nous avons dtmontrt la prtsence du dolichol dans le surnageant urinaire. La plus grande partie du dolichol prtsent dans le surnageant semble associt B des dtbris cellulaires ou des fragments membranaires. Chez les sujets en santt, la quantitt de ce stdiment correspond ttroitement au volume de l'urine. I1 est peut-&treillogique d'exprimer le dolichol urinaire par rapport au volume de I'urine, mais I'ttroite relation entre la quantitk de sediment et le volume d'urine justifie son emploi. fitant donnt la prhence d'une quantitt substantiellede dolichol dans le surnageant, il semble prtftrable d'utiliser I'urine totale non centrifugke comme tchantillon pour la mesure du dolichol. Mots clks : dolichol, urine, ddiment, surnageant. [Traduit par la redaction]
Introduction Dolicol and its derivatives are lipid intermediates participating in the synthesis of N-linked glycoprotein. Its clinical implication was not recognized until Wolfe reported elevated levels of free dolichol in the brains of patients with Alzheimer's disease (Wolfe et al. 1982). Since then, dolichol accumulation has been reported in the brains of patients with several neurodegenerative disorders (Ng Yink Kin and Wolfe 1982; Sakakihara et al. 1987), in the urine of chronic alcoholics and patients with ceroid-lipofuscinosis (Ng Ying Kin and Wolfe 1982; Wolfe et al. 1983, Pullarkat et al. 1984; Wolfe et al. 1986), and in the serum from patients with aspartylglucosarninuria (Salaspuro et al. 1990). ABBREVIATIONS: PBS, phosphate buffered saline; M,, relative mass. Printed in Canada / Im~rimbau Canada
