step in heparan sulfate biosynthesis and a prerequisite to subsequent N- and 0-sulfation. It has previously been shown that the sulfation of liver heparan sulfate.
THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 266,No. 14,Issue of May 15,pp. 8671-8674, 1991 D 1991 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U,S. A.
Communication
of HSPGs’ that occur in basement membranes and on cell surfaces. For instance, defective formation of HSPG is believed to perturb the electrostatic filtration barrier in the kidneys and thusleads to diabetic proteinuria (Kanwar et al., 1980, 1983;Deckert et al., 1989). It is therefore important to gain insight into the mechanism that regulates the incorporation of negative charge during HSPG biosynthesis. This process is initiated by the formation of a nonsulfated (Received for publication, February 1, 1991) (GlcA/3l,4+GlcNAc~ul,4-t), polymer that is subsequently Erik Ungerg, Inger Pettersson, Ulf J. Erikssonp, modified by several enzymes. The first modification step is Ulf Lindahlll, and LenaKjellen N-deacetylation of some of the glucosamine residues catalyzed From the Departmentof Veterinary Medical Chemistry, by glucosaminyl N-deacetylase. The resulting free amino Swedish University of Agricultural Sciences, the groups are sulfated by an N-sulfotransferase. The following §Department of Medical Cell Biology, University of modification reactions involve epimerization of D-GkA to LUppsala, and the llDepartment of Medical and IdoA units, 2-O-sulfation of hexuronic acid residues and 6-0Physiological Chemistry, University of Uppsala, the Biomedical Centre, Box 575, S-751 23 Uppsala, Sweden sulfation (more rarely 3-0-sulfation) of glucosamine residues (Lindahl et at., 1986; Lindahland Kjellen, 1987; Lindahl, N-Deacetylation is the initial polymer modification 1989). Since deacetylation is a prerequisite to N-sulfation and step in heparan sulfate biosynthesis and a prerequisite N-sulfate groups are required for substrate recognition by the to subsequent N- and 0-sulfation. It has previously GlcA C5-epimerase as well as by the 0-sulfotransferases, the been shown that thesulfation of liver heparan sulfate N-deacetylase has a key regulatory role in determining the is lowered in diabetes (Kjellen, L., Bielefeld, D., and overall structure and the charge density of the final product. Hijak, M. (1983) Diabetes 32, 337-342). To investiPrevious experiments showed that heparan sulfate from gate whether the reduced sulfation is the result of a diabetic rat liver was less sulfated than control heparan sullowered N-deacetylase activity, we have assayed this fate, whereas there was no difference in the ratio of N- to 0enzyme in hepatocytes from streptozotocin-diabetic sulfate groups (Kjell6n et al., 1983). These results suggested rats. In addition, the activity of the glucuronosyl C5- that N-deacetylation might be impaired in diabetes. We reepimerase, which catalyzesa modification reaction cently demonstrated that the expression of glucosaminyl Nsubsequent to N-sulfation, was measured. The deacetylase activity, expressed per microgram of cell pro- deacetylase activity in mouse mastocytoma tissue depends on tein, was about 40% lower in diabetic hepatocytes as the concerted action of (at least) two protein components, compared with control cells, whereas the epimerase one of which, a 110-kDa glycoprotein, has been purified to activity was unaffected. Recently, a 110-kDa glyco- homogeneity (Pettersson et d., 1991). Surprisingly, this protein turned out to possess N-sulfotransferase activity (Petprotein that carries N-sulfotransferase activity was identified as one of at least two protein components tersson et al., 1991) and is most likely the mouse counterpart required for N-deacetylation in mouse mastocytoma of the rat liver N-sulfotransferase (97 kDa) characterized by tissue (Pettersson,I., Kusche, M., Unger, E.,Wlad, H., Brandan and Hirschberg (1988). In thepresent paper,we demonstrate that hepatocytes from Nylund, L., Lindahl, U.,and Kjellen, L. (1991)J. Biol. Chem. 266,8044-8049). We therefore investigated if streptozotocin-diabetic rats express less N-deacetylase activthe lowered N-deacetylase activity in diabetes could ity than control cells. Furthermore, this deficiency can be be ascribed to a deficiency in either one of the corre- ascribed to the N-sulfotransferase component, whereas the sponding rat components. The results indicated that (i) other protein factor(s)required for N-deacetylase activity are the glycoprotein component ispresentinlimiting present in excess amounts in both control and diabetic cells. amounts in both control anddiabetic cells, (ii) diabetes results in a lowered activity of this component, and EXPERIMENTAL PROCEDURES (iii) excess amounts of the additional protein(s) needed for N-deacetylase activityare present in both control Materials and diabeticcells.
Decreased Activity of the Heparan Sulfate-modifying Enzyme Glucosaminyl N-Deacetylase in Hepatocytes from Streptozotocindiabetic Rats*
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Several of the complications of diabetes have been attributed to a lowered production and/or lowered charge density * This work was supported by Grants 2309, 6525, and 7475 from the Swedish Medical Research Council and Konung Gustaf V:s 80irsfond; the Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences; and Kabi AB, Stockholm. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence shouldbeaddressed. Tel: 46-18-174276; Fax: 46-18-150762.
Rats-Streptozotocin was a gift from The Upjohn Co. Female Sprague-Dawley rats (age -12 weeks) of the U and H substrains (Eriksson, 1988)wereused intheexperiments.Forinduction of diabetes, rats were given a single injection of streptozotocin (45 mg/ kg of body weight) that was dissolved in 0.01 M citrate buffer, pH 4.5, and injected into the tail vein 6-8 weeksbefore the experiment. Animals with a serum glqose concentration in excess of 20 mmol/ liter 1 week after the injectionwere considered to be diabetic. On the day of the experiment, all diabetic rats had serum glucose levels in excess of 20 mmol/liter, and all controlswere below 5 mmol/liter.
I The abbreviations used are: HSPG, heparan sulfate proteoglycan; HS, heparan sulfate; GlcA, glucuronic acid; IdoA, iduronicacid WGA, wheat germ agglutinin; MES, 2-(N-morpholino)ethanesulfonicacid; HEPES, N-2-hydroxyethylpiperazine-N’-2-ethanesulfonicacid PPO, 2,5-diphenyloxazole; POPOP, 1,4-bis[2-(5-phenyloxazolyl)] benzene.
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Cell Preparation.-Hepatocytes were isolated by collagenase perfusion and differential centrifugation according to a modification (Obrink, 1982)of the method described by Seglen (1972).This method has been previously shown to yield hepatocytes of >95% purity. The cells, pelleted by centrifugation a t 50 X g, were solubilized by vortex mixing for 1min with an equal volume of 1%Triton X-100 in 50 mM Tris-HC1, pH 8.0. After centrifugation a t 600 X g for 10 min, the supernatants were frozen in portions at -70 “C. Mouse MastocytomaN-Deacetyluse-Fractionation of mouse mastocytoma N-deacetylase was performed as described previously (Pettersson et al., 1991). Briefly, freshly dissected mouse mastocytoma tissue (Furth et al., 1957) was suspended in 50 mM Tris-HCI, pH 7.4, containing 1%Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 2 mM EDTA, 10 pg of pepstatin/ml and was then homogenized in a Potter homogenizer. After centrifugation at 100,000 X g for 1 h, the supernatant was rendered 0.15 M with regard to NaCl and applied to a column of WGA-Sepharose. The nonbinding proteins were collected, and the column was washed with 50 mM Tris-HC1, pH 7.4, containing 0.1% Triton X-100, 0.15 M NaCl, 20%glycerol,before elution with the same buffer supplemented with 0.3 M GlcNAc. Both the nonbinding fraction (denoted mouse F) and the proteins eluted with GlcNAc (denoted mouse E) were essentially devoid of N-deacetylase activity when assayed separately. However, after recombination, theactivity originally applied to thecolumn was largely restored. In theWGA-chromatography step, the active component of mouse E was purified -7-fold. The mouse E (-2.4 mgof protein/ml) and F (-13 mg of protein/ml) fractions were dialyzed against 50 mM TrisHC1, pH 7.4, 0.1% Triton X-100, 20% glycerol, before use in mixing experiments with solubilized rat hepatocyte proteins. Methods
500
1000
Hepatocyte protein added (pg) FIG. 1. Determination of the linear range of the enzyme assays. N-Acetylglucosaminyl N-deacetylase ( A ) and glucuronosyl C5-epimerase ( B ) activities were measured as described under “Experimental Procedures,” using a reference sample composed of solubilized hepatocyte protein in equal amounts from diabetic and control rats. Each value given is the mean of two determinations.
.)
Total protein was determined in a flat bottom microtiter plate essentially as described by Hartree (1971), using bovine serum albuA ‘ B min (Fraction V; Boehringer Mannheim) as a standard. Enzyme Assays-The N-acetylglucosaminyl N-deacetylase was assayed essentially as described (Navia et al., 1983), using N - a ~ e t y l - ~ H labeled Escherichia coli K5 capsular polysaccharide (specific activity, 50,000 cpm of 3H/pg of hexuronic acid) as a substrate (Pettersson et al., 1991). Samples were incubated with -10,000 cpm of substrate in 40 mM MES, 10 mM MnC12,pH 6.3, in a totalvolume of 200 pl. After incubation at 37 ‘C for 35 min, reactions were terminated by addition ” of 200 p1 of “stopping solution” (1 M monochloroacetic acid, 0.5 M C D C D NaOH, 2 M NaCl). The released [3H]acetate was determined by FIG. 2. Enzyme activities in hepatocytes obtained from scintillation counting in a biphasic system obtained by adding 5 ml streptozotocin-diabetic and control rats. N-Deacetylase (A) and of toluene, containing 0.5% (w/v) PPO, 0.03% (w/v) POPOP, and C5-epimerase ( B )activities were measured in solubilized hepatocytes 10% (v/v) isoamyl alcohol to the assay mixtures. Assays forD-glUCUrOnOSylC5-epimerase were conducted essentially obtained from 9 control (0)and 11 diabetic (0)rats. All determinaas described by Campbell et al. (1983) using N/O-desulfated, re-N- tions were made in duplicate. After calculating the enzyme activity as a substrate (specific activity, per microgram of protein for each sample, the mean values obtained sulfated, [he~uronosyl-5-~H]heparin 600-1000 cpm of 3H/pg of hexuronic acid). Samples were incubated for the N-deacetylase and C5-epimerase activities, respectively, in with -4,000 cpm of substrate in 0.05 M HEPES, pH 7.4,0.015 M control hepatocytes were set a t 100%.The solid horizontal lines depict the mean values obtained for the enzyme activities in each group, EDTA, 0.05 M KCl, and 1% Triton X-100 in a total volume of 200 disregarding the values within parentheses. pl. After incubation at 37 “C for 60 min, reactions were terminated by addition of 200 p1 of “stopping solution” (see above). The released :’H,O was determined by scintillation counting in a biphasic system ( p < 0.015). In contrast, assays for D-glucuronosyl C5-epiobtained by adding 5 ml of toluene containing 2% (w/v) PPO, 0.12% merase activity showed no apparent difference between con(w/v) POPOP, and 25% (v/v) isoamyl alcohol to the assay mixtures. The linear range of each enzyme assay was established using a trol and diabetic cells (Fig. 2 B ) . Studies on biosynthetic polymer modification in mouse reference sample composed of equal amounts of solubilizedhepatocyte protein from control and diabetic rats (Fig. 1). All subsequent assays mastocytoma tissue have shown that N-deacetylation and N were performed on 10-pl samples containing 115-185 pg ofhepatocyte sulfation both depend on the same component, a 110-kDa protein, well within the linear range of the assays. glycoprotein (Pettersson et al., 1991).While this protein alone
is sufficient for N-sulfotransferase activity, N-deacetylation requires (an) additional protein@.). Presumably, N-deacetyTo test the hypothesis that the reduced sulfation of liver lation in rat hepatocytes involves a similar mechanism. The HS in diabetes is caused by a lowered activity of glucosaminyl 110-kDa protein from mouse mastocytoma, here denoted N-deacetylase, the activity of this enzyme was quantified in “mouse E,” can be separated from the additional protein(s) hepatocytes obtained from 11 streptozotocin-diabetic and 9 needed for N-deacetylase activity, “mouse F,” by chromatogcontrol rats. For comparison, we measured another enzyme, raphy on WGA-Sepharose (see “Experimental Procedures” glucuronosyl C5-epimerase, involved in the biosynthesis of andPettersson et al. (1991)). To investigate whether the HS (Fig. 2). The mean N-deacetylase activity in cells from decreased N-deacetylase activity in hepatocytes from diabetic diabetic rats was significantly ( p c 0.001) reduced and rats was due to a defective N-sulfotransferase component, i.e. amounted to -60% of that found in control cells (Fig. 2 A ) . “rat E,” or to a lack of the other protein factor(s),“rat F,” we Calculations including the “odd outliers” (within parentheses took advantage of the fractionated mouse mastocytoma comin Fig. 2 . 4 ) gave a reduction to 70%, still clearly significant ponents. When mouse E, devoid of N-deacetylase activity, RESULTS ANDDISCUSSION
N - D e a c e t y h e Activity in Diabetes
8673
biosynthetic modification of the protein, such as thediabetesinduced nonenzymatic glycosylation (Kennedy, 1984). In the liver and other organs, insulin deficiency leads to a general depression of DNA transcription, hence a lowered protein synthesis (Jefferson et al., 1983). However, all proteins are not equally affected; for instance, in the diabetic liver, the production of albumin is preferentially decreased (Jefferson et al., 1983). Since the N-deacetylase activity was expressed as enzyme activity per microgram of total protein,the reduced activity of rat E is due to a specific reduction in theexpression - +E +F - +E +F of rat E. It is notable that the glucoronosyl C5-epimerase, FIG. 3. Effects of added mouse E andmouse F on hepatocyte which catalyzes one of the polymer-modification reactions N-deacetylase activity. N-Deacetylase activity was measured in subsequent to N-deacetylation in HS biosynthesis, appeared duplicate samples of hepatocyte protein from control ( A ) and diabetic ( B ) rats. The assays were performed on samples from four control not to be affected by the disease. The N-deacetylation reaction is a pivotal step in the bioand four diabetic rats without (-) or with the addition of mouse E (-0.17 pg/gg of hepatocyte protein) ( + E ) or mouse F (-1 pg/wgof synthetic polymer-modification process since it is a prereqhepatocyte protein) (+E'). After calculation of enzyme activity per uisite to all subsequent reactions in the formation of a HS (or microgram of hepatocyte protein, the mean value obtained for the N heparin) chain (see Introduction). In accord with prediction, deacetylase activity in control hepatocytes without addition was set Bame et aL3 found that Chinese hamster ovary cells with a at 100%. Thevalues given are means + S.D. genetically defined N-sulfotransferase deficiency produced a HS with decreased N- as well as 0-sulfate contents andwith a lower IdoA/GlcA ratio than the HS synthesized by wildtype Chinese hamster ovary cells. Similar differences revealed by structural analysis of HS synthesized by diabetic and normal rat hepatocytes (Kjell6n et al., 1983),4thus can be ascribed primarily to defective N-deacetylation. Administration of insulin to thediabetic animals restored normal glycosaminoglycan structure? Therefore, it seems reasonable to conclude that the defective N-deacetylation of the HS pre0.0 0.5 1.0 1.5 cursor chain in diabetes and hence the other structural aberMouse E added rations observed, are caused by insulin deficiency. In fact, an ( @ I & ! hepatocyte protein) independent study by A. Kofoed-Enevoldsen; which also FIG. 4. Titration of hepatocyte N-deacetylase with mouse E. Increasing amounts of mouse E were added to duplicate samples revealedlowered N-deacetylase activity in diabetic liver, of solubilized hepatocyte protein obtained from one control (0)and showed that the enzyme activity was restored to normal by one diabetic (0)rat. After calculation of N-deacetylase activity per insulin treatment of the animals. microgram of protein, the value obtained for N-deacetylase activity HS is a ubiquitous component on cell surfaces and in in control cells without any added mouse E was set at 100%. extracellular matrices (Kjell6n and Lindahl, 1991), anda derangement of normal HS structure thus may be expected was added to solubilized hepatocytes, the enzyme activities of to affect various functional properties of tissues and organs. both diabetic and control cells increased substantially (Fig. A number of the clinical complications associated with dia3). Hence, both diabetic and control cells seemed to contain betes appear to derive from structural/functional alterations excess amounts of rat F. In contrast, addition of mouse F of basement membranes (Deckert et al., 1989), andit is (even over a wide range of concentrations; not shown in detail) therefore significant that basement membrane HS (Cohen et had little or no effect on the N-deacetylase activity (Fig. 3), al., 1988), similar to liver HS (Kjell6n et al., 1983), is underindicating that ratE was present in limiting amounts in both sulfated in diabetes. The modified HS chains will beincapable types of cells. of normal interaction with other basement membrane comMouse E is separated from mouse F by chromatography on ponents such as laminin, collagen type IV, and fibronectin WGA-Sepharose at physiological ionic strength (Pettersson (such interactions are furtherimpaired due to non-enzymatic et al., 1991). Chromatography under mild conditions on blue glycation of the proteins (Tarsi0 et al., 1987, 1988)), potenSepharose or thiol-Sepharose can also be used for this pur- tially resulting in perturbed assembly/organization of the pose,' indicating that the affinity between the N-deacetylase basement membrane. Our finding of diabetes selectively afcomponents is low. It is therefore not surprising that we were fecting a key enzyme in the biosynthesis of HS may therefore unable to saturate rat Fwith increasing amounts of mouse E provide novel clues to theunderstanding of the pathogenesis (Fig. 4). However, control and diabetic hepatocytes seemed to of this disease. contain similar amounts of rat F, since their N-deacetylase REFERENCES activities at any given amount of added mouse E were increased to similar levels. Diabetes thus leads to a reduction Brandan, E., and Hirschberg, C. B. (1988) J. Biol. Chem. 263,2417in the N-deacetylase-related activity of rat E, whereas rat F 2422 seems to be present in nonlimiting amounts in both control Campbell, P., Feingold, D. S., Jensen, J. W., Malmstrom, A,, and Roden, L. (1983) Anal. Biochem. 131,146-152 and diabetic cells. Unfortunately, due to a large variability in the assay, we were unable to reliably quantitate N-sulfotrans- Cohen, M. P., Klepser, H., and Wu, V.-Y. (1988) Diabetes 37,13241327 ferase activity, i.e. the other activity ascribed to component Deckert, T., Feldt-Rasmussen, B., Borch-Johnsen, K., Jensen, T., E, in hepatocytes. The reduced activity of rat E could be due to a lowered K. J. Bame, K. Lidholt, U. Lindahl, and J. D. Esko (1991) J . Biol. expression of the protein or to an altered processing or post- Chem. 266,in press. Y
' I. Pettersson, unpublished data.
E. Unger, unpublished data.
' A. Kofoed-Enevoldsen, personal communication.
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icul Applications, pp. 159-189, Edward Arnold, London Lindahl, U., and Kjellen, L. (1987) Biology of Proteoglycans, pp, 59104, Academic Press, Orlando Lindahl, U., Feingold, D. S., and Roden, L. (1986) Trends Biochem. Sci. 11,221-225 Navia, J. L., Riesenfeld, J., Vann, W. F., Lindahl, U., and Roden, L. (1983) Anal. Biochem. 135, 134-140 Obrink, B. (1982) Methods Enzymol. 82,513-529 Pettersson, I., Kusche, M., Unger, E., Wlad, H., Nylund, L., Lindahl, U., and Kjellkn, L. (1991) J. Biol. Chem. 266,8044-8049 Seglen, P. (1972) Exp. Cell Res. 74,450-454 Tarsio, J. F., Reger, L. A., and Furcht, L. T. (1987) Biochemistry 2 6 , 1014-1020 Tarsio, J. F., Reger, L. A., and Furcht, L. T. (1988) Diabetes 37,532539
