Endosomal Proteolysis of Insulin by an Acidic Thiol Metalloprotease ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY (0 lQ94by The AmericanSociety for Biochemistry and Molecular Biology, Inc.

Vol. 269. No. 4, Issue of January 28, pp. 3010-3016.1994 Printed in U.S.A.

Endosomal Proteolysis of Insulin by an Acidic Thiol Metalloprotease Unrelated to Insulin Degrading Enzyme* (Received for publication, September 2, 1993, and in revised form, September 30, 1993)

Frangois AuthierSO, Richard A. Rachubinskill, Barry I. Posnerll ,and JohnJ. M. BergeronS** From the $Department of Anatomy and Cell Biology and the IlDepartment of Medicine, McGill University, Montreal, Quebec H3A 2B2, Canada and the VDepartment of Biochemistry, McMaster University, Hamilton,Ontario L8N 325, Canada

Although insulin is degraded as a consequence of receptor-mediated endocytosis, the location and nature of the responsible proteinase(s) remain controversial. Insulindegrading enzyme (IDE; EC 3.4.22.11), a mainly cytosolic neutral thiolmetalloendopeptidase of 110 kDa, has been proposed to be the main cellular clearance mechanism. However, endosomes concentrate and degrade internalized insulin demonstrating that IDE is unlikely to be the relevant enzyme for endosomal proteolysis of internalized insulin in liver parenchyma. In purified endosomal fractions insulin was actively degraded at acid pH and IDE was undetectable as evaluated by immunoblotting, immunoprecipitation, or chemical cross-linking procedures. Affinity purifiedendosomal acidic insulinase displayed a pH optimum of 4-6.6, a lack of inhibition by EDTA and N-ethylmaleimide, and a partial metal-ion requirement (forMna+)all of which distinguished it from IDE. A small but detectablepresence of IDE in particulate nuclear (N) and large granule (ML) fractions was observed by differential centrifugation. By analytical centrifugation, IDE cosedimented with the organelle containing the peroxisomal marker proteins catalase and thiolase(median density, 1.21 g.cm-’). By preparative centrifugation,highly purified peroxisomes were observed to be enriched inIDE. Since allcloned cDNAs of IDE (human, rat, andDrosophila) reveal a deduced classical peroxisomal targeting sequence A/SKL at their carboxyl termini this may account for the peroxisomal location of IDE. Taken together, our studies identify an insulin-degrading enzyme in endosomes which is distinct from IDE. The latter’s presence in peroxisomes suggests that itsphysiological substrate(s) in vivoare polypeptides other thaninsulin.

Insulin secreted from the pancreas is extracted from the portal circulation to the extent of >45% in a single pass through the liver (Jaspan et al., 1981). This is effected by a receptor-mediated mechanism (TerrisandSteiner, 1975, 1976) which involves the rapid internalization of surfacebound insulin into components of the endosomal apparatus (Posner et al., 1980; Bergeron et al., 1985). We and others

* This work was supported in part by a grant from the Medical Research Council of Canada (to B. I. P. and J. J. M. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18U.S.C. Section 1734 solelyto indicate this fact. 8 Recipient of a postdoctoral fellowship from the Medical Research Council of Canada. ** To whom correspondence should be addressed Dept. of Anatomy and Cell Biology, McGill University, Montreal, Quebec H3A 2B2, Canada. Tel.: 514-398-6351; Fax: 514-398-5047.

have concluded that endosomes are a major site of degradation of insulin (Pease et al., 1985,1987; Hamel et ul., 1988;Doherty et al., 1990; Backer et al., 1990). This conclusion is based on (i) the extraction from endosomes of insulin fragments corresponding to thei n vivo sites of hormone hydrolysis (Hamel et al., 1988); (ii) the demonstration in cell-free systems of an ATP-dependent degradation of endosomal insulin with the generation of hormone fragments cleaved at the same sites observed in vivo (Hamel et al., 1988; Doherty et al., 1990; Clot et al., 1990); (iii) the inhibition of endosomal insulin degradation by acidotropic agents (i.e. chloroquine) in vivo and in cell-free systems (Posner et al., 1982; Khan et al., 1985; Bergeron et al., 1986; Smith et al., 1989; Doherty et al., 1990); (iv) the insensitivity of endosomal insulin degradation to inhibitors of lysosomal proteases in cell-free endosomes (Doherty et al., 1990) and in hepatoma Fao cells (Backer et al., 1990); and(v)the demonstration of ligand specificity in endosomal degradation using cell-free systems with intact endosomes (Doherty et al., 1990). By contrast, enzyme purification of whole cell extracts has identified a thiol-metalloendopeptidase,insulin degrading enzyme (IDE),’ with a neutral pH optimum and a wide tissue distribution (Duckworth, 1988). IDE was observed to be cytosolic in location and to display broad substrate specificity including insulin, glucagon, insulin growth factor 11, and atrial natriuretic factor polypeptides (Duckworth and Kitabchi, 1974; Kirschner and Goldberg, 1983; Duckworth, 1988; Rose et al., 1988; Misbin and Almira, 1989; Muller et al., 1991, 1992). The cDNA for IDE has been cloned and sequenced from human (Affholter et al., 1988, 1990), rat (Baumeister et al., 1993)) and Drosophila (Kuo et al., 1990) with sequencerelated similarities identified. The present study has attempted to evaluate the relevance of cytosolic IDE to theendosomal processing of insulin. Using immunoblotting and cross-linking procedures we detect IDE in cytosolic as well as peroxisomal fractions but not inendosomes. Moreover an affinity purified endosomal insulinase was differentiated from IDE by virtue of its acid pH optimum for insulin binding and degradation, its insensitivity to EDTA and N-ethylmaleimide, and its partialmetal-ion requirement. EXPERIMENTALPROCEDURES

Ligand Radioiodination, Protein Determination, Enzyme Assays, Animals, and Materials-Porcine insulin (Sigma) was radioiodinated by the chloramine-?‘ method as described previously (Posner et al., 1982) to specific activities of 100-180 pCi/pg. The protein content of isolated fractions was determined by the method of Bradford (1976). The abbreviations used are: IDE, insulin degrading enzyme; EN, endosomal fraction; ENS, soluble endosomal extract; GAP, GTPaseactivating protein; PAGE, polyacrylamide gel electrophoresis; Per, peroxisomes from normal rats; Per-c, peroxisomes from clofibratetreated rats; Mit, mitochondria.

3010

Endosomal Acidic Insulinase Acid phosphatase was assayed as described previously (Bergeron et al., 1982). Catalase was assayed as described by Baudhuin etal. (1964). Male Sprague-Dawley rats weighing 180-200 g were obtained from Charles River Laboratory (St. Constant, Quebec) and were fasted for 18 h before sacrifice. '"I-Labeled goat anti-rabbit IgG was purchased from DuPont-New England Nuclear and '"I-labeled goat anti-mouse IgG from ICN. Mouse monoclonal antibody 9B12 directed against the human IDEwas a kind gift of Dr. R. Roth (Stanford, CA). Rabbit polyclonal antiserum 2BS directed against the human IDEwas a kind gift of Dr. M. Rosner (Chicago, IL). Anti-GAP monoclonal antibody GP-15 was provided by G. Y. Zhang (Mollat et al., 1992). All other chemicals were reagent-grade from Sigma or Fisher. Liver Subcellular Fractionation-Rat liver nuclear (N), large granule (ML), and cytosolic fractions (S) were isolated by differential centrifugation as previously described (Bergeron et al.,1986; Khan et al.,1986, 1989).The ML fraction was subfractionated by centrifugation in analytical sucrose density gradients by loading l ml of this fraction ontothe top of 13-ml gradients of 0.5 to 2.0 M sucrose. After centrifugation a t 285,000 X g., for 2 h, 18 fractions were collected and analyzed for enzyme activity. Purified peroxisomes were obtained from normal (Per) andclofibrate-treated rata (Per-c) asdescribed by 0.5-fold catalase Bodnar and Rachubinski (1991) with an 18.6 enrichment for peroxisomes isolated from control rats (Per).Purified mitochondria (Mit), enriched 7.5-fold over homogenate in cytochrome c oxidase, were obtained as described by Bodnar and Rachubinski (1991). Rough endoplasmic reticulum was prepared by the protocol of Walter and Blobel (1983) as described previously (Wada et al., 1991; Ou et al., 1992). Plasma membrane was prepared according to the method of Neville (1968) as described by Authier et al. (1990, 1992). Combined endosomes (EN) were collected at the0.25 to 1.0 M sucrose interface of discontinuous sucrose gradients according to the method of Khan etal. (1989). The soluble extract from the endosomal fractions (ENS) was isolated by freeze/thawing in 5 mM sodium phosphate, pH 7.4, and disruption in the same hypotonic medium with a small Dounce homogenizer (15 strokes with the tight Type A pestle) followed by centrifugation a t 300,000 X , . g for 30 min. The resultant extract was referred to as the soluble endosomal fraction (ENS). Immunoprecipitation-Cytosol (S), endosomes (EN), and soluble endosomal extracts (ENS)(1mg/ml) were treated with Triton X-100 (0.1% final concentration) for 30 min at 4 "C. After centrifugation a t 300,000 X g., for 30 min, the supernatant was incubated with 50 x lo-' to M monoclonal anti-human IDE 9B12 for 16 h at 4 "C in 100 pl of 50 mM sodium phosphate buffer, pH 7.4. The monoclonal antibody was precipitated by the addition of40 pl of protein-ASepharose coated with anti-mouse IgG. After rotating for 60 min at room temperature, the fractions were centrifuged for 5 min at 10,OOO X g ." and the resultant supernatants were diluted in 0.2 M citrate phosphate buffer, pH 5 or 7, and tested for insulin degrading activity (Doherty et al., 1990). Immunoblots-After SDS-PAGE, samples were transferred to nitrocellulose filters (0.45 pm) for 90 min at 380 mA in transfer buffer containing 25 mM Tris base and 192 mM glycine. The filters were incubated for 2 h in blocking buffer (10 mM Tris-HC1, pH 7.5, 300 mM NaCl, 5% skim milk, and 0.05% Tween 20) followed byincubation with the primary antibody in the same solution for 16 h at 4 'C (mouse monoclonal 9B12 anti-human IDE diluted 1500, mouse monoclonal anti-human GAP diluted 1:200, rabbit antisera against human IDE diluted 1:200, rat liver fatty acyl-CoA oxidase diluted 1:300, rat liver hydratase-dehydrogenase diluted 1:300, and rat liver 3-ketoacyl-CoA thiolase diluted 1:300). After incubation, the filters were washed 3 times in 10 mM Tris-HC1, pH 7.5,300 mM NaCl, 0.5% skim milk, and 0.05% Tween 20 over a period of 1 h at room temperature. Bound immunoglobulin was detected using 'Z61-labeled goat anti-rabbit I g G except for the monoclonal 9B12 anti-IDE and the anti-human GAP GP-15 for which '=I-labeled goat anti-mouse IgG was used. Cross-linkingStudies-A modified protocol of Shii et al. (1985)was followed. The cytosolic fraction (30 pg of protein) and soluble endosomal extract (ENS) (40 pgof protein) were incubated in a final volume of30 and 80 pl, respectively, of 50 mM sodium phosphate buffer, pH 5-8,75 fmol of '261-insulinfor 60 min a t 4 "C or 15 min at 21 "C in the presence of 1 mM 1,lO-phenanthroline. Cross-linking was performed by adding disuccinimidyl suberate (0.01 M in dimethyl sulfoxide; final concentration 0.3 mM) for 15 min at the same temperature and terminated by the addition of 2 pl of 1 M Tris-HC1, pH 7.4. The preparations were made 1% in SDS and 2% in 8-mercapto-

*

3011

ethanol, heated for 1 min a t 100 "C, and subjected to SDS-PAGE in a 10% acrylamide resolving gel. Affinity Binding of Endosoml Acidic Insulinase-The endosomal soluble extract (100 pg) was adjusted to pH 5, 6, or 7 with 50 mM citrate phosphate buffer (final volume 1ml) and incubated with 1ml of bovine insulin-agarose for 16 h at 4 "C. The columns were then washed with 16 ml of the same buffer after which bound endosomal acidic insulinase activity was eluted with 16 ml of 50 mM Na2C03, pH 10.7. Each fraction (30 drops, approximately 1.8 ml) was immediately neutralized with 1 M Hepes and evaluated for protein content and '"I-insulin degrading activity at pH 5. The peak of endosomal acidic insulinase, eluted from insulin-agarose at pH 5 (fractions 1014; see "Results"), was pooled, concentrated by Amicon concentrator (YM-30 membrane), and analyzed by SDS-PAGE (Laemmli, 1970). Hormone Degradation Assays-'261-Insulin degradation was measured by the loss of trichloroacetic acid-precipitable radioactivity. Approximately 20 fmol of '"I-hormone were incubated at 37 'C with various fractions in 100 pl of 0.2 M citrate phosphate buffer at the indicated pH. Reactions were stopped by adding 2 ml of 10% ice-cold trichloroacetic acid. After 15 min at 4 "C, the samples were centrifuged at 10,OOO X g., for 20 min a t 4 "C, and the supernatants and pellets evaluated for radioactivity in a 1282 LKB y-counter. Insulin degrading activity is expressed as the percentage of total insulin degraded within the linear range of the degradation curve. RESULTS

We have attempted to evaluate the contribution of IDE to endosomal insulin degradation by (i) affinity cross-linking procedures to detect IDE; (ii) quantitative immunodepletion of IDE; (iii) assessment of the content of IDE in endosomes using Western blot analysis; and (iv) partial purification of endosomal acidic insulinase by affinity chromatography.

Identification of IDE in the Cytosolic but Not in Endosomal Fractions by Cross-linking and Imrnunodepletion-Chemical cross-linking experiments were done at pH 5-8 using '"Iinsulin with disuccinimidyl suberate as cross-linker (Fig. 1A). A prominent iodinated complex of 110 kDa was identified when cross-linking was done at pH 6-8 with a cytosolic ( S ) fraction (lanes 1-4). This protein corresponded to IDE since it was immunodepleted by the well characterized monoclonal antibody (9B12 to human IDE)of Shii and Roth (1986) (lanes 9 and IO). Immunoprecipitation with control non-immune mouse IgG revealed no depletion of the '261-insulin.IDE complex (lanes 7 and 8). When endosomes were comparably evaluated, no 110-kDa protein was found (Fig. LA, lanes 5 and 6). Insulin degradation by the S fraction and by the soluble extract from endosomes (ENS)was evaluated as shown in Fig. 1B. Insulin degradation was greater at pH 7 than pH 5 for the S fraction, whereas the opposite was found for the endosomal extract (Fig. lB,insets). When immunoprecipitation with increasing amounts of monoclonal antibody 9B12 was performed on the S fraction, greater than 75% of the insulin degrading activity was removed at anantibody concentration of lo-' M (Fig. 1B). Incontrast, incubation of the endosomal extract over the same range of antibody concentrations had no discernible effect on insulin degradation even at neutral pH (Fig. 1B). Partial Purification of Endosomal Acidic InsulinaseSubfractionation of endosomes into a soluble extract and a membrane fraction revealed that about 85% of an acid insulin degrading activity was found inthesupernatant fraction (Table I, lysate supernatant). Addition of the soluble endosomal extract to insulin-agarose columns revealed binding at pH 5 of an insulinase activity whose activity was also maximal at pH 5 (Fig. 2 4 ) . Elution with sodium carbonate, pH 10.7, resulted in the release of about 40% of the total recovered acidic insulinase (Table I). When the soluble endosomal extract was added to insulin-agarose columns at pH 6 or 7, progressively less binding of this activity was observed (Fig.

Endosomal Acidic Insulinase

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9B12 antibody concentration(M) FIG. 1. Comparison of the content ofIDE between cytosoland soluble endosomal extract from rat liver homogenates. Panel

A, 'mI-insulin (75 fmol) was cross-linked at theindicated pH values to proteins from the S (1 mg/ml, lanes 1-4) and ENS fractions (0.5 mg/ ml, lunes 5 and 6 ) with 0.3 mM disuccinimidyl suberate and 1 mM 1,lO-phenanthroline according to the protocol of Shii et al. (19851, and the fractions were subjected to SDS-PAGE as described under "Experimental Procedures." Native cytosolic IDE was immunoprecipitated with monoclonal antibody 9B12 (lunes 9 and IO) or mouse IgG (lanes 7 and 8),and the supernatants were then subjected to cross-linking and analyzed on SDS-PAGE. The arrow on the left indicates the mobility of the cross-linked complexes (about 110 kDa). The radioactive band a t about 69 kDa corresponds to radioactive bovine serum albumin which is present in the iodination mixture. Panel B, proteins (1 mg/ml) fractions were immunodepleted of IDE inthe presence of 0.1% Triton X-100 using monoclonal antibody 9B12 at the from the S and the ENS indicated concentrations. After immunoprecipitation and centrifugation, the resultant supernatant was tested for the ability to degrade '=Iinsulin at pH 7 (W) and pH 5 (0).Control experiments were done with normal mouse IgG with no effect noted at any concentration. Results have been normalized (100%)to that seen with normal mouse IgG. The insets in each panel show the specific activities of insulin degradation in the absence of monoclonal antibody 9B12 at pH5 (white bars) and pH7 ( b h k bars) with data expressed as femtomoles of ligand degraded per min/mg of cell fraction protein. Identical results were obtained when all assays were carried out in the absence of Triton X-100.

TABLE I Partialpurification of the endosoml acidic insulinase from endosomnl fraction (EN) of rat liver homogenate The endosomal acidic insulinase was partially purified from 98 mg of EN fraction obtained from rat liver homogenate as described under "Experimental Procedures." After an hypotonic shock, the lysate supernatant from the 300,000 X g., X 30 min centrifugation (about 7% of proteins from the startingfraction) was loaded onto a bovine insulin-agarose affinity column (1.5 X 12.5 cm) at pH 5 andthe proteinase was eluted by increasing pH to 10.7 as shown in Fig. 2A. The active fractions were pooledand concentrated. At each purification step, the specific activity of the enzyme was determined at pH5 by the trichloroacetic acid precipitation assay. Purification step Volume Relative purification activity Yield Specific protein activity Total Total ml

78.2 2604.8

Purified EN Lysate supernatant 7.1 Insulin-aearose 0.08

27 23.5 4.6

mg %/min

fmol/min/mg

984.61

2A). By contrast, greater than 45% of the neutral pHinsulin degrading activity of the S fraction was bound to insulinagarose at pH values of 6 and 7 (resultsnot shown) as previously demonstrated by Duckworth et al. (1972). The elution of endosomal acidic insulinase bound at pH5 yielded about 400-fold purification over parent endosomes (Table I). Following elution of the endosomal proteins bound at pH 5,

6.68

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100

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1

evaluation by SDS-PAGE showed two major Coomassie Bluestained proteins of 80 and 66 kDa (Fig. 2 B ) . To characterize the partially purified endosomal acidic insulinase, its pH optimum and theeffects of various proteinase inhibitors were evaluated (Fig. 3 and Table 11).The endosomal activity showed a broad pH optimum (from 4.0 to 5.5) (Fig. 3) distinct from that previously shown for IDE (Shii et al.,

Endosomal Acidic Insulinase FIG.2. Affinity purification of endosomal acidic insulinase. PanelA, the soluble endosomal extract (ENS)was incubated for 16 h at 4 "C with bovine insulin-agarose in 50 mM citrate phosphate buffered at theindicated pH. The gels were then washed with 16 ml of the same buffer and bound endosomal acidic insulinase activity was eluted with the same volume of 50 mM NazCO3,pH 10.7 (indicated by arrows). Portions (50 pl) of each fraction were incubated with lZ6Iinsulin (20 fmol) in 0.2 M citrate phosphate buffer, pH 5 (D).Insulin degrading activity was evaluated by trichloroacetic acid precipitation. The protein concentration of each fraction is indicated (A). Pam1 B, the distribution of proteins in the parent endosomal fraction ( l a n e 1 ) and in the affinity purified endosomal acidic insulinase (lane 2 ) are shown by staining with Coomassie Blue. Approximately 10 pgof protein have been applied to lane 1. The mobilities of the molecular mass markers areindicated in kDa on the ktt.

A

80

3013 pH S

60

80 60

B 200

116.2 91.4

*80 66.2

+66

45

0

5

10

15

Fraction number

TABLEI1 Effectof proteinase inhibitors on the degradation of ~251]iodoinsulin by partially purified endosornal acidic insulinase Endosomal acidic insulinase was partially purified (see Fig. 2) and incubated in 0.2 M citrate/phosphate, pH 5.0, with ['251]iodoinsulin (20 fmol) in the absence or presence of the indicated compounds. With the chelator 1,lO-phenanthroline, the ability of divalent metal ions to restore proteolytic activity to metal-depleted proteinase was also examined. After 60 min at 37 "C, the amount of trichloroacetic acid-soluble radioactivity was measured and expressed as the percentage of trichloroacetic acid-soluble radioactivity observed in the absence of inhibitor. The results shown are the means f S.D.of three determinations. Addition

7.56.55.54.53.5

Inhibitor ['"I]Iodoinsulin concentrations degraded % of control

PH FIG. 3. Degradation of ['261]iodoinsulinby partially purified endosomal acidic insulinase as a function of pH. Samples of partially purified endosomal insulinase (see Fig. 2) wereincubated in 0.2 M citrate phosphate buffer at theindicated pH in the presence of ['261]iodoinsulin(20 fmol). These suspensions were incubated for 30 min at 37 'C. The percentage of insulin degraded during incubation was estimated by the amount of trichloroacetic acid-soluble radioactivity released. The results shown are the mean f S.D. of three determinations.

1986). At pH 5, proteolysis of insulin was partially inhibited by 1,lO-phenanthroline, a chelating agent, by para-hydroxymercuribenzoate, a thiolreagent, and by bacitracin, indicative of a thiol metalloproteinase. However, it was unaffected by EDTA, N-ethylmaleimide, and iodoacetamide, the latter two of which inhibited IDE activity (Shii et al., 1986). The low level of inhibition by pepstatin A is consistent with some cathepsin D-like contamination, previously identified in hepatic (Runquist and Havel, 1991) and alveolar macrophage endosomes (Diment et al., 1988). Endosomal insulinase activity was unaffected by serine protease inhibitors (phenylmethylsulfonyl fluoride and benzamidine) or by the lysosomal protease inhibitor, leupeptin. Adding certain divalent cations to metal-depleted endosomal insulinase (Mn2+ andCo2+)restored activity completely, while others (Ca2+, MgZ+, and Zn2+)were ineffective (Table 11). Higher concentrations of Zn2+ (50-100 mM) actually in-

None Phenylmethylsulfonyl fluoride Benzamidine N-Ethylmaleimide Chloroquine Leupeptin Pepstatin-A Bacitracin Iodoacetamide p-Hydroxymercuribenzoate EDTA 1,lO-Phenanthroline + 10 mM MnC12 + 10 mM CoCl2 + 10 mM CaC12 + 10 mM M g C 1 2 + 10 mM ZnC12

1 mM 1 mM 1 mM 1mM 86.010 pg/ml 10 pg/ml 5 mg/ml 1 mM 1 mM 1 mM 0.1 mM 0.1 mM 0.1 mM 0.1 mM 0.1 mM 0.1 mM

100 97.6 f 6.13 104.7 k 16.0 115.9 f 3.23 114.8 f 3.56 f 1.00 72.0 f 2.10 43.0 f 3.34 104.2 f 14.6 8.76 f 3.85 104.1 f 5.25 44.9 f 2.84 101.1 f 1.33 98.6 f 6.32 43.8 f 3.03 46.3 k 7.16 37.6 f 2.16

hibited endosomal acidic insulinase activity(data not shown). Identification of IDE in Peroxisomes-Hamel et al. (1991) observed IDE in endosomal fractions by Western blotting. We observed IDE in cell fractions but could not confirm an endosomal localization. A systematic evaluation by immunoblot analysis of subcellular fractions isolated by differential centrifugation revealed IDE in particulate (N and ML) fractions aswell as in a cytosolic (S) fraction (Fig. 4). We elected to pursue this observation to gain possible insight into the physiological role of IDE. The IDE content of equal amounts of protein from nuclear

Endosomal Acidic Insulinase

3014 N

ML S

PM

EN ENS

5B, panels 3-5) revealed the expected immunoreactivity in

peroxisomes but detectable reactivity in the S fraction was * 7 (110’

I

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1

found only for thiolase (panel 5 ) . Clofibrate induces the proliferation of liver peroxisomes and increases the protein content of peroxisomes (cf. Per uersus Per-c, panels 3-5). Such peroxisomes (Per-c) revealed lower quantities of catalase but increased levels of enzymes of the peroxisomal @-oxidationpathway such as fatty acylCoA oxidase, hydratase dehydrogenase, and thiolase (Rachubinski et al., 1984; Bodnar and Rachubinski, 1991). No increase in IDE was detected inclofibrate peroxisomal fractions (Per-c), however, the level of the protein was maintained.

..“r

(120)

DISCUSSION

The purification of insulin degrading activity from rat liver cytosol (Duckworth et al., 1972) and red blood cells (Shii et al.,1986) yielded a thiolmetalloendopeptidase (IDE orinsulin protease) optimal at neutral pH. The cDNA for this enzyme has been cloned (Affholter et al., 1988,1990). However, it has remained unclear as to how insulin would come into contact with this IDE since biochemical and morphological evidence has demonstrated that endocytosed insulin initially accumulates and concentrates in the endosomal apparatus of liver parenchyma and rathepatoma cells inside which a progressive loss of insulin integrityhas been demonstrated (Posner et al., 1980; Bergeron et al., 1985; Hamel et al., 1988; Doherty et al., (N), large granule (ML), cytosolic (S), plasmamembrane (PM), and endosomal (EN) fractions was evaluated with 1990; Clot et al., 1990; Backer et al., 1990).However, a either a polyclonal antibody 2BS (Fig. 4A) to human IDE cytosolic pathway for insulin degradation cannot be formally (Kuo et al., 1993) or a monoclonal antibody 9B12 (Fig. 4B) to excluded (Harada et al., 1993). Affholter et al. (1990) and Hamel et al. (1991) have proposed human IDE (Shii and Roth, 1986). The soluble (S) fraction contained the highest concentration of antigen (110 kDa), that IDE may also be found in endosomes. Indeed Hamel et although immunoreactivity was also found in the low speed al. (1991) proposed that endosomal insulin degradation is particulate ML andN fractionsespecially with the polyclonal effected at neutral pH via IDE. We were unable to confirm antibody 2BS. Endosomes (EN) and the soluble hypotonic the presence of IDE in endosomes by chemical cross-linking, extract from endosomes (ENS) showed no detectable immu- immunodepletion with monoclonal antibody to IDE, or by immunoblot analysis. In the study of Hamel et al. (1991), a noreactivity. Using anti-GAP monoclonal antibodyGP-15 very small level of IDE was detected by Western blotting of (Mollat et al., 1992), immunoreactivity was restricted to the S fraction with none detectable the in ML or N fractions (Fig. an endosomal fraction with the same monoclonal antibody 9B12).However, the 4C). Hence immunoreactivity in organelles sedimenting in used in the presentstudy(antibody amount of endosomal cell fraction protein evaluated was not the N or ML fractions was unlikely to be due to nonspecific reported by Hamel et al. (1991) nor was any comparison made adsorption of cytosolic proteins. To localize IDE within the ML fraction, the latter was with IDE present in other subcellular fractions including the subfractionated on sucrose gradients and IDE activity was cytosol. Hence it remains difficult to evaluate if the Western compared with that of marker enzymes (Fig. 5A). IDE, eval- blot analysis (Fig. 9 of Hamel et al., 1991) revealed a minor uated by immunoblotting with monoclonal antibody 9B12 or contaminant whose signal was amplified by the method of by the assessment of insulin degrading activity a t neutral pH, Western blot analysis employed in this study. The possibility of a particulate form of IDE, indicated by revealed a median density of 1.2099 g - ~ m - This ~ . coincided Hamel et al. (1991),was pursued in ourstudies. We confirmed with the peak of the peroxisomal marker catalase (median density of1.2095 g.cmm3)and with the distribution of the that IDE is associated with the N and ML particulate fracperoxisomal marker thiolase as evaluated by immunoblotting tions, as well as thecytosolic (S) fraction. In thelarge granule with polyclonal antibody (Fig. 5A). By contrast, acid phos- (ML) fractionIDE was detected biochemically (using an phatase activity sedimented with a lower median density of insulin proteolysis assay at neutral pH) as well as by immu1.1815 g - ~ m and - ~ total protein distributed with a median noblot analysis. The analytical approach (de Duve, 1975) demonstrated the coincident sedimentation (median density, density of 1.1731 g . ~ m (results -~ notshown). ~ )the organelle harboring IDE (as assayed by Subsequently organelles sedimenting in the ML fraction 1.21 g - ~ m - of (ie. endoplasmic reticulum, endosomes, peroxisomes, and mi- its insulinase activity optimal at neutral pH and immunotochondria (de Duve, 1975; Bergeron et al., 1986; Khan et al., reactivity using IDE-specific antibodies) and theperoxisomal 1986)) were purified using established procedures and evalu- marker enzymes catalase and thiolase (Fig. 5A). The localizaated for their contentof IDE by immunoblotting with mono- tion of IDE in peroxisomes was evaluated by generating a clonal antibody 9B12 (Fig. 5B,panel 1) or polyclonal antibody highly enriched peroxisome fraction (>go% purity; Bodnar 2BS (Fig.5B, panel 2). Rough endoplasmic reticulum and and Rachubinski, 1991). Such purified peroxisomes and theS mitochondrial fractions showed little and endosomes no de- fraction showed equal concentrations of IDE, whereas purified tectable immunoreactivity. Peroxisomal fractions revealed a mitochondria, endosomes, or rough endoplasmic reticulum concentration of IDE equivalent to that in the S fraction. contained low to negligible IDE immunoreactivity (Fig. 5B). The recent compilation of IDE sequences by Baumeister et Immunoblotting for the peroxisomal marker enzymes fatty acyl-CoA oxidase, hydratase dehydrogenase, and thiolase (Fig. al. (1993) has revealed at the extreme carboxyl termini of

FIG. 4. Assessment of IDE by-immunoblot analysis of subcellular fractions isolated from rat .liver homogenates. Panel A, evaluation with polyclonal antiserum 2BS to human IDE (kind gift of Dr. M.Rosner); Panel B, evaluation with monoclonal antibody 9B12 to human IDE (kind gift of Dr. R. Roth); Panel C, evaluation with monoclonal antibody to GAP. Fifty pg of cell fraction protein were applied to each lane. The designation N, ML, and S corresponds to the 1,500 X g., X 10-min pellet, 27,000 X g., X 10-min pellet, and 130,000 X g,, X 60-min supernatant, respectively. PM refers to the plasmalemma fraction; EN to the endosomal fraction; and ENS to the soluble extract from the parent ENfraction. The molecular mass of IDE is 110 kDa in both A and B.

3015

IOE

-

(110)

1

2

3

4

5

6

7

8

9

10

11 12 13 14 15

16

1718

FRACTlONS

FIG. 5. Association of D E activity with peroxisomal fractions. Panel A, a large granule (ML) fraction was isolated from control rata and subfractionated by centrifugation on linear sucrose density gradients as described under “Experimental Procedures.” In each subfraction, insulin degrading activity a t neutral pH and catalase activity were measured, and the content of IDE and thiolase ( 2 % ) were evaluated by immunoblotting with monoclonal antibody 9B12 and polyclonal antibody, respectively. Frequency refers to the percentage of total enzymatic activity recovered in each fraction. Panel B, the cytosolic ( S ) , rough endoplasmic reticulum ( E R ) , endosomal ( E N ) , peroxisomal (Per), andmitochondrial fractions ( M i t )were evaluated by immunoblotting for their contentof IDE with monoclonal antibody 9B12 (Panel 1 ) ; for their content of IDE with the polyclonal antiserum 2BS (Panel 2 ) ; for their content of fatty acyl-CoA oxidase (AOX) (Panel 3); for their content of hydratase dehydrogenase (HD)(Panel 4 ) ; and for their content of 3-ketoacyl-CoA thiolase (Th) (Panel 5). The fractions were prepared from rat liver homogenates as described under ”Experimental Procedures.” Peroxisomal fractions were also prepared from clofibrate-treated rats (Per-c). Twenty pgof protein were loaded onto each lane and gelswere subjected to SDS-gel electrophoresis, transferred to nitrocellulose, and immunoblotted with antibodies as described under “Experimental Procedures.”

human, rat, and Drosophila IDES a peroxisomal targeting motif A/SKL (Gould et al., 1989, 1990) sufficient for protein import into peroxisomes in yeasts, plants, insects, and mammalian cells (Gould et al., 1990). Furthermore, the chemical addition of the SKL motif onto nonperoxisomal proteins was sufficient to direct these entities into peroxisomes as evaluated by immunofluorescence following microinjection into mammalian cells (Gould et al., 1989; Walton et al., 1992). Therefore the peroxisome-IDE association is concluded to be physiological due to: (i) thecosedimentation of IDE recovered in the ML fraction with peroxisomal markers; (ii) the presence of IDE in highly purified peroxisomes; and (iii) the presence of a peroxisomal targeting motif at the correct position (extreme carboxyl termini) in deducedprimarystructures of IDE. In addition to the well established location of IDE in the cytosol, our studies indicatethat a small amount is peroxisomal (about 10% as based on estimates of peroxisomal recoveries (Leighton et al., 1968)). It is noteworthy that by subcellular fractionation we also observed the presence of peroxisomal thiolase in the S fraction (Fig. 5B). It is well known that peroxisomes are fragile and that35% or more of catalase is routinely liberated to the cytosolic fraction during homogenization (Amar-Costesec et al., 1974). This leakage is variable as demonstrated by Alexson et al. (1985) who showed great variability in the leakage of individual peroxisomal enzymes perhaps due to their organization within the matrix of peroxisomes. Our own studies support this view since thiolase was present a t higher levels in the S fraction than hydratase dehydrogenase and fattyacyl-CoA oxidase. However, none of these enzymes was released from peroxisomes to the same degree as IDE for which we would estimate that>90% of the activity had been liberated artifactually by homogenization. Hence, peroxisomal fragility may not be the sole explanation for the location of IDE in thecytosolic (S)fraction. Therefore, the localization of IDEinboth cytosol and peroxisomes cannot be formally excluded by our studies. Although our studies donot provide insight into thefunctional significance of peroxisome-associated IDE, a possible role of at least the

peroxisomal located enzyme in insulin processing can be excluded since endocytosed insulin in peroxisomes has not been observed (reviewed in Bergeron et al. (1985)). Endosomes represent the major site of internalization and degradation of insulin. The studies reportedhere demonstrate that the endosomal insulinase activity is distinct from IDE by its lack of cross-reactivity with specific IDE antibodies and its inability to be linked covalently to iodinated insulin at neutral pH. The incubation of intact endosomes containing internalized lZ5I-ligandsdemonstrated that a low pH is optimal for processing of insulin and glucagon (Doherty et al., 1990; Authier et al., 1990; Authier and Desbuquois, 1991). The contrasting report by Hamel et al. (1991) may have reflected incomplete neutralization of intraendosomal pH in their cellfree incubations. Consideration of the electrogenic regulation of endosomal acidification indicates the requirement of chloride counter ions(Fuchs et al., 1989) either to allow for proton leakage or toeffect ATP-dependent acidification. In our studies, the in vitro degradation of insulin by an endosomal fraction from rat liver was insensitive to some inhibitors of IDE, supportingthe contention that proteinases other than IDE may be involved (Pease et al., 1987). Using an affinity purification protocol at acidic pH, we purified partially the acid pH optimum insulinase present in endosomes. The broad low pH optimum, pH 4-5.5, and the sensitivity of the partially purified proteinase to a spectrum of inhibitors are comparable with that observed in previous studies using cell-free endosomes containing internalized insulin (Pease et al., 1987; Doherty et al., 1990), suggesting that the partially purified proteinase is likely responsible for the endosomal clearance of insulin in vivo. However, the partially purified endosomal insulinase differs from IDE in several respects: (i) anacidic pH was required for maximal proteolytic velocity; (ii) the endosomal insulinase was not inhibited by EDTA or N-ethylmaleimide; (iii) a partial metal-ion requirement was observed for its enzymatic activity. This explains its partial orcomplete lack of inhibition, respectively,by 1,lOphenanthrolineand EDTA. Endosomal acidic insulinase

Endosomal Acidic Insulinase

3016

seems to be a rare example of a thiolmetalloendopeptidase optimal at low pH. It is thislow pH optimum which explains the requirement for endosomal acidification in insulin degradation and the inhibition of insulin degradation i n vivo by acidotropic agents (Posner et al., 1982; Doherty et al., 1990). Besides distinguishing it from IDE, the observation is noteworthy since glucagon (another IDE substrate) is also degraded in endosomes (Authier et al., 1990; Authier and Desbuquois, 1991),but by an unrelated acid pH optimum proteinase which does not bind appreciably to an insulin-agarose column at acid pH.' Sequence information deduced from the major protein bands should enable the elucidation of the primary structure of the enzyme by cDNA cloning. The availability of specific antibodies to endosomal acidic insulinase should enable the elucidation of the significance of this enzyme in the regulation of insulin receptor bioactivity in normal and disease (insulinresistant) states. Furthermore, antibodies may permit visualization of the site of delivery of the proteinaseinto the endocytic pathway whether by access to endosomes from the cell surface (Peters et al., 19901, or by fusion of proteinasecontaining vesicles directly from the Golgi apparatus (Geuze et al.,1985; Griffiths et al., 1990). Backer et al. (1990) have confirmed that liver derived Fao cells harbor endosomal acidic insulinase but that Chinese hamster ovary cells do not. Hence, endosomal acidic insulinase expression maybe limited to insulin receptor-enriched target cells in vivo andthus mechanistically linked to thetermination of insulin receptor signal transduction in such cells (Doherty et al., 1990). Acknowledgments-We thank Dr. P. A. Walton and S. Dahan for help in immunofluorescence and cryoimmunolabeling. We thank Dr. R. Roth (Stanford University, CA) and Dr. M. R. Rosner (University of Chicago, IL) for kind gifts of monoclonal antibody 9B12 and polyclonal antibody 2BS, respectively, as well as Dr. G. Y. Zhang (McGill University, Montreal) for anti-GAP monoclonal antibody GP-15. We thank G. M. Di Guglielmo for assistance in these studies. We thank Dr. G . Shore (McGill University, Montreal) for valuable suggestions on the manuscript. REFERENCES Affholter, J. A., Fried, V. A., and Roth,R. A. (1988)Science 242. 1415-1418 Affholter, J. A., Hsieh, C-L., Francke, U., and Roth, R. A. (1990)Mol. Endocrinol. 4,1125-1135 Alexson, S. E. H., Fujiki, Y., Shio, H., Lazarow, P. B. (1985)J. Cell Biol. 101, 294-305 Amar-Costesec,A., Beaufay, H., Wibo, M.,Thin&-Sempoux, D., Feytmans, E., Robbi, M., and Berthet,J. (1974)J. Cell BWL 61,201-212 Authier, F., and Desbuquois, B. (1991)Biochem. J. 280,211-218 Authier, F., Janicot, M., Lederer, F., and Desbuquois, B. (1990)Biochem. J. 272,703-712 Authier, F., Desbuquois, B., and De Galle, B. (1992)Endocrinology 131,447457 Backer, J. M., Kahn, C. R., and White, M. F. (1990)J.Bioi. Chem. 266,1482814835 Baudhuin, P., Beaufay,H., Rahman-Li, Y., Selliger, 0. Z., Wattiaux, R., Jacques, P., and De Duve, C. (1964)Biochem. J. 92,179-184 Baumeister, H., Miiller, D., Rehbein, M., Richter, D. (1993)FEES Lett. 317, 250-254 Bergeron, J. J. M., Rachubinski, R. A., Sikstrom, R. S., Posner, B. I., and Paiement, J. (1982)J. Cell Blol 92, 139-146 Bergeron, J. J. M., CNZ, J.,Khan, M. N., and Posner, B. I. (1985)Annu. Reu. Physwl. 47,383-403

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