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Identification of Insulin Domains Important for Binding to and Degradation by Endosomal Acidic Insulinase*

Institut National de la Santé et de la Recherche Médicale U510 (F.A.), Faculté de Pharmacie Paris XI, 92296 Châtenay-Malabry, France; Health Care Discovery (G.M.D.), Novo Nordisk A/S, Novo Alle, DK-2880 Bagsvaerd, Denmark; Laboratoire de Spectrométrie de masse (M.K., G.B.), Faculté de Médecine, 59000 Lille, France; Institut National de la Santé et de la Recherche Médicale U30 (G.C.), Hôpital Necker Enfants-Malades, 75015 Paris, France

Address all correspondence and requests for reprints to: François Authier, INSERM U510, Faculté de Pharmacie Paris XI, 5 rue Jean-Baptiste Clément, 92296 Châtenay-Malabry, France. E-mail: francois.authier{at}cep.u-psud.fr

	Abstract

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The endosomal compartment of hepatic parenchymal cells contains an acidic endopeptidase, endosomal acidic insulinase (EAI), which hydrolyzes internalized insulin at a limited number of sites. Although the positions of these cleavages are partially known, the residues of insulin important in its binding to and proteolysis by EAI have not been defined. To this end, we have studied the degradation over time of native human insulin and three insulin-analog peptides using a soluble endosomal extract from rat liver parenchyma followed by purification of the products by HPLC and determination of their structure by mass spectrometry. We found variable rates of ligand processing, i.e. high ([Asp^B10]- and [Glu^A13,Glu^B10]-insulin), moderate (insulin) and low (the H2-analog). On the basis of IC₅₀ values, competition studies revealed that human and mutant insulins display nearly equivalent affinity for the EAI. Proteolysis of human and mutant insulins by EAI resulted in eight cleavages in the B-chain which occurred in the central region (Glu^B13-Leu^B17) and at the C-terminus (Arg^B22-Thr^B27), the latter region comprising the initial cleavages at Phe^B24-Phe^B25 (major pathway) and Phe^B25-Tyr^B26 (minor pathway) bonds. Except for the [Glu^A13,Glu^B10]-insulin mutant, only one cleavage on the A-chain was observed at residues Gln^A15-Leu^A16. Analysis of the nine cleavage sites showed a preference for hydrophobic and aromatic amino acid residues on both the carboxyl and amino sides of a cleaved peptide bond. Using the B-chain alone as a substrate resulted in a 30-fold increase in affinity for EAI and a 6-fold increase in the rate of hydrolysis compared with native insulin. A similar role for the C-terminal region of the B-chain of insulin in the high-affinity recognition of EAI was supported by the use of the corresponding B²²-B³⁰ peptide, which displayed an increase in EAI affinity similar to the entire B-chain vs. wild-type insulin. Thus, we have identified a highly specific molecular interaction of insulin with EAI at the aromatic locus Phe^B24-Phe^B25-Tyr^B26. Analytical subfractionation of a postmitochondrial supernatant fraction showed that a pulse of internalized [¹²⁵I]Tyr^A14-H2-analog, a protease-resistant insulin analog, undergoes a greater lysosomal transfer and lesser degradation than [¹²⁵I]Tyr^A14-insulin, confirming that endosomal sorting is regulated directly or indirectly by endosomal proteolysis.

	Introduction

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LIVER parenchyma has evolved endosomally located mechanisms to regulate the specificity of receptor tyrosine kinase signal transduction (1). One of these regulatory mechanisms is at the level of intraendosomal ligand degradation (2, 3, 4). Liver endosomes contain an acidic insulinase activity, endosomal acidic insulinase (EAI), which is not related to insulin-degrading enzyme (IDE) (5). In conjunction with endosome-associated phosphotyrosine phosphatase(s) directed against the tyrosine-phosphorylated insulin receptor (6), EAI limits the temporal window of activation and transduction of insulin receptor internalized into the endosomal compartment (7).

EAI showed a low pH optimum (pH 4–5.5) and was easily extracted from endocytic vesicles by hypotonic shock (5). However, the nature of the responsible endosomal proteinase remained undefined, and no study has been directed toward the identity of the insulin degradation products and proteolytic cleavage sites using native human insulin. However, elucidation of the cleavage sites in the human insulin molecule is essential to provide precise information toward a better understanding of the substrate recognition and cleavage site specificity of EAI.

Previous studies have identified the structural domains of insulin responsible for binding and affinity to its receptor (8). These studies have putatively defined the regions A⁸, B⁹-B¹² and B²⁴-B²⁶ as critical to the high-affinity interaction of insulin with its receptor (8, 9, 10). Thus, substitution of Phe^B25 by aspartic acid ([Asp^B25]-HI) caused an almost complete loss of affinity for the insulin receptor in HepG2 cells (11). [Leu^B25]-pork insulin had only approximately 1% receptor binding affinity relative to pork insulin (12) and D-[Phe^B25]-HI displayed only approximately 4% potency relative to HI (13). Val^B12 and residues located in close vicinity were also considered as a putative receptor binding region (14) which was in agreement with a 3-fold higher affinity of [Asp^B10]-HI toward the insulin receptor compared with authentic HI (15). In addition, Thr^A8 interfered with binding to receptors because substitution with His^A8 contributed substantially to the high affinity of chicken insulin (16). In the present study, we have selected two groups of insulin analogs with widely different kinetics of endosomal proteolysis (higher and lower rates of degradation by EAI compared with wild-type HI), to study the molecular mechanisms responsible for altered endosomal clearance. Because it was essential to preserve receptor-binding capacity while altering protease affinity, low-K_d analogs, such as [His^A8,His^B4, Glu^B10,His^B27]-HI (H2-analog), [Asp^B10]-HI and [Glu^A13,Glu^B10]-HI, which are considered as superpotent insulins (7, 11, 17) were used in this present study.

The data presented in this present study have attempted to: 1) study the time sequence degradation of native insulin and three insulin analogs using soluble endosomal extract (ENs) from hepatic endosomes followed by HPLC analysis; 2) elucidate the major sites of cleavage of insulin and insulin mutants by EAI; 3) determine the structure of the major insulin and insulin-analog intermediates; 4) examine whether the quantitative differences in the degradation state of the insulin analogs were due to an altered affinity of the insulin mutants for EAI and/or qualitative differences in the processing of the ligands; and 5) study the endosomal fate and lysosomal transfer of an insulin analog which displays a reduced rate of proteolysis within endosomes compared with authentic insulin.

	Materials and Methods

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Peptides, ligand radioiodination, protein determination, antibodies and materials
Human insulin (used as the reference insulin) and porcine glucagon were purchased from Sigma. [His^A8,His^B4, Glu^B10,His^B27]insulin (H2-analog), [Asp^B10]insulin and [Glu^A13,Glu^B10]insulin were produced and purified as previously described (17). A-chain, B-chain and the B-chain fragment Arg^B22-Ala^B30 from bovine insulin were purchased from Sigma. Human IGF-I was purchased from Intergen. Porcine glucagon was obtained from Sigma. Mouse EGF was purchased from Collaborative Biomedical Products. Human insulin, H2-analog and glucagon were radioiodinated by the lactoperoxidase method and purified by reverse-phase HPLC to specific activities of 80–380 µCi/µg as described previously (7, 18). The protein content of isolated fractions was determined by the method of Lowry et al. (19). Mouse monoclonal antibody 9B12 directed against the human insulin-degrading enzyme (IDE) (20) was a kind gift from Dr. R. A. Roth (Stanford University, Stanford, CA). Rabbit antirat procathepsin B 7183 (21, 22) and rabbit antirat cathepsin L R958 (22, 23) were obtained from Dr. J. S. Mort (Shriners Hospital for Crippled Children, Montreal, Québec, Canada). Rabbit antirat tripeptidylpeptidase II (TPP II) (24) was a kind gift from Dr. J.-C. Schwartz (U109 INSERM, Center Paul Broca, Paris, France). Sheep antirat Thimet Oligopeptidase (TOP) (25) was a kind gift from Dr. J.-M. Chen (The Babraham Institute, Cambridge, UK). HPLC grade acetonitrile and trifluoroacetic acid (TFA) were obtained from Baker Chemical Co. All other chemicals were obtained from commercial sources and were of reagent grade.

Animals and injections
Male Sprague Dawley rats weighing 180–200 g were obtained from Charles River Laboratories, Inc. (St. Aubin Les Elbeufs, France) and were fasted for 18 h before they were killed. [¹²⁵I]Tyr^A14-labeled human insulin and [¹²⁵I]Tyr^A14-H2-analog (30 x 10⁶ cpm, 9 pmol) were diluted in 0.3 ml of 0.15 M NaCl and injected within 5 sec into the penis vein under light ether anesthesia.

Isolation of subcellular fractions from rat liver
Subcellular fractionation was performed using established procedures (5, 7, 18, 22, 26, 27, 28). The endosomal fraction (EN) was isolated by discontinuous sucrose gradient centrifugation and collected at the 0.25 M to 1.0 M sucrose interface (5, 7, 18, 22, 26, 27, 28). The soluble extract from the endosomal fractions was isolated by freeze/thawing in 5 mM Na-phosphate pH 7.4, and disrupted in the same hypotonic medium using a small Dounce homogenizer (15 strokes with the tight Type A pestle) followed by centrifugation at 300,000 x g for 30 min as described previously (5, 7, 22, 28).

In vitro endosome-lysosome transfer reaction
Animals were killed 10 min after injection of the appropriate radiolabeled ligand and livers rapidly removed and minced in ice-cold isotonic homogenization buffer containing 0.25 M sucrose, 10 mM Tes, pH 7.4, and 7 mM MgCl₂. Intact cells, nuclei, and mitochondria were removed from the homogenate by centrifugation at 3,300 x g for 10 min. A cell-free transfer reaction was carried out as described previously (29, 30). The postmitochondrial supernatant (referred to as the LPS fraction) was incubated at 4 C or 37 C with 5 mM ATP, 1 mg/ml creatine kinase and 20 mM phosphocreatine. After cooling to 4 C, incubation mixtures were layered over 10 ml linear gradients prepared from 35% (mass/vol.) Nycodenz and 0.25 M sucrose containing 10 mM Tes pH 7.4 and 2 mM EDTA (density range, 1.06–1.16 g/ml). The gradients were centrifuged at 200,000 x g for 60 min in a Beckman Coulter, Inc. SW 41 rotor, following which 0.5 ml fractions were collected, densities determined and enzyme activity, radioactivity, and immunoblot analyses carried out. Components appearing at densities 1.065–1.11 and 1.11–1.145 g/ml were scored, respectively, as truly endosomal and lysosomal. In each fraction the integrity of radiolabeled peptides was assayed by precipitation with trichloroacetic acid (TCA), as reported previously (5, 7). Two hundred microliters of each subcellular fraction was mixed with 2 ml of ice-cold 10% TCA for 2 h at 4 C. The samples were then centrifuged at 10,000 x g for 20 min at 4 C, and the supernatants and pellets were evaluated for radioactivity using a Packard -counter.

Immunoblot studies
Electrophoresed samples were transferred as previously described (22, 28), and the blots were immunoblotted with polyclonal antirat procathepsin B diluted 1/600, and then with HRP-conjugated goat antirabbit IgG as previously described (22, 28).

In vitro proteolysis of native and [¹²⁵I]Tyr^A14-labeled peptides
Soluble endosomal extract (ENs) (1 ng) was incubated for varying lengths of time at 37 C with 5 x 10^-5 M wild-type insulin, the H2-analog, [Asp^B10]insulin, [Glu^A13,Glu^B10]insulin, insulin A chain, insulin B chain or insulin B chain fragment Arg^B22-Ala^B30 in 200 µl of 50 mM citrate-phosphate pH 4. The samples were then acidified with acetic acid (15%) and immediately loaded onto a reverse-phase HPLC column.

In vitro degradation of the [¹²⁵I]Tyr^A14-HI or -H2-analog using ENs was also studied. The radiolabeled ligands (75 fmol) were incubated with 10 µg of soluble endosomal protein and various concentrations of unlabeled human insulin or insulin analogs in 100 µl of 75 mM citrate-phosphate buffer pH 4. Following incubation at 37 C, the amount of radiolabeled insulin- and H2-analog-degraded was measured by TCA-precipitation as described above. In some experiments, ENs was immunodepleted for cathepsin B, cathepsin L, IDE, TPP II or TOP prior in vitro proteolysis of [¹²⁵I]Tyr^A14-HI and [¹²⁵I]-glucagon at pH 4. ENs was incubated with antibodies for 16 h at 4 C in 200 µl of 20 mM sodium phosphate buffer pH 7. The monoclonal and polyclonal IgGs were precipitated by the addition of 50 µl of protein G-Sepharose. After rotating for 2 h at 4 C, the fractions were centrifuged for 5 min at 10,000 x g, and the resultant supernatants were subjected to proteolysis of radiolabeled ligands at pH 4 using the TCA precipitation assay.

HPLC separation of human insulin and insulin analog peptides
Reverse-phase HPLC was performed on a Beckman Coulter, Inc. System Gold model 127 liquid chromatograph equipped with a Rheodyne sample injector fitted with a 500 µl loop and a µBondapak C18 column (Waters, 0.39 x 30 cm; 10 µm particle size). Samples were chromatographed using as eluent a mixture of 0.1% TFA in water (solvent A) and 0.1% TFA in acetonitrile (solvent B) with a flow rate of 1 ml/min. Elution was carried out using two sequential linear gradients of 0–15% solvent B (5 min) and 15–39% solvent B (32 min), followed by an isocratic elution of 39% solvent B (13 min). Eluates were monitored on-line for absorbance at 214 nm with a LC-166 spectrophotometer (Beckman Coulter, Inc.).

Characterization of endosomal acidic insulinase activity
The soluble endosomal lysate was loaded onto a TSK-GEL G3000 SW_XL HPLC column (Tosoh Corporation, 0.78 x 30 cm) equilibrated at 4 C with 50 mM Na-phosphate buffer, pH 6. The column was washed with 30 ml Na-phosphate buffer pH 6.0 using a flow rate of 0.5 ml/min. Eluates were monitored on-line for absorbance at 214 nm with a LC-166 spectrophotometer (Beckman Coulter, Inc.). Each fraction (0.5 ml) was immediately adjusted to pH 4 with 0.5 M citrate-phosphate buffer and evaluated for [¹²⁵I]Tyr^A14-HI and -H2-analog-degrading activity by the TCA-precipitation assay. Fractions eluted from the gel-filtration HPLC column that contained insulin-degrading activity with the highest specific activity (fractions 20–21; see Fig. 5A) were pooled and used for in vitro proteolysis of native HI, competition studies and determination of optimal pH for the degradation of the HI and H2-analog peptides. In some experiments, affinity purification of EAI was also performed as previously described (5). Partially purified EAI was then incubated at 37 C with 10^-5 M HI in 200 µl of 300 mM citrate-phosphate, pH 4. The samples were then analyzed by reverse-phase HPLC as described above.

View larger version (27K): [in this window] [in a new window]   Figure 5. Partial characterization of endosomal HI-degrading activity on a gel-filtration HPLC column and after an insulin affinity chromatography. Endosomes were disrupted by hypotonic shock, and the soluble endosomal extract (ENs) (150 µg) was applied to a TSK-gel G3000 HPLC column. A, Absorbance profile at 214 nm. Arrow indicates the elution of rat serum albumin (SA; 66-kDa) identified by SDS-PAGE followed by Coomassie Blue-staining (results not shown). Arrowhead indicates the peak of acidic insulinase activity (fractions 20–21). In Panel B, each fraction was tested for its ability to degrade 50 fmol of [125I]TyrA14-HI or -H2-analog for 15 min at 37 C and pH 4 using the TCA-precipitation assay. C, Fractions 20 and 21 were pooled and incubated at pH 4 with 10-5 M HI for 30 min, and the hydrolysis products generated were then analyzed by reverse-phase HPLC using the same elution gradient as that described in Fig. 1. D, EAI was partially purified using an affinity purification protocol at acidic pH according to (5 ), and then incubated in 200 mM citrate-phosphate pH 4 with 10-5 M HI. After 15 min at 37 C, the hydrolysis products generated were then analyzed by reverse-phase HPLC using the same elution gradient as that described in Fig. 1. The degradation products observed in Panels C and D and numbered 1–7 display retention times identical with those seen with ENs in Fig. 1.

HPLC-electrospray ionization (ESI)-mass spectrometry (MS) coupling
Samples (3–5 nmol in 100 µl) were analyzed by HPLC coupled with ESI-MS. Chromatographic separation was carried out by HPLC using an PE Applied Biosystems (Foster City, CA) 140B automated gradient controller pump module equipped with a Perkin-Elmer Corp. LC 290 detector operating at 214 nm, and its output was connected to a Perkin-Elmer Corp. R 100A integrator. A reverse-phase C18-column (2 x 150 mm, Chrompack, lichrospher 100–5, The Netherlands) was used. The injector was a Rheodyne valve with a 100 µl loop (Cotati, CA). Samples were chromatographed using as eluent a mixture of 0.05% TFA in water (solvent A) and 0.05% TFA in acetonitrile (solvent B) with a flow rate of 0.2 ml/min. Elution was carried out as described above (see HPLC procedure section). The column effluent was split 1:5 to give a flow-rate of 40 µl/min into the electrospray nebulizer. Total ion current plots of all masses (TIC) and ESI-MS spectra were stored on a quadra 700 Macintosh computer and Mac Spec 3.2 software was used to plot, acquire and treat the ESI-MS data. The contour plot of data from LC/MS (m/z vs. retention time) made characteristic series of masses evident at different elution times after substraction of the eluent spectra. The voltage of the extraction cone was adjusted to 70 V and the potential of the spray needle was held at +5 kV. Ionspray mass spectra were acquired in the TIC mode from m/z 400-2400 amu, with a step size of 0.2 amu and a dwell time of 0.5 ms.

	Results

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Identification and kinetic analyses of insulin and insulin-analog intermediates
Figure 1 shows the HPLC elution profiles, measured by UV absorbance at 214 nm, of the 0-min incubation of native peptides (left panels) and after incubating native peptides with the ENs for the indicated time at pH 4 (right panels). Major intermediate peptide peaks (6 for HI, the H2-analog and [Asp^B10]-HI, and 8 for the [Glu^A13,Glu^B10]-HI) were observed in addition to the undegraded peptides (peak 7), which had decreased in peak height with variable rates of processing (Fig. 1). The rate of hydrolysis of each peptide was accurately determined by following the disappearance of the parent peptide by quantitation of the peak areas (Fig. 2), thus cleavage at any single bond would be detected. HI degradation showed a faster initial hydrolysis than that of the H2-analog (t_1/2 of 19 and 44 min, respectively) (7). When [Asp^B10]-HI and [Glu^A13,Glu^B10]-HI were used as substrates, the rate of hydrolysis was faster than that of HI with t_1/2 of 10 and 9 min respectively (Fig. 2).

View larger version (21K): [in this window] [in a new window]   Figure 1. HPLC elution profiles of the degradation products resulting from the incubation of wild-type insulin and insulin analogs with a soluble endosomal extract. Endosomes were disrupted by hypotonic shock, and the soluble endosomal extract (ENs) (1 ng) was incubated with 10-6 M human insulin (HI), [HisA8, HisB4, GluB10, HisB27]-HI (H2-analog), [AspB10]-HI or [GluA13, GluB10]-HI for the indicated time at pH 4 and 37 C. The proteolytic reaction was stopped with acetic acid (15%) and the incubation mixtures were analyzed by reverse-phase HPLC (right panels) as described in Experimental procedures. All panels show absorbance profiles at 214 nm. Intact insulin, H2-analog, [AspB10]-HI and [GluA13, GluB10]-HI had elution times of 44, 46, 48, and 43 min, respectively (left panels). The endosomal proteins alone did not give any detectable peak (results not shown). The major peptide pools, numbered sequentially 1–7, were collected from the time course profile indicated. These pools were subjected to mass spectrometry analyses (see Table 1).

View larger version (19K): [in this window] [in a new window]   Figure 2. Kinetics of processing of wild-type insulin and insulin analogs by a soluble endosomal extract. The in vitro proteolysis of HI and the three insulin analog peptides was measured by HPLC analysis as described in Fig. 1. Results are expressed as the amount of peptide degraded (% of control) after a 15-, 30-, or 60-min incubation, and normalized (100%) to that seen in the absence of added endosomal protein. Results are the mean ± SD of three different experiments performed on endosomal fractions prepared from separate liver fractionations.

View larger version (9K): [in this window] [in a new window]   Figure 3. Peptide bonds cleaved and structure of intermediates generated by endosomal acidic insulinase activity. The number refer to the intermediates as identified in Fig. 1. The symbols + and - indicate, respectively, major and minor pathway of degradation. Peptide products 4a and 4b are not identified using [GluA13, GluB10]-HI as a substrate.

The degradation of the H2-analog, [Asp^B10]-HI and [Glu^A13,Glu^B10]-HI revealed a similar pattern (Table 1). However, using [Glu^A13,Glu^B10]-HI as a substrate, the degradation products have only been cleaved at the B-chain peptide bonds. Thus, the Glu^A15-Leu^A16 bond did not appear to be further degraded by EAI activity within the [Glu^A13,Glu^B10]-HI mutant.

We then evaluated the ability of native ligands to inhibit [¹²⁵I]Tyr^A14-HI degradation by ENs (Fig. 4). HI was found to inhibit [¹²⁵I]Tyr^A14-HI proteolysis by ENs in a dose-dependent manner similar to that of the H2-analog and [Glu^A13,Glu^B10]-HI, with an IC₅₀ of 2–4 x 10^-5 M (Fig. 4A). [Asp^B10]-HI was approximately 2 times more effective than HI at competing for proteolysis of radiolabeled HI (IC₅₀ of 10^-5 M). Thus, the insulin mutants and HI share a common binding site on the EAI enzyme. To test further the relative catalytic selectivity of EAI toward the insulin molecule, we evaluated the ability of various peptides of approximately 3.5–7.5 kDa to inhibit [¹²⁵I]Tyr^A14-HI degradation at a concentration of 5 x 10^-5 M (Fig. 4B). Among the glucagon, EGF and IGF-I peptides, only glucagon was able to inhibit radiolabeled HI degradation but less effectively than HI and the insulin analog peptides (22% of inhibition).

View larger version (23K): [in this window] [in a new window]   Figure 4. Competition of HI, the insulin mutants and other peptides for the degradation of [125I]TyrA14-HI by endosomal acidic insulinase activity. Endosomes were disrupted by hypotonic shock, and the soluble endosomal extract (ENs) (0.1 mg/ml) was incubated with [125I]TyrA14-HI (75 fmol) at 37 C and pH 4 with the indicated concentrations of unlabeled ligands (A) or with the indicated peptides at a concentration of 5 x 10-5 M (B). The amount of degraded radiolabeled insulin was determined by precipitation with trichloroacetic acid. Results, mean ± SD of three different experiments performed on endosomal fractions prepared from separate liver fractionation, are expressed as percentage of degradation determined in the absence of unlabeled added peptides.

View larger version (17K): [in this window] [in a new window]   Figure 6. Assessment of endosomal acidic insulinase activity after a gel-filtration HPLC protocol and immunodepletion procedures. Endosomal acidic insulinase activity was partially purified using a TSK-gel G3000 HPLC column (see Fig. 5, A and B) and incubated with [125I]TyrA14-HI (75 fmol) at 37 C and pH 4 with the indicated concentrations of unlabeled HI or H2-analog peptides (A). The amount of degraded radiolabeled HI was determined by precipitation with TCA, and results are expressed as in Fig. 4A. In Panel B, partially purified EAI was incubated at different pH values for 10 min with [125I]TyrA14-HI or -H2-analog, after which the amount of degraded radiolabeled ligand was determined by precipitation with TCA. C, ENs was immunodepleted of various proteases using independently rabbit antirat procathepsin B (CB), -rat cathepsin L (CL), -rat tripeptidylpeptidase II (TPP II), -rat Thimet Oligopeptidase (TOP), and mouse antihuman insulin-degrading enzyme (IDE) at a dilution of 1/500. After immunoprecipitation using protein G-Sepharose and centrifugation, the resultant supernatants were tested for the ability to degrade [125I]TyrA14-HI or [125I]-glucagon at pH 4 using the TCA-precipitation assay.

A- and B-chains of insulin as substrates of endosomal acidic insulinase
Two substrates of the same protease are expected to compete with each other for the enzyme site, and thus, one substrate will act as a competitive inhibitor of the other. We have used this strategy to find new substrates for EAI derived from the insulin molecule ( Figs. 7–9). In competition assays, both the insulin A-chain (Fig. 7C) and B-chain (Fig. 8C) were potent in competing with radiolabeled insulin for proteolysis by ENs. Although insulin A-chain (IC₅₀ of 2 x 10^-5 M) was able to inhibit the degradation of radiolabeled insulin comparable to that of intact insulin, insulin B-chain (IC₅₀ of 6 x 10^-7 M) was found to be 30 times more potent than intact insulin and A-chain.

View larger version (25K): [in this window] [in a new window]   Figure 7. Assessment of the proteolysis of insulin A-chain by endosomal acidic insulinase activity. Endosomes were disrupted by hypotonic shock, and the soluble endosomal extract (ENs) was evaluated for its ability to degrade intact A-chain at pH 4 and 37 C. In Panel A, ENs (1 ng) was incubated with 10-6 M insulin A-chain for 90 min. The proteolytic reaction was then stopped with acetic acid (15%) and the incubation mixture was analyzed by reverse-phase HPLC. Both panels show absorbance profiles at 214 nm. Intact A-chain (upper panel) had an elution time of 23 min. The endosomal proteins themselves did not give any detectable peak (results not shown). The major degradation products (lower panel), numbered sequentially 1–6, were subjected to mass spectrometry analyses (see Table 2). In Panel B, the rate of insulin A-chain (closed circles) and HI (dashed line) proteolysis was determined as described in Panel A by following the disappearance of the peak area corresponding to the parent peptide. Results are expressed as peptide degraded (% of control) and normalized (100%) to that seen in the absence of added endosomal protein. In Panel C, ENs (0.1 mg/ml) was incubated with [125I]TyrA14-HI (75 fmol) with the indicated concentrations of unlabeled A-chain (closed circles) or HI (dashed line). The amount of degraded radiolabeled insulin was determined by precipitation with TCA. Results are expressed as a percentage of the amount of radioactive insulin degraded in the absence of unlabeled peptide.

View larger version (26K): [in this window] [in a new window]   Figure 8. Assessment of the proteolysis of insulin B-chain by endosomal acidic insulinase activity. Endosomes were disrupted by hypotonic shock, and the soluble endosomal extract (ENs) was evaluated for its ability to degrade intact B-chain at pH 4 and 37 C. In Panel A, ENs (1 ng) was incubated with 10-6 M insulin B-chain for 2 min. The proteolytic reaction was then stopped with acetic acid (15%) and the incubation mixture was analyzed by reverse-phase HPLC. Both panels show absorbance profiles at 214 nm. Intact B-chain (upper panel and peak 9 in lower panel) had an elution time of 47 min. The endosomal proteins themselves did not give any detectable peak (results not shown). The major degradation products (lower panel), numbered sequentially 1–8 as well as intact B-chain (peak 9), were subjected to mass spectrometry analyses (see Table 3). In Panel B, the rate of insulin B-chain (closed circles) and HI (dashed line) proteolysis was determined as described in Panel A by following the disappearance of the peak area corresponding to the parent peptide. Results are expressed as peptide degraded (% of control) and normalized (100%) to that seen in the absence of added endosomal protein. In Panel C, ENs (0.1 mg/ml) was incubated with [125I]TyrA14-HI (75 fmol) with the indicated concentrations of unlabeled B-chain (closed circles) or HI (dashed line). The amount of degraded radiolabeled insulin was determined by precipitation with TCA. Results are expressed as a percentage of the amount of radioactive insulin degraded in the absence of unlabeled peptide.

View larger version (26K): [in this window] [in a new window]   Figure 9. Assessment of the proteolysis of insulin ArgB22-AlaB30 B-chain fragment by endosomal acidic insulinase activity. Endosomes were disrupted by hypotonic shock, and the soluble endosomal extract (ENs) was evaluated for its ability to degrade the insulin ArgB22-AlaB30 B-chain fragment at pH 4 and 37 C. In Panel A, ENs (1 ng) was incubated with 10-6 M ArgB22-AlaB30 fragment for 5 min. The proteolytic reaction was then stopped with acetic acid (15%) and the incubation mixture was analyzed by reverse-phase HPLC. Both panels show absorbance profiles at 214 nm. Intact ArgB22-AlaB30 peptide (upper panel and peak 5 in lower panel) had an elution time of 20 min. The endosomal proteins themselves did not give any detectable peak (results not shown). The major degradation products (lower panel), numbered 1–4 and 6, as well as intact peptide (peak 5), were subjected to mass spectrometry analyses (see Table 4). In Panel B, the rate of ArgB22-AlaB30 (closed circles) and HI (dashed line) proteolysis was determined as described in Panel A by following the disappearance of the peak area corresponding to the parent peptide. Results are expressed as peptide degraded (% of control) and normalized (100%) to that seen in the absence of added endosomal protein. In Panel C, ENs (0.1 mg/ml) was incubated with [125I]TyrA14-HI (75 fmol) with the indicated concentrations of unlabeled ArgB22-AlaB30 fragment (closed circles) or HI (dashed line). The amount of degraded radiolabeled insulin was determined by precipitation with TCA. Results are expressed as a percentage of the amount of radioactive insulin degraded in the absence of unlabeled peptide.

As indicated in Table 2, A-chain was cleaved at Gln¹⁵-Leu¹⁶ bond, thereby releasing the N-terminal peptide A^1–15 (the major peak 5) and the C-terminal peptide A^16–21 (peak 3). Cleavages in close proximity to this site at Leu^A13-Tyr^A14, Tyr^A14-Gln^A15, Leu^A16-Glu^A17 and Glu^A17-Asn^A18 were also observed as evidenced by fragments A^18–21 (peak 1), A^17–21 (peak 2), A^14–21 (peak 4), A^1–13 (peak 5), A^14–16 and A^1–14 (peak 6).

The fragmentation of the B^22–30 peptide by ENs as evaluated by HPLC revealed that the endosomal protease degraded the B^22–30 fragment two times faster than that observed for intact insulin (Fig. 9, A and B). Five major intermediates were produced from the parent B^22–30 peptide (peak 5), one of which (peak 4) accounted for more than 50% of the cleavage products, suggesting that it represents the major processed form of the C-terminus B-chain.

Mass spectrometry analysis (Table 4) revealed that the B^22–30 fragment was cleaved at the Phe^B24-Phe^B25, Phe^B25-Tyr^B26 and Tyr^B26-Pro^B27 bonds as was observed with the entire insulin molecule (see Fig. 3). We found three N-terminally truncated forms of the B^22–30 peptide (peak 1 (B^27–30), peak 2 (B^26–30), and peak 4 (B^25–30)], of which one, the B^25–30 product represented the major intermediate. Peaks 3 and 6 corresponded to the remaining N-terminal residues of the above peptide products.

View larger version (29K): [in this window] [in a new window]   Figure 10. Time course of transfer of [125I]TyrA14-HI and [125I]TyrA14-H2-analog from the endosomal to the lysosomal position on Nycodenz gradients. In Panel A, the postmitochondrial supernatant (LPS fraction) was isolated from control rats and immediately subfractionated on linear Nycodenz density gradients (open symbols), or incubated with ATP and an ATP-regenerating system at 37 C for 30 min followed by subfractionation on linear Nycodenz density gradients (closed symbols). Galactosyltransferase (GT; circles) and acid phosphatase (AP; squares) activities were determined and results expressed as a percentage of total enzymatic activity recovered. The inset shows the content of procathepsin B (p-CB) and mature cathepsin B (CB) evaluated by immunoblotting with polyclonal antirat procathepsin B. Twenty microliters of each subfraction were loaded onto each lane. In Panel B, LPS fractions were isolated 10 min after rats were injected with [125I]TyrA14-HI (2 x 107 cpm; open symbols) or -H2-analog (2 x 107 cpm; closed symbols). LPS fractions were incubated with ATP and an ATP-regenerating system at 4 C for 30 min (circles), or 37 C for 15 min (squares), or 37 C for 30 min (triangles). Results are expressed as percentages of the total radiolabel on the gradient. In Panel C, LPS fractions were isolated 10 min after rats were injected with [125I]TyrA14-HI (2 x 107 cpm; open symbols) or -H2-analog (2 x 107 cpm; closed symbols). LPS fractions were incubated with ATP and an ATP-regenerating system at 4 C for 30 min (circles) or 37 C for 15 min (squares), or 37 C for 30 min (triangles). The amount of degraded radiolabeled ligand was determined by precipitation with TCA.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
This is the first study using rat endosomal acidic insulinase (rat EAI) in which the products of native HI and HI-analog peptides have been isolated and the amino acid sequence of the portions of the A- and B-chains determined by mass spectrometry. Proteolysis of HI by EAI results in one cleavage in the A-chain and eight in the B-chain. The endosomal protease cleaves the A-chain at the Gln^A15-Leu^A16 bond. The eight B-chain cleavages occur in two main regions of the polypeptide, one comprising the C-terminus part Arg^B22-Thr^B27 (5 sites) and the second appearing in the central region Glu^B13-Leu^B17 (3 sites). The degradation steps proceed via an ordered sequential pathway: 1) two primary end products of HI degradation are generated resulting from a major cleavage at residues Phe^B24-Phe^B25 and a minor cleavage at residues Phe^B25-Tyr^B26; 2) this is followed by the sequential removal of the C-terminal Phe^B24 and Gly^B23 with the concomitant release of residues Ala^B14, Leu^B15 and Tyr^B16 from the central region of the B-chain; and 3) the A-chain is then processed by one cleavage, which occurs at Gln^A15-Leu^A16.

Comparison of the nine bonds in the HI molecule hydrolyzed by EAI reveals that three have aromatic (Phe and Tyr) and four have hydrophobic residues (Leu and Ala) on their carboxyl side. In contrast, the residues identified on the amino side of the hydrolyzed bonds (aromatic, hydrophobic, hydrophilic, basic and acid amino acids) do not reveal any definite structural requirement for the enzyme. However, the finding that HI is primarily cleaved at the most hydrophobic peptide bonds Phe^B24-Phe^B25-Tyr^B26 is still consistent with a preference for nonpolar aromatic amino acid residues on the amino side. Thus, endosomal insulinase might preferentially catalyze the hydrolysis of bonds that have an aromatic or hydrophobic amino acid residue on both the carboxyl and amino sides but it can also tolerate other residues on the amino side.

Previous studies described the use of monoiodinated insulins and radio-sequencing procedures to identify on the sequenator the tyrosyl residue at which the radioactivity came off (32, 33). However, some of the intermediates might not have had the radioactive label and therefore escaped detection. Moreover, the radioactive iodine on the molecule might have had an effect on the endosomal proteolytic system. The position of the radioactive iodine may also affect the relative degradation rates of different substrates because different monoiodinated insulins are degraded at different rates (34). From these studies (32, 33), it has been suggested that endosomal processing of insulin induces cleavages at A¹³-A¹⁴, B¹⁴-B¹⁵, and B²⁴-B²⁵ bonds. We have confirmed here that these peptide bonds were effectively cleaved within the HI molecule. However, using a more sensitive approach, we found nine cleavage sites, of which six have not been previously reported. Moreover, the application of mass spectrometry for such a study leads to elucidation, for the first time, of the entire structure of twelve insulin intermediates generated within hepatic endosomes. EAI activity also differs from the exopeptidase cysteine-protease cathepsin B that processes internalized EGF and glucagon within rat liver endosomes by inducing proteolytic modifications at their C-terminus and N-terminus regions (22, 28).

The substrate and cleavage specificity of EAI is an intriguing issue. The question as to which specific properties are shared by EAI substrates has not yet been answered. Exploiting the fact that two substrates competing for the same enzyme inhibit one another, we have found three new substrates of EAI genetically derived from the HI molecule. Using HPLC analysis in which hydrolysis was measured by the generation of degradation products from unlabeled peptides, we demonstrated variable rates of ligand processing, i.e. high ([Asp^B10]-HI and [Glu^A13,Glu^B10]-HI), moderate (HI) and low (H2-analog) (Fig. 2). However, mass spectrometry analyses revealed that the degradation products generated from wild-type HI and HI-mutants by endosomal insulinase were very similar. Thus, the altered endosomal clearance of the mutant insulins does not arise from qualitative differences of ligand processing. Additionally, the substituted amino acids in the insulin analogs did not correspond to primary sites of cleavage of HI within endosomes. Finally, on the basis of IC₅₀ values, competition studies revealed that wild-type and mutant insulins displayed nearly equivalent affinity for the endosomal protease.

The 10–40 µM IC₅₀ values for HI and insulin analogs do not preclude these peptides from being physiological substrates for EAI. However, although the IC₅₀ value of HI for EAI appears 100- to 1000-fold higher than for the other suggested insulin protease IDE (35), the endosomal concentration of free insulin could be relatively high and may well approach or exceed the observed IC₅₀ value due to the small volume of endosomes [10^-17 liters (36, 37)]. Moreover, Leu- and Met-enkephalin, which are known physiological substrates for neprilysin, exhibit kinetic K_m values of 47 and 13 µM, respectively (38). Alternatively, the high IC₅₀ value of HI for EAI and the high proteolytic activity and affinity of EAI for insulin B-chain and a B^22–30 insulin fragment may also suggest that EAI is a nonspecific peptidase activity whose physiological substrates within the endosomal apparatus, other than insulin, have yet to be defined. In support of this, there is evidence in the literature that nonspecific endosomal proteases are active toward a number of internalized polypeptides and growth factors (3). For instance, we have recently demonstrated that cathepsin B, a general cysteine protease, down-regulates internalized EGF and glucagon within endosomes by inducing proteolytic cleavages of these ligands (22, 28).

The availability of analogs with very high affinity for the insulin receptor enabled us to elucidate whether the structural determinants required for binding to the receptor and EAI were identical. The three insulin analogs used in this study all displayed an increase in binding affinity to receptors (between 327 and 687%) and an increase in the metabolic and mitogenic potencies (between 207 and 573%) as compared with HI (100%) (8, 11, 17). Thus, our results which showed variable rates of analog processing (from high to low) compared with insulin suggest a fundamental difference between the mechanisms by which the endosomal protease and the receptors recognized these molecules. This agreed with the finding that the insulin B-chain, which displayed a complete loss of receptor affinity, had one of the highest affinities for EAI and the highest rate of proteolysis by the protease. However, among the three regions of insulin important for binding of the ligand to its receptor (8) the B^24–26 region, which has been implicated in the high-affinity binding of insulin to its receptor (9, 10), appeared to also confer high-affinity binding to EAI.

The influence of endosomal ligand degradation on the steady-state distribution of internalized ligands and their transit times throughout the endo-lysosomal compartment has been previously inferred from a comparison of insulin and EGF internalization in liver parenchyma (7, 28, 29, 30). Whereas EGF was selectively resistant to endosomal dissociation and only partially degraded within hepatic endosomes by the cysteine protease cathepsin B (28), insulin was subject to rapid endosomal proteolysis (5, 7). Although it has been difficult to demonstrate, even in the presence of protease inhibitors, the accumulation of radiolabeled insulin in lysosomes of liver parenchyma (39), the targeting of EGF to lysosomes was easily observed using the in situ rat liver model (40). Other evidence has come from the differences in endosomal degradation of transforming growth factor- and EGF after internalization, which coincided with altered intracellular trafficking and was linked to the different biopotency observed for the two ligands (41).

As a consequence, we elected to study the in vitro endosome-lysosome transfer of a genetically engineered tool, the insulin analog H2 containing four amino acid substitutions, and which is a poor substrate for EAI activity [(7); this study]. The different properties of insulin and the H2-analog following binding to the insulin receptor now allowed for a test of the influence of ligand proteolysis on the endosomal sorting of ligands using a homologous receptor system. To the best of our knowledge, the present study is the first such direct test of this hypothesis using an in vivo model. Using cell-free assays originally developed for asialofetuin, a ligand of the asialoglycoprotein receptor (42), we have recently proposed an in vitro model for the transfer of endocytosed ligand-receptor complexes to lysosomes in hepatic tissue (29, 30). We have shown that in a liver cell-free system containing in vivo endocytosed insulin, glucagon, GalBSA and EGF, these ligands are transferred in vitro from endosomes to lysosomes (29, 30). In addition to these ligands, the cell-free transfer of the H2-analog molecule was observed in endosomes isolated at a late stage of endocytosis [time of killing, 10 min (7)] and required the addition of cytosol and an ATP-regenerating system (29, 30, 42). However, the endosomal sorting of the H2-analog differed from the other polypeptides in several respects: 1) whereas degradation products were largely recovered in the soluble cytosolic fraction during the endo-lysosomal transfer of HI [this study (29)], the H2-analog transfer, as with the EGF transfer (30), was not accompanied by a net increase in intermediate products recovered in the upper part of the gradient; 2) lysosomal recovery after a 10 min incubation at 37 C was low (<15%) in the presence of insulin [this study (29)], moderate (15–20%) with EGF (30) and glucagon (29), and high (20–25%) with the H2-analog (this study), GalBSA and asialofetuin (29). Thus, we suggest that endosomal proteolysis influences the steady-state distribution of internalized ligands throughout the endo-lysosomal compartment.

The difference in endosomal degradation of these ligands following internalization also coincided with altered receptor trafficking and receptor phosphotyrosine profiles in the endosome and lysosome organelles (1, 28, 30, 43). Thus, a low lysosomal transfer of the and ß subunits of the insulin receptor occurred during endosome-lysosome interaction (29), whereas the targeting of the EGF receptor for degradation in lysosomes was clearly demonstrated using the in situ rat liver model (40), the in vitro endosome-lysosome fusion system (30), and various cell lines (44). Studies are underway to determine whether, in response to the insulin analog H2, subsequent insulin receptor activation in the endosome (7) causes a translocation of the insulin receptor to the lysosome compartment.

	Acknowledgments

 
This paper is dedicated to the memory of our friend and collaborator Gédéon Le Dranac (Faculté de Pharmacie Paris XI, Châtenay-Malabry, France), who died on February 28^th, 2000. We thank Pamela H. Cameron (McGill University, Montréal, Québec, Canada) for reviewing the manuscript. We thank Dr. R.A. Roth (Stanford University, Stanford, CA), Dr. J. S. Mort (Shriners Hospital for Crippled Children, Montreal, Québec, Canada), Dr. J.-C. Schwartz (U109 INSERM, Center Paul Broca, Paris, France) and Dr. J.-M. Chen (The Babraham Institute, Cambridge, United Kingdom) for kind gifts of anti-IDE, -procathepsin B, -TPP II and -TOP antibodies, respectively.

	Footnotes

 
* This work was supported by grants from the Institut National de la Santé et de la Recherche Médicale and the Fondation pour la Recherche Médicale (Grant 10000320-01, to F.A.).

Received June 16, 2000.

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