The ordered and compartment-specfific autoproteolytic removal of the furin intramolecular chaperone is required for enzyme activation

Abstract

The propeptide of furin has multiple roles in guiding the activation of the endoprotease in vivo. The 83-residue N-terminal propeptide is autoproteolytically excised in the endoplasmic reticulum (ER) at the consensus furin site, -Arg(104)-Thr-Lys-Arg(107)-, but remains bound to furin as a potent autoinhibitor. Furin lacking the propeptide is ER-retained and proteolytically inactive. Co-expression with the propeptide, however, restores trans-Golgi network (TGN) localization and enzyme activity, indicating that the furin propeptide is an intramolecular chaperone. Blocking this step results in localization to the ER-Golgi intermediate compartment (ERGIC)/cis-Golgi network (CGN), suggesting the ER and ERGIC/CGN recognize distinct furin folding intermediates. Following transport to the acidified TGN/endosomal compartments, furin cleaves the bound propeptide at a second, internal P1/P6 Arg site (-Arg-Gly-Val(72)-Thr-Lys-Arg(75)-) resulting in propeptide dissociation and enzyme activation. Cleavage at Arg(75), however, is not required for proper furin trafficking. Kinetic analyses of peptide substrates indicate that the sequential pH-modulated propeptide cleavages result from the differential recognition of these sites by furin. Altering this preference by converting the internal site to a canonical P1/P4 Arg motif (Val(72) --> Arg) caused ER retention and blocked activation of furin, demonstrating that the structure of the furin propeptide mediates folding of the enzyme and directs its pH-regulated, compartment-specific activation in vivo.

FIG. 1

Furin constructs. Fur/f/ha, fur/fΔpro, fur/fD153N, fur/f/ha, V72R:fur/ f/ha, and R75A:fur/f/ha all have the FLAG epitope tag (diagonal bars) inserted directly after the propeptide cleavage site, such that the N terminus of the FLAG sequence is exposed upon excision. mAb M2 recognizes either the blocked or exposed forms of the FLAG epitope, whereas mAb M1 requires the FLAG epitope at the free N terminus. In fur/f/ha, pro/ha, V72R:fur/f/ha and R75A:fur/f/ha the HA epitope tag (vertical bars) was inserted directly after the signal sequence (black). The HA epitope is recognized by the mAbs 12CA5 and HA.11. The furin cytosolic domain is recognized by the antiserum PA1-062. The Ltilisin-like catalytic domain (hatch marks) and the transmembrane domain (stippled) are indicated. The catalytic triad residues (Asp, His, Ser) are indicated. “Lollipops” denote glycosylation sites. Thick vertical bars indicate propeptide cleavage sites. The propeptide excision and internal cleavage motifs are boxed.

FIG. 2

Expression and in vitro activity of fur/fΔpro. A, membrane preparations from mock infected BSC-40 cells, or BSC-40 cells infected with VV:wt, VV:fur/f/ha, or VV:fur/fΔpro were analyzed by SDS-PAGE followed by immunoblotting, using either mAb M1 or mAb M2. Profurin (arrow), propeptide-excised furin (arrowhead), and mature sialylated furin (bracket) are indicated. B, proteolytic activity of the same membrane preparations against Pyr-Arg-Thr-Lys-Arg-MCA. Each column represents the average of two samples assayed in duplicate. Bars indicate standard deviations. C, BSC-40 cells grown on coverslips were infected with VV:fur/f/ha or VV:fur/fΔpro and then fixed at 5 h post-infection and processed for immunofluorescence. Fur/f/ha and fur/f/Δpro mature domains were visualized with mAb M1, and the samples were double-labeled for propeptide (HA.11) or signal sequence receptor (SSR). D, BSC-40 cells were infected with VV:wt, VV:fur/f/ha, or VV:fur/fΔpro and incubated at 37 °C for 4 h. The cells were pulse-labeled with [³⁵S]Met/Cys for 30 min and chased for 3 h in complete medium with excess cold Met and Cys. Cells were then harvested in mRIPA, and furin was immunoprecipitated with PA1- 062. Immunoprecipitates were incubated in reaction buffer (50 mM sodium citrate pH 6.0, and 0.1% SDS) in the absence (-) or presence (+) of 2.5 milliunits of Endo H. The digested samples were resolved by 8% SDS-PAGE and processed for fluorography. Furin zymogen (black arrowhead), mature furin with an excised propeptide (white arrowhead), and sialylated furin (white asterisk) are indicated.

FIG. 3

Rescue of fur/fΔpro activity and localization by propep-tide in trans. A, replicate plates of BSC-40 cells grown on coverslips were infected with VV:fur/fΔpro and VV:wt or VV:pro/ha and mAb M1 (30 μg/ml) added to the medium to label mature furin cycling to the cell surface. At 5 h post-infection, the cells were then fixed, permeabilized, and incubated with mAb M2 to detect the remaining furin. mAb M1 and mAb M2 were visualized with isotype-specific secondary antibodies. B, replicate plates of BSC-40 cells were infected with either VV:mNGF alone or with additional recombinant viruses as indicated. The signal from the precursor (proβ-NGF) and product (β-NGF) were quantitated, and the ratio of product to precursor determined. Each reading represents the average of two separate samples. Bars indicate standard deviations.

FIG. 4

Retention of fur/fD153N in the early secretory pathway. A, membrane preparations from mock-infected BSC-40 cells or cells infected with VV:wt, VV:fur/f/ha, or VV:fur/fD153N were analyzed by Western blot with mAb M1 and mAb M2. B, proteolytic activity of the same membrane preparations. Data are the average of two samples assayed in duplicate. Bars indicate standard deviations. C, replicate plates of BSC-40 cells were infected with VV:wt, VV:fur/f/ha, or VV:fur/fD153N, pulse-labeled, and chased as described in Fig. 2, and the immuno-precipitates were analyzed by SDS-PAGE and fluorography. D, BSC-40 cells were infected with VV:fur/fD153N and incubated at 37 °C for 5 h. In some cases, brefeldin A and cycloheximide (both at 10 μg/ml) were added for the final 2 h prior to fixation. Following fixation, the cells were permeabilized and incubated with PA1- 062 (Furin) and mAb G1/93 (ERGIC-53) followed by visualization with species-specific secondary antibodies. Arrowheads mark examples of structures containing both fur/fD153N and ERGIC-53.

FIG. 5

Autoproteolytic, intramolecular activation of furin. BSC-40 cells were infected with either VV:wt or VV:fur/fΔtc-k. At 16-18 h post-infection the cells were harvested, and membrane preparations were resuspended in pH 6.0 or 7.5 activation buffer as indicated (10 mM bis-Tris, pH 6.0/pH 7.5, with 5 mM CaCl₂ and 1mM βME). In A, 200 μM α₁-PDX or 200 μM decanoyl-Arg-Val-Lys-Arg-CMK were added prior to incubation at 30 °C for 3 h as indicated. Following incubation, the samples were analyzed for the propeptide by Western blot using the anti-HA mAb 12CA5. The intact propeptide (arrow) and cleaved ∼6-kDa HA-tagged N-terminal propeptide fragment (arrowhead) are indicated. In B, samples were diluted in pH 6.0 activation buffer on ice and Lsequently incubated at 30 °C for 3 h. Following incubation, furin activity was determined against the pyr-Arg-Thr-Lys-Arg-MCA Lstrate. Background activity from VV:wt-infected cells was Ltracted from that of VV:fur/fΔtc-k infected cells, and relative activity was calculated. The amount of active furin in the membrane preparation was determined by titration with decanoyl-Arg-Val-Lys-Arg-CMK. The data points indicate the mean of five separate experiments. Bars indicate standard deviation. In C, BSC-40 cells were infected with VV:fur/f/ha or VV:R75A:fur/f/ha, incubated for 4 h at 37 °C, and then pulse-labeled for 30 min with 100 μCi each of [³H]Arg and [³H]Leu. The cells were refed with serum-free defined medium (MCDB202) (5) containing excess cold Arg and Leu and harvested after the indicated chase times in TX/Ca²⁺ buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% TX-100, and 1 mM CaCl₂) supplemented with 10 μM dec-Arg-Val-Lys-Arg-CMK. Furin was immunoprecipitated with mAb M1 and resolved on a 15% SDS-PAGE peptide gel (47) that was processed for autoradiography. Furin and propeptide bands were excised, dissolved in Solvable (Packard Biosciences), and counted in Hionic Fluor LSC mixture (Packard). The relative propeptide signal is shown as a percentage of the furin signal. The labeling and chase incubations were conducted either in the absence (closed circles) or presence (open circles) of BFA (10 μg/ml). The experiments were repeated either 10 or 3 times in the absence or presence of BFA, respectively, and error bars indicate standard error of the mean.

FIG. 6

Expression and activity of R75A:fur/f/ha. BSC-40 cells infected with VV:wt, VV:fur/f/ha, or VV:R75A:fur/f/ha were processed for Western analysis (A), in vitro activity assays (B), and pulse-chase analyses (C) as described in Fig. 2.

FIG. 7

Furin·propeptide localization and propeptide dissociation. BSC-40 cells grown on glass coverslips were infected with either VV:fur/f/ha or VV:R75A:fur/f/ha and incubated at 37 °C. Where indicated (CHX treated), cycloheximide (10 μg/ml) was added at 2 h post-infection. At 6 h post-infection, the cells were fixed, permeabilized, and incubated with mAb M1 to detect furin mature domain and with HA.11 to detect propeptide; they were visualized with isotype-specific secondary antibodies. In some samples, mAb M1 was added to the culture medium (30 μg/ml) and incubated for an additional hour to label furin molecules recycling from the cell surface prior to fixation (M1 Uptake).

FIG. 8

V72R:fur/f/ha expression, activity, and localization. BSC-40 cells infected with VV:wt, VV:fur/f/ha, or V72R:fur/f/ha were processed for Western analysis (A), in vitro activity assays (B), and pulse-chase studies (C) as described in Fig. 2. Profurin (arrow) and propeptide-excised furin (arrowhead) are indicated. D, BSC-40 cells infected with VV:V72R:fur/f/ha were fixed after 5 h of incubation at 37 °C and then permeabilized and incubated with mAb HA.11 to detect furin and also with an anti-signal sequence receptor antiserum (SSR).

FIG. 9

In vitro activation of furin versus V72R mutant. A, BSC-40 cells infected with VV:wt, VV:fur/f/haΔtc-k, or V72R:fur/f/haΔtc-k were harvested, and membrane preparations were resus-pended in activation buffer and incubated at 30 °C in the presence of trypsin (+Tryp.) or trypsin and soybean trypsin inhibitor (+Tryp./STI) as described (13). Following the activation step, furin activity in each sample was determined using peptide Lstrate. B, an equivalent aliquot of each membrane sample was analyzed by Western blot using mAb M2 to monitor expression and processing of the furin constructs.

FIG. 10

Summary of furin activation. Following translation and signal sequence removal, the propeptide acts as an IMC to facilitate folding of the unstructured catalytic domain (gray-striped circle) into the active conformation (black-striped oval). This process is blocked by deletion of the proregion (fur/fΔpro) or by introduction of a canonical P1/P4 Arg furin consensus sequence at the site of internal proregion cleavage (V₇₂R:fur/f/ha), resulting in accumulation of misfolded furin molecules unable to exit the ER. The mutated internal cleavage site apparently out competes the native Arg¹⁰⁷ excision site for binding to the catalytic center. However, the aberrantly bound propeptide fails to correctly fold the profurin molecule (inset). After the initial ER folding events, furin undergoes autoproteolytic intramolecular excision of the propeptide at Arg¹⁰⁷. The propeptide, however, remains associated with the mature domain functioning as a potent autoinhibitor in trans during transport to the late secretory pathway. Propeptide excision can be blocked by inactivating furin (fur/fD₁₅₃N) and results in accumulation of an apparent folding intermediate in the ERGIC/CGN. Following propeptide excision, the inactive furin·propeptide complex transits to late secretory compartments (TGN/endosomes) where the relatively acidic pH promotes autoproteolytic, intramolecular cleavage of the propeptide at a second, internal site, Arg⁷⁵. The Arg⁷⁵ cleavage is followed by a rapid dissociation of the propeptide fragments and disinhibition of furin. These final activation steps can be blocked by either preventing transport to late secretory compartments (BFA) or by eliminating the internal furin cleavage site (e.g. Arg⁷⁵ → Ala in R75A:fur/f/ha). Blocking the internal cleavage of the propeptide at Arg⁷⁵ results in stabilization of the furin·propeptide complex and prevents activation of furin without altering its trafficking in the TGN/endosomal system.