A Point Mutation in the Juxtamembrane Stalk of Human Angiotensin I-converting Enzyme Invokes the Action of a Distinct Secretase*

Marwan AlfalahDagger , Edward T. Parkin§, Ralf JacobDagger , Edward D. Sturrock, Reinhard Mentele||, Anthony J. Turner§, Nigel M. Hooper§**, and Hassan Y. NaimDagger

From the Dagger  Department of Physiological Chemistry, School of Veterinary Medicine, D-30559 Hannover, Germany, the § Proteolysis Research Group, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom, the  Department of Medical Biochemistry, University of Cape Town, Rondebosch 7700, South Africa, and the || Abteilung fur Klinische Chemie und Klinische Biochemie, Ludwig-Maximilians-Universitat, D-80336 Muenchen, Germany

Received for publication, January 16, 2001, and in revised form, March 8, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Angiotensin I-converting enzyme (ACE) is one of a number of integral membrane proteins that is proteolytically shed from the cell surface by a zinc metallosecretase. Mutagenesis of Asn631 to Gln in the juxtamembrane stalk region of ACE resulted in more efficient secretion of the mutant protein (ACENQ) as determined by pulse-chase analysis. In contrast to the wild-type ACE, the cleavage of ACENQ was not blocked by the metallosecretase inhibitor batimastat but by the serine protease inhibitor, 1,3-dichloroisocoumarin. Incubation of the cells at 15 °C revealed that ACENQ was cleaved in the endoplasmic reticulum, and mass spectrometric analysis of the secreted form of the protein indicated that it had been cleaved at the Asn635-Ser636 bond, three residues N-terminal to the normal secretase cleavage site at Arg638-Ser639. These data clearly show that a point mutation in the juxtamembrane region of an integral membrane protein can invoke the action of a mechanistically and spatially distinct secretase. In light of this observation, previous data on the effect of mutations in the juxtamembrane stalk of shed proteins being accommodated by a single secretase having a relaxed specificity need to be re-evaluated.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Angiotensin I-converting enzyme (ACE,1 EC 3.4.15.1) plays a key role in the control of blood pressure and fluid and electrolyte homeostasis (1). It exists primarily as a type I integral membrane glycoprotein, although a soluble form is present under normal conditions in blood plasma and other body fluids and is derived from the membrane-bound form through proteolytic cleavage in the juxtamembrane stalk region (2). The secretase responsible for the cleavage and secretion of ACE is a zinc metalloproteinase located at the cell surface (3-6). It is inhibited by hydroxamate-based compounds such as batimastat and displays a remarkably similar inhibition profile to that of the alpha -secretase that cleaves the amyloid precursor protein (7, 8). The precise site of cleavage in ACE has been determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry following enzymic fragmentation of the purified protein (9). This analysis revealed that the soluble somatic forms of human and porcine ACE were cleaved between Arg1203 and Ser1204, some 27 residues on the extracellular side of the transmembrane domain.

ACE is just one of several proteins that are post-translationally shed from the membrane through the action of secretases (also called sheddases or convertases) (10-12). Other proteins proteolytically cleaved from the membrane include tumor necrosis factor-alpha , transforming growth factor-alpha , amyloid precursor protein, and L-selectin. The secretases that cleave and release such proteins from the membrane have several properties in common including up-regulation by phorbol esters and inhibition by hydroxamate-based zinc metalloproteinase inhibitors. Numerous studies have investigated the sequence/structural requirements for recognition of an integral membrane protein by its cognate secretase (for example see Refs. 13-18). This usually has been investigated by mutating residues in the extracellular juxtamembrane stalk region and monitoring their effect on secretion of the expressed protein. Such studies, in which numerous mutations failed to abrogate cleavage and secretion of the membrane protein, have concluded that for each substrate protein there is a single secretase with a relaxed sequence specificity and that the critical parameter for cleavage efficiency is the relative conformation (possibly alpha -helical) of the stalk region. However, an alternative explanation for these observations is that the mutations in the juxtamembrane stalk invoked the action of other distinct secretases. The involvement of multiple secretases was suggested by Zhong et al. (19) when numerous point mutations in the stalk region of the amyloid precursor protein resulted in that protein being cleaved at multiple sites, although there was no experimental evidence (inhibition profile, cellular location) to support this suggestion.

Here we report that a single point mutation in the juxtamembrane stalk region of ACE invokes the action of a mechanistically and spatially distinct secretase. Asn631 in the single domain form of ACE (20) (corresponding to Asn1196 in human somatic ACE), 7 residues N-terminal to the normal secretase cleavage site, was mutated to Gln (Fig. 1). The mutant protein (ACENQ) was transport-competent and enzymatically active, indicating that the mutation has no general effects on the folding of ACE. Pulse-chase analysis revealed that ACENQ was more readily cleaved and secreted into the medium than the wild-type protein. Mass spectrometric analysis of the secreted protein and temperature block and inhibitor studies indicated that ACENQ was being cleaved between Asn635 and Ser636 in the endoplasmic reticulum (ER) by a serine protease and not between Arg638 and Ser639 at the cell surface by the batimastat-sensitive metallosecretase. In light of this observation, the conclusions of earlier studies investigating the sequence requirements of various secretases through analysis of the effect of mutations on the secretion of the substrate protein require re-evaluation and should not be interpreted solely in the context of a single protease activity having a relaxed specificity.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Construction of ACENQ-- The expression vector pECE containing a C-terminal fragment of human ACE in which the N-terminal signal peptide was fused with the C-terminal domain (pECE hACE) (20) was used. In this construct the membrane-proximal stalk region, transmembrane, and cytosolic domains are identical to those in human somatic ACE. Asn631 (Fig. 1) in this construct, which corresponds to Asn1196 in human somatic ACE, was the template for oligonucleotide-directed mutagenesis with the Quick ChangeTM in vitro mutagenesis system (Stratagene) using the following oligonucleotides: ACENQup (5'-TGG CCG CAG TAC CAA TGG ACG CCG AAC-3') and ACENQdo (5'-GTT CGG CGT CCA TTG GTA CTG CGG CCA-3'). The mutation of T to C was confirmed by sequencing, and the plasmid obtained was denoted pECE hACENQ.


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Fig. 1.   Schematic diagram of angiotensin I-converting enzyme. The single domain form of human ACE has an N-terminal signal peptide (diagonally hatched box) and a C-terminal transmembrane domain (black box) followed by a short cytoplasmic tail (20). The amino acid sequence of the juxtamembrane stalk region is shown and numbered below the sequence according to this single domain form of ACE. The batimastat-sensitive secretase cleavage site (Down-arrow ) and the DCI-sensitive cleavage site (down-arrow ) are indicated, and part of the hydrophobic transmembrane domain is in italics and underlined. The Asn residue (631) that was mutated to Gln is in bold and underlined.

Cell Culture-- The neuronal cell line IMR-32 (21) was cultured in Dulbecco's modified Eagle's medium/Ham's F-12 supplemented with 10% fetal bovine serum, penicillin (50 units/ml), streptomycin (50 mg/ml), and 2 mM glutamate (all from Life Technologies, Inc.). Cells were maintained at 37 °C in 5% CO2 in air, grown to 70% confluence in 25-cm2 flasks, washed once with Opti-MEM, and then transiently transfected using LipofectAMINE and 8 µg of DNA diluted in 2 ml of Opti-MEM. After 5 h, 3 ml of growth medium was added to the flasks, and the cells were incubated overnight. The LipofectAMINE/medium mix was then replaced with fresh growth medium. After another 24 h the cells were washed with Opti-MEM and incubated with either batimastat (provided by Dr. G. Christie, SmithKline Beecham Pharmaceuticals, Harlow, U.K.) or 1,3-dichloroisocoumarin (DCI, Sigma) for 7 h. The medium was then harvested and centrifuged at 1000 × g to remove cell debris. Cells were then washed in phosphate-buffered saline (PBS), scraped into PBS, centrifuged at 1000 × g for 5 min, and resuspended in PBS. After sonication and centrifugation at 5000 × g for 20 min to remove nuclei, the cell membranes were pelleted by centrifugation at 100,000 × g for 90 min.

Metabolic Labeling and Phase Separation in Triton X-114-- Transiently transfected IMR-32 cells were labeled 48-60 h post-transfection with 80 µCi of [35S]Met in Met-free Dulbecco's modified Eagle's medium containing 2% fetal calf serum, 50 units/ml penicillin, and 50 mg/ml streptomycin (denoted Met-free medium). In pulse-chase experiments, labeling was performed for 1 h at 37 °C followed by a chase with nonlabeled Met for different periods of time. The labeled cells were rinsed twice with PBS prior to harvesting. Cells were subjected to phase separation in Triton X-114 essentially as described by Wilson et al. (22).

Immunoprecipitation and SDS-Polyacrylamide Gel Electrophoresis-- Cells were solubilized with 1 ml/dish cold lysis buffer (25 mM Tris/HCl, pH 8.0, 50 mM NaCl, 0.5% Triton X-100, and 0.5% sodium deoxycholate), and the cell extracts were centrifuged at 10,000 × g for 15 min to remove nuclei and debris. Thereafter, the supernatants were incubated with the rabbit anti-ACE polyclonal antibody (RP183) (23) and precipitated with protein A-Sepharose. After immunoprecipitation, the protein A-Sepharose beads were washed three times with washing buffer A (0.5% Triton X-100 and 0.05% sodium deoxycholate in PBS) and three times with washing buffer B (500 mM NaCl, 10 mM EDTA, and 0.5% Triton X-100 in 125 mM Tris/HCl, pH 8.0) prior to analysis of the samples by SDS-polyacrylamide gel electrophoresis and fluorography as described previously (24). The digestion of 35S-labeled immunoprecipitates with endo-beta -N-acetylglucosaminidase (Endo) H and Endo F/glycopeptidase F was performed as previously described (24).

Activity Assays-- ACE activity in the medium and membrane samples was determined by incubation with 5 mM benzoyl-Gly-His-Leu in 0.1 M Tris/HCl, pH 8.3, 0.3 M NaCl, and 10 µM ZnCl2 at 37 °C. The released benzoyl-Gly was separated from the substrate and quantitated by reverse phase HPLC as described previously (25).

Isolation and Mass Spectral Analysis of Secreted ACE-- The medium was collected from IMR-32 cells transiently transfected with either pECE hACE or pECE hACENQ, centrifuged at 3000 × g for 5 min and then concentrated in a stirred ultrafiltration cell (Amicon) using a 10-kDa cut-off membrane. The samples were then dialyzed overnight against 10 mM Hepes/NaOH, 0.3 M KCl, and 0.1 mM ZnCl2, pH 7.5. ACE was then isolated by affinity chromatography on lisinopril-Sepharose as described previously (26). The enzyme was eluted from the affinity column with 0.1 M sodium borate, pH 9.5, dialyzed, and concentrated using Centricon 10-kDa cut-off filters (Vivascience, Cambridge, U.K.). Purified secreted wtACE and ACENQ proteins were reduced and protected with vinyl pyridine prior to digestion with endoproteinase Lys-C. The total digest was analyzed directly by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, or the digest was first fractionated by HPLC and the C-terminal peptide was identified by automated N-terminal peptide sequencing, before mass spectral analysis (27, 28).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

ACENQ Is Secreted from the IMR-32 Cells-- IMR-32 cells, which previously have been shown to cleave and release ACE in a batimastat-sensitive manner (8), were transfected with cDNA encoding either wtACE or ACENQ. Both constructs were expressed in the cells as determined by metabolic labeling with [35S]Met followed by immunoprecipitation from the cell lysate with the anti-ACE antibody (Fig. 2). wtACE was present in the medium, consistent with its release from the cell surface by the batimastat-sensitive secretase (Fig. 2). ACENQ was also detected in the medium from the cells, and indeed the relative ratio of ACE protein in the medium compared with the cell lysate appeared greater for the mutant as compared with the wild-type protein. Deglycosylation with Endo F revealed that both wtACE and ACENQ were N-glycosylated (Fig. 2). Deglycosylation revealed that there was a distinct difference in size between wtACE in the cell lysate as compared with that secreted into the medium, consistent with removal of the C-terminal transmembrane and cytosolic domains of the protein by the secretase (9). Interestingly, such a size difference was not apparent between the cell lysate and medium samples of ACENQ (Fig. 2).


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Fig. 2.   Secretion of ACE from IMR-32 cells. IMR-32 cells were transfected with either wtACE or ACENQ cDNA and biosynthetically labeled for 6 h with [35S]Met. ACE was then immunoprecipitated from the cell lysate (L) or medium (M) with the anti-ACE antibody, and the immunoprecipitate was incubated in the absence (-) or presence (+) of Endo F for 2 h at 37 °C. The samples were analyzed on a 6% polyacrylamide SDS gel and visualized by fluorography.

ACENQ Is Secreted into the Medium Faster than Wild-type ACE-- The rate at which ACENQ was secreted into the medium compared with that of wild-type ACE was assessed by pulse-chase labeling (Fig. 3). wtACE appeared in the medium after 4 h of chase, consistent with previous reports using other cell lines (27, 29). At 6 h of chase a considerable amount of wtACE was still present in the cell lysate. In contrast, ACENQ was detected in the medium from the cells after 30 min of chase. After 4 h of chase the majority of ACENQ had been secreted from the cell and was no longer present in the lysate sample. The apparent larger size of the medium form of the mutant protein compared with that in the lysate is because of complex glycosylation of the protein as it traffics along the secretory pathway. The ACENQ detected in the cell lysate is mannose-rich glycosylated as assessed by its complete sensitivity toward Endo H, whereas the ACENQ species secreted into the medium was Endo H-resistant, indicating that it has acquired complex-type glycans in the Golgi apparatus (data not shown).


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Fig. 3.   Transport kinetics of ACE. After transfection with either wtACE or ACENQ cDNA, IMR-32 cells were biosynthetically labeled with [35S]Met for 30 min and chased with cold medium as indicated. ACE was then immunoprecipitated from the cell lysate (L) or medium (M) with the anti-ACE antibody, and the immunoprecipitated samples were analyzed by SDS-polyacrylamide gel electrophoresis followed by fluorography.

ACENQ Is Not Cleaved by a Batimastat-sensitive Secretase-- The ability of the secretase inhibitor batimastat to block the release of wtACE and ACENQ from the IMR-32 cells was assessed (Table I). As compared with untreated cells, batimastat significantly inhibited (81%) the release of wtACE into the medium with a concomitant increase in the amount of activity detected in the membrane fraction, consistent with previous results (8). Surprisingly, no ACE activity could be detected in the membranes from the cells expressing ACENQ, and batimastat failed to significantly inhibit the release of this mutant form of the protein into the medium. No ACE activity was detected in the total cellular membrane fraction from the untreated cells expressing ACENQ even after an eight-times longer incubation period with the substrate benzoyl-Gly-His-Leu (data not shown). Incubation of the cells expressing ACENQ with the serine protease inhibitor DCI reduced the secretion of the protein into the medium by 75% with a concomitant increase in activity detected in the membrane sample. Interestingly, DCI seemed to stimulate the release of wtACE into the cell medium with a concomitant decrease in the membrane fraction. This is consistent with a previous observation (27), although the mechanism responsible for this increased shedding is not known.

                              
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Table I
Effect of inhibitors on the shedding of ACE and ACENQ
IMR-32 cells transfected with either wtACE or ACENQ cDNA were incubated for 7 h in the absence of inhibitor or in the presence of either 20 µM batimastat or 100 µM DCI. The medium was then harvested, and membranes were prepared as described under "Experimental Procedures," and the samples assayed for ACE activity with benzoyl-Gly-His-Leu as substrate. Results are the mean ± S.E. of three experiments.

ACENQ Is Cleaved in the ER-- The failure to detect ACENQ enzymic activity in the total cellular membrane fraction (Table I) suggested that this mutant form of ACE may be cleaved soon after synthesis in the ER. To investigate this, IMR-32 cells expressing ACENQ were incubated at 15 °C to prevent transport of the proteins beyond the ER (Fig. 4A). ACENQ was susceptible to deglycosylation with Endo H, indicating that it was in the high mannose form. Phase separation in Triton X-114 was used to determine whether ACENQ retained in the ER by the temperature block lacked the hydrophobic membrane-anchoring domain (Fig. 4B). Although a significant amount of newly synthesized ACENQ was detected in the detergent-rich pellet, possibly because the 15 °C treatment impaired the activity of the DCI-sensitive protease, a significant amount of a smaller form was also present in the detergent-poor supernatant, consistent with removal of the C-terminal membrane-anchoring and cytosolic domains by an activity within the ER.


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Fig. 4.   Analysis of the cellular compartment of ACENQ cleavage. After transfection with ACENQ cDNA, IMR-32 cells were biosynthetically labeled with [35S]Met at 15 °C for 4 h. A, cells were lysed in lysis buffer, and ACE was immunoprecipitated with the anti-ACE antibody prior to incubation in the absence (-) or presence (+) of Endo H for 2 h at 37 °C. B, the cells were lysed in the presence of Triton X-114 followed by centrifugation at 13,000 × g. Supernatant (S) and pellet (P) fractions were immunoprecipitated separately with the anti-ACE antibody and analyzed by SDS-polyacrylamide gel electrophoresis followed by fluorography.

ACENQ Is Cleaved at a Different Bond in the Juxtamembrane Stalk to wtACE-- The cleaved forms of both wtACE and ACENQ were purified from the conditioned medium of transfected cells by chromatography on lisinopril-Sepharose (25). The purified proteins were digested with endoproteinase Lys-C and subjected to matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Mass spectrometric analysis of wtACE secreted from the IMR-32 cells revealed a peak at m/z 1690.8 (Table II), identical to the calculated m/z for the peptide Leu625-Arg638. This is consistent with the normal secretase cleavage site between Arg638 and Ser639 and is in agreement with the site of cleavage seen in ACE secreted from other cells and in serum (9). In contrast, analysis of the HPLC fractionated Lys-C digest of ACENQ revealed a major peak at m/z 1406.3 (Table II), which likely represented the peptide Leu625-Asn635 (m/z 1406.3, calculated m/z 1390.5: the increase in mass is caused by the oxidation of Trp632). The identity of this peptide was confirmed by partial N-terminal sequencing. Thus, the major site of cleavage was at the Asn635-Ser636 bond, three residues on the N-terminal side of the normal Arg638 and Ser639 cleavage site (see Fig. 1). There was also evidence for secondary trimming of the C terminus of the soluble form of ACENQ to Trp632.

                              
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Table II
Observed [M + H+] ions of Lys-C peptides in wtACE and ACENQ


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The Asn to Gln point mutation in the juxtamembrane stalk of ACE clearly results in more efficient cleavage and secretion of the protein. At first sight this could be attributed to the mutation making ACENQ a better substrate for the batimastat-sensitive cell surface metallosecretase. However, closer inspection clearly shows that the shedding of ACENQ is caused by the action of a spatially and mechanistically distinct secretase. The serine protease cleaving ACENQ is acting in the ER and cleaves ACE at a different bond in the stalk region to the normal secretase. Interestingly a mutant form of ACE, ACE-JGL, in which the stalk region had been replaced with a Ser/Thr-rich sequence that was partially O-glycosylated, was observed to be secreted from Chinese hamster ovary cells more rapidly than wtACE, and its shedding was not blocked by the hydroxamic acid-based compound TAPI (27). Similar to ACENQ, the shedding of ACE-JGL was blocked by DCI; however, it was not determined whether cleavage of this mutant occurred intracellularly. The major serine proteases in the secretory pathway are the family of proprotein convertases, including furin, which are involved in the proteolytic processing of a variety of secreted and membrane-bound proteins (30). However, this family of proteases shows a specificity for cleaving after dibasic or monobasic sequences, and the cleavage site in ACENQ does not fit this specificity (see Fig. 1). Also, the active forms of the proprotein convertases are located primarily in the trans-Golgi network rather than in the ER.

The effect of the Asn631 to Gln mutation invoking a distinct secretase is in contrast to a recent report in which a different point mutation in the juxtamembrane stalk of somatic ACE, associated with a variation in the levels of soluble ACE in plasma, resulted in enhanced cleavage by a metallosecretase (31). In that case mutation of Pro1199 in human somatic ACE (equivalent to Pro634 in the ACE construct used in the present study; see Fig. 1) to Leu resulted in more efficient cleavage/secretion of the mutant protein. Although the cleavage of the P1199L mutant was blocked by the hydroxamate-based metallosecretase inhibitor compound 3, the precise site of cleavage was not determined. Those authors, Eyries et al. (31), proposed that a local conformational modification caused by the Pro to Leu mutation leads to better accessibility of the stalk region to the normal ACE secretase, resulting in the enhancement of the cleavage/secretion process. Secondary structure predictions of wild-type ACE using the on-line JPred server indicated that the region from Leu620 to Asp657 is predicted to be a flexible loop, bounded by an alpha -helix on the N-terminal side and the transmembrane alpha -helix on the C-terminal side. The mutation of Asn631 to Gln had no affect on the secondary structure prediction, suggesting that the observed difference in cleavage of ACENQ is not caused by a dramatic change in secondary structure. However, it is possible that the mutation promotes binding to the serine protease either directly (Gln is a larger side chain and may be able to make more favorable contacts with the enzyme) or indirectly (by allowing the loop to adopt a more favorable conformation for binding).

The residue mutated in the present study, Asn631, lies in a potential N-glycosylation sequon (Asn-Trp-Thr-Pro), raising the possibility that this residue is glycosylated such that its mutation to Gln prevents glycosylation and exposes a site, that is normally obscured by the glycan chain, to cleavage by a serine protease within the ER. However, this explanation seems unlikely on four counts. 1) Trp in the middle position of the N-glycosylation sequon has been shown to severely reduce the efficiency of glycosylation of the preceding Asn, whereas a Pro following the sequon prevents N-glycosylation altogether (32, 33). 2) Consistent with this, there is no evidence for glycosylation of this Asn residue in the mature form of either human somatic or testicular ACE (Refs. 27 and 34 and Table II). 3) In rabbit ACE this potential glycosylation site is absent, yet the enzyme is cleaved by a hydroxamic acid-sensitive metallosecretase (6). 4) Attempts to mimic the effect of the Asn to Gln mutation in wild-type ACE by incubation of cells with tunicamycin, which blocks formation of the core dolichol-linked oligosaccharide thus preventing N-glycosylation from taking place, failed to lead to secretion of wtACE in a batimastat-insensitive manner (data not shown).

An interesting feature of ACENQ is the dramatic increase in its transport kinetics and maturation rate along the secretory pathway as compared with the membrane-bound wild-type protein. In fact, attainment of complex glycans on ACENQ occurs at a rate that is almost three orders of magnitude greater than that of wtACE. In general it is unlikely that nonanchoring of a protein leads per se to a higher efficiency in its intracellular transport relative to its anchored counterpart. This view is supported by several examples of proteins from which the transmembrane domains have been eliminated. Membrane-bound intestinal sucrase-isomaltase, for instance, exhibits similar transport kinetics as its soluble anchorless isoform (35). Likewise, deletion of the transmembrane domain in the neurotrophin receptor has no implications on the intracellular transport of the mutant protein (36). Furthermore, there is no general rule that anchorless proteins are transported more rapidly along the secretory pathway than membrane-bound ones. Indeed, with certain proteins the reverse seems to be the case. For example, deletion of the membrane-anchoring domain from the murine prion protein results in the secreted form of the protein reaching the cell surface at a 4-fold slower rate than the wild-type protein (37), and the soluble form of the trypanosome variant surface glycoprotein is also trafficked at a slower rate than the membrane-bound form (38).

The main rate-limiting step in the transport of membrane and secretory proteins from the ER to the Golgi apparatus is their acquisition to a correct folding and transport-competent quaternary structure in the ER (for a review see Ref. 39). In this respect, oligomerization in the ER constitutes for many proteins a crucial criterion before they exit this organelle. This event has been shown to involve the transmembrane domains (40-45) (for a review see Ref. 46). Wild-type membrane-bound ACE does not dimerize in the ER (47), thus excluding a possible influence of dimerization and the transmembrane domain on its transport kinetics as compared with soluble ACE. One may speculate that the delayed intracellular transport of membrane-bound ACE versus ACENQ is caused by the existence of a structural motif in the cytosolic tail that promotes an interaction of ACE with soluble and/or membrane-bound factors in the cytosol and thus regulates its transport kinetics. As such, cleavage of ACENQ in the ER eliminates this motif and results in a more efficiently transported protein.

In conclusion, in previous studies the results of mutations at or near the secretase cleavage site in a membrane protein have usually been interpreted in the context of a single protease activity having a relaxed sequence specificity. However, the results of the present study reveal another explanation: such mutations may invoke the action of other distinct proteases. In the light of this and in the absence of such additional data, the conclusions of earlier studies investigating the sequence requirements of various secretases through analysis of the effect of mutations on the secretion of the substrate protein must be interpreted with caution and require re-evaluation.

    ACKNOWLEDGEMENTS

We thank Mhairi Nimick for help in purifying the secreted forms of ACE and David Westhead for assistance in the secondary structure predictions.

    FOOTNOTES

* This work was supported by grants from Deutsche Forschungsgemeinschaft (to H. Y. N.), the Medical Research Council of Great Britain (to N. M. H. and A. J. T.), the British-German Academic Research Collaboration (to N. M. H. and H. Y. N.), and the Volkswagen Foundation and South African Medical Research Council (to E. D. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** To whom correspondence should be addressed. Tel.: 44-113-233-3163; Fax: 44-113-233-3167; E-mail: n.m.hooper@leeds.ac.uk.

Published, JBC Papers in Press, March 23, 2001, DOI 10.1074/jbc.M100339200

    ABBREVIATIONS

The abbreviations used are: ACE, angiotensin I-converting enzyme; ACENQ, ACE mutant protein; ER, endoplasmic reticulum; DCI, 1,3-dichloroisocoumarin; PBS, phosphate-buffered saline; HPLC, high pressure liquid chromatography; wt, wild type; Endo, endo-beta -N-acetylglucosaminidase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Ehlers, M. R. W., and Riordan, J. F. (1989) Biochemistry 28, 5311-5318[Medline] [Order article via Infotrieve]
2. Hooper, N. M. (1991) Int. J. Biochem. 23, 641-647[CrossRef][Medline] [Order article via Infotrieve]
3. Oppong, S. Y., and Hooper, N. M. (1993) Biochem. J. 292, 597-603[Medline] [Order article via Infotrieve]
4. Ramchandran, R., Sen, G. C., Misono, K., and Sen, I. (1994) J. Biol. Chem. 269, 2125-2130[Abstract/Free Full Text]
5. Beldent, V., Michaud, A., Bonnefoy, C., Chauvet, M.-T., and Corvol, P. (1995) J. Biol. Chem. 270, 28962-28969[Abstract/Free Full Text]
6. Ramchandran, R., and Sen, I. (1995) Biochemistry 34, 12645-12652[Medline] [Order article via Infotrieve]
7. Parvathy, S., Oppong, S. Y., Karran, E. H., Buckle, D. R., Turner, A. J., and Hooper, N. M. (1997) Biochem. J. 327, 37-43[Medline] [Order article via Infotrieve]
8. Parvathy, S., Hussain, I., Karran, E. H., Turner, A. J., and Hooper, N. M. (1998) Biochemistry 37, 1680-1685[CrossRef][Medline] [Order article via Infotrieve]
9. Woodman, Z. L., Oppong, S. Y., Cook, S., Hooper, N. M., Schwager, S. L. U., Brandt, W. F., Ehlers, M. R. W., and Sturrock, E. D. (2000) Biochem. J. 347, 711-718[CrossRef][Medline] [Order article via Infotrieve]
10. Hooper, N. M., Karran, E. H., and Turner, A. J. (1997) Biochem. J. 321, 265-279[Medline] [Order article via Infotrieve]
11. Schlondorff, J., and Blobel, C. P. (1999) J. Cell Sci. 112, 3603-3617[Abstract/Free Full Text]
12. Primakoff, P., and Myles, D. G. (2000) Trends Genet. 16, 83-87[CrossRef][Medline] [Order article via Infotrieve]
13. Maruyama, K., Kametani, F., Usami, M., Yamao-Harigaya, W., and Tanaka, K. (1991) Biochem. Biophys. Res. Commun. 179, 1670-1676[Medline] [Order article via Infotrieve]
14. Sisodia, S. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 6075-6079[Abstract]
15. Deng, P., Rettenmier, C. W., and Pattengale, P. K. (1996) J. Biol. Chem. 271, 16338-16343[Abstract/Free Full Text]
16. Ehlers, M. R. W., Schwager, S. L. U., Scholle, R. R., Manji, G. A., Brandt, W. F., and Riordan, J. F. (1996) Biochemistry 35, 9549-9559[CrossRef][Medline] [Order article via Infotrieve]
17. Deng, P., Wang, Y.-L., Haga, Y., and Pattengale, P. K. (1998) Biochemistry 37, 17898-17904[CrossRef][Medline] [Order article via Infotrieve]
18. Althoff, K., Reddy, P., Voltz, N., Rose-John, S., and Mullberg, J. (2000) Eur. J. Biochem. 267, 2624-2631[Abstract/Free Full Text]
19. Zhong, Z., Higaki, J., Murakami, K., Wang, Y., Catalano, R., Quon, D., and Cordell, B. (1994) J. Biol. Chem. 269, 627-632[Abstract/Free Full Text]
20. Wei, L., Alhenc-Gelas, F., Corvol, P., and Clauser, E. (1991) J. Biol. Chem. 266, 9002-9008[Abstract/Free Full Text]
21. Christie, G., Markwell, R. E., Gray, C. W., Smith, L., Godfrey, F., Mansfield, F., Wadsworth, H., King, R., McLaughlin, M., Cooper, D. G., Ward, R. V., Howlett, D. R., Hartmann, T., Lichtenthaler, S. F., Beyreuther, K., Underwood, J., Gribble, S. K., Cappai, R., Masters, C. L., Tamaoka, A., Gardner, R. L., Rivett, A. J., Karran, E. H., and Allsop, D. (1999) J. Neurochem. 73, 195-204[CrossRef][Medline] [Order article via Infotrieve]
22. Wilson, J. M., Whitney, J. A., and Neutra, M. R. (1987) J. Cell Biol. 105, 691-703[Abstract]
23. Williams, T. A., Barnes, K., Kenny, A. J., Turner, A. J., and Hooper, N. M. (1992) Biochem. J. 288, 875-881[Medline] [Order article via Infotrieve]
24. Naim, H. Y., Sterchi, E. E., and Lentze, M. J. (1987) Biochem. J. 241, 427-434[Medline] [Order article via Infotrieve]
25. Hooper, N. M., and Turner, A. J. (1987) Biochem. J. 241, 625-633[Medline] [Order article via Infotrieve]
26. Hooper, N. M., Keen, J., Pappin, D. J. C., and Turner, A. J. (1987) Biochem. J. 247, 85-93[Medline] [Order article via Infotrieve]
27. Schwager, S. L. U., Chubb, A. J., Scholle, R. R., Brandt, W. F., Mentele, R., Riordan, J. F., Sturrock, E. D., and Ehlers, M. R. W. (1999) Biochemistry 38, 10388-10397[CrossRef][Medline] [Order article via Infotrieve]
28. Schwager, S. L. U., Chubb, A. J., Scholle, R. R., Brandt, W. F., Eckerskorn, C., Sturrock, E. D., and Ehlers, M. R. W. (1998) Biochemistry 37, 15449-15456[CrossRef][Medline] [Order article via Infotrieve]
29. Sen, I., Samanta, H., Livingston, W., and Sen, G. C. (1991) J. Biol. Chem. 266, 21985-21990[Abstract/Free Full Text]
30. Seidah, N. G., Day, R., Marcinkiewicz, M., and Chretien, M. (1998) Ann. N. Y. Acad. Sci. 839, 9-24[Free Full Text]
31. Eyries, M., Michaud, A., Deinum, J., Agrapart, M., Chomilier, J., Kramers, C., and Soubrier, F. (2001) J. Biol. Chem. 276, 5525-5532[Abstract/Free Full Text]
32. Kaplan, H. A., Welply, J. K., and Lennarz, W. J. (1987) Biochim. Biophys. Acta 906, 161-173[Medline] [Order article via Infotrieve]
33. Mellquist, J. L., Kasturi, L., Spitalnik, S. L., and Shakin-Eshleman, S. H. (1998) Biochemistry 37, 6833-6937[CrossRef][Medline] [Order article via Infotrieve]
34. Yu, C. X., Sturrock, E. D., Wu, Z., Biemann, K., Ehlers, M. R. W., and Riordan, J. F. (1997) J. Biol. Chem. 272, 3511-3519[Abstract/Free Full Text]
35. Jacob, R., Alfalah, M., Grunberg, J., Obendorf, M., and Naim, H. Y. (2000) J. Biol. Chem. 275, 6566-6572[Abstract/Free Full Text]
36. Yeaman, C., Le Gall, A. H., Baldwin, A. N., Monlauzeur, L., Le Bivic, A., and Rodriguez-Boulan, E. (1997) J. Cell Biol. 139, 929-940[Abstract/Free Full Text]
37. Walmsley, A. R., Zeng, F., and Hooper, N. M. (2001) EMBO J. 20, 703-712[Abstract/Free Full Text]
38. McDowell, M. A., Ransom, D. M., and Bangs, J. D. (1998) Biochem. J. 335, 681-689[Medline] [Order article via Infotrieve]
39. Hammond, C., and Helenius, A. (1995) Curr. Opin. Cell Biol. 7, 523-529[CrossRef][Medline] [Order article via Infotrieve]
40. Doyle, C., Sambrook, J., and Gething, M. J. (1986) J. Cell Biol. 103, 1193-1204[Abstract]
41. Gething, M. J., McCammon, K., and Sambrook, J. (1986) Cell 46, 939-950[Medline] [Order article via Infotrieve]
42. Roth, M. G., Doyle, C., Sambrook, J., and Gething, M. J. (1986) J. Cell Biol. 102, 1271-1283[Abstract]
43. Boulay, F., Doms, R. W., Webster, R. G., and Helenius, A. (1988) J. Cell Biol. 106, 629-639[Abstract]
44. Copeland, C. S., Zimmer, K. P., Wagner, K. R., Healey, G. A., Mellman, I., and Helenius, A. (1988) Cell 53, 197-209[Medline] [Order article via Infotrieve]
45. Naim, H. Y., and Naim, H. (1996) Eur. J. Cell Biol. 70, 198-208[Medline] [Order article via Infotrieve]
46. Ellgaard, L., Molinari, M., and Helenius, A. (1999) Science 286, 1882-1888[Abstract/Free Full Text]
47. Naim, H. Y. (1992) Biochem. J. 286, 451-457[Medline] [Order article via Infotrieve]


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