From the Department of Biochemistry and Molecular
Biology, The Pennsylvania State University College of Medicine,
Hershey, Pennsylvania 17033, § Department of Biochemistry,
University of Mississippi Medical Center, Jackson, Mississippi 39216, and ¶ Department of Chemistry, The Pennsylvania State University
Eberly College of Science, University Park, Pennsylvania 16802
Received for publication, March 26, 2001, and in revised form, April 10, 2001
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ABSTRACT |
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Meprin A secreted from kidney and intestinal
epithelial cells is capable of cleaving growth factors, extracellular
matrix proteins, and biologically active peptides. The secreted form of
meprin A is a homo-oligomer composed of Meprins are zinc endopeptidases that are secreted from or found in
brush border membranes of kidney and intestinal epithelial cells of
humans and rodents. They are members of the astacin family of
metalloproteases and the metzincin superfamily (1, 2). The expression
of meprins enables activation or degradation of bioactive peptides,
growth factors, cytokines, hormones, and matrix proteins by limited
proteolysis (3-5).
Meprins are multidomain, oligomeric glycoproteins with subunit masses
of 85,000-110,000 Da (1). The subunits, Meprin subunits, a multidomain protease of 582 amino acids coded for near the major histocompatibility complex of the mouse and human genome. Analyses of the recombinant homo-oligomeric form of mouse meprin A by gel filtration, nondenaturing gel electrophoresis, and cross-linking (with disuccinimidyl suberate or
N-(4-azido-2,3,5,6-tetraflourobenzyl)-3-maleimidylpropionamide) indicate that the secreted enzyme forms high molecular weight multimers, with a predominance of decamers. The multimers are composed
of disulfide-linked dimers attached noncovalently by interactions
involving the meprin, A5 protein, receptor protein-tyrosine phosphatase µ (MAM) domain. The active protomer is the noncovalently linked
dimer. Linkage of active protomers by disulfide-bonds results in an
oligomer of ~900 kDa, which is unique among proteases and distinguishes meprin A as the largest known secreted protease. Electron
microscopy revealed that the protein was present in two states, a
crescent-shaped structure and a closed ring. It is concluded from this
and other data that the covalent attachment of the protomers enables
noncovalent associations of the native enzyme to form higher
oligomers that are critical for hydrolysis of protein substrates.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
and
, may associate to
form either homo- or hetero-oligomeric proteins (6). Multimers
containing
subunits (both homo- and hetero-oligomers) are
designated meprin A (EC 3.4.24.18), whereas homo-oligomers of
are
designated meprin B (EC 3.4.24.63). The mouse
and
subunits are
~42% identical at the amino acid level, and their cDNA-deduced
primary sequences predict a similar arrangement of functional domains
(1, 5).
cDNA (Structure TI) encodes
a 760-amino acid protein with an NH2-terminal signal
sequence followed by a prosequence, the protease domain (containing the
zinc active site), a MAM1
domain, a MATH (meprin and TRAF homology; TRAF, tumor necrosis factor
receptor-associated factor) domain, followed by an AM (after MATH)
domain, an EGF (epidermal growth factor)-like domain, a putative
COOH-terminal transmembrane domain (TM), and a cytoplasmic (C) tail.
The
subunit contains a 56-amino acid domain (I) inserted between
the AM and EGF-like domains that is not present in the
subunit. The
I domain has been shown to direct COOH-terminal proteolytic processing
of the
subunit during biosynthesis (7). The mature
subunit
lacks the domains COOH-terminal to the AM domain, including the
transmembrane domain. As a result, meprin
homo-oligomers are
secreted, and meprin
is only found membrane-bound via interactions
with the
subunit (which does not undergo COOH-terminal proteolysis
and is a type I integral membrane protein).
View larger version (6K):
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Structure I.
Meprin cDNA deduced amino acid
domain structure.
Studies of membrane-bound meprins indicated that the subunits are
linked via disulfide bonds to form dimers (6-8). These dimers can
further associate via noncovalent interactions to form tetramers (8).
Meprins are unique among proteases in their oligomeric structure.
Because hetero-oligomer formation requires interactions between the subunits and membrane-bound
subunits, it was unclear whether the
secreted homo-oligomer, which contains only
subunits, could
form multimers of the same oligomeric state or even larger complexes.
Native gel electrophoresis of unpurified secreted meprin A followed by
Western blot detection of the enzyme demonstrated that it migrated with
a mobility larger than 700 kDa (9). Mutation of Cys-320, the cysteine
residue in the MAM domain involved in an intersubunit disulfide bond,
to an alanine (C320A) resulted in expression of a monomeric form of
meprin. The mutant had little or no ability to hydrolyze proteins,
although it retained activity against peptide substrates and showed
decreased thermostability and an increased susceptibility to tryptic
degradation (9). This work indicated that the oligomeric state is
critical for activity and stability of the enzyme but could not
determine whether the properties exhibited by the monomeric mutant were
due to lack of formation of the disulfide-linked dimer or the inability
of the subunits to associate noncovalently. Moreover, the oligomeric state of meprin could not be accurately determined by native gel electrophoresis of the unpurified protein because the band observed could have been due to meprin subunits in association with other proteins. Thus, the oligomeric structure of the purified meprin protein
was investigated, and the importance of this structure to function was examined.
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EXPERIMENTAL PROCEDURES |
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Purification of the Meprin A Homo-oligomer--
Recombinant
mouse meprin A was purified from the media of human embryonic kidney
293 cells (American Type Culture Collection) stably transfected with
full-length mouse meprin cDNA (10). Briefly, the purification
involved mono-Q and superose-12 fast protein liquid chromatography
column chromatography; the protein was activated by trypsin after
mono-Q chromatography (5, 10).
Photocross-linking of Meprin-- An 8 µM meprin solution was labeled with 10 molar equivalents of N-(4-azido-2,3,5,6-tetrafluorobenzyl)-3-maleimidylpropionamide (Molecular Probes) for 12 h at 4 °C in the dark. Excess label was removed by passing the mixture through a Sephadex G-25 column. Cross-linking was initiated by subjecting the protein to ultraviolet light for 7 min, as described elsewhere (11), and quenched with 5 µl of gel loading buffer.
Mapping the Location of the Biotin-labeled Cysteine
Residue--
Fifty µl of an 800 µM
N-biotinyl-N'-[6-maleimidiohexanoyl]-hydrazide
(biotin-maleimide) solution dissolved in dimethylformamide was added to
500 µl of an 8 µM meprin solution. The protein was incubated for 10 h at 4 °C, and excess label was removed with a
Sephadex G-25 column. The labeled protein was subsequently
deglycosylated with peptide-N-glycosidase F (PNGase F; New England
BioLabs), and proteins were separated from carbohydrates by gel
filtration. Deglycosylated, labeled protein was incubated with 50 µg
of trypsin at pH 8.5, 37 °C for 14 h. The resulting peptides
were separated by a reverse phase C-18 HPLC column (11), and fractions
were analyzed by reflector mode MALDI mass spectrometry using an
-cyano-4-hydroxycinnamic acid matrix.
Electron Microscopy and Image Analysis-- Samples from different preparations of meprin were diluted with deionized water to ~25 µg/ml and then negatively stained with 1% uranyl acetate using a double carbon method (12). Electron micrographs were obtained with a LEO 912AB microscope operated at 100 kV at an absolute magnification of 31,500. The spectrometer slit of the energy filter was used to remove inelastically scattered electrons. For preparation of figures, negatives were digitized at an optical resolution corresponding to 6.4 Å/pixel on an Agfa Duoscan flatbed scanner, and then composites were prepared using Adobe Photoshop. Statistics of particle measurements were compiled from enlarged prints on a digitizing tablet with SigmaScan (Jandel).
Tris-(2-carboxyethyl)phosphine Hydrochloride (TCEP) Reduction and Analysis of Meprin-- Eight µM meprin was reduced with 5 mM TCEP (Molecular Probes), pH 8.0, for 30 min at 4 °C. Excess reducing agent was removed by a Sephadex G-25 column, and the enzyme was subjected to native gel electrophoretic analysis to determine the dissociation of the oligomer and nonreducing SDS-PAGE to assess the amount of disulfide bond reformation. Proteolytic activity was determined with an internally quenched fluorescent bradykinin substrate analog, BK+ (2-aminobenzoyl-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Lys(Dnp)-Gly-OH, where Dnp is dinitrophenyl), and the protein substrate azocasein as described previously (5).
Thermostability and Proteolytic Susceptibility of Meprin--
An
8 µM solution of native or reduced meprin was incubated
at 47 °C for 5-35 min at 5-min intervals. The protein was cooled to
25 °C, and the activity toward BK+ was determined. Susceptibility to
trypsin digestion was assessed by mixing 2 µl of an 800 nM solution of either the native protein or the reduced
protein with trypsin at final concentrations of 20-100 ng/ml. After 15 min at 25 °C, the reaction was stopped by the addition of a 3-fold excess of soybean trypsin inhibitor, and the protein was subjected to
SDS-PAGE followed by Western blotting to determine the extent of proteolysis.
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RESULTS AND DISCUSSION |
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The Meprin A Homo-oligomer Is Capable of Forming High Molecular
Weight Structures--
When purified recombinant mouse meprin A
homo-oligomer was subjected to nondenaturing, nonreducing gel
electrophoresis, the native protein migrated as a species larger than
the 669,000 molecular weight marker (Fig.
1A). The monomeric molecular
weight of a meprin subunit is ~90,000, indicating that the
multimeric protein is capable of forming at least octamers.
Cross-linking the protein with disuccinimidyl followed by SDS-PAGE in
the presence of 2-mercaptoethanol resulted in dimers and higher order
oligomers (Fig. 1B). The largest cross-linked species
observed migrated above the 669,000 molecular weight marker, confirming
that meprin can form an octamer or even higher order complexes. When
the protein was subjected to analytical size exclusion chromatography
to obtain more precise molecular weight information, the elution volume
of meprin corresponded to a molecular weight of 905,000, indicating
that the protein is primarily decameric (Fig. 1C). The width
of the meprin peak was comparable to that of thyroglobulin (669 kDa),
the protein standard closest in mass. However, the meprin peak was more
asymmetric than any of the monodisperse standard proteins. As shown in
the elution profile (Fig. 1C), there appears to be some
trailing on the right side of the peak, indicating the presence of
smaller oligomeric forms.
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The large size of the complex distinguishes the meprin A homoligomer as the largest known secreted protease. The ability of the secreted protein to form decameric complexes, compared with the tetrameric structure of hetero-oligomeric meprins (8), may result from the lack of the potential conformational barriers imposed by being membrane-bound.
The Meprin A Homo-oligomer Is Composed of Disulfide-linked Dimers
that Associate Noncovalently--
The electrophoretic mobility of the
protein under both reducing and denaturing conditions corresponds to
the monomeric molecular weight (Fig.
2A). Under denaturing (in the
presence of SDS) but nonreducing conditions (no 2-mercaptoethanol), the
protein migrates as a dimer (Fig. 2B). When the enzyme was
reduced with 2-mercaptoethanol and then subjected to native gel
electrophoresis (no SDS), the protein migrated as a dimer of
noncovalently linked subunits (Fig. 2C). These results
indicate that the oligomer is composed of disulfide-linked dimers that
associate noncovalently to form the native structure. The interaction
between the covalently linked dimers does not appear to be strong
because only dimers, not higher order oligomers, are observed after
reduction of the S-S bond.
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The Functional Protomer of the Meprin A Homo-oligomer Is the
Noncovalently Linked Dimer--
The secreted form of meprin A contains
11 cysteine residues per subunit (Structure
TII).
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Previous work indicated that Cys-320 in the MAM domain is responsible
for the covalent interaction of subunits (9). Mutation of Cys-320 to
alanine (C320A) resulted in a meprin monomer that exhibited altered
proteolytic activity and stability (9). This mutant was incapable of
forming not only disulfide-linked dimers but noncovalently linked
oligomers as well.
To determine whether the changes seen in the mutant were due to the loss of the disulfide-linked dimer or instead to loss of interactions between the noncovalently linked subunits, we reduced wild type meprin with the phosphine TCEP and measured the activity and stability of the protein. TCEP was selected as a reducing agent because it does not contain a thiol group. Thiol-containing reagents such as 2-mecaptoethanol or dithiothreitol have been shown to inhibit meprin by either chelation of the active site zinc ion or reduction of conserved intrasubunit disulfide bonds in the protease, MAM, or AM domains (13). Reduction with TCEP resulted in dissociation of the oligomer into noncovalently linked dimers, as was the case with 2-mercaptoethanol (Fig. 2C); the disulfide bond did not reform when TCEP was removed, even after a period of days (data not shown). To determine the number of free thiols that are solvent-exposed, meprin protein (2.5 µM) was incubated with 2 mM 5,5'-dithio-bis(2-nitrobenzoic acid). 5,5'-Dithio-bis(2-nitrobenzoic acid) reacts with free thiols, releasing thionitrobenzoic acid, which can be quantified (14). The 5,5'-dithio-bis(2-nitrobenzoic acid) assay indicated that there was one free thiol per subunit before reduction and three additional thiols per subunit after reduction with TCEP; most likely one intrasubunit disulfide bond was reduced in addition to the intersubunit disulfide bridge.
When meprin was reduced with TCEP, subjected to Sephadex G-25 column chromatography to remove the reducing agent, and assayed for enzymatic activities, little or no activity loss was detected. The rate of hydrolysis of BK+ by the reduced enzyme was 95% that of the nonreduced form (5.95 × 103 versus 5.66 × 103 fluorescence units/s for the native and reduced form, respectively). This demonstrated that the reduction and formation of noncovalently linked dimers had not significantly altered the protease domain of the enzyme, which contains two intradomain disulfide bridges, and that the disulfide-linked dimer is not required for activity toward peptides. Similarly, the activity of the reduced enzyme toward the protein substrate azocasein was 90% that of the nonreduced protein, indicating that only the noncovalently associated dimer was necessary for hydrolysis of proteins.
The stability of the TCEP-reduced form of meprin was similar to that of the native enzyme, as assessed by heat inactivation and vulnerability to trypsin digestion. The thermostability was evaluated by incubating the proteins at 46 °C, removing samples at time intervals, and subsequently determining the ability of the protease to hydrolyze BK+. Unlike the monomeric C320A mutant (9), the noncovalent dimeric reduced form of meprin exhibited the same rate of heat inactivation as the native enzyme. Furthermore, both the wild type and TCEP-reduced forms of the enzyme were equally resistant to proteolytic digestion by trypsin. These results indicate that the changes exhibited by the C320A mutant were not due to the inability of the protein to form the disulfide-linked dimer but rather to an inability to form the noncovalent associations; thus, the noncovalently associated dimer is the functional protomer of the oligomer.
The MAM Domain Is Involved in the Noncovalent Association between
the Disulfide-linked Dimer--
Only 1 of the 11 cysteine residues
present in the secreted form of the meprin subunit is a free thiol that
is exposed to solvent as determined by 5,5'-dithio-bis(2-nitrobenzoic
acid) (data not shown). This cysteine residue was specifically labeled
with the thiol-reactive photocross-linker
N-(4-azido-2,3,5,6-tetrafluorobenzyl)-3-maleimidylpropionamide, and the protein was subjected to ultraviolet light to initiate cross-linking (Fig. 3A). In
the absence of cross-linker and 2-mercaptoethanol, the protein migrated
as a single band corresponding to dimer (Fig. 3A, lane 1).
In the absence of cross-linker but in the presence of reducing agent,
the mobility of the protein was consistent with monomer only,
indicating that the intersubunit disulfide bond was completely reduced
(Fig. 3A, lane 2). In the presence of cross-linker and the
absence of the reducing agent, a band corresponding to a cross-linked
tetramer was observed (Fig. 3A, lane 3). When both
cross-linker and reducing agent were present, a cross-linked protein
corresponding to the meprin dimer was observed (Fig. 3A, lane
4). This indicates that the cross-link must have occurred between
the noncovalently linked subunits because cross-linking between the
disulfide-linked dimers would result in only dimers after SDS-PAGE,
regardless of whether reducing agent was present or not.
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To determine the location of the cysteine residue that was labeled with
N-(4-azido-2,3,5,6-tetrafluorobenzyl)-3-maleimidylpropionamide and thus one of the sites of interaction between the noncovalently linked subunits, meprin was labeled with the thiol-reactive probe biotin-maleimide. The site of the modified cysteine residue was determined by subjecting the biotin-labeled protein to tryptic digestion followed by MALDI mass spectrometry to identify the labeled
peptide as described under "Experimental Procedures." A
monoisotopic mass of 1655.826 Da ([M+H]+) was
observed by reflector mode MALDI mass spectrometry, which is consistent
with the monoisotopic mass of the protonated fragment containing amino
acids 353-361 modified with the biotin probe (expected mass of
1655.870 Da) (Fig. 3B). The labeled cysteine residue,
Cys-355, resides in the MAM domain, which has been described as an
adhesion domain that mediates protein-protein interaction. For
instance, the MAM domains of receptor protein-tyrosine phosphatase µ and are required for specific homophilic cell-cell interactions in
transfected nonadherent insect cells (15). The studies herein demonstrate that the MAM domain plays an important role in maintaining both covalent and noncovalent interactions in the meprin multimer. Removal of the MAM domain by truncation or deletion mutagenesis results
in misfolding of the protein and subsequent degradation by the
proteasome, demonstrating the importance of this domain in folding and
assembly of the native protein (16).
Electron Microscopy Demonstrates that Meprin Forms Flexible Chains
and Rings--
To further characterize the high molecular weight
oligomers, meprin was imaged by electron microscopy. In negatively
stained samples, two distinct populations of oligomers were seen (Fig. 4). As shown in a typical field (Fig.
4A), these are linear, open structures that have the
flexibility to bend into a variety of crescent shapes and closed
circular particles.
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The open chains, which comprise the majority of multimers, consistently measure 8-10 nm in width. The length of the chains varies considerably. Whereas most measure between 30 and 60 nm, some are as small as 20 nm or as large as 90 nm. Two distinct views of the chains are visible. One is most common in crescent shapes and is characterized by a smooth outer face and a notched or toothed inner face (Fig. 4B, row 1). The second orientation of the chains is typical of the straighter and shorter oligomers (Fig. 4B, row 2). In this view, both sides have regularly spaced notches, and there are also distinct stain-filled holes along the midline.
The second population of meprin multimer structures is composed of rings. The majority of these structures measure ~80 nm in circumference (Fig. 4D; Fig. 4B, row 3). This indicates that they are formed from the longer chains, that is, when sufficient length is reached to allow interaction of the ends and closure of the crescents. The average measurement of the inner diameter of these rings is 13 nm. Occasionally, much larger circles are seen (Fig. 4B, row 4). These measure 100-120 nm in circumference and appear to be formed by end-to-end interactions of two chains.
Assembly of the complex may involve attaching subunits together to form a rigid structure, such that upon the addition of subunits, the curvature of the oligomer increases until enough subunits are added to close the ring. Formation of a ring from a chain containing lower numbers of subunits may be thermodynamically unfavorable due to the rigidity of the structure. Although ring closure would provide more protein-protein contact, the strain on the structure would make it less stable than the open form, requiring the association of a critical number of subunits for ring formation. Chains containing fewer subunits than this number would be unlikely to form a ring and would remain open, whereas those containing more subunits would favor ring formation.
A model of the multimer is proposed in which the protein is built from
disulfide-linked dimers associated noncovalently to form either
crescent-shaped structures or closed rings (Fig.
5). The subunit interactions are mediated
by the MAM domain, which is involved in both the covalent linkage and
noncovalent linkage of the subunits. Reduction of the rings with TCEP
results in dissociation into the active protomer, the noncovalently
linked dimer. Similarly, reduction of the open, crescent-shaped
multimer yields only noncovalently linked dimers. As shown
schematically in Fig. 5, reduction would yield monomers at the ends of
the chain, which, in the absence of the restriction imposed by the
quaternary structure, are free to associate noncovalently.
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Meprin may function to degrade large complexes of proteins in
vivo, which could require multiple copies of the active protomer in close proximity. The presence of multiple subunits may serve to
increase the efficiency of proteolysis. Linkage of these units together
in a specified geometry could serve to effectively digest proteins, in
a manner reminiscent of the proteasome (18). Meprin also shares
similarities to other proteases such as tripeptidyl peptidase II and
aminopeptidase I. These enzymes are large multimeric complexes composed
of homo-oligomers that function to digest polypeptides and play roles
in protein turnover within the cell (19-21). Yeast aminopeptidase I is
a dodecamer with a molecular mass of ~640 kDa (20). Tripeptidyl
peptidase II forms even larger complexes, between 5000 and 9000 kDa
(21). It is composed of subunits of 138 kDa that associate to form a
rod-shaped structure about 50 nm in length and 17 nm in diameter (21).
However, unlike these proteases, the meprin A homo-oligomer is secreted
from the cell and degrades extracellular proteins and peptides.
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ACKNOWLEDGEMENT |
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We thank Dr. A. Daniel Jones for assistance with the MALDI mass spectrometry analysis.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant DK 19691 (to J. S. B.) and a supplement to that grant (to F. T. I.).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: Dept. of
Biochemistry and Molecular Biology, The Pennsylvania State University College of Medicine, Hershey, PA 17033. Tel.: 717-531-8586; Fax: 717-532-7072; E-mail: jbond@psu.edu.
Published, JBC Papers in Press, April 11, 2001, DOI 10.1074/jbc.M102654200
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ABBREVIATIONS |
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The abbreviations used are: MAM, meprin, A-5 protein, receptor protein-tyrosine phosphatase µ; MALDI, matrix-assisted laser desorption/ionization; TCEP, Tris-(2-carboxyethyl)phosphine hydrochloride; PAGE, polyacrylamide gel electrophoresis; HPLC, high pressure liquid chromatography; BK+, 2-aminobenzoyl-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Lys(Dnp)-Gly-OH, where Dnp is dinitrophenyl.
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