From the Department of Biochemistry and Molecular
Biology, Pennsylvania State University College of Medicine, Hershey,
Pennsylvania 17033 and the ¶ Department of Biochemistry,
University of Mississippi Medical Center,
Jackson, Mississippi 39216
Received for publication, August 28, 2002, and in revised form, October 22, 2002
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ABSTRACT |
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Meprin A and B, metalloproteases
consisting of evolutionarily related Proteolytic enzymes are essential components of many cellular and
extracellular processes from maturation of proteins to cell death (1).
Their activities and localities, however, must be highly regulated
because of their destructive potential. Regulation of proteases is
accomplished through several mechanisms including zymogen formation,
inhibition, localization to specific compartments, and transcriptional
regulation. The structures of proteases themselves have revealed
mechanisms to regulate proteolytic activity, and this has been amply
demonstrated in the high molecular mass oligomeric structures of the
proteasome, tripeptidyl peptidase II, and the tricorn protease (2-4).
These multimeric serine and threonine proteolytic complexes are homo-
or hetero-oligomeric, have molecular masses of 0.7-9 MDa, and are
found intracellularly. The structures serve to restrict, localize, and
concentrate proteolytic activity. Proteases at the cell surface are
known to form transient oligomeric complexes, such as those between
membrane-type 1 matrix metalloproteinase, tissue inhibitor of
metalloproteinase 2, and matrix metalloproteinase 2 that lead to the
activation of matrix metalloproteinase 2 (5). However, stable
secreted, multimeric proteolytic complexes were not described until
recently, when homo-oligomers of meprin A were found to form multimers
of ~0.9 MDa (6). These observations established meprin A as one of
the largest known secreted proteases.
Meprins, zinc-dependent metalloendopeptidases of the
"astacin family" and "metzincin superfamily," consist of
multidomain, evolutionarily related In mammals, meprins A and B are highly concentrated in kidney and
intestinal brush border membranes, e.g. these proteins are estimated to compose 5% of the mouse brush border membrane of proximal
tubule juxtamedullary nephrons (16, 17). Further, membrane-bound and
secreted meprins are expressed in leukocytes and cancer cells,
implicating these enzymes in inflammation and tumor biology (17-19).
The homo-oligomeric form of meprin A is found in rodent urine and the
media of transfected cell lines and colon cancer cells (18, 20, 21).
Although meprin B and hetero-oligomeric meprin A are predominantly
membrane-bound proteins in vivo, there is some evidence that
the membrane-bound form of human and rat meprin Meprins can cleave diverse polypeptides including cytokines, basement
membrane proteins, growth factors, protein kinases, gastrointestinal
peptides, and peptide hormones (10, 24-27). Recent studies
demonstrated that the individual subunits have marked differences in
their peptide bond and substrate specificities and conditions for
optimal activity (26, 28). The meprin Although it is known that dimerization of meprin A is important for
stability and activity toward protein substrates, little is known about
the propensities of meprin subunits to oligomerize (21). The recent
finding that secreted mouse homo-oligomeric meprin A was primarily
decameric contrasted with previous observations that indicated
hetero-oligomeric meprin A isolated from mouse kidney was primarily
tetrameric (6, 9). Little is known about the structure of meprin B,
partially because only small amounts have been available. In addition,
to date there has not been an accurate determination of the molecular
masses of meprin subunits, or oligomeric forms. The work herein was
conducted to determine definitively meprin subunit masses and to
characterize the oligomeric forms of meprin A and B. For these studies,
truncated and histidine-tagged recombinant rat meprin subunits were
prepared, allowing for the secretion and subsequent large scale
production of meprins. The recombinant proteins were found to have
similar properties to the native enzymes (28).
Expression and Purification of Meprins--
Meprins were stably
expressed in human embryonic kidney 293 cells by transfecting cells
with pcDNA 3.1 (+) expression vectors containing meprin sequence
using the calcium phosphate precipitation method. Recombinant rat
meprin PAGE and Immunoblotting--
Protein samples, boiled in sample
buffer with SDS and 2-mercaptoethanol, were routinely subjected to
electrophoresis on 7.5% Ready gels (Bio-Rad) unless indicated. For
native PAGE, 3-8% NuPAGE Tris acetate gels in the absence of SDS and
reducing agent were used (Invitrogen). Individual meprin Collection of Rat Urine--
Two female rats were placed in
metabolic cages, and urine was collected for 6 h. Urine was kept
on ice. After collection, urine was filtered through a 0.2-µm
cellulose acetate filter to remove particles. The filtered urine (6 ml)
was buffer exchanged into 8 ml of 20 mM Tris-HCl, 150 mM NaCl, pH 7.5, using Econo-Pac 10 DG chromatography
columns (Bio-Rad) and then concentrated to 600 µl using a Centriplus
YM-50 concentrator (Millipore).
Mass Spectroscopy Determination of the Monomeric and Dimeric
Molecular Masses of Meprin A and B--
Purified recombinant rat
latent and active meprins in the presence or absence of 10 mM dithiothreitol were used for matrix-assisted laser
desorption ionization/time of flight (MALDI-TOF) mass spectroscopy studies. Molecular masses were determined using a Voyager-DE RP/Pro BioSpectrometry work station (PerSeptive Biosystems). The machine was
set in the linear, delayed positive mode. The accelerating voltage was
25,000 V, grid voltage 89%, guide wire 0.15%, and extraction delay
time 1200 ns. At least 32 laser shots were taken per spectra, and at
least 5 spectra were used to give the molecular mass. Proteins were
diluted in a saturated matrix solution of sinapinic acid (Sigma) in
30% acetonitrile, 0.3% trifluoroacetic acid in water and then spotted
on a stainless steel plate (PerSeptive Biosystems). Standards were the
Sequazyme bovine serum albumin and IgG1 mass standard kits (PerSeptive
Biosystems). The low mass gate was set at 10 kDa. Data were analyzed
using Data Explorer software version 4.0.0.0 (Applied Biosystems).
Determination of the Molecular Masses of Meprin Proteins in
Solution by Size Exclusion Chromatography Light Scattering
(LS)--
The molecular masses of native meprin proteins were
determined using SEC-LS in the HHMI Biopolymer Facility and W. M. Keck Foundation Biotechnology Resource Laboratory by Ewa Folta-Stogniew as described (29, 30). Briefly, a 100-µg sample of each form of
meprin in a 500-µl volume was filtered through a 0.22-µm Durapore membrane (Millipore). The filtrate was applied to a Superose 6 HR 10/30
column upstream of Dawn DSP LS (Wyatt Technology), model 733 variable
wavelength KRATOS UV (Applied Biosystems), and OPTILAB DSP refractive
index (Wyatt Technology) detectors. The column was equilibrated in 20 mM HEPES, 150 mM NaCl, pH 7.5, at a flow rate
of 0.3 ml/min. The weight average molecular masses
(Mw) of rat meprins were calculated at peak maxima
using three independent analyses, the two- and three-detector method,
and ASTRA analysis. A second order Berry fit was used for latent and
active homo-oligomeric meprin A, and a zero order Debye fit was used
for the other meprins. The Mw was estimated
throughout the entire eluting peak at 5-µl intervals using ASTRA
software. Computations were performed as described (29, 30). For
analysis of meprin molecular masses under different conditions, a
Superose 6 10/30 HR column was calibrated using the molecular mass data
obtained from the SEC-LS analyses. The column was equilibrated in 20 mM Tris-HCl, 150 mM NaCl, pH 7.5, and the flow
rate was 0.3 ml/min.
Electron Microscopy and Image Analyses--
For electron
microscopic analyses, samples of purified recombinant rat latent and
active hetero-oligomeric meprin A and homo-oligomeric meprin A and B
were diluted to 10 µg/ml with 20 mM Tris-HCl, pH 7.5, with or without 150 mM NaCl. Samples were negatively
stained with 1% uranyl acetate and imaged at absolute magnifications
of 50,000 or 63,000. For image analyses, micrographs were digitized on
an Agfa Duoscan flatbed scanner at an optical resolution on the image
scale corresponding to either 4.06 or 3.22 Å/pixel, respectively.
Composites of typical fields and galleries of individual images were
prepared using Adobe Photoshop. Statistics of individual particle
measurements were compiled from enlarged prints on a digitizing tablet
using SigmaScan (Jandel).
All image analysis was performed using the SPIDER/WEB software package
(31). For two-dimensional averaging, each data set of untilted images
was processed by reference-free alignment and hierarchical ascendant
classification using principal component analysis. Three-dimensional
volumes were calculated by iterative back projection. Those for
activated meprin B and latent hetero-oligomeric A were constructed
de novo from tilt pairs of micrographs. These volumes were
used as references for determining reconstruction angles by projection
mapping for the volumes of latent meprin B and activated
hetero-oligomeric A, respectively. The numbers of images in each data
set were: 7382 for latent meprin B, 5850 for active meprin B, 4366 for
latent hetero-oligomeric meprin A, and 4470 for active
hetero-oligomeric meprin A. In all instances, the number of images was
limited so that angular coverage would be as even as possible. There
were no significant areas of missing information for any of the
volumes. Resolution limits were determined from the 50% cut-off of the
Fourier shell coefficient between volumes of half data sets. Volume
surfaces were created using IRIS Explorer (Numerical Algorithms Group).
They are shown after filtering to their resolution limits and at the
threshold for 100% mass, as calculated using the molecular masses
shown in Table I and a partial specific volume of 0.71 g/cm3.2
Purification and Initial Characterization of Recombinant
Histidine-tagged Rat Meprins--
All forms of meprin proteins were
purified to homogeneity (Fig. 1,
left panel). The latent meprin
Quantitative Western analysis was employed to determine the ratio of
the meprin
The activation of meprin subunits by trypsin resulted in mobility
shifts by SDS-PAGE (Fig. 1, left panel). The
molecular mass losses were 3.4 and 9.4 kDa for the meprin
All six forms of recombinant meprins were subjected to PAGE in the
presence of SDS and absence of 2-mercaptoethanol to ensure that
histidine-tagged proteins were also able to covalently dimerize in a
similar manner to wild-type meprins (9, 33). All forms of meprins
migrated in a manner consistent with the formation of disulfide-linked
dimers (Fig. 1, right panel). The molecular masses of homo-oligomeric latent meprin A and B were 156 and 171 kDa,
respectively (Table I). The hetero-oligomeric form of latent meprin A
had a molecular mass of 166 kDa. The molecular mass and densitometry
data are consistent with a disulfide-linked heterodimer of meprin Native PAGE Demonstrates Evidence of Meprin
Oligomers--
Coomassie staining of native PAGE gels indicated that
the latent and active forms of homo-oligomeric meprin A had a molecular mass considerably greater than 669 kDa (Fig.
2). Therefore, the activated
homo-oligomer of recombinant rat meprin A forms high molecular mass
complexes, analogous to the mouse homologue (6). The latent
homo-oligomeric meprin A was not able to enter the gel, indicating that
it was larger than the active counterpart. PAGE in the presence of SDS
and absence of 2-mercaptoethanol yielded dimers (Fig. 1,
right panel). Therefore, the formation of the large complexes was dependent on noncovalent interactions. In contrast,
the latent and activated forms of homo-oligomeric meprin B had
electrophoretic mobilities that corresponded to molecular masses of
~200 kDa, consistent with the formation of dimers (Fig. 2).
Analytical ultracentrifugation studies also demonstrate that rat meprin
B exists as a dimer.2 Dimers were also formed under
denaturing conditions (Fig. 1, right panel).
Therefore, rat meprin The Oligomeric Size of Meprins in Solution--
SEC-LS was used to
obtain additional oligomeric state information. SEC separates
polydisperse mixtures of proteins before the determination of
Mw. A single peak with a large tail was evident after SEC of the latent form of homo-oligomeric meprin A (Fig. 3A). The peak contained
macromolecules with a Mw range between 1.5 and 8.0 MDa (Table II). The signal saturated the
LS detector at the peak maximum (7.9 ml). Nevertheless, it was clear
from the Mw distribution that homo-oligomeric meprin
A formed a polydisperse pool of various molecular mass macromolecules.
The large complexes were composed of up to more than 100 monomers,
based on a monomeric molecular mass of 77.7 kDa (Tables I and II). The
Mw at 7.8 ml, near the peak maximum (7.9 ml), was
6.1 MDa (Table II). This value is consistent with this form of meprin
existing as oligomers composed of ~39 dimers or 78 subunits (Tables I
and II). Using the two- and three-detector approach, a
Mw of 5.5 MDa was predicted for the peptide portion
of the protein, indicating that on average 82 monomers are involved in
the macromolecular structures. Berry analysis revealed that the
macromolecules had root mean square (r.m.s.) radii ranging from 20 to
55 nm and an average of 26 nm (data not shown).
The SEC UV trace of the active form of homo-oligomeric meprin A
indicated that this protein was less heterogeneous than the latent
protein (Fig. 3B). The active protein peak contained
macromolecules with Mw values ranging from 1.0 to
1.7 MDa as compared with 1.5 to 8.0 MDa for the latent protein (Table
II). The peak maximum was at 10.8 ml and had a Mw of
1.5 MDa. It was clear that the peak contained a polydisperse mixture of
macromolecules containing between 14 and 22 monomers with an average of
20 monomers (icosamers) based on a monomeric molecular mass of 74 kDa
(Tables I and II). The two- and three-detector approaches provided a
Mw of 1.3 MDa for the peptide portion of the
protein. The molecular mass of the peptide portion is predicted to be
64.1 kDa per subunit, indicating that this form of meprin exists as
icosamers on average. The macromolecules had r.m.s. radii of gyrations
ranging from 11 to 34 nm and an average of 17 nm (data not shown). The
molecular mass value of active homo-oligomeric meprin A reported here
of 1.5 MDa is higher than that reported for the mouse homologue, which
was 900 kDa (6). SEC-LS is a more accurate technique, as compared with approaches used in previous studies for the measurement of molecular mass. However, there may be differences in multimerization of meprins
from various species.
Single, well resolved peaks were evident when the latent and active
forms of homo-oligomeric meprin B were subjected to SEC. The peak
maxima of latent and active meprin B were at 14.7 and 14.9 ml,
respectively, indicating that meprin B was much smaller than
homo-oligomeric meprin A (Fig. 3, C and D).
ASTRA-computed Debye analysis revealed that the peaks contained
macromolecules with Mw values of 154-185 and
145-166 kDa, demonstrating that both forms of meprin B existed in
monodisperse dimeric states based on monomeric molecular masses of 85.5 and 76.1 kDa (Tables I and II). The heterogeneity probably arises from
differential glycosylation. The rat meprin
After SEC of the latent form of hetero-oligomeric meprin A, two peaks
were evident with maxima at 13.6 and 14.5 ml (Fig. 3E). The
13.6-ml peak contained macromolecules with Mw in the
range of 300 and 360 kDa. The maximum of the peak had a
Mw of 333 kDa by Debye analysis. The two- and
three-detector approaches predicted molecular masses of 268 and 269 kDa, respectively. Based on the dimeric molecular mass of a heterodimer
(166 kDa; Table I) and the polypeptide sequence-predicted molecular
mass for the equimolar mixture of monomeric forms of the two subunits
(140 kDa), this peak contained a tetramer, a noncovalent dimer of
disulfide-linked heterodimers (Table II). Debye analysis indicated that
the second peak, which eluted at 14.5 ml, contained macromolecules with
Mw values between 181 and 240 kDa. The maximum of
the peak had Mw values of 228, 170, and 171 kDa by
Debye, two-detector, and three-detector approaches (Table II). The
observed Mw distribution by Debye calculations indicated that this peak contained a heterodimer of meprin
Two peaks were evident during the SEC of the active form of
hetero-oligomeric meprin A with maxima at 13.8 and 14.8 ml (Fig. 3F). ASTRA-computed Debye analysis indicated that the
13.8-ml peak contained macromolecules with Mw values
in the range of 250-331 kDa. The Mw at the maximum
of the peak was 304 kDa by Debye analysis; by the two- and
three-detector approaches, Mw values were 245 and
246 kDa, respectively (Table II). Based on the dimeric molecular mass
of a heterodimer (152 kDa; Table I) and the polypeptide sequence-predicted Mw for the equimolar mixture of
monomeric forms of the two subunits (127 kDa), this peak contained a
tetramer, presumably a noncovalent dimer of disulfide-linked
heterodimers. The second peak eluted with a maximum at 14.8 ml and
contained macromolecules with a range of Mw values
between 140 and 200 kDa (Table II). The observed Mw
distribution indicated that this peak contained a heterodimer of meprin
The Effect of Meprin Concentration and Ionic Conditions on the
Formation of Higher Order Oligomers--
SEC data indicated that the
oligomeric states of homo- and hetero-oligomeric meprin A are dependent
on the concentration of meprin (summarized in Table
III). The molecular masses and therefore oligomeric states of meprins were estimated using the LS-SEC data rather than traditional protein standards to avoid erroneous results because of shape effects, interaction with the resin, and other problems associated with calibrations of this type. The multimeric state assigned to homo-oligomeric meprin A was based on the elution volume of the peak maximum. As the concentration of homo-oligomeric meprin A was increased, the elution volume at which the peak maxima appeared was lowered; therefore, the apparent molecular mass of the
protein complex increased. The peak maxima had apparent molecular masses that indicated, on average, oligomers formed that had between 16 and 78 subunits in 150 mM NaCl. However, it is clear that
the samples had a broad range of molecular masses and oligomeric
states. Thus, some oligomers exist that are composed of less than 16 monomers and some exist that have more than 78 monomers in the
multimer. The range of elution volumes seen with latent homo-oligomeric meprin A was much larger than that for active protein (Table III). This
implies that the propeptide is intimately involved in oligomerization. The average number of monomers involved in the oligomer at the peak
maxima ranged from 16 to 78 for the latent and from 16 to 20 for the
active protein. A high NaCl concentration (1 M) was able to
disrupt the noncovalent interactions in meprin A, resulting in smaller
macromolecules. In the presence of 1 M NaCl, the latent form of homo-oligomeric meprin A existed in tetrameric states on
average, whereas the active protein existed in hexameric states on
average.
The presence of homo-oligomeric meprin A has been reported in rodent
urine (20). It was of interest to determine whether urinary meprin A
(
The latent and active homo-oligomeric meprin B behaved in a completely
different manner from homo-oligomeric meprin A by SEC. The elution
volume was unaffected by meprin B concentration (Table III). In
addition, the oligomeric state appeared to be independent of the
concentration of NaCl from 150 mM to 1 M (data
not shown). Therefore, dimeric species existed under all conditions studied.
It was evident that hetero-oligomeric meprin A formed two oligomeric
species by SEC. These forms were dimers and tetramers based on the
SEC-LS data (Fig. 3, E and F; Table II). The
dimer-tetramer was in a dynamic equilibrium. The oligomeric state was
dependent on the concentration of meprin (Table III). The latent
protein primarily formed tetramers under the conditions used, except at low concentrations of meprin (20 nM) and with high amounts
of NaCl (1 M); under these conditions, the dimer was
preferred. The active protein behaved in a similar manner, although the
dimer was only preferred in the presence of 1 M NaCl.
Therefore, hetero-oligomeric meprin A behaves in a manner similar to
that for homo-oligomeric meprin A; however, a tetramer was the highest
oligomeric species observed under the conditions studied.
Ultrastructure of Meprin Oligomers--
Transmission electron
microscopy was used to visualize the structures of meprin oligomers.
Both latent and active homo-oligomeric meprin A formed multiple types
of structures (Fig. 4, A and
B). These included rings, crescents, and spiral chains that
are similar to those seen for the mouse homologue (6). However, much
longer chain lengths were observed for the latent form of the rat
enzyme. In low salt conditions, the latent form of homo-oligomeric
meprin A existed as chains typically ~90-100 nm in length, but they
could extend up to 400 nm (Fig.
5A). In the presence of 150 mM NaCl, fewer of the extremely long polymers were seen and
the majority measured 50-75 nm consistent with the SEC-LS data (Fig.
5B). As presented at higher magnification, the latent
protein not only existed as circles and crescents but also as novel
tube-like and long spiral-like structures (Fig.
6, rows 1-3). The
tubes measured 30-40 nm in length and ~30 nm in width (Fig. 5,
C and D). The tube width corresponded to the
diameter of the circular forms and indicated that these are stacks of
three to six rings or highly condensed spirals. The occurrence of
tube-like structures was dependent on the presence of 150 mM NaCl.
The latent and active forms of meprin B were observed to be much
smaller particles than homo-oligomeric meprin A (Fig. 4, C
and D). The predominant class of averaged images of the
latent form of meprin B measured ~11 × 12 nm, whereas that of
the active form was slightly smaller at 10 × 10 nm (Fig.
7, row A). Both forms are characterized by discrete areas of high density surrounding a
central cavity. In the particular orientation shown for the latent
form, a surface opening is apparent. The latent and active forms of
hetero-oligomeric meprin A have markedly different dimensions to
homo-oligomeric meprin A and B (Fig. 4, E and F).
The most populated averaged images of these particles clearly show a
distinct barrel shape with a symmetric arrangement of two protomers
connected by thin bridges (Fig. 7, rows B and
C). The largest dimensions are ~18 × 11 nm for
both the latent and active forms.
Surface representations of the three-dimensional volumes provide
additional support for the dimeric and tetrameric composition of meprin
B and hetero-oligomeric meprin A, respectively (Fig. 7, rows
D and E). The volumes of latent and active meprin
B measure ~10 × 11 × 11 nm and have two distinct domains
likely corresponding to the individual subunits (Fig. 7, row
D). When comparing latent and active meprin B, the mass
bridging the two halves of the latent form is no longer visible in the
active form. The three-dimensional volumes of latent and active
hetero-oligomeric meprin A (Fig. 7, row E)
measure ~9 × 11 × 18 nm. The mass distributions are consistent with the four-ringed barrels seen in the two-dimensional projection (Fig. 7, rows B and C) and
indicate a symmetric assembly of two protomers. Again, in the active
form, less bridging mass is seen and the central opening is larger.
A model for the oligomerization of rat meprins is proposed in Fig.
8. The simplest form is meprin B, a
disulfide-linked dimer of The multiple technologies used in these studies establish
definitively that the meprin The kidney and intestine have the highest levels of expression of
meprins in vivo, and under normal circumstances meprin B and
hetero-oligomeric meprin A are membrane-bound, whereas homo-oligomeric meprin A is secreted into the lumen of the proximal tubule and intestinal lumen. The concentration of secreted meprin A in these tissues has not been determined; however, our studies indicate that in
rat urine the concentration is ~20 nM, where it exists as
a multimer of ~16 subunits. Both protein and salt concentrations markedly change from the kidney proximal tubule to the urine as water
and solutes are reabsorbed. Osmolarity, for example, varies from
isotonic conditions in the proximal tubule fluid (300 mosmol/liter; equivalent to 150 mM NaCl) to
hyperosmotic in the descending loop of Henle and urine (1400 mosmol/liter) and hypo-osmotic in the ascending loop of Henle (80 mosmol/liter; Ref. 38). Thus, the multimeric structure of
homo-oligomeric meprin A will vary markedly throughout the nephron. The
activities of meprins are also salt-dependent (28). The
affects of salt concentration on homo-oligomeric meprin A activity are
minor in the range between 150 and 1000 mM
(kcat/Km specificity
constants 77 and 86 × 104
M The membrane-bound forms of kidney meprin are concentrated in the
juxtamedullary region of the cortex (16). Based on the purification
schemes of meprin (i.e. the total amount purified and the
yield), the concentration of meprin at the kidney brush border membrane
in vivo is estimated to be in the high micromolar range
(39). There is evidence that membrane-bound meprins associate with
other proteins at the membrane, including other proteases (e.g. angiotensin-converting enzyme and leucine
aminopeptidase) and amino acid transporters (24, 32, 39). The data thus far indicate that meprin It has become increasingly apparent that several key proteolytic
processes depend on the formation of very large multiprotein complexes.
For example, in apoptosis a central scaffold protein oligomerizes to
form the apoptosome (700-1,400 kDa) and recruits and activates
caspases (40). Additionally, the tricorn protease, an icosahedral
capsid of 5-9 MDa, is proposed to interact with other proteases such
as prolyl iminopeptidase and the 20 S proteasome (700 kDa) with its
four stacked heptamer rings to form giant proteolytic machines (4).
Large macromolecular enzymatic assemblies have probably evolved as a
means of autocompartmentalizing a reaction or series of reactions. The
close proximity of many active sites may act by engulfing large protein
substrates and degrading them in a concerted fashion. Homo- and
hetero-oligomeric meprin structures may represent novel molecular
scaffolds that prevent inadvertent proteolysis by confining proteolytic
active sites to localized regions.
The structure of meprin B and the hetero-oligomeric form of
meprin A were elucidated for the first time in the work herein. It is
clear that the structures of the covalently linked dimers and
tetrameric meprins have distinct overall shapes and subunit arrangements. Both latent and active dimeric meprin B are approximately square, and each contains one large central cavity. In contrast, hetero-oligomeric meprin A is rectangular and somewhat resembles the
barrel shape of the proteasome. Again, both latent and active forms are
characterized by extremely large continuous central cavities.
Activation of meprin B and hetero-oligomeric meprin A results in an
expansion of the overall structures, as indicated by decreases in
central densities observed in the two- and three-dimensional images.
The volume dimensions of dimeric meprin B were ~10 × 11 × 11 nm, and those for tetrameric hetero-oligomeric meprin A were ~9 × 11 × 18 nm. Thus dimeric and tetrameric meprins are
similar in size to the 20 S component of the proteasome, a 700-kDa
complex with dimensions of ~11 × 11 × 15 nm (2). The
locations and structural contributions of the multiple glycans within
the meprin proteases are as yet unknown, and they may contribute to the
large volume, saccharides having greater hydrodynamic volumes compared with polypeptides. Further, meprins appear to have a more open structure than the proteasome. The central cavity of the proteasome is
narrow and represents less than 10% of the entire volume. In contrast,
the cavities in meprins represent ~30% of the total volume.
It is likely that the oligomerization propensity of meprins lies in the
noncatalytic domains (e.g. MAM, MATH, and AM). None of the
other astacin family members contain these interaction domains, and
none of them are known to form dimers or multimers. Both MAM and MATH
domains are implicated in homo- and heterophilic protein-protein
interactions (6, 13, 14, 41). For example, the MATH domain in TRAF2 is
involved in trimerization events (42). TRAF domains have been
identified in a diverse set of proteins throughout eukaryotes (43).
These domains permit homo- and heterophilic interactions with other
proteins carrying TRAF domains or act as a scaffold on which other
types of protein recognition elements are displayed. TRAF domains have
been identified in a large number of intracellular proteins that
are associated with signal transduction. Meprins are the only
extracellular proteins identified thus far to contain TRAF domains and,
therefore, broaden the potential role of these domains in cell physiology.
The meprins have emerged as unique proteinases with features/domains
common to many other proteins. The differences between the
evolutionarily related meprin and/or
subunits,
are membrane-bound and secreted enzymes expressed by kidney and
intestinal epithelial cells, leukocytes, and cancer cells. Previous
work established that the multidomain meprin subunits (each ~80 kDa)
form disulfide-bridged homo- and heterodimers, and differ in substrate
and peptide bond specificities. The work herein clearly demonstrates
that meprin dimers differ markedly in their ability to oligomerize.
Electrophoresis, light scattering, size exclusion chromatography, and
electron microscopy were used to characterize quaternary structures of
recombinant rat meprins. Meprin B, consisting of meprin
subunits
only, was dimeric under a wide range of conditions. By contrast, meprin
homodimers formed heterogeneous multimers (ring-, circle-, spiral-, and tube-like structures) containing up to 100 subunits, with molecular
masses at protein peaks ranging from ~1.0 to 6.0 MDa. The size of the
meprin
homo-oligomers was dependent on protein concentration, ionic
strength, and activation state. Meprin
heterodimers tended to
form tetramers but not higher oligomers. Thus, the presence of meprin
, which has a transmembrane domain in vivo, restricts
the oligomerization potential of meprin molecules and localizes meprins
to the plasma membrane. By contrast, the propensity of secreted meprin
homodimers to self-associate concentrates proteolytic potential
into high molecular mass multimers and thus allows for
autocompartmentalization. The work indicates that different mechanisms
exist to localize and concentrate the proteolytic activity of
membrane-bound and secreted meprin metalloproteinases.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
subunits (7, 8). The
subunits are 42% identical at the amino acid level, highly
glycosylated, and form disulfide-linked homo- or heterodimers (7, 9). Meprin A (EC 3.4.24.18) is a hetero-oligomer of
and
subunits or
a homo-oligomer of
subunits; meprin B (EC 3.4.24.63) is a
homo-oligomer of
subunits (10). The nascent subunits each have a
signal peptide that directs the protein to the lumen of the endoplasmic
reticulum during biosynthesis, and a propeptide that inhibits activity
(11). Each subunit has an astacin-like catalytic domain, and several
protein interaction domains including a meprin/A5
protein/protein-tyrosine phosphatase µ (MAM),1 a meprin and tumor
necrosis factor receptor-associated factor (TRAF) homology (MATH), and
an "after MATH" (AM) domain (12). The MAM and MATH domains are
found in cell-adhesion superfamily proteins and in adapter proteins in
signal transduction, respectively, and permit homo- and heterophilic
selective associations with self and other proteins (13, 14). The MAM,
MATH, and AM interaction domains are essential for the biosynthesis of
active, stable meprins (12). The nascent meprin subunits are both
synthesized with COOH-terminal epidermal growth factor (EGF)-like
domains, putative transmembrane-spanning domains, and short cytoplasmic
tails. However, the nascent meprin
subunit, but not the
subunit, has a 56-amino acid inserted domain between the AM and
EGF-like domains, and the presence of this domain allows for a
proteolytic event during maturation that liberates the meprin
subunit from the membrane (15). Because of this COOH-terminal
processing, homo-oligomers of meprin A are secreted proteins, whereas
hetero-oligomers of meprin A and homo-oligomers of meprin B are
membrane-bound.
can be shed from
the cell surface (9, 22, 23).
subunit has an acidic pH
optimum, prefers low ionic strength, and has a distinct preference for
acidic residues flanking the scissile bond in substrates. In contrast,
the meprin
subunit has a neutral to alkaline pH optimum, prefers
small or hydrophobic residues flanking the scissile bond, and has a
stronger preference for proline residues proximal but not flanking the
scissile bond. These substrate and activity differences imply different
functions for the meprin isoforms.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
was truncated at the putative mature carboxyl terminus
(Arg-603 of the AM domain), and a histidine tag was added to the
carboxyl terminus. Meprin
was truncated at Lys-648, which is at the
EGF-transmembrane border, and a GGGS linker and histidine tag were
added to the carboxyl terminus (28). The truncation of meprin proteins
at Arg-603 and Lys-648 resulted in the secretion of the subunits into
the media, and the addition of histidine tags allowed for a facile
purification scheme. Nickel-nitrilotriacetic acid affinity
chromatography was used to purify meprins. Homo-oligomeric meprin A and
B were produced by transfecting cells with meprin
or
cDNA
alone. A third cell line was transfected with both cDNAs to allow
for the production of hetero-oligomeric meprin A. All proteins were
secreted into the media as proenzymes. Active forms of meprins were
produced by limited digestion of the purified latent meprin with
trypsin as described (28). Meprin was treated with a 1:20 w/w ratio of
trypsin to meprin, and meprin activity was monitored over time. Trypsin
was inhibited with a 20-fold excess of soybean trypsin inhibitor when
meprin activity no longer increased. Trypsin and soybean trypsin
inhibitor were subsequently removed by size exclusion chromatography
(SEC) using a Superose 6 column. Meprin protein was subjected to
SDS-polyacrylamide gel electrophoresis (PAGE), and gels were stained
with Coomassie Blue to assess purity and verify complete activation.
and
subunits were detected using the subunit-specific polyclonal
antibodies, HMC52 and PSU56, respectively, that were developed by our
laboratory. HMC52 and PSU56 antibodies did not cross-react with the
incorrect subunit with the amounts used in these studies.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
used in these
studies is predicted to be larger than meprin
because of the
presence of the EGF-like and a portion of the AM domain in meprin
,
and indeed it appeared to be larger by SDS-PAGE. Meprin bands were diffuse as previously published and as expected for highly glycosylated proteins (9). Rat meprin
has eight potential asparagine-linked glycosylation sites compared with six in the rat meprin
sequence; additional glycosylation in the meprin
subunit could contribute to
the higher molecular mass compared with meprin
as well as the
occurrence of the more diffuse band. The band for the latent hetero-oligomeric meprin A migrated between that of homo-oligomeric meprin A and B. In some instances two bands were visible under reducing
conditions corresponding to each subunit. However, the subunits in the
hetero-oligomeric protein did not resolve well on the gels (Fig. 1,
left panel). To obtain more accurate molecular masses, MALDI-TOF was employed (Table I).
Using MALDI-TOF the molecular masses of latent meprin
and
subunits were found to be 85.5 and 77.7 kDa, respectively.
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Fig. 1.
Characterization of purified recombinant rat
meprins by SDS-PAGE. Left panel, latent
( ) and active (+) meprins were subjected to
electrophoresis under reducing conditions (presence of
2-mercaptoethanol) on 7.5% SDS-PAGE gels. Active meprins were produced
by limited trypsin hydrolysis as described (28). Right
panel, latent and active meprins were subjected to
electrophoresis under nonreducing conditions on 7.5% SDS-PAGE. Protein
was stained by Coomassie Brilliant Blue R-250. Prestained standards
were
2-macroglobulin (190 kDa),
-galactosidase (115 kDa), fructose-6-phosphate kinase (85 kDa), pyruvate kinase (66 kDa),
and fumarase (59 kDa). Homo A, homo-oligomeric
meprin A; Homo B, homo-oligomeric meprin B;
Hetero A, hetero-oligomeric meprin A.
Monomeric and dimeric molecular masses of meprin A and B
and
subunits in the purified rat hetero-oligomeric meprin A (21). Subunit-specific antibodies were used to quantify the
amount of each subunit present. Known amounts of purified homo-oligomers were used as standards, run on the same gels, and calibration curves were constructed. The amount of each standard and
meprin subunit in each sample was determined by densitometry. The
protein consisted of an approximately equal amount of subunits; a 1:1.2
ratio of the meprin
and
subunits was calculated.
and
subunits, respectively, as determined by MALDI-TOF (Table I). Trypsin
treatment removes the propeptide of both subunits (11, 22, 32). The
greater loss of molecular mass in the meprin
subunit is probably
the result of the additional loss of amino acids within the EGF domain. Indeed, trypsin is used to remove meprin
subunits from brush border
membranes (32).
and
subunits as expected (33). Molecular masses of 148, 154, and
152 kDa were determined for the active forms of homo-oligomeric meprin
A and B and hetero-oligomeric meprin A, respectively (Table I).
subunits do not interact noncovalently to
form larger complexes under these conditions. The active form of meprin
B was slightly smaller than the latent form as assessed by native PAGE
as expected. For hetero-oligomeric meprin A, two bands were visible for
both the latent and active forms. The mobilities of the bands
corresponded to molecular masses of ~200 and 400 kDa. Thus, latent
and active hetero-oligomeric meprin A existed as a mixture of dimers
and tetramers under these conditions (Fig. 2). Hetero-oligomeric meprin
A existed as dimers in the presence of SDS and absence of
2-mercaptoethanol (Fig. 1, right panel). Thus,
noncovalent interactions were involved in the putative dimer to
tetramer transition. Interestingly, the active tetramer had a larger
apparent molecular mass than the latent form by this technique.
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Fig. 2.
Native PAGE of recombinant rat meprins.
The latent ( ) and active (+) forms of
recombinant homo-oligomeric meprin A (Homo A) and
B (Homo B) and hetero-oligomeric meprin A
(Hetero A) were subjected to electrophoresis on
nonreducing and nondenaturing gels (native PAGE). 3-8% NuPAGE Tris
acetate gels were used in the absence of SDS and reducing agent.
Meprins were stained with Coomassie Blue. Thyroglobulin (669 kDa),
ferritin (440 kDa), catalase (232 kDa), and lactate dehydrogenase (140 kDa) were used as standards. MW, molecular mass.
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Fig. 3.
Native molecular masses of recombinant rat
meprins by SEC-LS. Approximately 100 µg of each meprin was
subjected to SEC on a Superose 6 column. The downstream refractive
index, UV (Abs 280 nm, solid line)
and light scattering detectors allowed for the determination of the
weight average molecular mass (Mw,
dotted line) by ASTRA software using Debye
(latent and active homo-oligomeric meprin A) or Berry analysis (all
other forms of meprin) at 5-µl intervals. Every tenth datum point is
shown on the molecular mass line for clarity. Panels
A and B, latent and active homo-oligomeric meprin
A; panels C and D, latent and active
homo-oligomeric meprin B; panels E and
F, latent and active hetero-oligomeric meprin A.
Native molecular masses of meprins
subunit has 8 potential
asparagine glycosylation sites; thus, the dimer would have 16 potential
sites. The Mw values at the peak maxima for the
latent and active proteins were 181 and 161 kDa, respectively, by Debye
analysis; the two- and three-detector methods predicted
Mw values for the polypeptide portions of species to
be 139 and 123 kDa. Based on the polypeptide portions having predicted
monomeric molecular masses of 72 and 63 kDa, this is good evidence that
the meprin B homo-oligomer existed as dimers; all three independent
methods of light scattering analyses gave equivalent results.
and
subunits. The calculated values were probably higher than actual values
because of poor resolution during the SEC step between the dimer and
tetramer. The r.m.s. radii of these molecules could not be determined
by this method because the size fell below the accuracy limit of the machine.
and
subunits. The peak did not resolve well from the major peak
that eluted at 13.8 ml. The maximum of the 14.8-ml peak had
Mw values of 182, 145, and 146 kDa by the Debye,
two-detector, and three-detector approaches (Table II). Although these
values are higher than expected for dimers, the observed
Mw distribution indicates that this peak contained a
heterodimer of meprin
and
subunits. The estimated values are
probably higher than the actual values caused by poor resolution
between the dimer and tetramer in the size exclusion chromatography step.
Oligomeric states of meprin A and B: effects of meprin concentration
only) had the ability to form large oligomeric complexes.
Therefore, rat urine was subjected to SEC on a Superose 6 column. The
meprin in the injection sample was at a concentration of ~200
nM based on quantitative Western analysis. The elution volume was ~11.5 ml. Based on calibrations using the SEC-LS data, this elution volume corresponds to a molecular mass at the peak maximum
of ~1.6 MDa. Thus, native rat meprin A forms large oligomeric species
in a manner similar to that for the recombinant protein.
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Fig. 4.
Electron micrographs of rat meprins.
Representative fields of negatively stained samples of each meprin
isoform are shown. A and B, latent and active
homo-oligomeric meprin A, respectively; C and
D, latent and active homo-oligomeric meprin B, respectively;
E and F, latent and active
hetero-oligomeric meprin A, respectively.
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Fig. 5.
Histograms of size measurements of latent
homo-oligomeric meprin A structures. The dimensions of selected
latent homo-oligomeric meprin A structures are shown. A,
chain lengths in absence of salt; B, chain lengths in
presence of 150 mM NaCl; C, lengths of tubes;
D, widths of tubes.
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Fig. 6.
Multiple structures of latent homo-oligomeric
rat meprin A observed in negatively stained electron micrographs.
Row 1, closed rings and crescents; row
2, tubes; row 3, spirals.
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Fig. 7.
Images from negatively stained electron
micrographs illustrate dimeric and tetrameric ultrastructure of latent
and active homo-oligomeric meprin B and hetero-oligomeric meprin
A. The left column contains images of the
latent forms of the enzymes, whereas the right
column depicts activated enzymes. Row
A, the first and third
panels are the most populated image averages of meprin B. The number of images in each average is in the lower
right of the panels; the second and
fourth panels depict corresponding images with
density contour overlays where whiter areas show highest densities.
Row B, image averages of hetero-oligomeric meprin
A. Row C, corresponding images with density
contour overlays. Row D, three-dimensional
reconstructions of meprin B. Row E,
three-dimensional reconstructions of hetero-oligomeric A. All
three-dimensional volumes have been filtered to their resolution limits
of 22-25 Å and are shown at a threshold calculated to
correspond to 100% of the protein mass.
subunits. Thus, meprin
subunits are
limited to covalent interactions and lack the ability to associate with
other meprin
and
subunits by noncovalent interactions. A
disulfide-linked homodimer of
subunits is the building block of
homo-oligomeric meprin A complexes. These dimers are able to associate
further to produce the high molecular mass structures seen by
electron microscopy. Oligomerization of homo-oligomeric meprin A is in a dynamic equilibrium whereby dimers continuously associate and disassociate with others dimers via noncovalent interactions. Finally,
hetero-oligomeric meprin A predominantly consists of a disulfide-linked
heterodimer as the building block. The dimers are able to associate
with other dimers, via noncovalent interactions of
subunits to form
tetramers. This is the largest oligomer possible because the meprin
subunits reside on the outer surface of the oligomer.
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Fig. 8.
Diagrammatic representation of rat meprin
oligomerization. Meprin B exists as a disulfide-linked dimer.
Homo-oligomeric meprin A exists in many forms including long chains,
spirals, rings, and tubes. The large complexes form via noncovalent
interactions between disulfide-linked dimers. Hetero-oligomeric meprin
A exists in a dimer-tetramer equilibrium. It is postulated that the
noncovalent interface of the tetramer is from the meprin subunit
because of the inability of meprin
subunits to interact
noncovalently.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
subunits have vastly different propensities to form homophilic associations. It is clear that meprin B
exists as a disulfide-linked dimer under a wide range of concentrations
and has no tendency to form higher molecular mass complexes. This
information about meprin
provides a rationale for the observation
that the hetero-oligomeric meprin
dimer only forms tetramers
despite the fact that the meprin
subunits tend to self-associate
into larger multimers. Thus, the solubilized recombinant form of
hetero-oligomeric meprin A studied herein, as the meprin A isolated
from mouse and rat kidney brush border membranes, is limited in the
ability to multimerize because of the presence of meprin
(9, 34).
The recombinant rat meprin
homodimers associate into a polydisperse
mixture of high molecular mass complexes (millions of daltons), as
homo-oligomeric meprin A in rat urine and recombinant mouse
homo-oligomeric meprin A (6). The previous work demonstrating that
mouse homo-oligomeric meprin A formed multimers was conducted with
activated protein. The work with the rat enzymes herein establishes
that latent homo-oligomers of meprin A form even larger complexes than
activated forms, and the degree of multimerization is dependent on
protein and salt concentration. We propose that the function of these
very large latent proteolytic complexes is to enable the movement of
these metalloproteinases through extracellular spaces in a
nondestructive, concentrated "particle" to areas where they are
activated, for example, to destroy foreign material or undesirable
protein complexes. The self-association of the latent proteases would
allow the delivery of a high concentration of proteolytic activity only
at specific sites containing activating proteases. Sites of
inflammation and metastasizing cancer cells, for example, are known to
be associated with the presence of many types of trypsin-like proteases
that are capable of activating meprins (35-37).
1 s
1, respectively), whereas
the differences in oligomerization are marked (oligomer contains 16-20
subunits at 150 mM and 6 at 1000 mM). Thus,
higher order oligomerization does not correlate with altered specific activity.
is the subunit involved in heterophilic interactions, in contrast to meprin
, which is clearly involved with
homophilic interactions. Heterophilic proteolytic complexes at the
plasma membrane could coordinately degrade proteins to produce small
peptides, dipeptides, and amino acids that can then directly feed to
the associated amino acid transporters. This would be an efficient link
for proteolysis and recycling of protein building blocks.
and
subunits will provide insights into elements or motifs that drive oligomerization, and this
will no doubt have relevance to many protein-protein interactions and
physiologic and pathological processes.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Bruce Stanley for assistance with the MALDI-TOF analyses and Dr. Faoud Ishmael for conducting the ultracentrifugation studies. SEC-LS was conducted in the Howard Hughes Medical Institute Biopolymer Facility and W. M. Keck Foundation Biotechnology Resource Laboratory by Ewa Folta-Stogniew. The collection of electron microscopic data by Karen Labat and the participation of Sheetal Bhan in image computations are also gratefully acknowledged.
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FOOTNOTES |
---|
* This work was supported by American Heart Association Predoctoral Fellowship 9910075U (to G. P. B.) and National Institutes of Health Grants DK 19691 and DK 54625 (to J. S. B.).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.
§ Present address: Clearant, Inc., Gaithersburg, MD 20879.
To whom correspondence should be addressed: Dept. of
Biochemistry and Molecular Biology, H171, Pennsylvania State University College of Medicine, Hershey, PA 17033-0850. Tel.: 717-531-8586; Fax: 717-531-7072; E-mail: jbond@psu.edu.
Published, JBC Papers in Press, October 23, 2002, DOI 10.1074/jbc.M208808200
2 F. Ishmael, personal communication.
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ABBREVIATIONS |
---|
The abbreviations used are: MAM, meprin, A5 protein, and protein-tyrosine phosphatase µ; MATH, meprin and tumor necrosis factor receptor-associated factor homology; TRAF, tumor necrosis factor receptor-associated factor; AM, after MATH; EGF, epidermal growth factor; SEC, size exclusion chromatography; MALDI-TOF, matrix-assisted laser desorption ionization/time of flight; LS, light scattering; Mw, weight average molecular mass; r.m.s., root mean square.
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