From the Departments of Pathology, ¶ Molecular
Biology and Biochemistry, and
Microbiology
& Molecular Genetics, College of Medicine, University of California,
Irvine, California 92697-4800 and the
Division of Allergy and
Pulmonary Medicine, Department of Pediatrics, Washington University
School of Medicine, St. Louis, Missouri 63110
Received for publication, October 16, 2002, and in revised form, December 4, 2002
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ABSTRACT |
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The bactericidal activity of mouse
Paneth cell The release of endogenous antimicrobial peptides by mammalian
epithelial cells contributes to innate mucosal immunity (1, 2). In the
small intestine of most mammals, Paneth cells that reside at the base
of the crypts synthesize and secrete microbicidal In mouse small intestine, MMP-7 is expressed only by Paneth cells (17),
where the enzyme activates all In this study, cryptdin biosynthesis was investigated by analyzing the
processing of recombinant pro-Crps by MMP-7 in vitro. We
focused on Crp4, because it is the most bactericidal mouse Animals and Tissue Preparation--
All procedures on mice were
performed with approval and in compliance with the policies of the
Institutional Animal Care and Use Committees of the University of
California, Irvine (UCI), and Washington University School of Medicine.
Six-week-old adult male C57BL/6 mice were purchased from Charles River
Breeding Laboratories, Inc. (North Wilmington, MA). MMP-7 null mice
were 6-8-week-old males backcrossed for 10 generations onto the
C57BL/6 background. Mice were housed in a specific pathogen-free
facility under 12 h cycles of light and dark and had free access
to standard rat chow and water.
Purification of Recombinant Mouse Pro-Crp4--
Recombinant
pro-Crp4 and pro-Crp4 variants with mutated MMP-7 recognition sites
were prepared using the pET-28a expression system to produce
NH2-terminal His6-tagged fusion proteins
(Novagen, Madison, WI). By PCR amplification, a Met-coding
trideoxynucleotide was incorporated 5' of codon 20 in the Crp4
precursor cDNA and cloned in-frame with the amino-terminal
His6 in the EcoRI/SalI sites of
pET-28a. For cloning pro-Crp4, forward primer pETPCr4-F (5'-GCGCGAATTCATGGATCCTATCCAAAACACA) was paired with
reverse primer SLpMALCrp4R
(5'-ATATATGTCGACTGTTCAGCGGCGGGGGCAGCAGTACAA), corresponding to
nucleotides 104-119 and 301-327 in prepro-Crp4 cDNA (20). Reactions were performed using the GeneAmp PCR Core Reagents (Applied Biosystems, Foster City, CA) by incubating the reaction mixture at
95 °C for 5 min, followed by successive cycles at 60 °C for 1 min, 72 °C for 1 min, and 94 °C for 1 min for 40 cycles. The underlined codon in the pETPCr4-F primer denotes a Met codon that was
introduced immediately upstream of the pro-Crp4
NH2-terminal Asp residue to incorporate a CNBr cleavage site.
Following PCR amplification, samples (25 µl) of individual reactions
were gel purified using 2% agarose gels, and extracted using QIAEX II
(Qiagen Inc., Valencia CA). In most cases, amplification products were
cloned in pCR2.1-TOPO, sequenced, digested with EcoRI and
SalI, and gel-purified EcoRI/SalI
inserts were ligated into EcoRI and SalI-digested
pET-28a plasmid DNA, and transformed into both
Escherichia coli XL-2 Blue and BL21(DE3)
Codon Plus cells (Stratagene Cloning Systems, Inc., La Jolla, CA).
Recombinant proteins were expressed for 6 h at 37 °C in
E. coli BL21(DE3) Codon Plus cells growing exponentially in
Terrific Broth medium by induction with 0.2 mM
isopropyl-1-thio-
Recombinant precursor fusion proteins were purified by
nickel-nitrilotriacetic acid (Ni-NTA, Qiagen) resin affinity
chromatography and recovered from fusions after CNBr cleavage.
His-tagged fusion proteins were eluted from Ni-NTA resin with 2 column
volumes of buffer consisting of 6 M guanidine HCl, 1 M imidazole, and 100 mM Tris-HCl (pH 6.0).
Fusion proteins, containing an NH2-terminal His6 tag, a 26-amino acid spacer, and pro-Crp4, were
dialyzed against 5% acetic acid and lyophilized. Lyophilized proteins
dissolved in 80% formic acid were adjusted to 10 mg/ml CNBr in 80%
formic acid and incubated under N2 overnight at room
temperature. Cleavage was terminated by addition of 10 volumes of
H2O, proteins were lyophilized, dissolved in 5% acetic
acid, and purified by C-18 RP-HPLC by eluting peptides over 120 min
with an 20-40% acetonitrile gradient. The identities of recombinant
pro-Crp4 molecules were verified by MALDI-TOF MS at the UCI Biomedical
Protein and Mass Spectrometry Resource Facility and by acid-urea PAGE
(21).
Site-directed Mutagenesis of Pro-Crp4--
Mutations were
introduced into recombinant pro-Crp4 molecules by PCR. In reaction 1, the mutant forward primer, e.g. pc4I44Dfor, containing the
mutant codon flanked by three natural codons was paired with
SLpMALCrp4R, the normal reverse primer at the 3'-end of the desired
sequence. In reaction 2, the mutant reverse primer, e.g.
pc4I44Drev, the exact complement of the mutant forward primer, was
paired with the normal forward primer pETPCr4-F at the 5'-end of the
desired sequence, and sequences were amplified from the pET-28a
pro-Crp4 construct as described above: 95 °C for 5 min, followed by
successive cycles at 60 °C for 1 min, 72 °C for 1 min, and
94 °C for 1 min for 40 cycles. Products from reactions 1 and 2 were
purified electrophoretically, and 0.5-µl samples of gel-purified DNA
were combined as templates in PCR reaction 3, using normal external
primers, SLpMALCrp4R and pETPCr4-F, as amplimers. The full-length,
mutated pro-Crp4 product of reaction 3 was cloned sequentially into the
vectors pCR2.1-TOPO and pET-28a as noted above, and all mutations were
verified by DNA sequencing prior to expression.
The following forward and reverse internal primers were used to
introduce mutations into pro-Crp4. To eliminate the
Ser43 Purification of Recombinant Mouse Chimeric Pro-CC--
The
pro-CC construct for production in the baculovirus expression system
has been described (18). Briefly, prepro-Crp15 cDNA was amplified
using a forward primer derived from sequence encoding the signal
peptide and a reverse primer that changed a Met residue in the COOH
terminus of the mature peptide to a Thr (characteristic of Crp1). In
addition, the COOH-terminal Arg residue of the precursor was converted
to Pro-Arg-Arg-His-His-His-His-His-His; Pro-Arg-Arg is the Crp4
COOH-terminal sequence. The amplified sequence was cloned into the
transfer vector pVL1393 and transfected into Sf9 insect cells
along with BaculoGold DNA (BD Pharmingen, La Jolla, CA) to produce
recombinant baculovirus. Following a 4-5-day infection of HighFive
insect cells (Stratagene) with recombinant baculovirus, cells were
harvested by centrifugation and stored at Site-directed Mutagenesis of Pro-CC--
As with pro-Crp4,
mutations were introduced into recombinant pro-CC by PCR. To generate
the L59S mutation, the forward primer used originally to
construct pro-CC, CRPBam-F
(5'-GCGGATCCTCCTGCTCACCAATCCTCCA-3') was paired with the
mutant reverse primer CRP-L59S
(5'-ACCAGATCTCTCGACGATTCCTCT-3'), where the underlined
sequence corresponds to BamHI and BglII
restriction sites, respectively. Conditions for amplification from the
pro-CC template were as follows: 94 °C for 5 min, 30 cycles of
94 °C for 1 min, 53 °C for 1 min, 72 °C for 2 min, and a final
extension at 72 °C for 15 min. The 220-bp product was cloned into
the pCR 2.1-TOPO vector (Invitrogen); the
BamHI-BglII fragment from this plasmid construct
was used to replace the BamHI-BglII fragment in
the pro-CC cDNA in pGEM-7Zf(+) (Promega). The full-length
(L59S)-pro-CC cDNA, flanked by BamHI and
EcoRI sites, was then transferred to the pVL1393 baculovirus
expression vector using these two restriction enzymes. To add the L54S
mutation, pGEM7-(L59S)-pro-CC was used as a template for forward primer
CRPBam-F and mutant reverse primer CRPL54/59SBglII-R containing a
BglII restriction site (underlined) (5'-AGATCTCTCGACGATTCCTCTTGACTAGAAGAGCC-3'). Amplification
parameters were similar to those used for (L59S)-pro-CC except that the
annealing temperature was increased to 65 °C and the number of
cycles was reduced to 25. The 220-bp product was subcloned into pVL1393
as described above for pro-CCL59S. To eliminate only the
Ser43 Purification of Crp4 and Pro-Crp4 Variants from C57BL/6 and
MMP-7-null Mice--
A naturally existing Acid Urea-Polyacrylamide Gel Electrophoresis--
Peptide
samples were lyophilized, dissolved in 20 µl of 5% acetic acid
containing 3.0 M urea, and electrophoresed on 12.5% AU-PAGE for 1 h at 100 V and for 3.5 h at 250 V (21).
Resolved proteins were visualized by staining with Coomassie R-250
after fixation in formalin-containing acetic acid/methanol. Crp4
and pro-Crp4 were identified by co-migration with authentic mouse Crps
and pro-Crps in AU-PAGE (>0.6 × RF of methyl
green dye) as described (22) and confirmed by MALDI-TOF-MS and
NH2- terminal sequencing.
Protein Analysis by Mass Spectrometry--
For reduction and
alkylation, recombinant peptides dissolved at 500 µg/ml in 6 M guanidine HCl, 100 mM Tris-HCl (pH 8.0) were reduced with dithiothreitol at 50 °C for 3-4 h using 5 mol of dithiothreitol per mol of polypeptide cysteine. After cooling, a 3-fold
mass excess of iodoacetic acid to dithiothreitol was added to the
reduced peptide solution, incubated for 10 min, and residual iodoacetic
acid was reacted with excess dithiothreitol. The native and alkylated
peptides were purified on C-18 RP-HPLC, and the molecular masses were
determined by MALDI-TOF MS, followed by NH2-terminal
sequencing in the UCI Biomedical Protein and Mass Spectrometry Resource Facility.
MMP-7 Cleavage of Mouse Pro-Crps in Vitro--
Recombinant
pro-Crp4 and pro-CC molecules were digested with MMP-7, analyzed by
AU-PAGE and SDS-PAGE, and samples of the proteolytic digests were
analyzed by NH2-terminal sequencing. Samples (1 µg) of
pro-Crp4 and all variants, as well as pro-CC and corresponding variants, were incubated with activated recombinant human MMP-7 (0.3-1.0 µg) catalytic domain (Calbiochem, La Jolla, CA, or Chemicon International, Inc., Temecula, CA) in buffer containing 10 mM HEPES (pH 7.4), 150 mM NaCl, 5 mM CaCl2 for 18-24 h at 37 °C. Samples of
pro-Crp4 digests were analyzed by AU-PAGE, and ~200 ng quantities of
complete digests were subjected to 5 or more cycles of
NH2-terminal peptide sequencing at the UCI Biomedical Protein and Mass Spectrometry Resource Facility. Pro-CC digests were
analyzed by Tris-Tricine SDS-PAGE (15% polyacrylamide), and bands were
visualized using GelCodeTM Blue staining reagent (Pierce).
For NH2-terminal sequencing by Edman degradation, 3 µg of
precursor incubated with MMP-7 were separated by Tris-Tricine SDS-PAGE
and transferred to and visualized on mini-ProBlott polyvinylidene
difluoride membranes (Applied Biosystems) according to the
manufacturer's instructions. Also, samples of the Crp1 prosegment
corresponding to residues 19-58 in prepro-Crp1 (23, 24) were digested
with MMP-7 and sequenced. The Crp1 proregion consisting of the
primary structure, DPIQNTDEETKTEEQPGEDDQAVSVSFGDPEGTSLQEES, was
synthesized by Quality Controlled Biochemicals, Inc., (Hopkinton, MA).
The composition and properties of the synthetic prosegment has been
reported previously (5).
Bactericidal Peptide Assays of Pro-Crp4 Activated by
MMP-7--
The activation of pro-Crp4 was assayed by conducting
bactericidal peptide assays with pro-Crp4 following digestion with
MMP-7 under conditions of quantitative cleavage at Leu59
(above). Samples consisting of exponentially growing bacterial cells
(~1 × 106 colony forming units (CFU)/ml) were
incubated with 0-20 µg/ml Crp4 or pro-Crp4 with or without
MMP-7-mediated proteolysis (above). After 60 min at 37 °C, 20 µl
of each incubation mixture was diluted 1:1000 with 10 mM
PIPES (pH 7.4), and 50 µl of the diluted samples were plated on
trypticase soy agar using a Spiral Biotech Autoplate 4000 (Spiral
Biotech Inc., Bethesda, MD). Surviving bacteria were quantitated as
colony forming units on plates after incubation at 37 °C for 12 h.
MMP-7-mediated Cleavage of Pro-Crp4--
Pro-Crp4 is cleaved by
MMP-7 at sites corresponding to those identified previously in natural
pro-Crps (5). Recombinant pro-Crp4 molecules expressed in E. coli were purified to homogeneity by RP-HPLC (Fig.
1). Because the pro-Crp4 protein lacks
methionine, CNBr provided a means for quantitative chemical cleavage of
affinity purified fusion proteins from which the pro-Crp4 component
could be separated from the His-tagged fusion partner by sequential RP-HPLC fractionation. The mass of the pro-Crp4 molecule was verified by MALDI-TOF-MS to be 8231 atomic mass units, and its
homogeneity was judged by analytical RP-HPLC, AU-PAGE, and
NH2-terminal sequencing. The sequence DPIQNT ... , the
consensus for mouse pro- Specificity of in Vitro Pro-Crp-4 Cleavage by
MMP-7--
Previously, Paneth cell
NH2-terminal peptide sequence analysis of MMP-7 digests of
pro-Crp4 showed that pro-Crp4 contains cleavage sites common to natural
pro-Crps (5, 19). Consistently, four NH2 termini were
detected in the digests: DPIQ ... , ISFG ... , LHEKS, and
LRGLL_Y_ ... , where the underscores denote empty
sequencing cycles characteristic of Cys residues. These NH2
termini correspond to Asp20 at the pro-Crp4 NH2
terminus, Ile44, Leu54, and Leu59
in the polypeptide chain (Fig. 2). These NH2 termini result
from MMP-7 cleavage of pro-Crp4 peptide bonds at
Ser43 MMP-7 Cleavage of Pro-Crp4 Activates Crp4 Bactericidal
Activity--
To test whether MMP-7 mediated pro-Crp4 proteolysis
results in the production of functional Crp4 peptide, bactericidal
peptide activity assays were performed on MMP-7-digested pro-Crp4.
Salmonella typhimurium PhoP( Disulfide Bonds Protect the Crp4 Peptide from MMP-7 Proteolysis
during Pro-Crp4 Activation--
The formation of disulfide bonds
within the pro-Crp4 molecule confers protection of the Crp4 peptide
from MMP-7-mediated proteolysis. To determine whether the specificity
of MMP-7 recognition and hydrolysis would be modified by disrupting the
disulfide array of the Crp4 peptide region in pro-Crp4, the MMP-7
digestion products of native, reduced, and reduced and alkylated
pro-Crp4 were characterized ("Experimental Procedures"). The extent
of Cys alkylation in reduced and alkylated pro-Crp4 was confirmed by
MALDI-TOF-MS, which showed that the mass of reduced and alkylated
pro-Crp4 was 350.22 atomic mass units greater than that of native
pro-Crp4 (theoretical increase = 348 atomic mass units), an
indication that the 6 Cys residues in pro-Crp4 had been acetylated. The
specificity of proteolysis at MMP-7 sites within the prosegment was the
same for native pro-Crp4 and reduced and alkylated pro-Crp4 (Fig 2).
However, MMP-7 also cut reduced pro-Crp4 and reduced and alkylated
(L54D)-Crp4 substrates at Leu62 Effects of Loss-of-function Mutations in Pro-Crp4 and Pro-CC on
MMP-7 Processing--
To test whether specific MMP-7-catalyzed
proteolysis within the proregion was a requirement for the activating
cleavage step at Ser58
The activating pro-Crp4 cleavage step at
Ser58
The effects of Ser mutations at Leu54 and Leu59
on MMP-7 recognition of the upstream Val44 site
(Ile44 in pro-Crp4) were tested further by analyzing the
MMP-7 digestion products of additional chimeric recombinant pro-Crp
molecules. For example, pro-CC is a chimeric pro-Crp derived from
pro-Crp15, which differs from pro-Crp1 at one amino acid position in
the proregion and at 3 residue positions in the mature Mutation of the Ser58
Two novel Crp4-related peptides purified from C57BL/6 mouse small
intestine were characterized by NH2-terminal peptide
sequencing. The NH2 terminus of the abundant
peptide, Crp4(B6a), was LHEKSSRDLI_Y_RKGG_NRGEQVYGT_ ...
, where the underscore characters denote deduced Cys residues (Fig.
6C). Analyses of Paneth cell secretory granules partially purified from mouse crypts showed that Crp4(B6a) was present at the
same relative concentration as in extracts of intact small bowel,
consistent with the localization of all known
Crp4(B6a) and Crp4(B6b) peptides both arise from Crp4-related genes
with loss-of-function mutations at the MMP-7 cleavage site near the
Crp4 peptide NH2 terminus. In the case of Crp4(B6a), an
L59S substitution, similar to that introduced into pro-CC (Fig. 5),
eliminates in vitro MMP-7 cleavage of pro-Crp4(B6a) near the peptide NH2 terminus (Fig. 6D). The consequence
of the mutation in vivo in C57BL/6 small bowel is the
accumulation of the abundant Crp4(B6a) peptide, which terminates at
Leu54 instead of Leu59 as a product of
MMP-7-mediated pro-Crp4(B6a) cleavage at
Ala53
Because the MMP-7 knockout is on the C57BL/6 genetic background (26)
the Crp4(B6a) precursor was purified from MMP-7-null mouse small bowel
("Experimental Procedures") (5). The identity of pro-Crp4(B6a) was
deduced by co-migration with pro-Crp4 in AU-PAGE and by MALDI-TOF MS,
which showed that the mass of the peptide was 8556.1, very close to the
mass of pro-Crp4(B6a) predicted from the AK008107 cDNA sequence
(Fig. 6C). As NH2-terminal sequence analysis of
MMP-7 pro-Crp4(B6a) digests showed, pro-Crp4(B6a) was cleaved only at
Ser43
The Crp4(B6b) peptide also harbors an L59S mutation that would abrogate
MMP-7-mediated proteolysis at Ser58 Loss-of-function Mutation at Ser58 In mouse small intestinal Paneth cells, pro-Crp activation is
mediated intracellularly by MMP-7, which processes 60-70% of pro-Crps
prior to secretion (5). The studies reported here show that the
processing reactions include cleavage steps at two conserved sites in
the proregion and a site at or near the Crp NH2 terminus.
Despite the specificity of MMP-7 proteolysis at these positions,
loss-of-function mutations in the two proregion cleavage sites had no
effect on the activating cleavage step at the NH2 terminus
of the mature Crp4 or Crp15 chimeric peptide, pro-CC. Cleavage of the
Ser43 One objective of these studies was to distinguish (a)
whether the activation cleavage at the peptide NH2 terminus
depends on partial degradation of the proregion, (b) whether
prosegment degradation depends on the activation cleavage step, or
(c) whether the different cleavage steps are independent
events. From the data in Fig. 5, we infer that prosegment proteolysis
does not regulate Crp peptide activation. Instead, it appears that the proregion remains intact until the activating cleavage event at the Crp
peptide NH2 terminus or near that site at
Ser53 In mature human and rabbit phagocytes, The molecular details of human Paneth cell The identification of the Crp4(B6a) variant peptide with a
loss-of-function L59S mutation (Fig. 6) shows that mouse populations can accumulate defective pro-Crps. These two variants of Crp4 represent
the only Crp4 variants identified in inbred populations and revealed
that the Crps from the C57BL/6 strain differ markedly from those of
129/SvJ, C3H/HeJ, BALB/cJ, and outbred Swiss mice (Fig. 6A,
and not shown). This observation should be considered before
extrapolating from C57BL/6 genomic DNA and cDNA sequences to other
strains of mice, at least with respect to Paneth cell gene products.
One consequence of the L59S mutation in vivo is an abundance
of the Crp4(B6a) peptide in C57BL/6 small bowel (Fig. 6), a molecule
that has less bactericidal peptide activity and is especially
attenuated against the two Gram-negative bacterial species tested (Fig.
7). Thus, one possibility is that the production of alternative
cryptdin peptides in different strains of mice may contribute to
variation in susceptibility to enteric pathogens. If mutations at MMP-7
cleavage sites can persist in mouse populations and if the mutated
processing intermediates can accumulate as abundant, attenuated peptide
variants, it seems likely that comparable defects could disrupt HD5 or
HD6 processing in human Paneth cells. Such mutations could predispose
certain individuals to increased susceptibility to enteric bacterial
infections, a notion that is speculative but testable.
defensins, or cryptdins, is dependent on processing
of cryptdin precursors (pro-Crps) by matrix
metalloproteinase-7 (MMP-7) (Wilson, C. L., Ouellette,
A. J., Satchell, D. P., Ayabe, T., Lopez-Boado, Y. S.,
Stratman, J. L., Hultgren, S. J., Matrisian, L. M., and
Parks, W. C. (1999) Science 286, 113-117). To investigate
the mechanisms of pro-Crp processing by this enzyme, recombinant
pro-Crp4, a His-tagged chimeric pro-Crp (pro-CC), and site-directed
mutant precursors of each were digested with MMP-7, and the cleavage products were analyzed by NH2-terminal peptide sequencing.
Proteolysis of pro-Crp4 with MMP-7 activated in vitro
bactericidal activity to the level of the mature Crp4 peptide by
cleaving pro-Crp4 at Ser43
Ile44 and
Ala53
Leu54 in the proregion and near the
Crp4 peptide NH2 terminus between Ser58
Leu59. Because the Crp4 NH2
terminus occurs at Gly61, not Leu59,
MMP-7 is necessary but insufficient to complete the processing of Crp4.
Crp activating proteolysis at S58
L59 was unaffected by I44S/I44D or
L54S/L54D loss-of-function mutations in pro-Crp4, and a (L59S)-pro-CC
mutant was cleaved normally at Ser43
Val44
and Ser53
Leu54 sites but not at the peptide
NH2 terminus. C57BL/6 mice contain an abundant (L59S)-Crp4
mutant peptide with Leu54 at its NH2 terminus
resulting from Ala53
Leu54 cleavage and
loss-of-function at the Ser58
Ser59 cleavage
site. Thus,
-defensins resulting from mutations at MMP-7 cleavage
sites exist in mouse populations. A pro-CC substrate containing both
L54S and L59S mutations resisted cleavage at
Ser43
Val44 completely, showing that cleavage
at one or both downstream sites must precede proteolysis at
Ser43
Val44. These findings show that MMP-7
activation of pro-Crps can occur without proteolysis of the proregion,
and prosegment fragmentation depends, at least in part, on the release
of the Crp peptide from the precursor.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-defensins, termed
cryptdins in mice, as components of apical secretory granules (3-6).
The granules are released by Paneth cells in response to cholinergic
agonists or when exposed to bacterial stimuli (7-10). Cryptdin
peptides constitute ~70% of the bactericidal peptide activity
released by mouse Paneth cells, and cryptdin concentration at the point
of secretion is at least 1000 times greater than the minimal
bactericidal concentration of the peptides (9). The production of
functional
-defensins involves proteolytic processing of inactive
precursor forms by mechanisms that differ between mice and humans (5,
11).
-Defensins are processed from inactive proforms by specific
proteolytic cleavage steps. Both neutrophil and Paneth cell
-defensins derive from ~10-kDa prepropeptides that contain
canonical signal sequences, acidic proregions, and a ~3.5-kDa mature
-defensin peptide in the COOH-terminal portion of the precursor.
Most pro-
-defensins are fully processed in mature phagocytic
leukocytes (12, 13), and processing of myeloid
-defensin precursors
occurs within 4-24 h after synthesis by apparently sequential events
that produce major intermediates of 75 and 56 amino acids (12-14).
Deletions in the prosegment adjacent to the proregion-defensin junction impaired post-translational processing of human neutrophil
pro-
-defensins when expressed heterologously in mouse 32DCL3 cells
(13). The anionic propeptide segments also appear to neutralize the
cationic COOH-terminal defensin peptides, as suggested by the
inhibition of in vitro
-defensin bactericidal activity
when intact proregions are added in trans (5, 14, 15). Human
Paneth cells store HD-5
-defensin in precursor form that is
converted rapidly by trypsin to the mature HD-5 peptide after secretion
(10, 11, 16), but mouse Paneth cell pro-Crps are processed and
activated by matrix metalloproteinase-7 (matrilysin,
MMP-7,1 EC 3.4.24.23) by
intracellular processing events that precede secretion (5).
-defensins from 8.4-kDa proforms
(18). Previously, both procryptdin-1 (pro-Crp1) and a COOH-terminal
His6 tagged pro-Crp chimera (pro-CC) containing sequence
from pro-Crp1, pro-Crp4, and pro-Crp15 were shown to be activated to
3.5 kDa
-defensins in vitro by MMP-7-catalyzed cleavage
at conserved sites in the proregion and at the junction of the
propeptide and the NH2 terminus of the mature cryptdin peptide (5, 18). In those studies, MMP-7 was found to cleave between
Ser43
Val44 in the prosegment and at
Ser58
Leu59, where Leu59 is the
NH2-terminal residue for all known mouse cryptdins except Crp4 and Crp5 (5). Additional preliminary evidence showed that MMP-7
cleaved pro-Crp4 at Ala53
Leu54 (5), a site
that corresponds to processing intermediates isolated from mouse small
intestine (19). Thus, mouse pro-Crps contain conserved sites within the
precursor proregion that MMP-7 recognizes and cleaves, but their role
in pro-Crp activation is uncharacterized.
-defensin, and comparisons of pro-Crp4 cleavage with cleavage of the
pro-Crp chimera pro-CC allowed the generality of MMP-7 site usage to be
assessed. The products of in vitro pro-Crp cleavage by MMP-7
and the effects of eliminating the proregion processing sites by
site-directed mutagenesis have been determined. The results show that
cryptdin activation by cleavage of the peptide bond at the peptide
NH2 terminus is independent of proteolysis within the
prosegment; however, cleavage upstream at
Ser43
Val44 is dependent on cleavage
downstream at the Ser58
Leu59 or
Ala/Ser53
Leu54 sites. Also, C57BL/6 mice
accumulate Crp4-related processing intermediates that result from
loss-of-function mutations at MMP-7 processing sites.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside under kanamycin
selection. Bacterial cells were harvested by centrifugation and stored
at
20 °C. Cells were lysed in 6 M guanidine HCl, 100 mM Tris-Cl (pH 8), and clarified by centrifugation in a
Sorvall SA-600 rotor at 30,000 × g for 30 min at
4 °C. Fusion proteins were purified immediately after lysate clarification.
Ile44 MMP-7 cleavage site, the
following mutant constructs were prepared: (I44D)-pro-Crp4 using
forward primer pc4I44Dfor (5'-CAGGCTGTGTCTGACTCCTTTGGAGGC) and reverse
primer pc4I44Drev (5'-GCCTCCAAAGGAGTCAGACACAGCCTG); (I44S)-pro-Crp4
using forward primer pc4I44Sfor (5'-CAGGCTGTGTCTTCCTCCTTTGGAGGC) and
reverse primer pc4I44Srev (GCCTCCAAAGGAGGAAGACACAGCCTG);
(V42D-S43E-I44D-S45E-F46D)-pro-Crp4 ((DEDED)-pro-Crp4) using forward
primer petPC4DEDEDfor (5'-GAGGACCAGGCTGACGAAGACGAAGACGGAGGCCAAGAA) and
reverse primer petPC4D EDEDrev
(5'-TTCTTGGCCTCCGTCTTCGTCTTCGTCAGCCTGGTC CTC). To eliminate the
MMP-7 cleavage site at Ala53-Val54, the
following mutant pro-Crp4 constructs were prepared: (L54D)-pro-Crp4, using forward primer pc4L54Dfor (5'-GAAGGGTCTGCTGA CCATGAAAAATCT) and reverse primer pc4L54Drev (5'-AGATTTTTCATGGTCAGCAGACCCTTC); (L54S)-pro-Crp4, using forward primer pc4L54Sfor
(5'-GAAGGGTCTGCTTCCCATGAAAAATCT) and reverse primer pc4L54Srev
(AGATTTTTCATGGGAAGCAGACCCTTC). To ablate both the
Ser43-Ile44 and
Ala53-Leu54 MMP-7 cleavage sites,
(DEDED)-pro-Crp4 was used as template for mutagenesis of the
Ala53-Leu54 site using the (L54D)-pro-Crp4
primers pc4L54Dfor and pc4L54Drev described above to produce
(DEDED/L54D)-pro-Crp4.
20 °C. Pellets were
thawed and lysed in 6 M guanidine HCl, 100 mM
sodium phosphate (pH 8), and 10 mM Tris-HCl (pH 8). Lysates were passed several times through an 18-gauge needle and centrifuged at
4 °C in a Sorvall SS-34 rotor at 12,000 × g.
Supernatants were incubated batchwise at room temperature with Ni-NTA
resin (Qiagen) for 1-3 h with mixing. Bound His-tagged precursor was
eluted with 8 M urea, 100 mM sodium phosphate
(pH 4.5), 10 mM Tris-HCl (pH 4.5), and 1% Triton X-100 in
0.5-1.0-ml fractions. Peak fractions were pooled and dialyzed against
50 mM NaCl, 20 mM Tris-HCl (pH 7.5). Protein
concentration and purity were assessed by reducing Tris-Tricine
SDS-PAGE and GelCode Blue (Pierce) staining. If further purification
was required, proteins were concentrated to 0.6 ml using Centricon YM-3
centrifugal filter devices and subjected to gel filtration
chromatography on a Superdex 75 HR/10/30 column connected to an
ÄKTAFPLC system (Amersham Pharmacia Biotech).
Val44 and
Ser53
Leu54 MMP-7 cleavage sites in pro-CC, a
two-step process was used. First, the forward primer CRPBam-F was
paired with the mutant reverse primer CRPL54SBglII-R
(5'-AGATCTCTCAACGATTCCTCTTGACTAGAAG AGCC-3'), where the
BglII restriction is underlined. The PCR product was
subcloned and transferred to pGEM7-pro-CC as described above. In
the second step, this new construct, pGEM7-(L54S)-pro-CC, was used as a
template for the mutant forward primer CRPV44SbbsI-F (5'-GAAGACGACCAGGCTGTGTCTTCCTCTTTTGGAGAC-3'), where the
BbsI restriction site is underlined, and a normal downstream
primer containing a PstI restriction site (underlined),
CRPPstI-R (5'-CTGCAGGTCCCATTTATGTGT-3'). The 130-bp
product was cloned into pCR2.1-TOPO; the
BbsI-PstI fragment from this construct was used
to replace the BbsI-PstI fragment in
pGEM7-(L54S)-pro-CC. Full-length cDNA from the resulting plasmid,
pGEM7-(V44SL54S)-pro-CC, was subcloned into the BamHI and
EcoRI sites of pVL1393. All mutations were verified by DNA sequencing prior to expression.
-defensin that has a L59S
mutation was purified from C57BL/6 mice, and the corresponding
precursor to that Crp4(B6a) variant was isolated from MMP-7 null mouse
small intestine by extraction with 30% acetic acid as described (5, 18). Protein samples were applied to analytical C-18 RP-HPLC columns
(Vydac 218TP54) in aqueous 0.1% trifluoroacetic acid and eluted at
~35 min using a 10-45% acetonitrile gradient developed over 55 min.
Protein fractions containing apparent pro-Crp4 were analyzed by acid
urea-(AU)-PAGE as described (18, 21), and their identities were deduced
from a combination of NH2-terminal peptide sequencing,
MALDI-TOF-MS, and comparisons with the corresponding cDNA sequence
in RIKEN (accession number AK008107). Purification of the Crp4 mutant
and corresponding pro-Crp was completed subsequently by C-18 reverse
phase-HPLC using a 120-min, 20-40% acetonitrile gradient, from which
cryptdin precursors eluted between 23 and 30% acetonitrile. Peptide
concentrations were determined using bicinchoninic acid assay (Pierce,
Rockford, IL).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-defensins, was the only NH2
terminus detected. Also, cleavage of recombinant pro-Crp4 with MMP-7
in vitro produced a single evident product that migrated
only slightly slower than Crp4 in AU-PAGE gels and was comparable with
Crp4 in bactericidal activity (Fig. 1).
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Fig. 1.
Recognition and cleavage of mouse pro-Crp4 by
MMP-7. A 1-µg sample of recombinant pro-Crp4 ("Experimental
Procedures") was incubated overnight without (left lane)
or with (right lane) 2 µg of MMP-7. Proteins in the
samples were resolved by AU-PAGE and stained with Coomassie Blue (21).
Electrophoretic mobilities of individual components are noted at the
right.
-defensin precursor substrates
isolated from MMP-7 null mouse small intestine were cleaved in
vitro with MMP-7, identifying cleavage sites at
Ser43
Val44 and
Ser58
Leu59, the latter site corresponding to
the NH2 terminus of the known mouse cryptdins except for
Crp4 and Crp5 (5, 24). In addition, MALDI-TOF-MS analyses of partially
purified mouse intestinal proteins had identified an apparent pro-Crp
processing intermediate with Leu54 as its amino terminus
(19). Because Crp4 is the most potent of the mouse
-defensins, and
the Crp4 proregion differs from other pro-Crps at several positions at
or proximal to the predicted MMP-7 cleavage sites (Fig.
2), we tested whether MMP-7 processed pro-Crp4 in vitro and whether the products would correspond
to those produced by hydrolysis of pro-Crp1.
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Fig. 2.
MMP-7 cleavage sites in mouse pro-Crps.
Samples consisting of 2 µg each of natural pro-Crp6, synthetic Crp1
prosegment, recombinant pro-Crp4, and reduced and alkylated pro-Crp4
were incubated overnight with 0.5 mol eq of MMP-7, and digests were
analyzed by 5 cycles of NH2-terminal peptide sequencing.
Cleavage sites disclosed by protein sequencing are noted by
downwards arrows ( ) that interrupt the individual
sequences. Numerals below the primary structures
refer to residue positions at the beginning of detected
NH2-terminal sequences, numbered with the initiating Met
residue in prepro-Crps as residue position 1. The mature Crp4 and Crp6
peptide sequences are shown as bold underlined text.
Ile44 and
Ala53
Leu54 in the proregion and at
Ser58
Leu59 near the Crp4 peptide
NH2 terminus. When natural pro-Crp6 purified from
MMP-7-null mouse small intestine was analyzed similarly after MMP-7
digestion, the NH2 termini detected were DPIQNT ... , VSFGDP ... LQEES, and LRDLV ... , the same positions
identified in pro-Crp4 and in previous reports (19) (Fig. 2)
(5). No other MMP-7 cleaved sites were apparent in these
studies. Also, MMP-7 digested the 39-residue, synthetic Crp1 proregion
at Ser43
Val/Ile44 and
Ser/Ala53
Leu54, the same positions cleaved
in pro-Crp4 and pro-Crp6 (Fig. 2). This finding shows that the
specificity of MMP-7 cleavage of proregion sites is independent of the
COOH-terminal presence of an
-defensin. Because the
NH2-terminal amino acid of natural Crp4 is
Gly61, not Leu59, MMP-7 proteolysis is not
sufficient to catalyze complete activation of pro-Crp4 to the fully
mature Crp4 peptide. The Arg60
Gly61 cleavage
step does, however, require prior hydrolysis at
Ser58
Leu59 by MMP-7, because MMP-7 null mice
lack mature Crp4 with Leu59 or Gly61
NH2 termini (5, 18). The enzyme that removes the
Leu59-Arg60 dipeptide at
Arg60
Gly61 is unknown but may be a trypsin-
like aminodipeptidase.
) cells were combined with
0-3 µM Crp4 or pro-Crp4 that had been digested with a
0.2 mol eq of MMP-7 or incubated without enzyme ("Experimental
Procedures"). No bacterial cell killing was detected when cells were
exposed to MMP-7 alone (not shown) or to intact pro-Crp4 (Fig.
3). Conversely, quantitative Ser58
Leu59 hydrolysis near the Crp4 peptide
NH2 terminus activated pro-Crp4 to a bactericidal peptide
activity level that was equivalent to equimolar quantities of mature
Crp4 with the Gly61 NH2 terminus (Fig. 3).
Consistent with the biochemical evidence of pro-Crp4 proteolysis by
MMP-7 (Figs. 1 and 2), MMP-7 cleavage produces a functional Crp4
peptide from its inactive precursor. These findings provided rationale
for testing whether structural determinants of those cleavage events
exist in the precursor molecule.
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Fig. 3.
Activation of Crp4 bactericidal activity by
MMP-7 proteolysis. The indicated micromole quantities of
recombinant pro-Crp4 were incubated overnight with or without 0.5 mol
eq of MMP-7, and samples were combined with exponentially growing
E. coli DH5 cells in 10 mM PIPES (pH 7.4),
1% Trypticase soy broth for 1 h at 37 °C ("Experimental
Procedures"). Following exposure, bacteria were plated onto TSA
plates, incubated for 16 h at 37 °C, and surviving bacteria
were quantitated as colony forming units per ml (CFU/ml);
CFU/ml values below 1 × 103 indicate that no CFU were
detected. Symbols:
, pro-Crp4;
, pro-Crp4 after digestion with
MMP-7;
, Crp4.
Leu63,
Cys64
Tyr65, and
Phe87
Leu88, all sites within the polypeptide
backbone of the Crp4
-defensin moiety (Fig. 2). From the relative
recovery of individual NH2 termini, the
Phe87-Leu88 peptide bond in reduced and
alkylated pro-Crp4 appears to be more susceptible to MMP-7 proteolysis
than the Leu62
Leu63 and
Cys64
Tyr65 cleavage sites that were cleaved
to similar extents (data not shown). In more than 20 MMP-7 digests of
native pro-Crp4, pro-Crp4 variants, and other pro-Crps isolated without
reduction, MMP-7 cleavage events in the Crp4 peptide had not been
detected (Figs. 2, 4-6). These findings
are evidence that the Crp4 tridisulfide array protects the peptide from
MMP-7-mediated proteolysis during pro-Crp4 processing.
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Fig. 4.
MMP-7 proteolysis of pro-Crp4 variants with
site-directed mutations in MMP-7 processing sites. Panel A,
recombinant pro-Crp4 peptides were mutagenized, expressed, and purified
as described ("Experimental Procedures") and judged to be
homogeneous by detection of only one terminus by
NH2-terminal peptide sequencing (data not shown) and by
nonreducing AU-PAGE analysis. Samples containing 2 µg of protein/lane
were electrophoresed in AU-PAGE and stained with Coomassie Blue. Lanes:
1, pro-Crp4; 2, (I44D)-pro-Crp4; 3,
(I44S)-pro-Crp4; 4, (L54D)-pro-Crp4; 5,
(L54S)-pro-Crp4; 6, (DEDED)-pro-Crp4; 7,
(DEDED/L54D)-pro-Crp4; 8, Crp4. Upper arrow at
the right indicates pro-Crp4 and pro-Crp4 variants;
lower arrow points to a mature Crp4 peptide standard.
Panel B, samples consisting of 2 µg each of pro-Crp4 and
pro-Crp4 variants were incubated overnight with 0.5 mol eq of MMP-7
(Fig. 1), and digests were analyzed by 5 cycles of
NH2-terminal peptide sequencing. Cleavage sites disclosed
by protein sequencing are noted by downwards arrows ( )
that interrupt the individual sequences. Numerals above the
(I44D)-Crp4 primary structure refer to residue positions at the
beginning of each detected NH2 terminus, numbered with the
initiating Met residue in prepro-Crp4 as residue position 1. Filled, inverted triangles are positioned
above positions with site-directed mutations, and
substituted residues are denoted in bold underlined
typeface. The mature Crp4 peptide sequence is shown in bold
italic type. Asterisks above the reduced
(L54D)-pro-Crp4 sequence denote sites of proteolysis in the Crp4
peptide that are not cleaved when the Crp4 tridisulfide array is
intact.
Leu59, a series of
pro-Crp4 molecules with mutations at Ile44,
Leu54, and Leu59 were prepared as substrates
for MMP-7 (Fig. 4A, "Experimental Procedures").
Substituting residues 44 or 54 with Asp or Ser (Asp/Ser) ablated MMP-7
cleavage at those positions completely but only at the mutagenized
site, because cleavage at unaltered
Ala53
Leu54 and
Ser58
Leu59 sites proceeded normally (Fig.
4B). Curiously, although the I44D/I44S substitutions
abrogated cleavage at Ser43
Ile44, MMP-7 cut
(I44D/S)-pro-Crp4 variants at an alternative
Ser45
Phe46 site that is not cleaved in
wild-type pro-Crp4. To eliminate alternative sites for proteolysis at
or near Ser43
Ile44, (DEDED)-pro-Crp4 was
prepared ("Experimental Procedures"), and MMP-7 cleaved that
substrate only at the Ala53
Leu54 and
Ser58
Leu59 positions (Fig. 4B).
Similarly, the L54D/L54S substitution eliminated cleavage at
Ala53
Leu54, but MMP-7 hydrolyzed
(L54D/S)-pro-Crp4 normally at Ser43
Ile44 and
Ser58
Leu59 (Fig. 4B). Note that
reduced and alkylated (L54D)-pro-Crp4 also was cleaved at the same
sites within the Crp4 moiety as wild-type reduced and alkylated
pro-Crp4 (Figs. 2 and 4B).
Leu59 does not require cleavage to
occur at either MMP-7 recognition site in the proregion. Because MMP-7
processed the Ser58
Leu59 site when the
Ser43
Ile44 or
Ala53
Leu54 sites were mutagenized
individually (Fig. 4B), pro-Crp4 lacking both prosegment
cleavage sites was prepared by introducing an L54D mutation in
(DEDED)-pro-Crp4. The resulting (DEDED/L54D)-pro-Crp4 molecule was
digested quantitatively by MMP-7 at
Ser58
Leu59 (Fig. 4B). Sequencing
MMP-7 digests of (DEDED/L54D)-pro-Crp4 failed to detect new
NH2 termini that might have been produced by alternative
cleavage events associated with the mutagenesis. Thus, even though
MMP-7 cleaves the pro-Crp4 proregion with specificity, activation of
Crp4 in vitro is independent of the processing steps in
the prosegment.
-defensin peptide (Fig. 5A) (18, 24).
The molecule also contains a COOH-terminal His6 tag linked
to a PRR tripeptide sequence that was introduced immediately following
the COOH-terminal cysteines in the Crp15 sequence ("Experimental
Procedures," Fig. 5A). In previous studies, the patterns
of pro-CC and pro-Crp1 cleavage by MMP-7 were found to be similar, in
that cleavage at Ser58
Leu59 yielded mature
peptides for both precursors (18). Recombinant pro-CC molecules
containing an L59S mutation, (L59S)-pro-CC, or both L54S and L59S
mutations, (L54S/L59S)-pro-CC (Fig. 5A), were produced in
the baculovirus expression system and affinity purified from insect
cell lysates ("Experimental Procedures"). As shown by SDS-PAGE and
NH2-terminal sequencing, MMP-7 cleaved (L59S)-pro-CC at
Ser53
Leu54 but not at
Ser58
Ser59 (Fig. 5B).
Furthermore, MALDI-TOF analyses of the digests confirmed cleavage in
the proregion at Ser43
Val44 in both the
pro-CC and (L59S)-pro-CC substrates. However, when both
Leu54 and Leu59 were converted to Ser in the
(L54S/L59S)-pro-CC molecule, the Ser43
Val44
site no longer was digested by MMP-7 (Fig. 5C). The
(L54S/L59S)-pro-CC double mutant resisted cleavage by MMP-7 completely,
and no alternate sites to Ser43
Val44
proteolysis could be detected. Consistent with analysis of
MMP-7-digested (DEDED/L54D)-pro-Crp4 (Fig. 3B), V44S and
L54S mutations in the upstream sites of the pro-CC proregion did not
affect the activating cleavage step at Leu59 in pro-CC as
determined by NH2-terminal sequencing of MMP-7 digests (data not shown). Thus, MMP-7 hydrolysis of the
Ser43
Val44 site requires (a) that
cleavage events at Leu54, Leu59, or both,
release a nearly full-length prosegment from pro-CC and (b)
that those cleavage steps precede hydrolysis of the
Ser43-Val44 peptide bond.
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Fig. 5.
Cleavage of pro-CC serine mutants by
MMP-7. A, the amino acid sequence of the pro-Crp
chimera, pro-CC, is shown compared with mutants (L59S)-pro-CC and
(L54/59S)-pro-CC, which contain one and two Leu to Ser substitutions,
respectively. Numbering of the residues below the sequence
follows the same scheme as in Fig. 3. The residues
underlined in the pro-CC sequence are those that differ
between pro-CC and pro-Crp1. The arrows indicate the sites
of cleavage by MMP-7, with cleavage at the third site in pro-CC
liberating the mature peptide (as defined by NH2 termini of
small intestinal peptides isolated from outbred Swiss mice (24)). The
Ser mutations in (L59S)-pro-CC and (L54/59S)-pro-CC are shown in
bold. The arrows in (L59S)-pro-CC indicate the
sites of MMP-7 cleavage as determined in B and by protein
sequencing. B and C, samples (1 µg) of either
pro-CC and (L59S)-pro-CC (B) or (L54/59S)-pro-CC
(C) were incubated overnight with (+) or without ( ) 2 µg
of active human MMP-7, and digests were resolved by SDS-PAGE (15%
polyacrylamide) and stained with GelCode Blue. The position of bands
corresponding to MMP-7, as well as each precursor
(Procryptdin) and its major MMP-7-cleaved form
(Cryptdin), are noted at the right. The molecular
mass of protein markers is indicated in kDa to the left.
Note that the largest prosegment fragment (DPIQ ... QAVS) derived
from MMP-7 cleavage is not visible on these gels because its highly
negative charge appears to preclude staining by anionic dyes (5).
Leu59 Processing
Site in C57BL/6 Mouse Pro-Crp4 Variants--
Paneth cells in C57BL/6
mice contain a Crp4 variant peptide with an L59S mutation that
abrogates cleavage at that position by MMP-7. Comparative AU-PAGE
analyses of intestinal protein extracts showed that the abundant
apparent
-defensins of C57BL/6 mice do not co-migrate with Crps from
inbred or outbred strains of mice (Fig.
6A). To investigate the
structural basis for the differences, the distinctive C57BL/6 Crp
peptides were isolated by RP-HPLC and identified as potential Crps by
co-migration with Crp markers in AU-PAGE and by MALDI-TOF MS (Fig.
6B). Comparisons of selected peptides before and after
reduction and alkylation with iodoacetamide identified molecules with 6 Cys residues as peptides with masses that increased by 344.9 (theoretical value = 348 atomic mass units when acylated).
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Fig. 6.
Loss-of-function mutation in an MMP-7
cleavage site in C57BL/6 mice. Panel A, samples consisting
of 500 µg of total protein extracted from full-length small
intestines of several inbred strains of mice were analyzed by AU-PAGE
under standard conditions ("Experimental Procedures"). Extracts
were from the following mice: lane 1, outbred Swiss;
2, BALB/cJ; 3, C3H/HeJ; 4, FvB/N;
5, C57BL/6; 6, MMP-7-null (C57BL/6 background);
7, 2 µg of Crp3. The boxed region denotes the
cryptdin-containing portion of the gel, the large arrow at
the right indicates the position of the Crp4(B6a) peptide,
and lower arrow shows the position of Crp3. Note that the
C57BL/6 cryptdins do not co-migrate with those of the other strains.
Panel B, samples consisting of 2 µg of the four purified
peptides shown and 500 µg of total protein extracted from C57BL/6
mouse small intestine were analyzed by AU-PAGE under standard
conditions ("Experimental Procedures"). Lanes: 1,
pro-Crp4; 2, Crp4; 3, pro-Crp4(B6a);
4, Crp4(B6a), extract from C57BL/6 mouse small bowel.
Upper arrow shows the position of Crp4 and Crp4(B6a)
precursors, the large middle arrow indicates the position of
Crp4(B6a) peptide, and the lowest arrow shows the position
of Crp4. Panel C, NH2-terminal sequences
obtained for Crp4(B6a) and Crp4(B6b) peptides purified from C57BL/6
small intestine are shown aligned with their predicted precursors
deduced from their corresponding cDNAs. Filled,
inverted triangles indicate sites of loss-of-function mutagenesis
in Crp4(B6a) and apparent introduction of an alternative MMP-7
recognition site in Crp4(B6b). Peptide sequences are shown in
bold type, and peptide sequences determined by
NH2-terminal protein sequencing are shown
underlined. Panel D, samples consisting of 2 µg
each of pro-Crp4 and pro-Crp4(B6a) purified from MMP-7 null mice were
incubated overnight with 0.5 mol eq of MMP-7 (Fig. 1), and digests were
analyzed by 5 cycles of NH2-terminal peptide sequencing.
Cleavage sites disclosed by protein sequencing are noted by
downwards arrows ( ) that interrupt the individual
sequences.
-defensins in mouse
small intestine. In contrast to the abundance of Crp4(B6a), the
Crp4(B6b) peptide was recovered only at very low levels from C57BL/6
mouse small bowel, and the NH2-terminal sequence of the peptide was LSRDLI_L_RNR ... (Fig. 6B). A BLASTP search
revealed that both sequences corresponded to Crp4-related small
intestinal cDNAs in the RIKEN data base (accession numbers AK008107
and AK008266, respectively). Alignment of the peptide sequences, their
deduced pro-Crps and pro-Crp4 identified the peptides as Crp4 variants.
The Crp4(B6a) and Crp4(B6b) proregions resemble the Crp4 prosegment
more closely than other Crp prosegments (Fig. 6C), and both
peptides lack 3 amino acid residues between the fourth and fifth
cysteine residue positions. That 3-codon deletion displaces amino acids
that are present in all other
-defensins, and the deletion, found
only in Crp4, would shorten the loop formed by the
Cys3-Cys5 disulfide bond (23-25). Previously,
extensive sequencing of intestinal cDNAs and genomic clones from
outbred Swiss mice, and C3H/HeJ, BALB/cJ, and 129/SvJ inbred mouse
strains had not detected variant Crp4 coding sequences (Refs. 20 and
23-25 and data not shown).
Leu54. Furthermore, both Crp4(B6a) and
Crp4(B6b) contain a Asp61 substitution for
Gly61, indicating that other processing sites are subject
to mutation.
Val44 and at
Ala53
Leu54 (Fig. 6D). Thus, the
Crp4(B6a) peptide derives from an inactivating L59S mutation at the
Ser58
Leu59 MMP-7 cleavage site.
Leu59. In
Crp4(B6b), however, an additional S57L mutation appears to have
provided an alternative MMP-7 cleavage site at
Lys56
Leu57 that leads to the appearance of
Crp4(B6b), although at very low levels. The order in which the S58L and
L59S mutations appeared cannot be inferred from these data, but the
prospect that the S58L change may have rescued an earlier
L59S loss-of-function substitution is an attractive but speculative
notion. Whether Crp4(B6b) levels are low because MMP-7 does not cleave
pro-Crp4(B6b) efficiently at Lys56
Leu57 is
not known. Prepro-Crp4(B6b) mRNA levels are very low relative to
those of other
-defensin mRNAs (data not shown).
Leu59
Is Associated with Attenuated Bactericidal Activity--
To test
whether L59S inactivation of the
Ser58
Leu59 MMP-7 cleavage site has potential
effects on innate immunity, the bactericidal activities of
Crp4(B6a) and Crp4 were compared against four species of
bacteria. Relative to Crp4, Crp4(B6a) was less active against all
bacterial species tested (Fig. 7).
Against Staphylococcus aureus and
Listeria monocytogenes, both peptides had equivalent bacterial cell killing activities at concentrations of 5 µg/ml or
greater (Fig. 7B), but Crp4(B6a) had markedly lower activity against Gram-negative E. coli and the defensin-sensitive
S. typhimurium PhoP(
) strain (Fig. 7A).
Although these experiments show that Crp4(B6a) is less potent than
Crp4, the differences in activity cannot be attributed solely to the 5 additional amino acids at the peptide NH2 terminus. Crp4
and Crp4(B6a) are different at 7 residue positions and the composition
and length of their COOH termini also differ greatly. Nevertheless, the
findings show that mutations at MMP-7 cleavage sites exist in mouse
populations and that the mutated processing intermediates can
accumulate as abundant peptide variants.
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Fig. 7.
Relative bactericidal activities of Crp4 and
Crp4(B6a). Exponentially growing bacterial cells were exposed to
the indicated concentrations of Crp4 or Crp4(B6a) for 1 h, and
surviving bacteria were quantitated as described in the legend to Fig.
3. In both panels, open symbols denote Crp4 data points, and
filled symbols represent Crp4(B6a) data points. Symbols in
panel A: , Crp4 versus E. coli
ML35;
, Crp4(B6a) versus E. coli ML35;
,
Crp4 versus S. typhimurium PhoP(
);
, Crp4(B6a) versus S. typhimurium PhoP(
);
symbols in panel B:
, Crp4 versus L. monocytogenes 104035;
, Crp4(B6a) versus L. monocytogenes 104035;
, Crp4 versus S. aureus 710a;
, Crp4(B6a) versus S. aureus
710a.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Ile44 and
Ser53
Leu54 sites in the synthetic cryptdin-1
prosegment alone by MMP-7 occurred without the
-defensin moiety
being present, and thus the Crp peptide does not play a critical role
in making the Ser43
Val44 and
Ser53
Leu54 sites accessible to the
processing enzyme. However, the forced retention of CC in the proform
by mutagenizing both Ser53
Leu54 and
Ser58
Leu59 sites (Fig. 5) eliminates
cleavage at the Ser43
Val44 position.
Leu54 is completed. Possibly, MMP-7
catalyzed proteolysis in the proregion at
Ser43
Val44 eliminates the ability of the
proregion to inhibit Crp bactericidal activity (5) by fragmenting the
39- and 34-amino acid prosegment fragments that are released from
pro-Crps by proteolysis at Ser58
Leu59 or
Ser53
Leu54, respectively. To summarize,
proregion fragmentation depends on Crp peptide activation rather
than the converse.
-defensins predominantly
exist in the fully processed state, which is mediated by analogous
cleavage events (12, 13, 15). Over a 4-24-h period, three primary
cleavage events generate HNP-1 major intermediates of 75 and 56 amino
acids, as well as the 30-residue mature peptide, in cells of myeloid
origin (12, 13, 15). Extensive amino acid deletions from the
COOH-terminal region of the pro-HNP-1 propeptide, but not from the
NH2-terminal region of the prosegment, impaired pro-HNP-1
processing (13). Both sets of observations are consistent with MMP-7
processing of pro-Crps in that mutations at the COOH terminus affect
cleavage upstream. Our results suggest that the Crp prosegment is
removed nearly intact then proteolyzed subsequently, whereas pro-HNPs
are sequentially truncated at the NH2 terminus;
nevertheless, the end result is the same. Although the enzymes that
mediate pro-
-defensin processing differ depending on the system, the
overall scheme of
-defensin processing is similar in myeloid and
epithelial cells. The anionic prosegments of both cryptdins and HNPs
inhibit
-defensin bactericidal activity in vitro (5, 15),
and thus may confer cytoprotection during granulogenesis until
processing is complete (14).
-defensin processing
provide interesting contrasts and comparisons with the biology of
pro-Crp activation in the mouse. Mouse Paneth cells secrete mature,
3.5-kDa cryptdins as components of secretory granules, because
MMP-7-mediated pro-Crp processing takes place intracellularly before
secretion (5). On the other hand, human Paneth cells release the
-defensin HD5 precursor, pro-HD5-(20-94), into the small
intestinal lumen (11), and pro-HD5(20-94) is processed rapidly after
secretion by anionic and meso isoforms of trypsin and not by MMP-7,
which human Paneth cells lack (10,
11).2 A trypsin cleavage site
in pro-HD5-(20-94) at Arg62
Ala63 gives rise
to the major form of the mature peptide found in washes of the small
intestinal lumen (11). However, other processing intermediates,
resulting from hydrolysis within the prosegment, also have been
identified, suggesting that alternative sites may be used. For example,
an HD5 peptide with the NH2 terminus Gly37 has
been isolated from supernatants of human small intestinal crypts
stimulated to release Paneth cell granules with carbamyl choline (10).
The extensive differences between the processing of
-defensins by
mice and humans suggest that it is the capacity for releasing mature,
microbicidal
-defensins that is conserved, and that Paneth cells
from mice and humans evolved differing mechanisms to ensure the
delivery of functional peptides into the lumen.
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ACKNOWLEDGEMENTS |
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We thank Thomas Broekelmann of Washington University for expert peptide sequencing and analysis of pro-CC. Khoa Nguyen, Vungie Hoang, Victoria Rojo, and Amy Schmidt provided excellent technical assistance.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants DK10184 (to D. P. S), DE14040 (to C. L. W.), and DK44632 (to A. J. O.).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.
§ Current address: The Dow Chemical Company, Biocides R&D, Buffalo Grove, IL 60089.
** To whom correspondence may be addressed: Division of Allergy and Pulmonary Medicine, Dept. of Pediatrics, Washington University School of Medicine, St. Louis, MO 63110. Tel.: 314-286-2861; Fax: 314-286-2894; E-mail: wilson_c@pcfnotes1.wustl.edu.
§§ To whom correspondence may be addressed: Dept. of Pathology, College of Medicine, University of California, Irvine, CA 92697-4800. Tel.: 949-824-4647; Fax: 949-824-1098; E-mail: aouellet@UCI.EDU.
Published, JBC Papers in Press, December 13, 2002, DOI 10.1074/jbc.M210600200
2 C. L. Wilson and C. L. Bevins, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: MMP-7, matrix metalloproteinase-7; Crp, cryptdin; pro-Crp, procryptdin; MALDI-TOF MS, matrix-assisted laser desorption ionization-time of flight mass spectrometry; RP-HPLC, reverse-phase high performance liquid chromatography; AU-PAGE, acid urea-polyacrylamide gel electrophoresis; Ni-NTA, nickel-nitrilotriacetic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PIPES, 1,4-piperazinediethanesulfonic acid; CFU, colony forming units; pro-CC, chimeric pro-Crp.
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