(Received for publication, August 4, 1995; and in revised form, September 25, 1995)
From the
Profilaggrin, an insoluble precursor of the intermediate
filament-associated protein filaggrin, contains multiple internal
repeats (PIRs). At terminal differentiation of epidermis, proteolytic
processing within a ``linker'' region of each PIR releases
soluble filaggrin in a two-stage process. The first stage
endoproteinase (PEP1, profilaggrin endoproteinase 1) cleaves mouse
profilaggrin at a subset of the linkers, yielding processing
intermediates consisting of several filaggrin repeats. An epidermal
endoproteinase that cleaves the requisite linker subset has been
purified 4,966-fold from mouse epidermal extracts. SDS-polyacrylamide
gel electrophoresis demonstrated a band of molecular mass of 29.5 kDa
that correlated with the activity. Labeling with
[H]diisopropylfluorophosphate identified PEP1 as
a serine protease; inhibitor studies suggest that it is similar to
chymotrypsin, as expected from previous in vivo studies. The
purified PEP1 cleaved a peptide derived from profilaggrin (P1) at three
residues within and adjacent to a multiple tyrosine sequence,
consistent with the in vivo processing sites. No exopeptidase
activity was detected. PEP1 is only active toward insoluble
profilaggrin, resulting in partial solubilization, consistent with a
role in dispersal of profilaggrin during terminal differentiation. In
contrast to the specific cleavage of mouse profilaggrin, PEP1 cleaved
all linker regions of rat profilaggrin. Studies with phosphorylated P1
suggest that PEP1 specificity may be partly regulated by profilaggrin
phosphorylation.
Aggregation of keratin filaments into a tight, orderly array is
a prominent feature of terminal differentiation of mammalian cornified
epithelia, such as epidermis. Evidence is accumulating that keratin
structure is intimately involved in tissue architecture and tensile
strength (reviewed in (1) ). The reorganization of keratin
filaments at terminal differentiation involves filaggrin, a protein
that is synthesized as a large, highly phosphorylated precursor,
profilaggrin (reviewed in (2) ). Profilaggrin contains multiple
internal repeats (profilaggrin internal repeat (PIR)()) that
are almost identical with one another in rodent, although they vary
widely in sequence in human profilaggrin. PIRs vary in size (26 kDa in
mouse, 42 kDa in rat) and sequence (30-42% identity between mouse
and human) between different species, as well as in number, even
between strains (typically 10 to 30 PIRs are
present)(3, 4, 5, 6, 7, 8, 9, 10) .
The size of the PIR is similar to that of filaggrin, and it is often
assumed that each PIR represents a filaggrin to be released; however,
the boundaries of the PIRs do not correspond to the termini of
filaggrin. The offset between the PIR and filaggrin may represent an
evolutionary holdover (5) or may play some unknown functional
role. Profilaggrin contains a large Ca
-binding
amino-terminal domain (7, 8) and may also contain a
short atypical sequence at the carboxyl
terminus(5, 6, 9, 10) . Synthesis of
profilaggrin involves phosphorylation by several kinases at up to 21
sites in each PIR(11) . Mature profilaggrin is insoluble and
stored in non-membrane-bound cytosolic keratohyalin granules. At
terminal differentiation, the granule disperses; at the same time,
profilaggrin undergoes dephosphorylation by phosphatase 2A (PP2A) and
at least one other phosphatase (12) and proteolytic processing
by two endoproteinases and at least two exopeptidases (13, 14, 15) to release the soluble
filaggrin. The regulation of granule dispersal and the interaction of
the processing products with keratins are not understood, although it
seems likely that release is tightly regulated in order to prevent
premature collapse of the keratin network.
Human, rat, and mouse
PIRs show limited areas of sequence identity that are most extensive
around the site of proteolytic processing(5, 10) .
Proteolytic processing occurs within a region referred to as the linker
region and involves an initial cleavage at a hydrophobic site within
this linker region, followed by carboxypeptidase and aminopeptidase
cleavages at the new termini to produce the final amino and carboxyl
termini of filaggrin(13, 15) . The actual amount of
sequence removed during release of filaggrin varies in different
situations, so that a specific linker sequence cannot be defined.
Processing occurs in two independently regulated stages. Little is
known about the regulation of the first stage, although the second
stage is regulated by Ca influx through
nifedipine-sensitive channels (14) . The first stage in mouse
epidermis or cultured rat keratinocytes produces dephosphorylated
intermediates consisting of several copies of filaggrin; for example,
2DI (two domain intermediate) refers to an intermediate consisting of
two filaggrin domains joined by a linker segment(16) . In mouse
profilaggrin, there are two different primary sequences at the linker
regions; these two variants differ by the presence of an FYPVY insert (4, 13) and are referred to as
and
. The first stage of processing involves cleavage of
only the
linker regions. In rat profilaggrin, there
are no differences in the primary sequence of the PIRs(15) ,
requiring some other explanation to account for the two-stage
processing observed in cultured rat keratinocytes(14) .
Inhibitor studies with cultured rat keratinocytes indicate that the first stage endoproteinase (PEP1 = profilaggrin endoproteinase 1) is chymotryptic-like and that the second endoproteinase is a leupeptin-sensitive enzyme, most likely calpain(14) . Earlier studies identified an endoproteinase activity in epidermal extracts which cleaved mouse profilaggrin at the linkers(13) . This endoproteinase activity was inhibited by chymostatin, which also inhibited in vivo processing, indicating that this endoproteinase was indeed PEP1. We now report purification of this enzyme from mouse epidermal extracts and demonstrate that it has the properties expected for PEP1.
Profilaggrin was purified as described
previously(11, 15) . PEP1 activity was detected with
an SDS-PAGE assay whereby profilaggrin was deposited as a thin film. To
test for evenness of the film on the surface (rather than formation of
a firm pellet at the bottom of the tube), the film was stained with
Coomassie Blue dye in water; after destaining in water, the evenness of
the protein deposit could be judged by visual examination. Assays for
protease activity were carried out in 0.5-ml Eppendorf tubes by
precipitation of 5 µl of profilaggrin (stock solution in 9 M urea, 50 mM Tris, pH 8) with 95 µl of 5 mM ZnCl at 0 °C for 30 min, followed by
centrifugation in an Eppendorf centrifuge for 5 min at 4 °C. After
the supernatant was removed, 100 µl of reaction mixture (2 mM dithiothreitol, 0.01 mg/ml pancreatic trypsin inhibitor (Trasylol,
Sigma), 50 mM Tris, pH 8.0, and various column fractions or
buffer alone) was added. After a timed incubation at 38 °C,
reactions were stopped by adding 33 µl of hot 4
Laemmli
sample buffer and boiling for 5 min. Half of each sample was analyzed
on SDS-PAGE gradient gels as described previously(13) ,
staining with Coomassie Blue. Products were quantified by densitometric
scanning of the stained gel. Reaction rates were then determined by
fitting the data to a straight-line equation using Enzfitter software
(excluding the zero time point). For analysis of solubility of the
products, the assay tubes were respun after the timed incubation, and
the supernatants were removed. Supernatants and pellets were analyzed
separately.
PEP1 was purified from an epidermal extract, prepared as
described previously(13) . The extract was chromatographed in
DE52 in 2 mM dithiothreitol, 0.1 mM EDTA, 50 mM Tris, pH 8.0, developed with a gradient 0-0.4 M NaCl, followed by gel filtration on Sephacryl S-300 (1.0
100 cm) in 2 mM dithiothreitol, 0.1 mM EDTA, 100
mM Tris, pH 8.0. After diluting with equal volumes of 2 mM dithiothreitol, 0.1 mM EDTA, the active fractions were
applied to phenyl-agarose (Sigma P9533) in the same buffer, using a
gradient up to 1 M NaCl to elute the bound proteins.
For
kinetic studies with phosphorylation isoforms of P1, the
unphosphorylated, monophosphorylated, and diphosphorylated P1 peptides
were purified by HPLC on Synchropak C18 (4.6 mm 240 cm) in
trifluoroacetic acid/acetonitrile as described previously(15) .
Digestion of the profilaggrin peptide P1 by PEP1 was carried out in the
same assay buffer as for digestion of profilaggrin; the reaction
mixtures were analyzed by Synchropak C18 HPLC. Fractions were collected
during the HPLC analyses, lyophilized, and resuspended in 0.1% formic
acid, 50% MeOH for mass analysis and sequencing by tandem mass
spectrometry as described previously(15) . On-line mass
spectrometric analysis of sites of proteolysis of profilaggrin was
accomplished by treatment of profilaggrin with PEP I as usual, except
that the pancreatic trypsin inhibitor was left out of the reaction
mixture; the PEP I was then inhibited with TPCK, followed by digestion
of the products with 2% TPCK-treated trypsin. The resulting tryptic
peptides were then analyzed by HPLC with the HPLC column directly
coupled to a mass spectrometer, as described
previously(11, 15) .
The effect of inhibitors was determined by preincubating the PEP1 with each inhibitor for 20 min at room temperature before assaying. Gas-phase sequence analysis was accomplished by transferring the protein to PVDF, followed by gas phase microsequencing directly from excised Coomassie-stained bands(18) .
Figure 1: Assay of PEP1 activity on Swiss-Webster mouse profilaggrin, comparing the effect of adding PP2A along with the PEP1. Reaction mixtures were analyzed by SDS-PAGE as described under ``Materials and Methods.'' The times of incubation are indicated above each lane. Activity of PEP1 produces specific cleavage of the linker regions, yielding processing products that have masses that correspond to one, two, three, etc. filaggrin units, as indicated at the left (the filaggrin unit had an apparent mass during SDS-PAGE of 30 kDa for products derived from mouse profilaggrin). The profilaggrin in this case did not behave well on SDS-PAGE, producing a smear rather than a discrete band. The specific enzymes added are indicated at the top. The arrowheads at the left indicate the number of filaggrin units in each proteolytic product. Molecular mass markers are indicated on the right (in kDa).
The linker region of rodent profilaggrin
contains several adjacent tyrosines that might be cleaved
nonspecifically by a number of known proteinase contaminants of
epidermal extracts. To determine the amount of nonspecific cleavage,
the differential cleavage of and
linker regions by PEP1 was exploited. Balb/c mouse profilaggrin
has a distribution of
linkers which should not
produce filaggrin-sized (30-kDa) PEP1 products. This allowed us to
distinguish PEP1 from contaminating endoproteinases that would produce filaggrin-sized fragments (see detailed discussion below).
A comparison of the results with the two profilaggrin substrates is
shown in Fig. 1(using Swiss-Webster profilaggrin) and Fig. 2(using Balb/c profilaggrin). Swiss-Webster profilaggrin
was much larger than Balb/c profilaggrin (we estimate that there are
more than 30 PIRs in Swiss-Webster profilaggrin, and 13 or 14 in Balb/c
profilaggrin), leading to difficulty in resolubilizing the
Swiss-Webster profilaggrin after precipitation. To test for recovery of
the proteolytic products, profilaggrin which had not been precipitated
was analyzed on the same gel. Quantitation of the stained bands by
densitometric scanning showed mass recoveries of 85-103% (5
trials), when comparing a nearly complete digest (such as that produced
at 60 min in Fig. 1) with the intact, unprecipitated
profilaggrin.
Figure 2:
Activity of PEP1 from the phenyl agarose
activity peak (Fig. 3B) toward Balb/c mouse
profilaggrin. Assays were carried out as described in Fig. 1.
The time of incubation in minutes is shown above each lane. The numbers at the right indicate the number of PIRs in each
product (the unfilled arrows indicate missing components that
would be expected if there was random cleavage of the linkers; these
sizes were determined by chymotryptic cleavage of the Swiss-Webster
profilaggrin). The masses of the products labeled 2 and 3 agree with
those predicted for 2 or 3 filaggrin units. However, no product is
observed for the mass predicted for the 4 or 5 filaggrin units.
Instead, a product that corresponds to an abnormally large 5 is
observed. In the higher mass range, only a few of the possible products
are seen, suggesting an ordered disassembly of this profilaggrin. The 5b and 6b products from the Balb/c profilaggrin are
not multiples of the PIRs; they probably reflect the presence of the
amino and carboxyl termini of profilaggrin on those products. Note that
at early times there appears to be specific cleavage of only a few of
the linker sites, suggesting that PEP1 is discriminating among the
linkers.
Figure 3: Purification of PEP1 from rat epidermis. Silver-stained gels of column fractions from DE52 chromatography (A) and phenyl-agarose (B) chromatography. Chromatography conditions are given under ``Materials and Methods.'' The activity toward Swiss-Webster profilaggrin and Balb/c profilaggrin is indicated below the fraction (only those having significant activity are shown). The bands at 50-65 kDa in B are contaminants of the buffers.
Once a relatively pure PEP1 was available, kinetic studies were undertaken to find a way to quantify activity. The kinetic complexity for production of the smaller intermediates (which would require at least two proteolytic events for their release) precluded use of normal enzyme units; however, densitometry indicated that the product containing two filaggrin units (2DI) gave the most reproducible recovery in assays conducted on several days using both Swiss-Webster and Balb/c profilaggrin. When the amount of 2DI was determined by densitometry of the SDS-PAGE gel, a short lag in the production of the smaller products (consisting of one, two, or three filaggrin domains) was revealed, as was expected because two cleavage events are required. To quantify activity, the enzyme solution was diluted so that the rates of 2DI release after 5 min was linear with enzyme concentration, although normal Michaelis-Menten kinetics were not obtained when varying the amount of profilaggrin in the precipitate. This assay was then used to quantify the activity of the protease for estimation of yield during the enzyme purification (Table 1).
The usefulness of the two profilaggrin substrates in evaluating the presence of a contaminant is demonstrated in the DE52 chromatography. Below each lane of the DE52 profile, the optical density of several products is compared. Examination of 2DI produced from either Swiss/Webster or Balb/c profilaggrin indicated the presence of a broad activity peak 10 fractions wide. However, only the first half of this broad peak yielded a 30-kDa product from Balb/c profilaggrin. These data indicated that a contaminating proteinase eluted just before PEP1, but could not be resolved completely until the phenyl-agarose step; the contaminating proteinase did not bind to this resin. Inhibitor sensitivity of the contaminating proteinase indicated it was similar to, if not identical with, mast cell chymase; this identity was supported by amino-terminal microsequencing after electroblotting to polyvinylidene difluoride(17) .
In the most highly purified PEP1 fractions, a 29.5-kDa band could be correlated with activity (Fig. 3B). Two 18-19-kDa contaminants were present in amounts that varied in different preparations. The elution of PEP1 from Sepharose S-300 indicated a mass of about 30 to 35 kDa (assuming a globular configuration), suggesting that the 29.5-kDa PEP1 did not have associated subunits. Besides the small contaminants, some minor bands could be detected after longer silver stain development, so that this PEP1 preparation appeared to be about 50% pure. Comparison of the 29.5-kDa band with the silver-stained standards indicated that approximately 1.5 µg of PEP1 was obtained from the epidermis of 40 newborn mice. Several attempts at further purification resulted in complete loss of the protein, which was not surprising in view of the small amount present. An attempt to microsequence PEP1 after electroeluting to polyvinylidene difluoride did not give any signal above background; this may reflect a blocked amino terminus, although it is more likely that there was simply too little material for sequencing.
Conditions that solubilize profilaggrin, such as high salt(13) , also inhibited PEP1. The pH profile of PEP1 showed activities that were fairly constant from pH 6.5 to 8.5, with a rapid decrease in activity below pH 6.5 and complete inhibition by pH 5.0. A small decrease in activity (about 20%) was seen at pH 9.0. This pH profile is consistent with identification of PEP1 as a cytoplasmic enzyme.
Figure 4:
Sequence of proteolytic products produced
by action of PEP1 on the a linker mouse profilaggrin (A) or peptide P1 from rat profilaggrin (B). Each panel includes the sequence of the linker region, where each
amino acid is represented by the one-letter code; * indicates
a phosphorylated residue. The partial sequences below the main sequence
in A are the amino-terminal sequences of the intact
two-filaggrin unit product and three-filaggrin unit product, as
determined by gas-phase microsequencing after transfer to
polyvinylidene difluoride. The sequences in B were determined
by MS/MS analysis of HPLC-resolved peptide subfragments produced after
incubation of peptide P1 with PEP1. Both the amino and carboxyl termini
of the products could be identified, providing clearer evidence of
processing events than that provided by the amino-terminal
sequencing.
In the second experiment, a peptide containing the proteolytic processing sites of rat profilaggrin (P1, see (15) ) was used as a substrate for PEP1. The products were resolved by reverse-phase HPLC, their masses were determined by mass spectrometry (MS), and their sequence was determined by tandem mass spectroscopy (MS/MS) as described previously(11, 15) . Five products were recovered from the reverse-phase HPLC chromatography; their observed masses were 3089.8, 2763.6, 1165.6, 1328.5, 1491.5 Da/e. Cleavage of P1 at three sites as shown in Fig. 4would give predicted masses of 3090.1, 2763.8, 1165.5, 1328.6, 1491.7, using monoisotopic masses below 1500 Da/e and average masses above 1500 Da/e. The MS/MS spectra resembled those previously reported for some of the amino termini of filaggrin (15) and confirmed the cleavage site. In two cases, both of the products of the cleavage were observed; in the third case, only one product was observed (this was a low yield product, and the other subfragment was probably lost in handling). The cleavage of rat P1 peptide occurred at sites similar to those observed with mouse profilaggrin; however, it was now clear that some cleavage occurred after the YYY as well (Fig. 4B). An adjacent valine was not cleaved, nor was the only other tyrosine in the PIR, which is 35 residues away from the multiple tyrosine site (both of these sites were cleaved by the contaminating proteinase). It thus appears that PEP1 can cleave at any tyrosine in the linker peptide.
Analysis of filaggrin amino and carboxyl termini produced in vivo had previously revealed that processing involved exopeptidase action after an initial endoproteinase cleavage(15) . In order to eliminate the possibility that PEP1 had exopeptidase activity, a time course of the reaction was examined for recovery of the various products (which were separated on reverse phase-HPLC). Product peak heights increased linearly with no lag until the P1 peptide was completely digested. There was no evidence for conversion of the larger fragments to smaller fragments, even up to 20 min after the complete digestion of the P1 peptide and after addition of a second aliquot of PEP1 (not shown). Because an exopeptidase should convert larger fragments to smaller, these data indicate that PEP1 did not have exopeptidase activity.
Because the results with the P1 peptide indicated that PEP1 activity
might be modulated by the presence of phosphate in the linker region,
activity of PEP1 on profilaggrin treated with phosphatase 2A (PP2A) was
examined, which is capable of nearly complete removal of both
phosphates on P1. ()Both mouse and rat profilaggrin were
examined. Addition of PP2A and PEP1 together had no effect on
proteolytic processing of mouse profilaggrin, compared to PEP1
alone (Fig. 1). To ensure that the phosphatase was active under
these conditions, parallel experiments with
P-labeled
mouse profilaggrin showed that 45% of the radiolabel was removed by the
end of the experiment. In fact, when PEP1 plus PP2A was compared with
PP2A alone, there was a 2-fold enhancement of release of radiolabel
from mouse profilaggrin (not shown). These data indicate that PEP1
processing of mouse profilaggrin is directed exclusively by the primary
sequence, although variable phosphorylation in the PIRs may explain the
ability of PEP1 to discriminate among the apparently identical
linkers observed with Balb/c profilaggrin (Fig. 2).
On the other hand, PP2A added with PEP1 markedly enhanced the rate of proteolytic cleavage of rat profilaggrin, compared to PEP1 alone (particularly obvious at 7 and 15 min in Fig. 5), although the extent of proteolysis at longer times was the same in both cases. In this case, the phosphate content of the peptides derived from rat profilaggrin was determined by directly coupling the HPLC eluate to a mass spectrometer(17) , rather than radiolabeling. No significant difference in the phosphate content of the profilaggrin peptides was detected when comparing PP2A plus PEP1 to PP2A alone (not shown). However, PP2A efficiently removed all phosphate from the P1 peptide derived from rat profilaggrin under these conditions.
Figure 5: Effect of PP2A on PEP1 processing of rat profilaggrin. The wells marked + have PP2A added, while those marked - have only PEP1. The numbers above each pair of lanes indicate the time of incubation. This profilaggrin is particularly hard to resolubilize (see ``Results''). However, the proteolytic products clearly show that the presence of PP2A markedly enhances profilaggrin processing by PEP1. Furthermore, PP2A affects the mobility of the products as well. Parallel experiments indicated that the maximum extent of dephosphorylation was no more than 45% at the longest time point. Molecular mass markers are at the left and arrows at the right indicate the number of filaggrin units (rat filaggrin unit is 42 kDa).
The difference in behavior of rat and mouse profilaggrin
toward PP2A and PEP1 may be due to extended structural features of the
substrate. A well characterized example of this is found in peptide
hormone processing, which occurs at dibasic sites situated in, or next
to, -turns(18) . Although structure prediction programs
show that profilaggrin has little
structure(5, 9, 10) , there is predicted
structure in the linker region, with a
structure just before and
an
helix following (the residues predicted to have these
structures are all included within the sequence of the P1 peptide). The
two phosphorylation sites of P1 occur four residues apart at one end of
the predicted
helix. It seems plausible that in rat profilaggrin,
where the
structure is not as hydrophobic, perturbation of the
helix by these adjacent phosphates would have more of an effect
than in mouse profilaggrin. Indeed, dephosphorylation in rat
profilaggrin has a more profound effect on rat profilaggrin structure
than on mouse profilaggrin structure, as reflected in the altered
mobility of the PEP1 rat profilaggrin products on SDS-PAGE (Fig. 5), while no alteration in mobility was observed with
mouse profilaggrin PEP1 products (Fig. 1).
However, regulation of proteolysis by phosphorylation at the P1 sites does not explain adequately the complete absence of filaggrin-sized products in first-stage processing in cultured rat keratinocytes. It is tempting to speculate that more extensive structural features may be relevant to the specific proteolysis of a subset of the linker regions in rat profilaggrin, possibly induced by association with keratin, while mouse profilaggrin cleavage is nearly completely determined by the primary structure adjacent to the proteolysis sites.
Figure 6: Solubilization of smaller profilaggrin processing products. PEP1 was incubated with Swiss-Webster profilaggrin for the times indicated at the top of each pair of lanes. The enzyme reaction mixtures were removed, and the reaction was stopped by boiling in Laemmli sample buffer. The protein remaining bound to the tube was solubilized with boiling Laemmli sample buffer. Both fractions were analyzed by SDS-PAGE. P indicates the material remaining in the tube, while S indicates the proteins solubilized into the reaction mixture. The molecular mass markers are on the right, and the number of filaggrin units in each band is indicated on the left.