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(Received for publication, June 29, 1995; and in revised form, August
21, 1995) From the
Loricrin is the major protein of the cornified cell envelope of
terminally differentiated epidermal keratinocytes which functions as a
physical barrier. In order to understand its properties and role in
cornified cell envelope, we have expressed human loricrin from a
full-length cDNA clone in bacteria and purified it to homogeneity. We
have also isolated loricrin from newborn mouse epidermis. By circular
dichroism and fluorescence spectroscopy, the in vivo mouse and
bacterially expressed human loricrins possess no
Terminal differentiation in the epidermis involves the
expression of a number of specific proteins that ultimately fulfill
different structural roles in the cornified, dead stratum corneum cell.
One set of proteins is the keratin intermediate filaments and the
interfilamentous matrix protein
filaggrin(1, 2, 3) . A second set of proteins
is used to construct the cornified cell envelope (CE), ( In particular, a variety of
data have suggested that loricrin comprises about 75% of the total CE
protein mass (reviewed in (9) ), or 85-95% of the
cytoplasmic two-thirds of the CE. In fact, amino acid sequencing of
many peptides recovered by the proteolysis has now provided rigorous
support for this idea(7) . About 90% of the molar mass of
peptides from the cytoplasmic two-thirds of the CE consisted of
loricrin-loricrin cross-links, as well as smaller amounts of SPR1 and
SPR2 proteins, which appear to serve as cross-bridging proteins among
the loricrin. Thus loricrin appears to function as a major
reinforcement protein for the CE on the cytoplasmic face of the
structure. Presumably, loricrin admixed with the SPRs, is deposited
over a scaffold of elafin, cystatin Yet only anecdotal data are available on the structure of loricrins
and how they may be cross-linked by TGases. Loricrins are unusual in
their high contents of glycine, usually configured as tandem inexact
peptide repeats(5, 10, 11, 12) ,
that are predicted to have little organized structure(6) .
Based on their unusual flexibility properties, however, we have
proposed that these sequences adopt a novel glycine loop motif (13) . Whatever their structure, these sequences are flanked by
lysine- and glutamine-rich terminal sequences and interrupted by
glutamine-rich domains that recent sequencing analyses of CE peptides
showed are involved in isodipeptide
cross-linking(6, 7) . Of these, the terminal lysine
(Lys To date, direct biochemical and biophysical
experiments on isolated native loricrins to explore their structure and
these hypotheses have not been reported. An early study (20) described the isolation of granules from newborn rat
epidermis that are likely to be the loricrin-containing L granules of
that tissue(21) , but no further data have been reported. One
major problem is that loricrins are thought to be cross-linked into
large oligomers immediately after synthesis(14) . Accordingly,
in this paper, we have expressed full-length human loricrin in bacteria
and used it to study its structural and biochemical properties. We show
here that while it can function as a complete substrate in vitro for the three TGases known to be active in the epidermis, the
enzymes function differently, which is likely to have profound
implications for the assembly of the CE.
Loricrin purification was monitored on 8-16%
or 4-12% SDS-polyacrylamide gradient gels (Novex) and by Western
blotting using an established rabbit anti-human loricrin
antibody(14) , followed by staining with the horseradish
peroxidase method (Bio-Rad). Loricrin was also easily monitored by
autoradiography of
For in vitro cross-linking studies using the
recombinant human loricrin as a complete TGase substrate, the purified
unlabeled or
Figure 1:
Purification of recombinant human and in vivo mouse loricrins. The loricrin-enriched supernatants
were chromatographed on a mono-S column, from which pure loricrin (arrow) was eluted by 0.2 M NaCl. Inset, SDS
gels of purified loricrins; M, molecular mass markers of size
shown; lanes 1 and 2, recombinant human loricrin; lanes 3 and 4, in vivo mouse loricrin. Lanes 1 and 3, Coomassie-stained gels; lanes 2 and 4, Western blots using the loricrin
antibody.
By both Coomassie staining and Western blotting of SDS
gels, human loricrin consisted of a single band of M
Figure 2:
Spectroscopic properties of recombinant
human and in vivo mouse loricrins. a, UV absorption
spectrum. The extinction coefficient measured at 280 nm is
Figure 6:
Utilization of glutamines and lysines in
recombinant loricrin by TGases 1 (a) and 3 (b). These
data are calculated from the molar yields of each in vitro cross-linked peptide listed in Table 1and Table 2. In panel c, the utilization of loricrin residues in cross-links
recovered from isolated foreskin epidermal CEs (7) is shown for
comparison.
Measurements of the CD spectra of the loricrins (Fig. 3a) were done to evaluate their secondary structures.
As predicted(6) , they have little organized secondary
structure in solution at 20 °C; there is essentially no
Figure 3:
Circular dichroism spectra of recombinant
human and in vivo mouse loricrins. a, spectra of
recombinant human (solid line) and in vivo mouse
loricrins (squares) at 20 °C and pH 7.4. b,
spectra of recombinant human loricrin at 20 °C (solid
line), 37° (dashed line), 50 °C (dotted
line), or after a 20-50-20 °C temperature transition (diamonds). Inset shows the same transition in the
aromatic region. Data for in vivo mouse loricrin were
identical. c, spectra of recombinant loricrin in the presence (circles) or absence (solid line) of 4 M guanidine hydrochloride. Data for in vivo mouse loricrin
were identical.
Figure 4:
Three epidermal TGases cross-link loricrin in vitro differently. Equivalent amounts of each enzyme
activity of full-length recombinant human TGase 1 (a), guinea
pig liver TGase 2 (b), or guinea pig epidermal TGase 3 (c) were reacted with
Pilot experiments with the three TGases using the small
amounts of available in vivo mouse loricrin gave almost
identical results (data not shown).
Figure 5:
Characterization of in vitro cross-linked loricrin peptides formed by TGases 1 and 3.
Preparative reactions as in Fig. 4cross-linked by TGase 1 (b) or TGase 3 (c) were subjected to proteinase K
digestion, and the products were resolved by HPLC chromatography. In
comparison with a sample of uncross-linked loricrin (a),
several new peaks were identified, recovered, and sequenced. The 10 (b) or 16 (c) cross-linked peptides are listed in Table 1and Table 2,
respectively.
In the case of the TGase 1 reaction, 10 peptides were
identified (Fig. 5b) and sequenced (Table 1),
with a total yield of cross-link of about 1.2 mol/mol. This means that
>85% of the cross-link was recovered; the unfound 0.2 mol/mol
(<15%) were peptides too small to be resolved in the HPLC. Peptides
1, 7, 9, and 10 involve likely interchain cross-links because adjacent
Gln and Lys residues were used on the same sequences. Peptides 6 and 8
also may involve interchain cross-links between the beginning of one
chain and the end of another. Peptides 2-5 may involve either
inter- or intrachain cross-links. Thus two-thirds (molar basis) involve
interchain cross-links, in support of the pattern of oligomerization of
loricrin by the TGase 1 enzyme (Fig. 4a). The
interchain cross-linking by TGase 1 involved predominantly residues
Gln In the case of the TGase 3 reaction, 16
peptides were identified (Fig. 5c) and sequenced (Table 2), with a yield of 2.0 mol/mol (85% of cross-link
recovered). Peptides 2, 3, 5, 7, 8, 14, 15, and 16 involved the same
sequences as those formed by the TGase 1 enzyme. Peptides 14-16
involve obligatory interchain cross-links, and peptides 4, 5, 8, and 13
involve possible interchain cross-links through terminal sequences.
However, together these amount to only 25% of the molar total. Thus
Comparative kinetic constant data on the TGase 1, 2, and 3
enzymes are shown in Table 3, and reveal that recombinant
loricrin is a very efficient substrate for all three TGases. For
example, by way of comparison with previous data, the kinetic
efficiency (k From the double displacement kinetic
mechanisms involved, we calculated the K
However, the loricrins have only a limited degree of structure in
solution at physiological pH, as predicted by conventional algorithms (6) . Based on an unusual motif present in loricrins and
several other types of proteins, we proposed a glycine loop
hypothesis(13) , in which long sequences highly enriched in Gly
residues were folded into loops by the interaction of occasional
interspersed aromatic residues. Our present data now provide support
for this hypothesis. We show that what little structure is present in
loricrins involves the Tyr residues, which associate in a hydrophobic
environment (Fig. 2).
Interestingly, the TGases 1 and 3 utilized
loricrin as a complete substrate in different ways. First, they used
different Gln and Lys residues in quantitatively different amounts.
TGase 1 used mostly Gln The striking observation of the use of only
certain residues for inter- and intrachain cross-linking in vitro prompted us to compare these data with the previous in vivo cross-linking data for loricrin obtained from CEs (Fig. 6c)(7) . In the in vivo data
set, most of the available Gln and Lys residues were utilized in both
intra- and interchain cross-linking, although certain preferential
residues, Gln In
the absence of more information, the in vivo data set cannot
provide estimates of the percentage of intra- and inter-chain
cross-linking, although both reactions had clearly occurred. However,
we can now explore this question by estimation of the relative roles of
the TGases 1 and 3 in the cross-linking of loricrin in vivo,
based on the patterns of specificity of residue usages seen in
vitro. For example, visual inspection reveals that the molar usage
rate of Lys Accordingly, these
considerations indicate a hitherto unrecognized important role for the
TGase 1 enzyme in the cross-linking of loricrin to the CE in
vivo.
Volume 270,
Number 44,
Issue of November 3, 1995 pp. 26382-26390
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
or 
structure but have some organized structure in solution associated with
their multiple tyrosines and can be reversibly denatured by either
guanidine hydrochloride or temperature. The transglutaminase (TGase) 1,
2, and 3 enzymes expressed during epidermal differentiation utilized
loricrin in vitro as a complete substrate, but the types of
cross-linking were different. The TGase 3 reaction favored certain
lysines and glutamines by forming mostly intrachain cross-links,
whereas TGase 1 formed mostly large oligomeric complexes by interchain
cross-links involving different lysines and glutamines. Together, the
glutamines and lysines used in vitro are almost identical to
those seen in vivo. The data support a hypothesis for the
essential and complementary roles of both TGase 1 and TGase 3 in
cross-linking of loricrin in vivo. Failure to cross-link
loricrin by TGase 1 may explain the phenotype of lamellar ichthyosis, a
disease caused by mutations in the TGase 1 gene.
)a
15-nm-thick layer of protein deposited on the inner surface of the cell
periphery, which serves as a physical barrier for the
epidermis(4, 5) . The CE proteins are rendered
insoluble by cross-linking by both disulfide bonds and the N-(
-glutamyl)lysine isopeptide bond
formed by the action of one or more of the three known epidermal
transglutaminases (TGases)(4, 5, 6) . Several
proteins have now been documented as CE constituents by direct
sequencing analyses of cross-linked peptides(7) , including
loricrin, small proline-rich proteins 1 and 2 (SPR1 and SPR2), elafin,
keratins, filaggrin, and desmoplakin. The proteins involucrin and
cystatin
are also likely constituents, but direct sequencing of
cross-linked peptides involving these proteins has not yet been
reported (reviewed in (8) )., involucrin, and possibly
other as yet unidentified proteins(7, 8, 9) . ) and two internal glutamines (Gln
and
Gln
) account for
75% of the cross-links(7) .
The loricrins of three species sequenced so far differ only in the
sizes of the glycine motifs; the flanking cross-linking sequences have
been highly
conserved(6, 11, 12, 14) . Thus, it
was postulated that loricrins adopt a compact mesh-like array that
provides insolubility yet flexibility to the CE of normal epidermis.
Nor are data available on the nature of the TGase(s) responsible for
cross-linking loricrins. Extant models have suggested that TGase 1 (
)is involved in initial or scaffold assembly steps of the
CE involving involucrin and perhaps cystatin and elafin and that
TGase 3 is responsible for the final reinforcement steps of loricrin
accretion(4, 5, 8, 9) . However,
both TGase 1 (15, 16, 17, 18) and
TGase 3 (17, 19) are expressed in the epidermis
essentially coincidentally with loricrin, so this scheme awaits more
rigorous study.
Expression of Loricrin in pET-11a System
A
full-length cDNA clone encoding human loricrin (6) was
configured into the pET-11a bacterial expression vector (Novagen,
Madison, WI) by the addition of suitable linkers to the existing EcoRI ends of the cDNA clone(22) . The 5`-untranslated
nucleotide sequence of the cDNA sequence TCCTCAC was modified to
TCTTCAT in order to reduce the free energy to enable more efficient
association with bacterial
ribosomes(22, 23, 24) . After transformation
into the host Escherichia coli B strain BL3/DE3 (Novagen),
cultures (0.1-5-liter volume) (in LB broth supplemented with 50
µg/ml of ampicillin) were grown to A of
0.6, and protein expression was induced with 1.0 mM
isopropyl-1-thio-
-D-galactopyranoside for 3 h. In some
cultures, L-[S]cysteine (0.5
µCi/ml) was added immediately prior to induction.
Purification of Recombinant Loricrin
Following
induction, fresh or previously frozen (-70 °C) bacterial
pellets (10,000 
g for 30 min) were lysed in a buffer
of 50 mM Tris-HCl (pH 8.0), 0.1 M NaCl, 1 mM dithiothreitol, 1 mM EDTA, and the protease inhibitors
leupeptin (1 mM), 4-(2-aminoethyl)benzene-sulfonyl fluoride
(0.2 mM), calpain inhibitor (10 µM), and
aprotinin (0.1 unit/ml) and pelleted at 10,000 g for
30 min. The bacterial lysate was dialyzed against three changes of
100-fold volume of 25 mM citrate buffer, pH 3.6, containing 1
mM dithiothreitol and 1 mM EDTA. At each change of
dialysis buffer, the precipitated bacterial proteins were removed by
centrifugation at 10,000 g for 10 min. Loricrin was
then purified from this supernatant by chromatography on a 0.5
5-cm Mono-S column (Pharmacia Biotech Inc.) using a Pharmacia fast
protein liquid chromatography system and a gradient of 0-1.0 M NaCl in the citrate buffer. Loricrin eluted at about 0.2 M salt.S-cysteine-labeled protein since
bacterial proteins contain very little cysteine.
Isolation and Purification of Monomeric Mouse
Loricrin
Freshly prepared newborn mouse epidermis was extracted
by homogenization (5 ml/tissue) in the above citrate buffer containing
8 M urea, freed of urea by dialysis, and chromatographed on
the Mono-S column as above.Cross-linking of Loricrin as a Complete Substrate by
TGases in in Vitro Assays
Human full-length TGase 1 was
expressed in bacteria and purified from the lysate (soluble) fraction
as described previously (22) . Guinea pig liver TGase 2 (Sigma) (25) and guinea pig epidermal TGase 3 (19) were also
used.S-loricrin was equilibrated by dialysis into
a buffer of 50 mM Tris-HCl, 50 mM NaCl, 1 mM
dithiothreitol, 1 mM EDTA, pH 7.5. The solutions were made to
5 mM CaCl
to initiate reaction at 37 °C. In
analytical cross-linking experiments, 25 µg (1 nmol) of S-labeled loricrin (about 8
10
dpm)
was utilized in a 100-µl reaction volume. In order to standardize
the reactions for comparisons of the TGases, the same amount of enzymic
activity was used for each enzyme. These activities were measured by
[
H]putrescine (Amersham Corp., specific activity
26 Ci/mmol) incorporation into succinylated casein, and the amount of
TGase 1, 2, or 3 that incorporated 0.45 pmol/min into the casein was
used. This corresponds to 79 nM for TGase 1 and TGase 3 and 4
nM for TGase 2. Aliquots were stopped by the addition of EDTA
(7 mM final concentration). The cross-linked products were
separated on 4-12% polyacrylamide gels and analyzed by
autoradiography. Selected bands were quantitated by scanning in a
computing densitometer with ImageQuant software, version 3.0 (Molecular
Dynamics). In preparative experiments with TGase 1 or 3, 4-10
nmol of loricrin were reacted in a volume of 250 µl for 2 h. In
these cases, an excess of enzyme (equivalent to about 1 pmol/min of H-putrescine incorporation into casein) was used to drive
the reaction to completion, as judged in control experiments.Kinetic Studies of Putrescine Incorporation into
Loricrin
Kinetic reactions were carried out using full-length
bacterial expressed TGase 1 and guinea pig TGase 2 and 3 in the same
buffer used for in vitro assays. Five concentrations of
unlabeled loricrin (2.5, 3, 4, 5, and 7 µM), three
different concentrations of [H]putrescine (0.07,
0.10, and 0.14 mM), and an appropriate amount of enzyme were
used in a final volume of 100 µl for 10 min at 37 °C. For each
enzyme, the amount of activity utilized was standardized with
putrescine incorporation into casein as above. The reactions were
stopped by spotting 25 µl of the initial mixture on 3MM filter
paper (Whatman) and washing the filter sequentially, for 10 min, in 20,
10, and 5% cold trichloroacetic acid and finally 95% ethanol. Filters
were dried, and the radioactivity was counted(26) . These
reactions utilized a large molar excess of putrescine in order to
achieve linearity of reaction kinetics(22) , so as to favor
TGase attachment of only one amine group of the putrescine to loricrin
rather than TGase cross-linking of loricrin by putrescine or
cross-linking of loricrin to itself. The data for initial velocity of H-putrescine incorporation into loricrin conformed to a
modified double displacement mechanism as described previously for
TGase-catalyzed reactions(27) . Kinetic constants were
calculated as described(22) .Isolation and Quantitation of the Isodipeptide
Cross-link
Aliquots of loricrin cross-linking reactions (200
pmol) were subjected to total proteolytic digestion with proteinase K
(Promega), leucine amino peptidase (Sigma), and carboxypeptidase Y
(Boehringer Mannheim)(6, 7, 28) . Control
reactions consisted of enzymes only or of loricrin before
cross-linking. The N-(
-glutamyl)lysine
isodipeptide (Accurate Biochemical Corp.) elutes near methionine at
28.6 min on a Beckman 6300 amino acid analyzer.
Peptide Mapping and Amino Acid Microsequencing
The
2-h preparative cross-linking reactions of loricrin with TGases 1 and 3
were digested with proteinase K (3% enzyme to loricrin protein by
weight) for 3 h at 37 °C(7) . Peptides were resolved by
HPLC using a reverse phase ultrasphere ODS C18 column (4.6 250
mm) with a gradient of 0-100% acetonitrile containing 0.08%
trifluoroacetic acid. A control sample of uncross-linked loricrin was
digested and resolved similarly. New peaks that appeared in the former
reactions with respect to the latter were collected, concentrated, and
covalently attached to a polyvinylidene difluoride solid support
(Sequelon-AA, Millipore Corp.). The peptides were sequenced to
completion in an LF-3000 (Porton) gas-phase sequencer. Released
phenylthiohydantoin-derivatized amino acids were resolved and
quantitated by on-line analytical HPLC (Beckman Instruments, using
System Gold software). Where possible, the amounts of the cross-linked
peptides were quantitated by amino acid analysis prior to sequencing.
In other cases, a good estimate of amounts were possible based on the
size of the A trace from the HPLC column and
the amino acid content from the sequence.
Biophysical Assays
Loricrin solutions
(0.1-0.4 mg/ml) were equilibrated into the same buffer as used
for in vitro TGase cross-linking reaction. Steady state
fluorescence excitation and emission spectra were recorded on a
photon-counting spectrofluorometer (Fluoromax Instruments, Paris,
France). The bandwidths of excitation and emission monochromators were
in the range of 2-4 nm. In all fluorescence experiments, spectra
were corrected for possible Raman contributions by buffer-base line
subtraction. Absorption and circular dichroism (CD) measurements were
carried out using a Jasco Uvidec 650 spectrophotomer and Jasco 600
spectropolarimeter, respectively. In both cases, 0.1-cm quartz cuvettes
were used. The sample holders were thermostated before and during
measurements, using external circulation. In the CD stability studies,
the loricrin solutions were maintained at 50 °C for 1 h and
reversed to 20 °C for 1 h before measurement.
Expression and Purification of Recombinant Human
Loricrin
The purpose of the present work was to undertake a
detailed study of this major CE structural protein of the epidermis. In
this study, we have expressed human loricrin in bacteria using the
pET11a system(22) . The loricrin was very soluble in 25 mM pH 3.6 sodium citrate buffer, which enabled a simple two-step
recovery from the bacterial proteins; >90% of bacterial lysate
proteins were insoluble in this buffer and were removed by
precipitation following dialysis, concentrating the loricrin by 2
orders of magnitude in one step without loss, enabling final
purification by fast protein liquid chromatography (Fig. 1). The
maximal yield was about 5 mg/liter. Several unsuccessful attempts were
made to improve this yield, such as by growing the bacteria to higher
density before induction of protein expression, making adjustments to
the culture nutrients, changing the length of time of
isopropyl-1-thio--D-galactopyranoside induction, and
using an enriched culture medium (Terrific Broth, Life Technologies,
Inc.). The lack of improved yields suggests that the loricrin is a
toxic protein that kills the bacteria shortly after expression.
Consistent with this idea, the loricrin was not retained within
bacterial inclusion bodies but rather was entirely present in the
bacterial lysate supernatant fraction, which, happily, simplified its
purification.
30 kDa (Fig. 1, lanes 1 and 2), which is 15% high, based on its known amino acid
sequence(6) . Its amino acid composition, as determined
following total enzymic digestion, was exactly as predicted from its
deduced amino acid sequence, including high contents of Gly, Ser, and
Cys (data not shown). In addition, we found that whereas the
recombinant loricrin was very soluble at pH 3.6 (>20 mg/ml), its
solubility at physiological pH was limited to about 0.4 mg/ml.
Isolation of Monomeric Loricrin from Newborn Mouse and
Human Epidermis
Based on an early report (20) and on the
ease with which we could purify recombinant human loricrin, we next
tested the possibility of extraction of native loricrin with citrate
buffers from mouse and human epidermis. By inclusion of 8 M
urea to the citrate buffer used above, the living layers, but not
stratum corneum layers, were dispersed as expected(29) . Upon
removal of the urea by dialysis, the keratins and profilaggrin were
quantitatively precipitated(30) , whereas the loricrin remained
in solution. The supernatants from both newborn mouse and human
foreskin epidermis were then resolved on the Mono-S column. A peak of in vivo mouse loricrin of 65 kDa was eluted in exactly
the same location as recombinant human loricrin (Fig. 1, lanes 3 and 4). This value is about 35% high (the
size determined from its complete amino acid sequence is 48
kDa(14) ), and as for human loricrin, this may be due to the
anomalous binding of SDS due to the unusually glycine-rich sequences.
The yield of
4 µg of loricrin/epidermis was recovered in
freshly excised tissue of 0-1-day-old pups only; no loricrin
could be recovered in tissue from older mice or from tissue that had
been stored for >2 h. The yield of in vivo human loricrin
from foreskin epidermis in identical experiments, however, was <0.1
µg/tissue. As these yields represent only a trace of the total
loricrin expressed in living epidermis, based on its abundance in
CEs(7, 8, 9) , loricrin is apparently rapidly
cross-linked after synthesis, as proposed earlier(14) .
Spectroscopic Properties Show That in Vivo Mouse and
Recombinant Human Loricrins Have the Same Structures
The
fluorescence spectra of in vivo mouse and recombinant human
loricrins measured at pH 7.4 were found to be identical. The absorption
spectrum (Fig. 2a) shows a maximum peak at 275 nm,
indicating that the Tyr and Phe residues present play a relevant role
in the absorption of light below 290 nm. The single Trp residue is
responsible for the shoulder at 282 nm. Structured spectra are visible
by steady state fluorescence excitation (Fig. 2b). The
fluorescence signal observed through an emission monochromator at 303
nm (Fig. 2b, line 1) shows a maximum shifted
to lower wavelengths, which still reveals absorption of the Tyr
residues. This implies that the Tyr residues occupy a distant position
with respect to the Trp environment, because the energy transfer
between them is negligible. In order to investigate whether the Tyr
residues occupy a hydrophobic environment, the loricrins were denatured
in 6 M guanidine hydrochloride (final concentration). The
results showed only a 10% decrease in the 305-350-nm fluorescence
emission ratio when excited at 280 nm (data not shown). Since it is
highly unlikely that the distance between the Trp and Tyr residues
should decrease with unfolding, the data indicate that denaturation
shifts the bulk of the Tyr residues toward a more polar environment.
These data are consistent with the glycine loop
hypothesis(13) , in which the aromatic residues are thought to
associate in a hydrophobic core to anchor loops of Gly residues. To
explore the properties of the Trp residue alone, an emission
monochromator at 350 nm yielded much broader unstructured spectra (Fig. 2b, line 3). The steady state emission
spectra for both Tyr residues and Trp (excited at 280 nm) and for Trp
alone (excited at 295 nm) are shown in Fig. 2c, lines 1 and 2, respectively. The selective excitation
of Trp at 295 nm shows an emission spectrum centered at 356 nm,
suggesting that this residue is exposed to the solvent and thus located
on the external surface of the protein. These data are consistent with
the cross-linking data (see below): Trp (human) (or
Trp
(mouse)) is located three residues from the terminal
Lys, the most used Lys residue in in vivo and in vitro cross-linking reactions (see Table 2and Table 3and Fig. 6).
. b, steady state
fluorescence excitation spectra of loricrins at
= 303 nm (1, broken line),
= 325 nm (2, solid line), or
= 350 nm (3, dotted line). c, steady state fluorescence emission spectra to measure
tryptophan and tyrosines (
= 280 nm, line
1) or tryptophan alone (
= 295 nm, line 2).
or
structure present. However, the spectra of the recombinant human
and in vivo mouse loricrins were superimposable (Fig. 3a), establishing that the recombinant protein
had refolded into the native configuration of mouse loricrin.
Interestingly, these loricrin CD spectra are very similar to those of
bovine pancreatic trypsin inhibitor (31) and filaggrins (32) which have unusually small amounts of secondary structure.
In order to ascertain that the low degree of order was not due to
denaturation during purification or to inappropriate folding following
expression in bacteria, the stability of the overall protein structure
was measured as a function of temperature and guanidine hydrochloride.
Whereas the loricrins were unfolded by heating to 50 °C, the CD
signals were normalized when returned to 20 °C, indicating
refolding of the protein structure (Fig. 3b).
Denaturation in 4 M guanidine hydrochloride increased the CD
signal at 225 nm, implying some unusual secondary structure, but this
was reversed on removal of the reagent, indicating protein refolding (Fig. 3c).
Recombinant Human Loricrin Is a Complete Substrate for
Epidermal TGases
A variety of data have documented that
loricrins are a major substrate for TGases in the
epidermis(6, 7, 14) , but there are no data
on which TGase is responsible for the cross-linking. The reason is
simply that TGases 1 and 3 are largely co-expressed in the epidermis (15, 16, 17, 18, 19) , so
that it has been difficult to dissect the individual roles of these
enzymes. Therefore, we have used S-labeled recombinant
human loricrin as a substrate for three TGases in in vitro reactions. Fig. 4shows that all three TGases cross-link
loricrin into oligomers. This means that the loricrin functions as a
complete substrate in the sense that it provides both glutamines and
lysines for cross-linking. However, reaction with equal amounts of
TGase activities revealed differences in the amount of cross-links
inserted and the nature of the products formed. For all three enzymes,
the amount of activity used and the length of reaction (2 h) was
determined in control reactions to allow complete reaction. The
reactions resulted in the formation of 1.4, 0.2, and 2.3 mol of
isopeptide cross-link/mol of loricrin for TGases 1, 2, and 3,
respectively (Fig. 4, a, b, and c,
respectively); use of more enzyme or increased incubation times did not
alter the 2-h cross-link patterns shown nor the yields of
cross-link/mol. In each reaction, a significant proportion of the
loricrin in a time-dependent manner (25, 10, and 60%; TGases 1, 2, and
3, respectively) remained as a monomer that migrated somewhat faster
than unreacted loricrin. Judging from Coomassie staining, Western
blotting, and amino acid analyses, this shift was not likely due to
degradation (data not shown). A more likely possibility is the
insertion of intrachain cross-links; i.e. the monomeric
loricrin had became more compact and migrated faster in the SDS field.
Also, in each case, some of the loricrin remained uncross-linked (10,
80, and <5%; TGases 1, 2, and 3, respectively), since it retained
the same apparent molecular size as the control. In the cases of TGase
1 and 2, some loricrin (65 and 10%, respectively) was oligomerized into
aggregates too large to enter the SDS gel (Fig. 4, a and b). In the case of TGase 3, however, most of the
cross-linked products were only dimers, trimers, and tetramers, each of
which themselves increased in migration rate suggestive of the
formation of intrachain cross-links. No detectable protein formed large
oligomers (Fig. 4c). Thus the three enzymes treat the
loricrin substrate differently. TGase 2 cross-links the loricrin
relatively poorly, and about two-thirds of the cross-linking by TGase 1
(
1 mol/mol) involves interchain cross-links to form very large
oligomers; but most of the cross-linking by TGase 3 (>2 mol/mol)
involves intrachain cross-links. This pattern suggests that these two
TGases may cross-link loricrin in a complementary manner. To test this
idea, Fig. 4d shows that following an initial
cross-linking by TGase 3 (2.4 mol of cross-link/mol of loricrin),
subsequent reaction with TGase 1 resulted in further cross-linking (a
total of 3.3 mol of cross-links/mol) into oligomers too large to enter
the SDS gel.
S-labeled recombinant
loricrin for 2 h as described under ``Materials and
Methods,'' fractionated on a 4-12% gradient SDS gel, and
autoradiographed. In panel d, following a 2-h reaction with
TGase 3, a similar amount of TGase 1 activity was added for a second
2-h period. In each case, lane C represents reaction with
enzyme in the presence of 7 mM EDTA and lanes 1-5 represent incubation times of 10, 20, 30, 60, and 120 min,
respectively. Protein size markers are shown (as in Fig. 1, inset). Arabic numbers (e.g. 1), monomeric
loricrin; Arabic prime numbers (e.g. 1°, etc.),
intrachain cross-linked monomer, etc.
Identification of Cross-links Formed in Recombinant
Loricrin in Vitro by TGase 1 and TGase 3
To characterize the
nature of the cross-links formed in the above in vitro reactions, we performed preparative experiments involving
4-10 nmol of loricrin. After the 2-h reaction, the protein was
digested with proteinase K in order to recover cross-linked peptides
suitable for direct sequencing (7) . The peptides were resolved
by HPLC, and the profile was compared with a sample of unreacted
loricrin (Fig. 5a) to identify shifted peaks likely to be
cross-linked.
, Gln
, Lys
, Lys
,
and Lys, whereas the intrachain cross-linking involved
mostly residues Gln
, Gln
, and Lys
(Fig. 6a).
75% of the molar total involve intrachain cross-links, again in
support of the pattern of cross-linking by the TGase 3 enzyme (Fig. 4c). In this case, the interchain cross-linking
by TGase 3 involved predominantly residues Gln
,
Gln, Gln
, Gln
, and some
Lys
, but the bulk of the likely intrachain cross-linking
involved residues Gln
, Gln
,
Lys
, and Lys
(Fig. 6b).
Kinetics of TGase 1, 2, and 3 Cross-linking of
Recombinant Loricrin in Vitro
Given that TGase 1 and TGase 3 can
both utilize recombinant loricrin in vitro as a complete
substrate, albeit with different behavior, we wanted to obtain more
quantitative information on the rates of reaction. Many investigators
have explored cross-linking reactions in vitro by various
TGases of target substrate Gln(s), using model simple amines such as
putrescine for example (25) or onto target substrate Lys(s)
using Gln-containing peptides(33, 34, 35) .
However, to date, no kinetic data have been reported on the
cross-linking of a complete TGase substrate protein in which
both Gln and Lys are used simultaneously for intra- and/or interchain
cross-linking. Thus a complexity arises because the TGases can use
either the putrescine as the amine donor or the protein's own
Lys(s) as the amine donor(s). A second complexity arises in the present
study because, unlike involucrin in which mainly only one Gln is used
in in vitro cross-linking reactions(36) , loricrin can
provide multiple Gln and Lys residues for cross-linking simultaneously,
and the preferred residues used differ between the TGase 1 and TGase 3
enzymes (Fig. 6; Table 1and Table 2). To circumvent
these complexities, we have used high concentrations of putrescine in
the in vitro reactions, 1-2 orders of magnitude higher
than the concentration of available Gln residues in the loricrin
substrate. In this way, we have suppressed the propensity for
intra-/interchain cross-linking of loricrin to itself and the
likelihood of putrescine oligomerization of loricrin in order to
measure only the incorporation of a single amine of putrescine onto one
of the favored Gln residues of loricrin. Control experiments showed
that no more than 5% of the substrate of the lowest concentrations was
consumed, and there was no cross-linking of loricrin either to itself
or by putrescine during the reaction. The data calculated are not true K
values but represent ``average'' K values for the multiple Gln residues in
loricrin./K
) of TGase 1
for loricrin is 10-fold higher (0.29
min
µM) than
for succinylated casein (0.029
min
µM)(21) . While the
average K value for TGase 1 (17
10M) is similar to that for TGase 2 (16
10M), it is 3 times higher than
the average K
value for TGase 3 (5
10M). This means that TGase 3 is more
efficient in cross-linking recombinant loricrin. Presumably, this
reflects the greater proficiency of usage of Gln
and
Gln
by TGase 3 than TGase 1 or of Gln
and
Gln
by TGase 1. This is also supported by our earlier
work(22) : the TGase 1 enzyme shows a greater affinity for
recombinant loricrin (1.7
10M)
than either succinylated casein (5
10M) or synthetic loricrin and SPR1 peptides (12
10M). In addition, the V
values for TGases 1 and 3 are higher than for TGase 2 (Table 3). This confirms the observation (Fig. 4b) that the participation of TGase 2 in loricrin
cross-linking is quite weak. values
for putrescine, which show a higher value for TGase 3 (Table 3).
This means that TGase 3 greatly prefers to use the Lys of loricrin as
the amine donor rather than the exogenous putrescine. We conclude that
this enzyme, after binding preferentially to the Gln or
Gln
residues, induces conformational changes in loricrin,
producing a more compact form enabling intrachain cross-linking to the
preferred Lys
residue.
Recombinant Human and in Vivo Mouse Loricrins Have the
Same Structures in Solution
By fluorescence spectroscopy (Fig. 2) and CD (Fig. 3) experiments, we show here that
the structures of recombinant human and in vivo mouse
loricrins are indistinguishable. The identical reversible structural
changes induced by either temperature or guanidine hydrochloride
denaturation support the notion that the recombinant human loricrin had
refolded into a native conformation. These observations have allowed us
to use the recombinant loricrin with confidence to explore its
substrate properties by the three TGases active in the epidermis. Loricrin Is a Complete TGase Substrate in Vitro and in
Vivo
The data of Fig. 4and Fig. 5show that
loricrin can function in vitro as a complete substrate for the
TGases 1 and 3 (and 2 weakly); that is, Gln and Lys residues on the
same protein chain are recruited for cross-linking. These data are in
agreement with our earlier in vivo CE cross-linking
data(7) , which showed that loricrin, as well as SPR1, SPR2,
and elafin, are also complete TGase substrates in vivo. Thus,
our in vitro cross-linking paradigm can be used with
confidence to ascertain the complex processes of cross-linking of these
various CE substrates., Gln
,
Gln
, Lys
, Lys, and
Lys
; TGase 3 used mostly Gln
,
Gln, Gln
, Gln
,
Gln
, Gln
, Lys
, Lys
and Lys
(Fig. 6). Second, the predominant
reaction of TGase 1 was interchain cross-linking (
1 mol/mol)
through the preferential use of Gln
, Gln
,
Gln
, Lys
, with some intrachain
cross-linking (<0.5 mol/mol) with Gln
,
Lys
, and Lys
(Table 1, II).
Conversely, the predominant reaction of TGase 3 was intrachain
cross-linking (>2 mol/mol) through preferential use of
Gln
, Gln
, Gln
,
Gln
, Lys
, and Lys
with some
interchain cross-linking (<0.5 mol/mol) with Gln
,
Gln, Lys
(Table 1, Table 2).
This means there is a direct relationship between the types of
cross-linking (inter- versus intrachain) and the specific
residues utilized and the dual and complementary roles of these two
TGases in this process.
, Gln, Gln
,
Gln
, Gln
, Lys
,
Lys, Lys
, and Lys
, accounted
for >90% of the total. Significantly, these same residues were used
preferentially by TGases 1 and/or 3 in the present in vitro data. This strongly supports the conclusion from the biophysical
measurements that the recombinant loricrin must have adopted a native
structure. By way of contrast, in vitro cross-linking of
denatured or proteolysed involucrin used multiple Gln residues, whereas
essentially only one was used in native involucrin (36) .
seen in vivo would require 72%
cross-linking by TGase 3 and 28% by TGase 1. Likewise, the in vivo Lys
data would require 71% cross-linking by TGase 1
and 29% by TGase 3. Thus, least squares fitting (9) of the
summed fractions for each of the residue positions Lys
,
Lys, Lys
, Lys
,
Gln
, Gln, Gln, Gln
,
Gln
, Gln
, Gln
, and
Gln
show that 35 ± 7% of the total cross-links
formed in vivo are likely inserted by the TGase 1 enzyme and
65 ± 6% by the TGase 3 enzyme. Residue positions Lys
and Gln deviate by 1-2 standard deviations
from these means, and residues Gln
, Gln
,
and Gln
were not used at all in vitro, but since
these five residue positions were used infrequently in vivo,
their weighted contributions to the mean are low.
A Model for the Complementary Cross-linking of Loricrin
in Vivo
Based on protein expression patterns, extant models on
CE assembly (4, 5, 7, 8, 9) suggest
that the TGase 1 enzyme cross-links such soluble proteins as involucrin
and cystatin to form a scaffold, onto which the TGase 3 enzyme
deposits loricrin in a final stabilization event. These events must be
very complex because the TGase 3 enzyme is a soluble (cytosolic)
enzyme(18, 19) , whereas the TGase 1 enzyme system
consists of membrane-associated (15, 16, 18) and multiple soluble
forms(18) . Our new data strongly suggest that both TGase 1 and TGase 3 are involved in loricrin deposition in
vivo and in different ways. We propose that the TGase 1 enzyme
cross-links the loricrin to the CE scaffold to form a large polymeric
structure through interchain cross-linking with the preferential use of
certain Gln and Lys residues. Subsequently, or possibly simultaneously,
the TGase 3 enzyme further cross-links the attached loricrin by mostly
intrachain cross-links using primarily different Gln and Lys residues
to form a more compact structure. We cannot exclude an alternative
model in which the soluble TGase 3 enzyme first cross-links the
loricrin into small compact oligomers, which are then glued together
and to the CE by interchain cross-links with the soluble and/or
membrane-associated forms of the TGase 1 enzyme. Both models are
consistent with our present recovery of trace amounts of soluble
monomeric loricrin from mouse epidermis and with pulse-chase
experiments in which it was shown that newly synthesized loricrin in
organ cultured epidermis is rapidly cross-linked into a form too large
to enter an SDS gel(14) .Implications for Lamellar Ichthyosis
This
realization for the first time of the probable in vivo roles
of both TGases 1 and 3 in loricrin assembly onto the CE has important
implications for the autosomal recessive disease lamellar ichthyosis.
This disorder of cornification of the epidermis manifests as
generalized plate-like scales with pathology showing disruption in the
uppermost differentiating and stratum corneum layers of the
epidermis(37) . Two papers have reported reduced expression of
the TGase 1 enzyme and abnormal loricrin
deposition(38, 39) . More recent
reports(40, 41, 42) have shown that
mutations in the TGM1 gene resulting in a defective or
inactive TGase 1 enzyme are the cause of this disease. Our new data
suggesting an essential role for the TGase 1 enzyme in cross-linking
loricrin and CE formation offers a new insight into the mechanism of
pathogenesis of this disease.
))
We thank Dr. Soo-Youl Kim for advice on the assembly
of the loricrin expression construct and its use in the pET-11a system.
Will Idler kindly assisted in much of the bacterial protein expression
work. During the course of this work, we enjoyed stimulating
conversations with Drs. Valeria Catani, Alessandro
Finnazzi-Agrò, John Folk, and Ulrike Lichti.
George Poy synthesized the oligonucleotides used in this study.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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