(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 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.
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), ()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) ).
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 , involucrin, and possibly
other as yet unidentified proteins(7, 8, 9) .
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) 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.
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 S-cysteine-labeled protein since
bacterial proteins contain very little cysteine.
For in vitro cross-linking studies using the
recombinant human loricrin as a complete TGase substrate, the purified
unlabeled or 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.
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
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.
Figure 2:
Spectroscopic properties of recombinant
human and in vivo mouse loricrins. a, UV absorption
spectrum. The extinction coefficient measured at 280 nm is
. 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).
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 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).
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 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.
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, Gln
, Lys
, Lys
,
and Lys
, whereas the intrachain cross-linking involved
mostly residues Gln
, Gln
, and Lys
(Fig. 6a).
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
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).
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/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
10
M) is similar to that for TGase 2 (16
10
M), it is 3 times higher than
the average K
value for TGase 3 (5
10
M). 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
10
M)
than either succinylated casein (5
10
M) or synthetic loricrin and SPR1 peptides (12
10
M). 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.
From the double displacement kinetic
mechanisms involved, we calculated the K 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.
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, 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.
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, 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) .
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 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.
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.