From the Laboratory of Skin Biology, NIAMS, National Institutes of Health, Bethesda, Maryland 20892-2752
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ABSTRACT |
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Small proline-rich 1 (SPR1) proteins are
important for barrier function in stratified squamous epithelia. To
explore their properties, we expressed in bacteria a recombinant human
SPR1 protein and isolated native SPR1 proteins from cultured mouse keratinocytes. By circular dichroism, they possess no A large body of recent amino acid sequencing data has demonstrated
that members of the three known classes of small proline-rich (SPR)1 proteins serve as
constituents of the cornified cell envelope (CE) of stratified squamous
epithelia (1-5), which is a specialized structure essential for
barrier function (6-9). The SPR1 (two members), SPR2 (8-11 members)
and SPR3 (one member) are assembled from a common plan (10-14). Their
amino (head) and carboxyl (tail) domains are enriched in Gln and Lys
residues and consist of sequences that have been conserved between each
member of an SPR class but differ between classes. These flank a
central domain consisting of a series of Pro-rich peptide repeats of
sequences that likewise have been conserved between members of a class
but vary between classes. The SPRs become cross-linked to themselves
and other CE structural protein constituents by both disulfide
bonds and N Examination of the way in which the SPRs were cross-linked to protein
partners in human and mouse CE preparations revealed several novel
features of their properties and functions (1-5). First, only head and
tail domain Gln and Lys residues were used for cross-linking. Indeed,
multiple adjacent residues were often used simultaneously on the same
protein to form a complex interchain, and perhaps intrachain,
cross-linked network (3-5). Each SPR molecule participated in as many
as four cross-links (4). Second, the SPRs were found to be cross-linked
to many protein partners, and indeed, they were consistently found to
function as cross-bridgers between themselves or between loricrin,
involucrin, etc. (4, 5). Fourth, as initially suggested from expression
studies by earlier investigators, our sequencing data directly revealed that the amounts of SPRs present in the CEs from various tissues varied
widely; while human foreskin epidermal keratinocyte CEs contained about
5% SPRs (2), the CEs of the mouse forestomach contained about 22%
SPRs (5). Further modeling analyses revealed that the amounts of SPRs
in the epidermal CEs from trunk, lip, and footpad correlate well with
the degree of physical and mechanical trauma to which the tissues are
normally subjected (5). Thus, we have proposed that cross-bridging SPRs
play an important role as modulators of the biomechanical properties of
the CEs and the entire epithelium in which they are expressed (5).
In order to further explore these functions of the SPRs, we need to be
able to express large quantities of the proteins for study. Since their
cross-bridging role is mediated to a substantial degree through TGase
cross-linking, we also need to know which enzymes are responsible for
their cross-linking in vivo and how this is done. In an
initial study, we described the preparation and some properties of a
member of the human SPR2 class, and showed that it is cross-linked
almost entirely by the TGase 3 enzyme in vitro and in
vivo (32). In this paper, we have extended this work to the study
of a member of the human SPR1 class. We show here that its
cross-linking is much more complex, since it requires at least two
TGases (3 and 1) operating in a sequential manner using different Gln
residues for cross-linking to its partners as seen in
vivo.
Bacterial Expression and Purification of Human SPR1--
A
full-length cDNA clone encoding human SPR1 (clone 15B of Ref. 10)
was obtained as a generous gift from C. Backendorf. Following the
addition of appropriate linkers, it was inserted into the pET-11a
bacterial expression vector (Novagen, Madison, WI) and transformed into
the host Escherichia coli B strain BL 21/DE3 (Novagen).
Protein expression was induced in the presence or absence of
L-[35S]cysteine (2 µCi/ml) as described
previously (32, 33). Fresh or previously frozen pellets of bacteria
were lysed and dialyzed against several changes of 100 volumes of 25 mM sodium citrate (pH 3.6), 1 mM
dithiothreitol, 1 mM EDTA, and a mixture of protease inhibitors as described (32, 33). While most of the bacterial proteins
precipitated, the SPR1 protein remained soluble. Purification to
homogeneity was achieved using an Amersham Pharmacia Biotech fast
protein liquid chromatography system on a 0.5 × 5 cm Mono-S column equilibrated in the citrate buffer with a 0-1.0 M
NaCl gradient and was eluted with 0.18 M salt.
The fractions were analyzed on 4-20% SDS-polyacrylamide gels
(Novex, San Diego, CA) with Coomassie stain or by Western
blotting using a polyclonal antibody broadly reactive against both
mouse and human SPR1 proteins (14). The enhanced chemiluminescence detection was performed with the Super Signal CL-HRP Substrate System
(Pierce). Alternatively, the SPR1 proteins were monitored by autoradiography.
Isolation of Native Mouse SPR1a/b Proteins--
Mouse primary
keratinocytes were grown to confluency in low calcium (0.05 mM) medium. After 6 days, they were transferred to medium
containing high Ca2+ (1.4 mM), 10 nM staurosporin (to induce SPR1 expression (34)), 100 µM LTB-2 (transglutaminase inhibitor) (35) (Syntex
Research), and 0.5 µC/ml [35S]cysteine. After 24 h, the cells were harvested in phosphate-buffered saline, lysed by
polytron homogenization, and centrifuged (10,000 × g,
10 min, 4 °C). The cytosol was then dialyzed against the citrate
buffer used above, under which conditions the bulk of the keratin
proteins precipitated, leaving the SPR1a/b proteins highly enriched.
Their final purification was done following the method for the
bacterial expressed proteins, and the proteins were eluted by 0.2 M NaCl.
Isolation of TGase Forms for Cross-linking--
Three TGase
enzymes were used. Human full-length TGase 1 was expressed in
baculovirus, and the particulate fraction containing the unprocessed
full-length but active TGase 1 form was isolated as described (36).
This has a specific activity of about 6 pmol of putrescine
incorporation into succinylated casein/h/pmol of TGase 1 protein (36),
as measured using 14C-putrescine (Amersham Pharmacia
Biotech; specific activity, 118 mCi/mmol) and remains fully active for
at least 1 day. A typical yield was 5-10 nmol of TGase 1/liter of
insect cell culture medium. We also isolated the several TGase 1 isoforms present in the cytosolic and membrane-bound compartments of
normal human epidermal keratinocytes (NHEK) grown in high
Ca2+ medium in the presence of
[35S]methionine for 4 days after reaching confluency,
exactly as described previously (37, 38). These included the
membrane-bound full-length 106-kDa enzyme with a specific activity of
about 5 (as assayed using the above units); the 67/33/10-kDa complex
released from membranes by use of Triton X-100 with a specific activity of about 1000; the cytosolic full-length enzyme with a specific activity of about 50; the cytosolic 67/33-kDa complex with a specific activity of about 500; and the cytosolic 67-kDa form with a specific activity of about 250. We have noted that several of these isoforms begin to lose activity at 4 °C within about 6 h of isolation
(37, 38), presumably because of unfolding and/or destabilization (36).
The established method of using 14C-iodoacetamide for
measurement of TGase 1 molar amounts recovered in each culture
experiment takes about 1 day (39), and thus to perform experiments
using constant molar amounts of enzyme isoforms is problematic because
of loss of enzyme activity. However, we have found that when NHEK cells
are cultured under consistent conditions, the amounts of
[35S]methionine specific activity incorporated into the
TGase 1 isoforms (as measured after iodoacetamide titrations) are
reproducible and are 21 ± 2 dpm/pmol for the cytosolic 67-kDa
form and 40 ± 4 dpm/pmol for all other active forms. (This is to
be expected based on the methionine contents (37).) Accordingly, we
used the amount of 35S label to estimate the molar amount
of each TGase 1 isoform recovered from fast protein liquid
chromatography columns. In a typical culture of normal human epidermal
keratinocytes in a 100-mm dish, we recovered 100-200 pmol of TGase 1, or 15-100 pmol of each of the above membrane-bound or cytosolic isoforms.
Guinea Pig Liver TGase 2 Was Obtained Commercially (Sigma).
TGase 3 proenzyme, which has no measurable activity, was isolated and
purified from guinea pig skin. It was activated by dispase as described
previously (40). The amount used for reactions was quantitated by amino
acid analysis.
Cross-linking of SPR1 Proteins--
For in vitro
cross-linking studies using the SPR1 proteins as complete TGase
substrates and measurement of isodipeptide cross-link formation, the
purified SPRs were equilibrated into a buffer of 50 mM
Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM
dithiothreitol, and 1 mM EDTA. Reactions contained 10 µg
of 35S-labeled recombinant human or native mouse SPR1
proteins (about 7500 dpm) and about 12.5 pmol of TGase enzyme (~500
nM enzyme concentration; 250-500 dpm) in a 25-µl volume.
Reactions were initiated by the addition of Ca2+ to the
mixture (5 mM final concentration) and incubated at
37 °C. Timed aliquots of 1.5 µl each were removed and subjected to total enzymic digestion for measurement of the amount of cross-link formed during the reactions (41). In some experiments, the total reactions were analyzed by SDS-polyacrylamide gel electrophoresis on
4-20% gradient gels, blotted onto nitrocellulose membranes, and
analyzed by autoradiography. In preparative experiments, 100 µg of
unlabeled recombinant human SPR1 were reacted in a volume 250 µl. In
this case, we used the baculovirus membrane-bound full-length TGase 1 form and activated TGase 3 at concentrations of 700 nM for
18 h. Experiments showed that the cross-linking had proceeded to
completion (see Fig. 3). We also performed a preparative experiment with the high specific activity keratinocyte membrane-bound
67/33/10-kDa isoform at 50 nM enzyme concentration, a
reaction that was completed in 2 h.
Kinetic constants of the three TGases were determined for the
recombinant human SPR1 protein exactly as described before (32, 42).
Protein Chemical Methods--
The concentrations and purity of
the purified SPR1 proteins and TGase enzymes were determined by amino
acid analysis. Uncross-linked or cross-linked SPR1 proteins were
digested with trypsin (1:30 by weight; Sigma; sequencing grade) for
6 h at 37 °C, and the peptides were resolved on a Phenomex ODS
reverse phase HPLC column (2.1 × 250 mm) containing 0.08%
trifluoroacetic acid and with a gradient of 5-65% acetonitrile over
70 min (32, 33). The peaks were collected and sequenced on a Porton
LF3000 gas phase sequencer as described previously (32, 33, 42, 43). In the case of the reactions with the TGase 3 enzyme, or TGase 3 followed
by TGase 1, peptides were poorly resolved due to extensive cross-linking; in these experiments, 1-min fractions were collected across the peak for sequencing (32). Circular dichroism spectra on the
intact SPR1 proteins or synthetic peptides (see below) were performed
as before (32, 33, 43).
Use of Synthetic Peptides--
The following peptides based on
published human SPR1 sequences (13) were synthesized and purified by
HPLC: human SPR1 head A domain, SSQQQKQPCIPPP; human SPR1 head B
domain, PPPQLQQQQVKQPCP; human SPR1 head A + B domain,
SSQQQKQPCIPPPQLQQQQVKQPCQ; human SPR1 tail domain, SIVTPPPAQQKTKQK;
human SPR1 central domain repeat, (PKVPEPCQ)2,
(PKVPEPCQ)4, and (PKVPEPCQ)6. Kinetic constants
using the head and tail domain peptides as substrates for putrescine incorporation were determined exactly as described before (32, 33, 42).
Recently, we described the preparation of a recombinant human SPR2
protein and demonstrated that it is a favored substrate of the TGase 3 enzyme in vitro and in vivo (32). In the present study, we have repeated these experiments using recombinant human and
native mouse SPR1 proteins. We show here that both the TGase 3 and 1 enzymes are likely to be essential for SPR1 cross-linking in
vivo, and in this consecutive order of reaction.
Expression and Purification of the Recombinant Human SPR1
Protein--
Following expression in bacteria using the pET11a system,
the human SPR1 protein was enriched from bacterial lysates by dialysis into 25 mM citrate buffer, pH 3.6 (in which it was very
soluble), and purified by chromatography on a Mono-S fast protein
liquid chromatography column (Fig. 1).
The maximal yields were of the order of 2-5 mg/liter, typical of the
CE proteins loricrin (33) and SPR2 (32) studied previously. As expected
from its unusually basic pI value, the expressed SPR1 protein migrated
on SDS gels with an apparent molecular mass of ~15 kDa, somewhat
higher than the 10-kDa size calculated from its known sequence (Fig. 1,
inset).
Isolation of Native SPR1a and SPR1b from Cultured Mouse Epidermal
Keratinocytes--
For the purposes of comparison of biochemical and
structural properties, we isolated native mouse SPR1 proteins from
cultured keratinocytes. Under the conditions used, mouse keratinocytes express high levels of the SPR1 proteins, which consist of two gene
products, termed SPR1a and SPR1b, that differ primarily by the presence
of 13 or 14 central domain peptide repeats, respectively (14). By
dialysis of phosphate-buffered saline extracts of keratinocytes into
the pH 3.6 citrate buffer, the vast majority of epidermal proteins
precipitated, thereby enriching for the SPRs, which were then purified
to homogeneity on the Mono-S column. They were separated by cutting out
bands from SDS gels. Their total yield was ~1 mg/108
confluent cells. These data indicate that a significant amount of the
SPR1a/b proteins remain soluble in the keratinocytes cultured under
these conditions.
Circular Dichroism Spectra of Native and Recombinant SPR1
Proteins--
CD spectra were performed to evaluate the secondary
structures of the recombinant human and native mouse SPR1 proteins.
They have a limited degree of organized structure in phosphate-buffered saline (data not shown) or the TGase enzyme assay buffer at 20 °C,
but there was essentially no Epidermal TGases Use Recombinant Human and Native Mouse SPR1
Proteins as Complete Substrates but in Different Ways--
Three TGase
enzymes that are expressed in stratified squamous epithelia were used
to cross-link in vitro the recombinant SPR1. We used the
same molar amount (typically 500 nM) of each of the several
isoforms of TGase 1, the only known active form of TGase 2, and
inactive or activated forms of TGase 3, in order to make direct
comparisons of their reactions (44, 45).
First, we measured the amount of isopeptide cross-link formed in the
reactions and showed (Fig. 3) that most
of the enzymes inserted measurable amounts into recombinant human SPR1,
indicating that each enzyme uses this protein as a complete substrate.
However, the extent and rates of reactions varied widely. The TGase 2 enzyme inserted relatively little (<0.2 mol/mol) cross-link (Fig.
3B); the activated TGase 3 enzyme inserted the most (3.1 mol/mol) (Fig. 3C); and, as expected, the proenzyme form of
TGase 3 was inactive. A maximal amount of 1.1-1.2 mol/mol of
cross-link was inserted by each of the five TGase 1 isoforms employed,
but at different rates (Fig. 3A). The reaction was completed
within 5 min with the highest specific activity form (67/33/10-kDa
complex recovered from the membrane fraction of NHEK cells), but 15-18
h was required for the lowest specific activity form (full-length
membrane-bound forms expressed in NHEK cells or baculovirus). Previous
work has shown that there is about a 200-fold difference in specific
activity between the intact and activated forms (38), which is
reflected in these observed reaction rates. In general, the times
required for reaction completion for each of the five isoforms mirror
accurately their specific activities as previously measured against a
succinylated casein substrate. These data support the conclusion that
the various TGase 1 forms all use the SPR1 and casein substrates the
same way.
Second, we resolved the cross-linking reactions by SDS-polyacrylamide
gel electrophoresis and then performed autoradiography. By subsequent
excision of the 35S-labeled bands from the SDS gels, we
were able to obtain quantitative information. With each of the TGase 1 isoforms, 85-92% of the SPR1 protein remained as a monomer (Fig.
4A for 67/33/10-kDa complex recovered from the NHEK membrane fraction, cytosolic 67-kDa form, and
baculovirus intact form; data for other isoforms not shown); only
traces of protein were oligomerized into apparent dimers or larger
species trapped at the top of the gel. Similarly, only 8% of the SPR1
protein was oligomerized by the TGase 2 enzyme (Fig. 4A).
However, Western blotting analyses with a specific TGase 2 antibody
revealed that this was not due to SPR-SPR oligomerization but rather
mostly due to autocatalytic cross-linking to TGase 2 itself (data not
shown), a phenomenon described previously for this (32, 47, 48) and the
related factor XIIIa TGase enzymes (49). On the other hand, the TGase 3 enzyme cross-linked the proteins, readily giving rise to short
oligomeric products so that 88-98% of the SPR1 protein had been
oligomerized (Fig. 4A). These data thus reveal fundamentally
different reaction processes between the three TGases.
Third, we cross-linked in the same way samples of native mouse SPR1a/b
proteins using the baculovirus full-length form of TGase 1, as well as
TGases 2 and 3 (Fig. 4B). The pattern of cross-linking was essentially identical to that shown above. However, in these cases,
the monomer and dimer bands of mouse SPR1a/b appeared to be broader,
and some protein had migrated at a slightly faster rate than in the
EDTA controls. Based on Coomassie staining, Western blots, and amino
acid analyses (data not shown), this shift was probably due to
intrachain cross-linking (i.e. some monomer and dimer SPR1
proteins had become more compact and migrated faster). A similar
observation has been made previously in the in vitro cross-linking of loricrin (33). Moreover, these data mean that the ways
in which the various TGases use the SPR1 proteins are independent of
the numbers of repeating peptide motifs of the central domain.
Kinetics of TGase 1, 2, and 3 Cross-linking of Recombinant SPR1
Proteins--
We determined the kinetic constants of the cross-linking
of the recombinant human SPR1 protein with the baculovirus-expressed full-length TGase 1, TGase 2, and activated TGase 3 enzymes (Table I). By use of high concentrations of
putrescine, we suppressed putrescine oligomerization of SPR1. Estimates
of kinetic values are complicated by the fact that the SPR1 protein
serves as a complete substrate and that multiple Gln residues are
utilized (see below), so that the values obtained represent average
data for the multiple Gln residues used. The data show that there are significant differences in the kinetic efficiencies
(kcat/Km) of the reactions.
As suggested by the autoradiograms of Fig. 4, the TGase 3 enzyme
cross-linked SPR1 2-4 times more efficiently than TGases 1 or 2. These
data are very similar to those for recombinant loricrin (33) and SPR2
(32). From the double displacement mechanisms involved, we also
calculated the Km values for putrescine in the human
SPR1 reaction, which show a much higher value for the TGase 3 enzyme.
These data suggest that this enzyme greatly prefers to use the Gln
residues of SPRs for cross-linking.
We also determined kinetic parameters for the TGase 1 and 3 enzymes
using as substrates synthetic peptides of sequence corresponding to the
head and tail domains of human SPR1 (Table I). While the kinetic
efficiency values were all less than for the intact protein, the values
for the tail domain peptide were only about 2-fold less. However, there
were wide variations for the head domain sequences. We note that there
are two Gln/Lys regions encompassing residues 1-13 and 14-25, which
we have termed the head A and head B domains delineated by a triple Pro
motif (see Fig. 5D). We found that the TGase 1 enzyme used the head B domain sequences >100 times
more efficiently than the head A domain sequences, but the TGase 3 enzyme used the head A domain 18 times more efficiently than the head B
domain. Thus, there are apparent sequence-specific preferences for
these two TGases.
Amino Acid Sequencing Analyses of Cross-linking Reactions By The
TGase 1 and 3 Enzymes in Vitro--
Next, we obtained more specific
information on the residues used for cross-linking in the recombinant
SPR1 by the TGase 1 and 3 enzymes. Due to very low degrees of reaction
(Figs. 3 and 4), we could not obtain useful information for TGase 2 cross-linking. Samples of uncross-linked or cross-linked protein were
digested to completion with trypsin, and the peptides were separated by HPLC. Amino acid sequencing of shifted, reduced, or new peaks provided
quantitative information on the utilization of every Gln and Lys
residue of the protein.
In the case of the reaction with the baculovirus full-length TGase 1 (Fig. 5B), the peptide peaks eluted at 23 min (residues 38-43), 30 min (residues 44-51), 36 min (residues 52-67), and 37 min
(residues 22-36) containing central domain repeats and were not
visibly reduced. (Note that Lys59 was reproducibly only
partially cleaved by trypsin). However, the peptide eluted at 22 min
containing residues 1-6 was reduced by about 20%; those eluted at 39 min containing residues 68-84 and at 41 min containing residues 7-21
were reduced by about 60%. Several smaller new peptides appeared late
in the chromatogram that contained cross-links involving Gln and Lys
residues of these peptides. Also, we observed in these peaks about a
5% utilization of Lys43, indicating a minor degree of
cross-linking of the central repeating domain (Fig. 5D,
upper numbers). Note that tryptic peptides
containing the terminal residues 85-86 and 87-88 were not resolved by
this HPLC program. However, these residues were encountered in the unresolved peak eluted between 42-48 min, so that it was possible to
estimate the relative percentages of these and all other Gln and Lys
residues used for cross-linking as summarized in Fig. 5D
(upper numbers). The total amounts used
corresponded to the appearance of equivalent amounts of isodipeptide.
The total loss of these residues suggested that the TGase 1 enzyme had
inserted about 1 mol/mol of cross-link, which is consistent with the
amount isolated directly.
Furthermore, samples that had been cross-linked by the high specific
activity 67/33/10 kDa TGase 1 form recovered from the membrane fraction
of NHEK cells gave similar data on residue usage (not shown). Together
with several foregoing data, it appears that the forms of TGase 1 all
treat the SPR1 substrate the same way and validate use of the
baculovirus-expressed full-length enzyme, which is more stable and
available in quantity.
In the case of cross-linking with the TGase 3 enzyme (Fig.
5C), the peptides eluted at 23, 30, 36, and 37 min
containing central domain sequences were reduced by about 10%,
suggesting a minor degree of usage of Lys37,
Lys43, and Lys59 for cross-linking. However,
peptides containing head (residues 1-6 at 22 min and residues 7-21 at
41 min) and tail (residues 68-84 at 39 min) domain sequences were
quantitatively shifted to an unresolved peak late in the chromatogram.
By sequencing 1-min time aliquots across this peak, it was possible to
estimate the extents of usage of each Gln and Lys residue of the end
domains (Fig. 5D, lower numbers),
although as many as four sequences were running simultaneously. These
accounted for >95% of about 3.1 mol/mol of inserted isodipeptide.
Samples that had been cross-linked by these TGase 1 or TGase 3 enzymes
for only 6 h yielded nearly identical data on Gln/Lys residue
usage, except that the amounts used were less because of incomplete
reaction, indicating that there were no time-dependent preferences for cross-linking.
Correlation of in Vitro and in Vivo Cross-linking of SPR1
Proteins--
In Fig. 6, we have
summarized the present in vitro data for TGases 1 and 3, and
compared them with the in vivo utilization of Gln and Lys
residues of SPR1 proteins observed in our previous studies on CEs
isolated from either human foreskin (1, 2) and cultured epidermal
keratinocytes (Fig. 6B)
(4)2 or mouse forestomach
epithelia (Fig. 6C) (5). Comparisons of mouse and human data
are relevant, since the numbers, locations, and sequences around the
head and tail domain Gln and Lys residues are very similar (14).
Several points emerge. First, there were no cases of usage of central
domain Gln or Lys residues in the in vivo studies, which
compares well with their summed <5% utilization for cross-linking
in vitro. Second, overall there are remarkable similarities
in the patterns of utilization of the head A and B and tail domain
sequences: for virtually every residue, the relative profiles were
retained. The most significant difference appears to be
Gln5 of human SPR1, which comparatively was used about
twice more in vitro than in vivo. Third, these
data suggest that the recombinant SPR1 protein is treated in a
generally very similar way by the TGase 1 and 3 enzymes cumulatively
in vitro as occurs in vivo in the foreskin
epidermis or forestomach epithelium and thereby offer further evidence
that the recombinant protein had adopted its native configuration
in vitro. Moreover, fourth, it becomes clear that
both the TGase 1 and 3 enzymes are required for the appropriate cross-linking of the SPR1 proteins in vivo. In
view of the differences in the usage of some Gln residues of the head domain in vivo compared with the summed in vitro
data for TGases 1 and 3, we cannot however exclude the possibility that
another TGase enzyme(s) may also contribute to the effective
cross-linking of the SPR1 proteins in vivo (50). We conclude
that the TGase 2 enzyme is only weakly involved, if at all (Figs. 3 and
4). Similar conclusions were made for another major CE structural
protein, loricrin (33), but these data differ significantly from those of SPR2, for which essentially only the TGase 3 enzyme was used (32).
However, we observed some asymmetry in the distribution of usage of
head and tail domain sequences in vitro and in
vivo (Fig. 6). The majority of the Lys residues used are located
on the tail domain, while most Gln residues used are located on the
head domains. Since the first step of a TGase cross-linking reaction
involves recognition of a donor Gln residue (44-46), our in
vivo and in vitro observations strongly suggest that
TGase substrate specificities are governed primarily by the head domain
sequences of the SPRs. Consistent with this conclusion, we observed
that the patterns of in vitro cross-linking of recombinant
human SPR1 and native mouse SPR1a/b proteins, which have highly
homologous head domain sequences, were essentially identical (Fig.
4D), although they contain six or 13/14 central domain
peptide repeats, respectively. Also, each of the Gln and Lys residues
of the tail domain sequences was used relatively approximately equally
by the TGase 1 and 3 enzymes, although 3-fold less by TGase 1 (Figs. 4
and 5).
Differential Functional Use of Head A and B Domains by TGases 3 and
1, Respectively--
However, there were clear differences in
utilization of different head domain sequences. Examination of the
kinetic data for synthetic peptides of Table I and the cross-linking
data of intact recombinant SPR1 of Figs. 5 and 6 reveal that the head A
domain sequences were used almost exclusively (>90%) by TGase 3, and those of head B mostly (~85%) by TGase 1. Furthermore, in the case
of the TGase 3 enzyme, more than 3 mol/mol of isopeptide cross-link
were inserted, and ~90% of the protein was oligomerized into dimers
and tetramers (Fig. 4). It is therefore likely that head A domain
sequences were used by the TGase 3 enzyme in vitro largely
for interchain head-to-head, or head-to-tail cross-linking. However, we
cannot rule out the possibility of significant intrachain cross-linking
as well. The reason for this uncertainty is that it was necessary to
proteolyze the in vitro cross-linked products (Fig. 5) in
order to elucidate sequencing information; thus, only fragments of
sequences were obtained joined by isopeptide bonds, so that it remains
unclear whether the cross-links were interchain or whether some were
intrachain as well (4). On the other hand, the TGase 1 enzyme inserted
1.1-1.2 mol of isopeptide into the recombinant human and native mouse
SPR1 proteins using almost exclusively the head B domain sequences (see
Fig. 5B). Since only ~11% of the protein was
oligomerized, it appears that most of the cross-linking in
vitro involved intrachain links within individual SPR1 protein
molecules, as inferred from the mobility shifts seen in Fig. 4. We note
that there was a minor degree of overlap in these two enzyme functions,
since 5-10% of TGase 3 cross-linking involved head B domain
sequences, and ~15% of the TGase 1 reaction involved interchain
cross-linking as well as head A domain sequences (Fig. 6).
The present conclusion that the head A and B domains are used in
vitro for cross-linking by the TGase 3 and TGase 1 enzymes, respectively, is entirely consistent with our previous data for the
recombinant human SPR2 protein (32). Only interchain cross-linking was
observed with both enzymes, and moreover, TGase 3 inserted >10 times
more cross-links than TGase 1 (32). In this regard, it is noteworthy
that the SPR2 protein contains the equivalent of head A domain
sequences only, since a second Gln/Lys-rich region following a
triple Pro motif is absent (13, 32). These differences imply important
structural-functional differences in their utilization in epithelia.
Analysis of in Vivo Cross-links--
SPRs were found to be
cross-linked in vivo to many other proteins, including to
themselves. Our data base of in vivo cross-links is based on
extensive proteolysis procedures in order to obtain informative
peptides for sequencing and includes 234 occurrences of human (4) and
121 mouse (5) SPR proteins, of which 38 involved direct SPR-SPR
linkages. Since the present work on SPR1 and the previous study on SPR2
proteins explored the cross-linking of the SPR proteins to themselves,
it is now possible to predict which TGase enzymes may have formed the
human and mouse cross-links in vivo (Table
II). Some peptides clearly resulted from
interchain cross-linking between separate SPR protein molecules, but in
many cases, the short piece of SPR sequence recovered after proteolysis precluded assignation of the type of cross-linking. Of the 38 SPR-SPR
cross-linked peptides, eight involved head A-head A sequences of which
most were interchain, and 11 involved head A-tail linkages. We can now
conclude that these were likely inserted by the TGase 3 enzyme. A group
of seven involved head A-head B linkages and seven tail-tail linkages
might have been inserted by either the TGase 1 or 3 enzymes. We predict
that the TGase 1 enzyme could have inserted four head B-tail
cross-links and one head B-head B linkage.
However, reconstruction of the potential TGase enzymes used for the 97 single SPR-protein (X) or 220 SPR-X-SPR linkages is less certain. We
note that the head A domain Gln residues were used three times more
than head B domain Gln residues (4), which based on the present
observations, suggests that the TGase 3 and 1 enzymes preferentially
use head A and head B domain sequences, respectively. Thus, we propose
that 140 could have been inserted by TGase 3, 61 by TGase 1, and 116 by
either enzyme (Table II).
SPR Cross-linking with Loricrin--
Previously, we have
demonstrated that loricrin is likewise cross-linked in vivo
by both the TGase 1 and 3 enzymes using different Gln and Lys residues
(33), but in this case, there was more overlap between the various
residues used and enzymes (about one-third). We have documented 38 SPR1-loricrin and 19 SPR2-loricrin peptides in human epidermal CEs as
well as 58 SPR1-loricrin and 26 SPR2-loricrin peptides from mouse
forestomach CEs (4). Accordingly, now we have reexamined these 141 data
elements to ascertain which Gln and Lys residues were joined in an
attempt to correlate the TGase enzymes used (Table
III). The analyses show that SPR1 head A
domain or SPR2 head domain sequences, predicted to be used by the TGase 3 enzyme, were preferentially cross-linked to loricrin Gln and Lys
residues that themselves were predicted to be utilized rather specifically by the TGase 3 enzyme. Likewise, we find a very high correlation of cross-linking of head B domain SPR1 sequences to loricrin at sites predicted to be used preferentially by the TGase 1 enzyme. These data strongly infer a common highly coordinated mechanism
for the cross-linking of loricrin and SPRs together in
vivo.
Recombinant SPR1 Is Cross-linked Sequentially by the TGase 3 Followed by the TGase 1 Enzymes to Form Very Large Oligomers--
In
view of the above considerations that both the TGase 1 and 3 enzymes
are required for the correct cross-linking of SPR1 proteins in
vivo using different Gln/Lys residues of the head B and A domains,
respectively, we wondered whether the two enzymes operate
simultaneously or at different times. To test this, we repeated the
in vitro reactions using 35S-labeled recombinant
human SPR1 in which we first separately cross-linked to completion with
either TGase 1 or TGase 3 and then performed a second cross-linking
reaction on the products with TGase 3 or TGase 1. When
baculovirus-expressed full-length TGase 1 was used to cross-link the
short oligomeric products of an initial complete TGase 3 reaction,
there was a major shift to very large material that could not enter the
gel: excision of 35S label revealed that within 12 h,
>90% had been thus converted (Fig.
7A). Similar data were
obtained when the high specific activity 67/33/10-kDa complex was used
for 1 h (not shown). In both cases, a total of 4.2 mol of
isodipeptide cross-link had been inserted per mol of SPR1 protein, or
about 1 mol/mol more by TGase 1, thus implying that additional Gln/Lys
residues had been recruited. Following trypsin digestion and HPLC
fractionation, a very broad unresolved peak of highly cross-linked
material was observed, not unlike that for TGase 3 enzyme alone (Fig.
5, compare E with C). Sequencing 1-min aliquots
across the peak revealed significant increases in the amounts of head B
Gln residues used for cross-linking, but those of head A domain
sequences were essentially unchanged (Fig. 5G,
upper numbers; compare with Fig. 5D,
lower numbers). Thus, the additional
cross-linking performed in the secondary TGase 1 reaction involved
mainly head B sequences. We also repeated these experiments in the
reverse order of cross-linking by TGase 3 following initial maximal
TGase 1 cross-linking (Fig. 7B). In this case, however,
there was not a major change in the cross-linking pattern. The
secondary TGase 3 enzyme reaction transformed about 20% of the
monomeric sized TGase 1 products into short oligomers and 5% into very
large oligomers that could not enter the gel, but most remained as the
monomer. Also, only about an additional 0.5 mol of cross-link/mol was
inserted. Sequencing analyses revealed minor changes in the degrees of
utilization of head A domain residues by the TGase 3 enzyme (Fig. 5,
F and G, lower
numbers).
Together, these in vitro data strongly imply an obligatory
temporal order of the cross-linking of the SPR1 proteins in
vivo by the TGase 3 enzyme first, followed later by TGase 1. For
apparent spatial and/or structural reasons that are not clear, it
appears the head A domain sites must be utilized first by the TGase 3 enzyme before TGase 1 can efficiently recognize the head B domain sites, whereas occupancy of head B sites first interferes with a
subsequent TGase 3 reaction. Accordingly, further attempts to obtain
detailed structural information on the SPR1 proteins seem desirable.
A Model for Cross-linking of SPRs and Loricrin Together in
Vivo--
Any model must account for several established aspects of
SPR and loricrin expression in epithelia. First, when co-expressed in
epithelia, SPR expression always precedes that of loricrin (21, 51).
Second, we note that SPRs are very soluble and are deposited in both
the nucleus and cytoplasm (21, 51), and antibodies elicited against
tail domain epitopes used for cross-linking (Figs. 4 and 5; Table I)
yield a bright cytoplasmic staining reaction in tissues such as footpad
epidermis or rodent forestomach, where they are abundantly expressed
(14, 21). Third, on the other hand, loricrin is insoluble in
physiological conditions (33). Fourth, a peripheral staining by the SPR
antibodies is observed in more differentiated cells coincidentally with
initial loricrin expression (21, 51). Fifth, antibodies against both SPRs and loricrin no longer react in fully cornified epithelia, indicating lost epitopes due to cross-linking (14, 21, 41, 52). Sixth,
the activated TGase 3 enzyme is soluble (cytosolic) (40, 53), whereas
the most highly active forms of TGase 1 in keratinocytes are
membrane-bound (37, 38, 54, 55). Finally, any model must conform to the
phenotypic observations of the TGM1 gene
Our data allow the following model, which is consistent with all of the
extant observations. We propose that SPR proteins might be expressed
early because more time is required to gather them together by an
active (3) or passive transport system near the cell periphery than is
required for loricrin. Then juxtaposed loricrin and SPRs at or near the
cell periphery are first cross-linked together to form small oligomers
by the cytosolic TGase 3 enzyme using most of the head A domain and
some tail sequences of the SPR1 and -2 proteins and favored internal
Gln and head/tail Gln/Lys residues of loricrin. Subsequently, the
highly active TGase 1 enzyme form(s) anchored to the membrane attach
this loricrin-SPR1/2 complex to the growing CE structure by further
cross-linking using available SPR1 head B domain and remaining tail
sequences of SPR1/2 to the additional available head/tail sequences of
loricrin. These temporal differences may be explained in part by TGase
enzyme availability as well as in part by the particular conformation of the SPR head domain sequences. It is conceivable that the SPR1/2 head A-like domain sequences that are homologous to those of loricrin (57) evolved so as to favor initial cross-linking by TGase 3 because it
is a cytosolic enzyme. Utilization of SPR1 head B domain residues or
other more internal loricrin residues by TGase 1 can occur only
subsequently (Fig. 7). Because the vast majority of TGase 1 activity is
membrane-bound presumably at or very near the site of final protein
deposition of the CE, this enzyme is used for the final reinforcement
stages of CE assembly.
Furthermore, this model is readily consistent with the pathophysiology
of lamellar ichthyosis disease caused by absent TGase 1 activity.
Because the TGase 3 enzyme cross-links the proteins with high
specificity at specific sites different from those of the TGase 1 enzyme, the TGase 3 enzyme cannot adequately compensate or supplement
for the lost TGase 1 enzyme activity. Thus, the reinforcement stage of
CE assembly occurs only poorly, resulting in the devastating loss of
barrier function seen in human lamellar ichthyosis patients. Similarly,
this model is entirely consistent with the observed morphological and
histological features and severe loss of barrier function in the mouse
A similar scenario should be applicable to the CEs of those epithelia
that express much less or no loricrin and where the SPRs instead are
mostly cross-linked to themselves and other scaffold proteins.
Furthermore, the second most frequent partner to which SPRs have been
found cross-linked in vivo is involucrin (4). Again, both
head A and head B domain sequences were commonly used, suggesting the
involvement of both TGases 1 and 3 (4). However, comparable analyses of
SPRs and involucrin cross-linking are not yet possible, since only
limited data on favored residues used by TGases are available for
involucrin (58).
Conclusions--
Altogether, these studies indicate that the
cross-linking of SPR proteins to the CE for effective barrier function
is a complex process, involving multiple TGases operating through
numerous Gln and Lys residues, and in multiple well coordinated steps. The availability of recombinant loricrin and SPR proteins now will
permit further experiments to explore how they function together to
affect the biomechanical properties of the CE and the epithelium in
which they are expressed (5). Such experiments may also shed light on
the reason why there are multiple apparently functionally very similar
yet differentially expressed SPR1 and SPR2 gene products.
or
structure but have some organized structure associated with their
central peptide repeat domain. The transglutaminase (TGase) 1 and 3 enzymes use the SPR1 proteins as complete substrates in
vitro but in different ways: head domain A sequences at the amino
terminus were used preferentially for cross-linking by TGase 3, whereas
those in head domain B sequences were used for cross-linking by TGase
1. The TGase 2 enzyme cross-linked SPR1 proteins poorly. Together with
our data base of 141 examples of in vivo cross-links
between SPRs and loricrin, this means that both TGase 1 and 3 are
required for cross-linking SPR1 proteins in epithelia in
vivo. Double in vitro cross-linking experiments
suggest that oligomerization of SPR1 into large polymers can occur only
by further TGase 1 cross-linking of an initial TGase 3 reaction.
Accordingly, we propose that TGase 3 first cross-links loricrin and
SPRs together to form small interchain oligomers, which are then
permanently affixed to the developing CE by further cross-linking by
the TGase 1 enzyme. This is consistent with the known consequences of
diminished barrier function in TGase 1 deficiency models.
INTRODUCTION
Top
Abstract
Introduction
References
-(
-glutamyl)lysine or
N1,N8-bis(
-glutamyl)spermidine
isopeptide bonds formed by transglutaminases (TGases), resulting in an
insoluble macromolecular protein complex ideal for barrier function
(6-9). Moreover, for reasons that are not yet understood, individual
SPR family members are differentially expressed in highly variable ways
in many different types of epithelia (10-31).
MATERIALS AND METHODS
RESULTS AND DISCUSSION
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Fig. 1.
Purification of recombinant human SPR1 and
native mouse SPR1a/b. The SPR-rich citrate supernatants were
chromatographed on a Mono-S column, from which the pure proteins were
recovered. This profile is for the recombinant human SPR1 protein; the
arrow indicates the position of elution of the native mouse
SPR1a/b proteins. Inset, SDS gels and Western blots of
purified proteins. Lane 1, Coomassie staining of
purified recombinant human SPR1; lane 2, Western
blot of lane 1 using the broadly reacting SPR1
antibody (14); lanes 3 and 4,
Coomassie staining of native mouse SPR1a and SPR1b, respectively.
Inset, molecular mass markers in kDa are as shown.
or
structure present (Fig. 2A, lines 1 and
2, for recombinant and native proteins, respectively). However, the signal strength of native mouse SPR1a/b was reproducibly greater than that of recombinant human SPR1. Mouse and human SPR1 proteins have highly homologous head, tail, and central eight-residue peptide repeat sequences but differ by containing 13 versus
6 central repeats, respectively. We performed additional experiments with synthetic peptides. First, peptides corresponding exactly to the
head or tail domains of human SPR1 proteins generated only very weak CD
signals, suggesting little significant structure (Fig. 2B,
lines 1 and 2, respectively). On the
other hand, peptides containing two (line 3),
four (line 4), or six (line
5) peptide repeats characteristic of the eight-residue
central domain of SPR1 proteins generated CD spectra that correlated
with peptide size. Next, we assessed the overall structural properties
of the SPR1 proteins as a function of temperature and guanidine
hydrochloride. The recombinant human SPR1 (Fig. 2C,
line 1) protein was at least partially unfolded
by heating at 40 °C (line 2) and 60 °C
(line 3), but the signals were normalized when
returned to 20 °C (dotted line), indicating
refolding of the protein structures. Similarly, recombinant human (Fig.
2D, solid line 1) or native
mouse (dotted line 1) SPR1 proteins
could be reversibly denatured upon the addition of (lines
2) and then renatured upon the subsequent removal of (dashed lines) 4 M guanidine
hydrochloride. Together, these data suggest that only the central
peptide repeat domains of SPR1 proteins contain organized structures
and that the signal strength of the CD spectra correlates to some
degree with the numbers of peptide repeats. Moreover, the recombinant
human protein possesses an organized structure similar to that of the
native mouse SPR1a/b proteins of cultured keratinocytes. From this we
conclude that the recombinant human SPR1 protein had folded into a
native configuration upon isolation from the bacteria and is therefore
appropriate for use in further biochemical experiments.
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Fig. 2.
Circular dichroism spectra of SPR1
proteins. A, spectra of recombinant human SPR1
(line 1) and native mouse SPR1a/b proteins (line
2), measured at 20 °C. B, spectra of synthetic
peptides corresponding in sequence to human SPR1 of the 25-residue head
domain (line 1), the 15-residue tail domain (line
2), or of two (line 3), four (line 4), or
six (line 5) repeats of the eight-residue central repeating
motif, measured at 20 °C. C, spectra of recombinant human
SPR1 measured at 20 °C (line 1), 40 °C (line
2), or 60 °C (line 3) or after a 20-60-20 °C
temperature transition (dotted line). D, spectra
of recombinant human SPR1 (solid lines) or mouse SPR1a/b
(dotted lines) in the absence (lines 1) or
presence of (lines 2) or after removal of (dashed
lines) 4 M guanidine hydrochloride, measured at
20 °C.
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Fig. 3.
TGases cross-link recombinant SPR1 to varying
degrees. Equimolar amounts (~500 nM) of five
isoforms of TGase 1 (A), TGase 2 (B), and
inactive proenzyme as well as dispase-activated TGase 3 (C)
were used to cross-link 10 µg of SPR1 for varying times as shown.
Aliquots were removed and digested to completion with proteases, and
the products subjected to amino acid analysis to measure the amount of
isodipeptide cross-link. The data are the averages of 2-4 separate
experiments.
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Fig. 4.
TGases cross-link SPR1 proteins in varying
patterns. A, recombinant human SPR1; B,
native mouse SPR1a/b. Reaction conditions are the same as those of Fig.
3. Four forms of native TGase 1 harvested from NHEK were used, of which
two are shown in A, as well as the recombinant
membrane-bound (mb) TGase 1 from baculovirus. The
SDS-polyacrylamide gels were examined by autoradiography of
35S-labeled proteins. Note that in this case the mouse
SPR1a and -b proteins were not resolved but yielded a broad monomer
band. The times of reactions are shown. C, control reactions
performed for 18 h in the presence of 10 mM EDTA. The
sizes of standards are shown on the left. The
asterisk marks the position of the 67-kDa component of the
TGase 1 enzymes used. After exposure to x-ray film, bands were cut out
for quantitation of 35S label.
Kinetic parameters of cross-linking of recombinant SPR1 and synthetic
peptides by TGases
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Fig. 5.
Cross-linking in vitro of
recombinant human SPR1 by TGases 1 and 3. Tryptic digests of
protein before (A) or after cross-linking by the TGase 1 (B) or TGase 3 (C) enzymes are shown. The
products were resolved by HPLC as described under "Materials and
Methods." D, sequence of human SPR1 listing the percentage
of utilization of Gln and Lys residues: TGase 1 enzyme
(upper row); TGase 3 enzyme (lower
row). Similar experiments were done for double enzyme
reactions of TGase 3 followed by TGase 1 (E) or TGase 1 followed by TGase 3 (F), together with the estimates of
residue usage (G).
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Fig. 6.
Comparative utilization of Gln and Lys
residues for cross-linking in vitro and in
vivo. A, cross-linking of recombinant SPR1
in vitro (present work). B, summarized data on
cross-linking of human SPR1 proteins in vivo generated from
amino acid sequencing of isolated CEs from several sources of human
epidermal keratinocytes (1, 2, 4).2 C, summary
of data for mouse SPR1 proteins in vivo generated from amino
acid sequencing of isolated forestomach CEs (5). Red
bars, Gln residues; blue bars, Lys
residues; open bars, TGase 1 cross-linking;
closed bars, TGase 3 cross-linking. Note that
unlike human SPR1a/b, mouse SPR1a/b contain different numbers of
central domain peptide repeats, which affects the numbering of terminal
domain residues. Thus, tail sequence positions in C are
numbered from the conserved terminus, denoted as E.
Predicted TGase enzymes used for SPR cross-linking in human and mouse
CEs in vivo
Cross-links between loricrin and SPRs: Predictions of TGases used
. Numbers of occurrences are shown in parenthesis. Cross-links
involving tail domain sequences are not listed, since there was no
apparent TGase enzyme specificity.
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Fig. 7.
Double reaction of recombinant SPR1 reveals
an obligatory temporal order of cross-linking by TGase 3 followed by
TGase 1. Reactions as in Figs. 3 and 4 were performed with ~700
nM of recombinant baculovirus TGase 1 and activated TGase
3. In A, the products of an 18-h TGase 3 reaction were
reacted again with TGase 1 for the times shown. In B, an
18-h TGase 1 reaction was reacted again with TGase 3 for the times
shown.
/
mouse (56)
and the natural human knockout of the TGM1 gene in lamellar ichthyosis.
/
model.
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ACKNOWLEDGEMENTS |
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We thank Dr. Claude Backendorf for providing cDNAs for human SPR proteins, Dr. Ulrike Lichti for expert advice, and Dr. Soo-Youl Kim for assistance in the initial construction of the expression vector.
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FOOTNOTES |
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* 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.
To whom correspondence should be addressed: National Institutes of
Health, Bldg. 6, Rm. 425, 9000 Rockville Pike, Bethesda, MD 20892-2752. Tel.: 301-496-1578; Fax: 301-402-2886; E-mail: pemast{at}helix.nih.gov.
2 L. N. Marekov and P. M. Steinert, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: SPR, small proline-rich (also, for example, to indicate the SPR1 protein); CE, cornified cell envelope; HPLC, high pressure liquid chromatography; TGase, transglutaminase; NHEK, normal human epidermal keratinocyte(s).
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REFERENCES |
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