Lamellar ichthyosis is a congenital recessive
skin disorder characterized by generalized scaling and
hyperkeratosis. It is caused by mutations in the TGM1
gene that encodes the transglutaminase 1 (TGase 1) enzyme, which is
critical for the assembly of the cornified cell envelope in terminally
differentiating keratinocytes. TGase 1 is a complex enzyme existing as
both cytosolic and membrane-bound forms. Moreover, TGase 1 is
proteolytically processed, and the major functionally active form
consists of a membrane-bound 67/33/10-kDa complex with a myristoylated
and palmitoylated amino-terminal 10-kDa membrane anchorage fragment. To
understand better how point mutations, deletions, and truncations found
in lamellar ichthyosis disease affect the structure and function of
TGase 1, we have expressed in baculovirus and keratinocytes a number of
reported TGase 1 mutants. The structural implications of these
mutations were examined using a homology-derived three-dimensional
model of TGase 1 generated from the known x-ray structure of the
related coagulation factor XIIIa enzyme. The present studies
demonstrate that loss of TGase 1 activity is not restricted to
mutations that directly affect the enzymatic activity. We report a new
class of mutations that impair the subsequent post-synthetic processing of the protein into its highly active functional forms.
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INTRODUCTION |
Congenital recessive ichthyoses
(CRI)1 describe a clinically
heterogeneous class of disorders of cornification that primarily affect
the epidermis and hair (1-6). The phenotype appears at birth often
presenting as a translucent collodion membrane encasing the newborn but
later progresses to a variety of clinical findings ranging between fine
white scaling, often combined with erythroderma, to large plate-like
brown scales without erythema. Other clinical findings include
palmar-plantar hyperkeratosis, eclabium, ectropion, scarring alopecia,
and diminished skin barrier function. Recent studies have revealed
marked biochemical (7, 8) as well as genetic variations in CRI patients
as follows: to date, in certain families, linkage has been described to
chromosome regions 14q11 (9, 10), 2q33-35 (11), or to neither
(11-13). However, all cases of the large brown plate scaling phenotype
have been linked to 14q11, and several different mutations in the
TGM1 gene located at this site have been identified (10, 11,
14-15). The term lamellar ichthyosis (LI), the former umbrella name of the CRI group of diseases, should now be used exclusively for the CRI
disease involving mutations in this gene (13).
The TGM1 gene encodes transglutaminase 1 (TGase 1) which is
a member of a class of enzymes that form
N
-(
-glutamyl)lysine or mono- or
bis(
-glutamyl)spermidine isopeptide bond cross-links between
proteins, thereby forming stable, insoluble macromolecular assemblies
(16-19). TGase 1 is one of at least three such enzymes known to be
involved in the formation of barrier function in stratified squamous
epithelia, in particular in the formation of the cornified cell
envelope, which is a 15-nm thick layer of protein deposited just inside
the cell periphery (18, 20-23). The cornified cell envelope is formed
when TGases 1 and 3 (and perhaps TGase 2) cross-link a variety of
structural proteins including desmosomal proteins, involucrin, the
small proline-rich proteins, and loricrin in the epidermis,
trichohyalin in the inner root sheath of the hair follicle, as well as
other undefined proteins in the hair fiber cuticle. In vitro
studies have indicated that some substrates including loricrin and
involucrin require both enzymes for apparent sequential cross-linking
to form normal stabilized structures (22, 24). This requirement may
explain the phenotype of LI disease; the TGase 3 enzyme cannot replace
the essential cross-linking performed by the absent TGase 1 enzyme. The
TGase 1 enzyme itself shows a complex pattern of activities since it exists in multiple cytosolic and membrane-bound forms in terminally differentiating keratinocytes (25, 26). However, whereas much of the
enzyme remains in the intact, very low specific activity (zymogen) form
of about 106 kDa, most activity resides in a proteolytically processed
form of 67/33/10-kDa chains that are held together by secondary
interactions while bound to the membrane through acyl myristate and
palmitate adducts on the 10-kDa portion (26, 27).
Mutations in the TGM1 gene that produce a defective enzyme
due to truncations, point substitutions, etc. are located on a number
of sites along the protein. Patients can be either homozygous for a
single mutation or compound heterozygous for two different mutations
(10, 11, 14-16). However, it is not yet clear how these changes affect
the structure and function of TGase 1. To explore this, we have
expressed in baculovirus and keratinocytes a number of reported mutant
TGase 1 forms to study their activity and processing properties. Also
we have examined the likely structural consequences of the mutations by
comparison with the known three-dimensional structure of the related
factor XIIIa (fXIIIa) enzyme.
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MATERIALS AND METHODS |
Construction of the Recombinant Transfer Vectors and Recombinant
Baculoviruses in Insect Cells--
All recombinant DNA technology was
done according to standard procedures (28, 29). A full-length wild type
human TGase 1 cDNA (30) was cloned into the baculovirus vector
pVL1392 (PharMingen) by insertion at the EcoRI sites. This
was then used as a template for the eight mutant forms (see Table I)
that correspond to mutations seen in LI patients selected in this
study. The TransformerTM Site-directed Mutagenesis Kit
(CLONTECH) based on the method described by Deng
and Nickoloff (31) was used for introduction of mutations.
The TGase 1 cDNAs in the pVL1392 vector were under the
transcriptional control of the strong baculovirus polyhedrin promoter. The nine TGase 1 recombinant virus clones (wild type and eight mutant
forms) were obtained by co-transfection of each vector with the
modified Autographa californica nuclear polyhedrosis virus
DNA (BaculoGold DNA, PharMingen). BaculoGold DNA carries a lethal
deletion and does not code for viable virus particles by itself.
Co-transfection of this DNA with a complementing plasmid construct
rescues the lethal deletion of this virus DNA and reconstitutes, by
homologous recombination, viable virus particles inside the transfected
insect cells (32). Insect cells (Sf9, PharMingen) were grown in
Grace's insect medium supplemented with 10% fetal calf serum (Life
Technologies, Inc.).
In the co-transfection experiments, 2 × 106 cells
were plated in 60-mm dishes. Two µg of each of the nine vectors was
separately mixed with 0.5 µg of linearized viral BaculoGold DNA and
incubated for 10 min at 23 °C. After incubation, 1 ml of
transfection buffer (25 mM HEPES, pH 7.1, 125 mM CaCl2, 140 mM NaCl) was added to each tube and mixed. The DNA/transfection buffer mixtures were added to
plates that contained 1 ml of regular medium and incubated at 27 °C
for 3-4 h. At the end of the transfection, the medium was replaced
with fresh Grace's insect medium, and the cells were kept for 5 days
at 27 °C. The medium containing the nine recombinant virus clones
was amplified to produce a high titer stock solution, and the titers
(1-8 × 108 plaque-forming units/ml) were evaluated
by end point dilution. For protein production, cells were maintained
and infected, either as monolayer or in suspension, in a rotary shaker,
with 1-4 × 108 plaque-forming units/ml of culture at
a density of 1.5-2 × 106 cells (>95% viability)
per ml of culture. Expression of the nine recombinant TGase 1 clones
was confirmed by Northern blot analysis. Protein production was
detected by Western blot using a specific polyclonal TGase 1 antibody
(33). For lipid modification experiments, Sf9 cells were
metabolically labeled (1 mCi each/ml of medium) with
[14C]palmitate or [14C]myristate (NEN Life
Science Products, 650 Ci/mol). The labels were added 4 h prior to
harvesting.
Construction of Mammalian Expression Vectors--
The wild type
and four mutant TGase 1 cDNA clones (R141H, R322Q, S41Y, and R314L)
were excised at the NotI and XbaI sites from
pVL1392 vector and directly subcloned into the mammalian expression
vector pCMV (CLONTECH), under the control of the
human cytomegalovirus immediate enhancer/promoter. In addition, a tag of histidine residues had been attached to the carboxyl-terminal end of
the wild type and the two active mutant TGase 1 clones (S41Y, R314L).
We generated these clones by polymerase chain reaction amplifying with
standard conditions (34) from TGase 1 cDNA vectors used for
baculovirus expression. The polymerase chain reaction primers were
designed with an extension of 16 nucleotides containing NotI
site at the 5'-end (plus primer,
5'-ATAAGAATGCGGCCGCATGGATGGGCCACGTTCCGATGTGGGCCGTTG-3') and an
extension of 18 nucleotides that encode for six histidine residues
followed by a termination codon TGA and BamHI site at the
3'-end (minus primer,
5'-TGCTCTAGACTAATGATGATGATGATGATGAGCTCCACCTCGAGATGCCATAGGGA-3'). The modified clones were sequenced to verify amplification
efficacy and then assembled into the mammalian expression vector pCMV
for transfection studies. These cultures were labeled with
[35S]cysteine/methionine as described below since we
found that the polyhistidine monoclonal antibody (Sigma) also
immunoprecipitated processed forms of profilaggrin and filaggrin that
are expressed in cultured NHEK cells in differentiating conditions;
these proteins do not contain cysteine or methionine (35).
Cell Culture, Transfections,
-Galactosidase Activity, and
Protein Assay in Keratinocytes--
Cryopreserved normal human
epidermal keratinocytes (NHEK) were obtained from
CLONTECH (San Diego, CA) and grown in calf skin collagen (Sigma)-coated dishes in serum-free keratinocyte growth medium
(KGM, Clonetics) at 0.05 mM Ca2+, supplemented
with 60 µg/ml bovine pituitary extract. Third passage cells were used
for transfection experiments. Transient transfections were performed in
triplicate using Lipofectin reagent (Life Technologies, Inc.), as
described previously (36). Briefly 2-3 × 105 cells
were plated in each 35-mm well of six-well culture plates 16-20 h
before transfection. Transfections were done when cultures had reached
70% confluency. Transfection efficiencies were always monitored by use
of a cytomegalovirus
-galactosidase construct (pCMV-
,
CLONTECH). Cells were washed once at 37 °C with
phosphate-buffered saline and then preincubated for 30 min at 37 °C
with keratinocyte serum-free medium. For each well, 1.5 µg of
reporter plasmid and 0.5 µg of pCMV-
were mixed with 6 µg of
Lipofectin and incubated for 20 min at 23 °C. The lipid/DNA mixture
was then added into each well and incubated for 3-4 h. After that,
medium was removed, and to improve transfection efficiencies, cells
were shocked with 15% glycerol in keratinocyte serum-free medium for 3 min. Medium containing either 0.05 or 1.2 mM
Ca2+ was then replaced. Some transfection experiments were
performed in 60- or 100-mm dishes, with proportional increase in
seeding densities, amounts of plasmids, and Lipofectin. In some cases cells were harvested 50-120 h post-transfection, and in labeling experiments 6 h prior to harvesting, 2 µCi/ml of a mixture of [35S]cysteine and [35S]methionine (Amersham
Pharmacia Biotech, 14.3 mCi/ml) was added. In one set of experiments to
measure half-lives, this amount of isotope was added to the cultures
immediately following transfection (25-27) for 6 h, and then the
medium was replaced with high Ca2+ medium, and at different
time points cells were harvested.
Cells were lysed in 0.2 ml of ice-cold lysis buffer (50 mM
Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM
dithiothreitol, 1 mM EDTA, and a mixture of protease
inhibitors (Boehringer Mannheim) that included 0.2 mM
4-(2-aminoethyl)benzenesulfonyl fluoride, 5 µg/ml benzanidine, 5 µg/ml pepstatin, 5 µg/ml aprotinin), and the suspension was
homogenized by sonication. The samples were centrifuged for 15 min at
14,000 × g at 4 °C to separate the cytosolic and
particulate fractions. The particulate fractions were washed with lysis
buffer, and then membrane-bound proteins were extracted into lysis
buffer containing 0.1% Triton X-100 in ice for 30 min. After
centrifugation, solubilized proteins were separated by membrane debris
and washed with lysis buffer to remove the detergent (37).
Aliquots of the cytosolic and membrane-bound fractions were used for
TGase assay, TGase immunoprecipitation, column chromatography, and for
protein quantification, using methods described previously (25-27).
The
-galactosidase activity was assayed by using a commercial enzyme
assay system (Promega). Where necessary, TGase activities were
normalized by protein content and
-galactosidase activity. The final
data are the average of at least three independent experiments, each
with duplicate samples.
Cross-linked cell bodies were harvested following exhaustive extraction
in SDS and 2-mercaptoethanol as for the preparation of cornified cell
envelopes (20, 21), resuspended in 1 ml of phosphate-buffered saline,
and light scattering at 310 nm was measured.
In Vitro Cross-linking Experiment--
Membrane-bound TGase 1 was isolated from the particulate fraction of Sf9 cells exactly
as described above. Recombinant 35S-loricrin was used as a
complete substrate as described previously (24). In the cross-linking
experiment 25 µg of labeled loricrin (about 8 × 104
dpm) was utilized in a 100-µl reaction volume, and the amount of
recombinant baculovirus TGase 1 that incorporated 0.7 pmol/min [14C]putrescine (Amersham Pharmacia Biotech,, 118 mCi/mmol) into casein was used. The reaction was initiated by the
addition of 10 mM CaCl2. Aliquots were stopped
by addition of 10 mM EDTA after 15, 30, 60, and 120 min.
The cross-linked products were separated on a 4-12% polyacrylamide
gels and analyzed by autoradiography.
Generation of the TGase 1 Model--
The three-dimensional model
of human TGase 1 was generated on the basis of the known atomic
structure of human factor XIIIa (fXIIIa) as present in the Protein Data
Base (38) entries 1GGT and 1FIE (39, 40). By using the modeling
packages WHATIF (41) and INSIGHT-II (38, 42), the sequence of TGase 1 residues 107-789 was inscribed onto the structural frame provided by
fXIIIa residues to 43-729. By using loop-search and debumping options, the resulting model for TGase 1 was then optimized at positions where
deletions (Ser125, Gly187, and
Pro505) or non-conservative substitutions occurred,
respectively. Residues Gly96-Gly106 of TGase 1 including the 97NAAGDG102 hexapeptide were
modeled manually onto residues Asn18-Glu30 of
the fXIIIa activation peptide. Pictures were generated with the
INSIGHTII software.
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RESULTS |
Mutant TGase 1 Forms Studied--
The purpose of the present work
was to explore the structural-functional consequences of some of the
known mutations of the TGM1 gene seen in LI. Data from protein
expression and transfection studies were combined with structural
information from a predicted three-dimensional model of the TGase 1 enzyme based on the known crystal structure of the related factor XIIIa
enzyme. The TGase 1 mutations studied in the present work are listed in
Table I, and as our data show, they offer
a spectrum of alterations affecting the enzyme and afford new insights
into the molecular basis of LI disease.
Expression and Biochemical Characterization of Recombinant Human
Wild Type and Mutant Forms of TGase 1--
We expressed in the
baculovirus system the human wild type TGase 1 cDNA and the eight
mutant forms that have been reported in LI which are listed in Table I.
Protein expression was evident from 48 h postinfection and reached
a maximum at 72 h (Fig. 1, A and B). Western blot analyses using a specific
polyclonal anti-human TGase 1 antibody revealed that the recombinant
enzymes were not proteolytically processed in this system and remained
as the full-length protein with an apparent molecular mass of 97 kDa
(Fig. 1B). The mutation M422stop generated a truncated
protein of size about 55 kDa (Fig. 1, A and B).
As has been observed in NHEK cells, the baculovirus-expressed TGase 1 (wild type and mutant forms) was present in both the cytosol and
membrane-bound fractions. About 90% of each of eight recombinant
proteins was retained in the latter (data not shown), which is very
similar to the distribution of the TGase 1 enzymes in proliferating
basal epidermal keratinocytes or stationary NHEK cells grown in low
Ca2+ submerged liquid cultures (26). The one exception was
mutant form S41Y, in which about half of the protein was present in the cytosol (see Fig. 4F). The recombinant baculovirus-expressed
wild type TGase 1 was also modified by lipids (Fig. 1C). The
labeling of the cytosolic and membrane-bound enzyme forms with
myristate indicates the protein was constitutively
N-myristoylated as in keratinocytes (27). The weak
incorporation of palmitate also implies partial S-acylation
by myristate and palmitate. Similar data were obtained for the R141C
and R314L mutants (data not shown), suggesting their distributions onto
the membrane fraction were due to normal wild type lipid modification.
However, in the case of the S41Y mutant, there were only traces of
lipid incorporation, consistent with increased amounts in the cytosolic
fraction. Furthermore, the recombinant wild type enzyme was capable of
cross-linking in vitro its natural substrate loricrin (Fig.
1D) with a pattern of polymerization similar to that
described previously for the bacterially expressed enzyme (24).
Together, these data indicate that the wild type TGase 1 enzyme
expressed in baculovirus has properties very similar to the native
enzyme expressed in NHEK cells. Moreover, we found that the wild type
recombinant baculovirus enzyme has an activity half-life of about
12 h (data not shown). Thus it is much more stable than the
enzymes isolated from bacteria (34) or keratinocytes (24-26, 44) and
is well suited for in vitro cross-linking studies.

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Fig. 1.
Expression and characterization of wild type
and mutant forms of TGase 1. A, Coomassie staining of the 30 µg of total protein from Sf9 cells infected with wild type
baculovirus (CTRL) and recombinant baculoviruses bearing
wild type TGase 1 and the eight mutants cDNAs as shown.
B, Western blot using a specific polyclonal antibody for
TGase 1; the asterisks denote the TGase 1 protein forms of
about 97 or 55 kDa. C, autoradiography of Sf9 cells
infected with wild type baculovirus (ctrl) or recombinant
baculovirus bearing wild type TGase 1 labeled with
[14C]palmitic and [14C]myristic acid in the
cytosolic and membrane-bound fractions. D, in
vitro cross-linking of 35S-labeled human loricrin by
membrane-bound recombinant baculovirus TGase 1, in the presence of EDTA
(ctrl) of after reaction for the times shown. As for the
bacterially expressed enzyme (24), both oligomerization and intrachain
cross-linking occurs.
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Specific Activities of Recombinant Wild Type and Mutant Forms of
Human TGase 1--
Table II lists the
specific activities of the cytosolic and membrane-bound forms of the
wild type and eight mutant recombinant enzymes. The very low specific
activities of the wild type enzyme were almost identical to those of
the native intact (106-kDa form) enzyme expressed in NHEK cells or
isolated foreskin keratinocytes (26). As expected, most analyzed
mutants showed reduced enzymatic activities. Both membrane-bound and
cytosolic TGase 1 activities were reduced to 0-20% of the wild type
control for mutations R141H, R142H, R141C, and L268
. The specific
activity of R322Q was also reduced. The truncated enzyme generated by
the premature stop codon (M442stop) was inactive, as expected from
earlier deletion cloning experiments (34). Unexpectedly, two mutant
forms (S41Y and R314L) showed 2-4-fold increases in specific
activities in comparison to the wild type enzyme (Table II).
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Table II
Specific activities of wild type and mutant TGase 1 forms expressed in
baculovirus
Specific activities are expressed as picomoles of
[14C]putrescine incorporated into succinylated casein/h/pmol
of TGase 1 protein. TGase 1 amounts are estimated by active site
titriation with [14C]iodoacetamide (34). The results are
averages ± S.D. of 3-5 measurements.
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Transfections of Wild Type and Mutant Forms into NHEK
Cells--
The full-length wild type and four mutant cDNAs (two of
low or zero activity, R141H and R322Q, and two of higher activity, S41Y
and R314L) were transfected into NHEK cells. Transfection efficiencies
within the range of 15-30% were obtained in these experiments as
estimated by co-transfection with a
-galactosidase control vector
(data not shown; and see Fig. 3C). Each activity value was
standardized for units of
-galactosidase in order to compare the
different transfection experiments with the different constructs. These
experiments were performed under proliferating (in low
Ca2+, 0.05 mM) and differentiating (in high
Ca2+, 1.2 mM) conditions (Fig.
2, A and B,
respectively). As expected for NHEK cells (26, 27), in both cases most
of the transfected TGase 1 enzyme was directed to the membrane
fraction. In low Ca2+ media (Fig. 2A), while the
activity level of the wild type transfectant cultures increased
commensurate with the transfection efficiency, the activities of the
R141H and R322Q mutants were similar to those of the untransfected or
sham-transfected NHEK cells, indicating they are not functional, as
observed in baculovirus (Table II). Likewise, the two active mutants
(S41Y and R314L) generated total levels of enzyme activity that were
higher than for the wild type construct and commensurate with the
transfection efficiencies, indicating they were also as functional in
NHEK cells as in baculovirus. However, in high Ca2+ media,
the level of activity of each of the mutant constructs was at
background level and lower than for the wild type construct (p < 0.01) (Fig. 2B). Similar results were
obtained from the measurement of the formation of cross-linked cell
bodies in the same cells in that the data paralleled the activity
levels (data not shown).

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Fig. 2.
Transfections of wild type and mutant TGase 1 forms into NHEK cells grown in low and high calcium. NHEK cells
were transfected in low calcium (0.05 mM) and grown in low
calcium (A) or high calcium (1.2 mM)
(B) media for 48 h, using the wild type TGase 1 and the
four indicated mutant forms. Total TGase activity was measured for
membrane-bound (closed bars) and cytosolic (stippled
bars) fractions and standardized for units of -galactosidase
activity. Lane C indicates the endogenous TGase 1 activity
(keratinocytes were sham-transfected only with the -galactosidase
control vector). All data are the averages (±S.D. of 3-5
experiments).
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The S41Y and R314L Mutant TGase 1 Forms Are Not Proteolytically
Processed in Terminally Differentiating NHEK Cells--
To explore
further these differences, new constructs of the wild type and S41Y and
R314L mutant TGase 1 forms were assembled in a mammalian expression
vector system in which a tag of six histidine residues had been
attached to the carboxyl-terminal end. This was done in order to
distinguish between the endogenous and mutant TGases following
transfection into NHEK cells.
In initial experiments, we tested whether the presence of this tag
interferes with the expression and/or processing of the wild type TGase
1 protein in differentiating NHEK cells grown in the presence of
[35S]methionine/cysteine. The total (combined cytosolic
and membrane-bound) TGase 1 forms present in the cells including the
transfected and endogenous enzymes were immunoprecipitated with the
polyclonal anti-TGase 1 antibody (Fig. 3,
A and B) and resolved by fast protein liquid
chromatography into two major peaks that contained the full-length
TGase 1 protein of about 106 kDa (fraction 84) and the processed
complex of 67/33/10-kDa bands of TGase 1 (fraction 94), respectively.
Essentially all activity was attributable to the latter, as described
previously (26, 27) (Fig. 3B). Other minor peaks of activity
in fractions 36, 66, and 74 contain the cytosolic 67-, 106-, and
67/33-kDa complex components, whereas fraction 120 contains the
inactive 33-kDa band (which is poorly precipitated by the TGase 1 antibody (25, 33)). Immunoprecipitation of the same cultures with the
polyhistidine antibody generated identical data (Fig. 3, C
and D), except that the amount of label precipitated was
about 28% that with the TGase 1 antibody, which thereby provides an
estimate of the transfection efficiency. Moreover, the specific
activities of the highly active processed forms of fractions 94 were
the same (1050 ± 180 and 980 ± 150 pmol of
[3H]putrescine/h/pmol TGase 1 protein, respectively) and
are almost 200-fold greater than that of the intact 106-kDa form (Table
II; Ref. 26). Samples of the immunoprecipitates of both antibodies were
resolved on gradient SDS-PAGE gels (Fig.
4, B and E). The results obtained were the same and demonstrated that in both cases about half of the total TGase 1 protein was processed into the 67-, 33-, and 10-kDa forms. Thus the presence of the histidine tag on the
carboxyl terminus does not interfere with TGase 1 processing or
activity.

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Fig. 3.
The S41Y and R314L mutant forms of TGase 1 are not proteolytically processed in terminally differentiating
keratinocytes: chromatography study. Mammalian expression vector
constructs containing the His-tagged wild type and the two mutant TGase
1 forms shown were transfected into NHEK cells and grown in high
calcium media for 4 days. Following a 6-h pulse with
[35S]methionine/cysteine prior to harvesting, the total
(cytosolic and membrane-bound) TGase 1 forms were immunoprecipitated
with either the TGase 1 (A and B) or
polyhistidine (C-H) antibodies, chromatographed
by fast protein liquid chromatography, and assayed by measurement of
either 35S dpm (left columns: dpm/fraction) or
TGase activity (right columns: dpm of
[14C]putrescine incorporated into succinylated
casein/fraction).
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Fig. 4.
SDS-PAGE gels reveal absence of proteolytic
processing of S41Y and R314L mutant forms of TGase 1. Cultures
similar to above were harvested at the times indicated, processed as in
Fig. 3, but analyzed by SDS-PAGE on 10-20% gradient gels and
autoradiographed for 4-16 days. Immunoprecipitated with the following:
A-C, TGase 1 antibody;
D-G, polyhistidine antibody. A and
D, non-transfected NHEK cultures; B and
E, transfected with the wild type TGase 1 construct;
C and F, transfected with the S41Y construct; and
G, transfected with the R314L construct. The sizes of the
processed TGase 1 protein components are shown. Note that in these
experiments, much of the 10-kDa band (arrowhead) was lost by
apparent degradation.
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These experiments were repeated with NHEK cells transfected with the
constructs containing the His-tagged S41Y and R314L mutant TGase 1 forms. By using identical culture conditions as above, chromatography
(Fig. 3, E-H, respectively) and SDS-PAGE gels
(Fig. 4, F and G, respectively) revealed that the
products immunoprecipitated by the polyhistidine antibody correspond to
the intact membrane-bound 106-kDa form. Therefore, for both mutants,
only trace amounts had been proteolytically processed under these
terminally differentiating conditions.
In pulse-chase experiments we estimated the half-lives of the intact
wild type and S41Y and R314L mutant forms. Whereas the half-life of the
membrane-bound wild type protein was about 30 h as expected (27),
the half-lives of the S41Y and R314L mutants were clearly greater (50 and 60 h, respectively) (Fig.
5A). Similarly, the cytosolic
forms are more stable (40 and 60 h, respectively, versus 20 h for the wild type) (Fig.
5B).

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Fig. 5.
The S41Y and R314L mutant TGase 1 forms are
more stable than the wild type enzyme. Cultures similar to Fig. 3
were pulsed for 6 h with high calcium media containing
[35S]methionine/cysteine following transfection, washed,
and then grown for up to 4 days in high calcium media. Membrane-bound
(A) and cytosolic (B) proteins were
immunoprecipitated with the polyhistidine antibody, resolved by
SDS-PAGE as in Fig. 4, and exposed for quantitation by scanning
densitometry.
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Together, these data demonstrate that although these two mutants are
stably expressed and retained in the NHEK cells, they remain as the
intact very low specific activity (zymogen) proteins. They are not
processed into the highly active functional enzymes as for the wild
type protein.
Predicted Structural Model of TGase 1--
In order to explore the
structural consequences of the mutant forms studied in this paper, we
constructed a three-dimensional model of human TGase 1 (Fig.
6) using as the basis the known crystal structure of the related human fXIIIa (39). The high sequence conservation and few deletions provide a realistic model of the TGase 1 molecule. This includes four main domains that have been designated the
amino-terminal
-sandwich, the central core domain containing the
active site (catalytic core domain), and
-barrels 1 and 2 (39). In
the case of fXIIIa, an activation peptide precedes the
-sandwich
domain. In the case of TGase 1, a
90-residue sequence that is
involved in membrane anchorage and substrate recognition or specificity
(26) precedes the
-sandwich domain. We note that when TGase 1 is
proteolytically activated to generate the 10/67/33-kDa complex form,
the sites of cleavage are located near the junction of the membrane
anchorage and
-sandwich domains and to the junction of the catalytic
core domain and
-barrel 1 domain, respectively (Fig.
7). Since both the full-length 106-kDa TGase 1 and the 67/33/10-kDa complex normally purify in monomeric form
(Refs. 25-27; see Fig. 3), only one monomer of the dimeric fXIIIa
structure was used in the modeling study, omitting the part of the
fXIIIa activation peptide preceding Asn18, the part
mediating the intermolecular inhibition (Figs. 6 and 7) (39).

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Fig. 6.
Three-dimensional model of TGase 1. View
of the entire model with the four main structural domains and proposed
interaction between the 10-kDa membrane anchorage fragment with the
body of TGase 1. Most mutations described here are located at the
interface between the amino-terminal -sandwich domain and the
catalytic core domain. Spheres indicate the positions of the
mutated residues. TGase 1 residues 95-106 are highlighted in
orange. The 97NAAGDG102 hexapeptide,
tightly associated with the -sandwich and catalytic domain, is
magenta. The association of the 10-kDa membrane anchorage
fragment with the main body of the enzyme is proposed to occur in the
region between the -sandwich domain and the active site around
Cys375, in a conformation that inhibits TGase 1 activity.
On proteolytic cleavage at Arg92 and formation of the
active 10/67/33-kDa complex, the 10-kDa fragment undergoes structural
re-arrangements but remains attached through specific
interactions.
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Fig. 7.
Alignment of the amino-terminal portions of
fXIIIa and TGase 1. The two TGase 1 hexapeptide motifs
(55NAADDD60 and
97NAAGDG102) are highlighted by gray
boxes. The major proteolytic cleavage site in TGase 1 (Arg92, large arrowhead) precedes the
amino-terminal boundary of the -sandwich domain by 14 residues. The
corresponding proteolytic activation site for fXIIIa is shown by the
small arrowhead and is located in a portion of sequence
(lowercase letters) that was not observed in the crystal
structure (39). The fXIIIa sequences preceding Asn18 are
shown in italicized lowercase letters. Black and
white boxes delineate -helix and -strand secondary
structure features, respectively. The location of the S41Y
substitution, acyl lipid attachment sites, and other sites of sequence
identity are indicated.
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Following proteolytic activation at Arg92, most of the
membrane-bound TGase 1 remains associated with the 10-kDa membrane
anchorage domain (26, 27) to form a 67/33/10-kDa complex. In the
crystal structure of Ca2+-activated and thrombin-cleaved
fXIIIa, the activation peptide remains associated with the body of the
enzyme and adopts a practically identical conformation as compared with
the fXIIIa zymogen (39, 40). In particular, we note a high homology
between two hexapeptide segments of this domain of TGase 1, 55NAADDD60 and
97NAAGDG102 (Fig. 7), with a region of the
fXIIIa activation peptide, 20NAAEDD25, that
tightly interacts with the
-sandwich domain and the catalytic core
domain (39). This interaction is mediated by fXIIIa Asp243
and Arg252, both conserved in TGase 1 (Asp305
and Arg314). This suggests that the high overall structural
similarity between these two enzymes also includes parts of the
activation peptide of fXIIIa and the membrane anchorage domain of TGase
1. Based on the observed phenotype for the R314L mutant, we predict the second 97NAAGDG102 hexapeptide motif is
associated with the amino-terminal
-barrel domain in the unprocessed
full-length 106-kDa form of the enzyme. The second motif,
55NAADDD60, might represent an alternative mode
of binding, for example to modulate substrate specificity after
cleavage at Arg92 (26, 34). Only the part of the fXIIIa
activation peptide starting from Asn18 (corresponding to
TGase 1 Gly95) was therefore included in the TGase 1 model.
In the loop region following the 97NAAGDG102
segment, small local re-arrangements centered around
106GG107 were sufficient to close the loop
leading to the first
-strand of the
-sandwich domain.
Mutant R322Q and Inactive Mutants R141C, R141H, and R142H--
As
is evident from Fig. 6, most of the substitutions cluster around the
interface between the amino-terminal
-sandwich domain and the
central catalytic core domain. An exception is R322Q near the surface
of the catalytic domain far from the active site (Fig. 8A), consistent with its
milder phenotype. Consequently, no particular role in either substrate
recognition or enzymatic activity can be attributed to this residue.
While this in principle is also true for positions Arg141
and Arg142, these residues, conserved in all members of the
TGase family (30), are an integral part of a chain of hydrogen bonds
connecting the amino-terminal
-sandwich domain through
Asp253 in the linker region to the catalytic core domain
(Fig. 8B). Since both mutants showed expression levels and
lifetimes comparable to that of the wild type enzyme in the baculovirus
expression system, overall protein stability seems not to be affected.
A conformational change affecting the relative orientation between the
amino-terminal domain and the catalytic core domain is therefore the
most likely cause for their enzymatic inactivity, as the amino-terminal domain is indispensable for substrate recognition and processing (26).

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Fig. 8.
Stereo images of the effects of the
Arg141, Arg142, Arg314, and
Arg322 mutations in the local environment. A,
local environment around Arg314 and Arg322.
Whereas Arg322 is exposed on the surface,
Arg324 is buried in the domain interface and mediates
(through Asp305) tight interactions between the catalytic
domain and initial part of the 97NAAGDG102
hexapeptide motif (magenta). TGase 1 residues 95-96
preceding this motif are orange. Main chain functional
groups involved in hydrogen bonds are shown in black.
B, close view of the chain of hydrogen bonds connecting the
amino-terminal -sandwich and the catalytic domains through
Arg141, Arg142, and Asp253.
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Deletion Mutants L268
and M442stop--
The absence of
structural integrity, a prerequisite for most enzyme activity, readily
explains the complete absence of catalytic activity in case of the
M442stop mutation. Truncation of the polypeptide chain at this position
removes a considerable part of the catalytic core domain (residues
443-572), presumably creating a misfolded central domain unable to
assemble a functionally active site. Previous deletion cloning
experiments documented loss of activity when these sequences were
removed (34). As before for the R141H and R142H mutations, proper
function of the enzyme will be destroyed due to the absence of a
correctly oriented amino-terminal
-sandwich domain.
Active Mutants S41Y and R314L--
Both of these mutants exhibited
an increased basal activity of the unprocessed 106-kDa polypeptide and
do not undergo activation by proteolytic cleavage at Arg92
in differentiating keratinocytes (Figs. 3 and 4). The relationship between the two phenotypes seems at first sight to be unrelated. Arg314 is located in the interface between the
amino-terminal
-sandwich domain and the catalytic core domain and
mediates (through Asp305) specific interactions to the
initial part of the hexapeptide motif of the 10-kDa fragment (Fig.
8B). Substitution of Arg314 into Leu would
abolish these interactions and potentially destabilize the preceding
loop region, as found for an analogous mutation in fXIIIa (R252I),
which resulted in undetectable protein levels presumably due to reduced
protein stability (45). However, the presence of Pro310 in
the preceding loop region is likely to significantly increase the local
conformational stability in TGase 1. At the corresponding position of
fXIIIa, an aspartic acid (Asp248) is instead present.
Presumably also, this increased stability, as reflected by a very long
half-life (Fig. 5), prevents or interferes with proteolytic activation
at the nearby Arg92.
For the S41Y mutation, no direct prediction can be derived from our
model, since this residue is located in the membrane anchorage segment
which could not be modeled. We suggest that this substitution promotes
a conformational change in the 10-kDa fragment, which as for R314L,
results in a net reduction of the inhibitory capacity of the 10-kDa
fragment domain.
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DISCUSSION |
The TGase 1 enzyme exists in keratinocytes in multiple forms due
to post-translational proteolytic processing (25-27). In proliferating or stationary cells, most protein exists as the intact form of low
specific activity (zymogen) and is tightly bound to cellular membranes.
(This form retains some activity and is not "inactive" as
previously thought (26)). Only minor (<5-10%) amounts cycle into the
cytosol. However, in terminally differentiating keratinocytes, up to
50% of the membrane-bound TGase 1 is proteolytically processed by
cleavage at sites that correspond very closely to inter-domain junctions of its predicted structure. The resulting 67/33/10-kDa complex shows a 200-fold increase in specific activity and is responsible for most of the TGase 1 activity in keratinocytes during
terminal differentiation (26).
In the present paper, we have explored the structure-function
relationship for several mutants of the TGase 1 enzyme observed in LI
disease. We have combined biochemical data obtained for expressed forms
of the mutants with structural information from a homology-derived
model of TGase 1. The mutant form M442stop results in large deletions
of sequences known to be essential for activity. Apart from mutant
R322Q, which has only a modestly reduced activity, most other mutants
caused by point mutations (R141C, R141H, R142H, and L268
) resulted
in loss of function due to the failure to establish key ionic and
hydrogen bond interactions (Fig. 8, A and B).
These changes are likely to cause partial protein misfolding and/or
domain rearrangements with resultant loss of activity, that is LI
disease was caused by the greatly reduced enzyme activity in the
keratinocytes.
Identification of a Novel Type of Mutant: Interference with
Postsynthetic Proteolytic Activation--
For the last two mutants,
S41Y and R314L, a more detailed characterization was necessary in order
to understand why they should cause LI disease. At first glance they
appear to involve gain of function; both mutant enzymes were
significantly more active than wild type TGase 1 when expressed in
baculovirus or transfected keratinocytes grown under proliferating
conditions, in situations where only the unprocessed 106-kDa form of
the enzyme is observed (Fig. 2 and Table II). Further experiments in
terminally differentiating NHEK cells showed, however, that both
variants could not be proteolytically processed into the functional
highly active form (Figs. 3 and 4). In view of the large differences in
specific activities between the unprocessed 106-kDa and the
proteolytically activated 67/33/10-kDa complex (26), the net
consequence of the mutant forms was therefore the same as for the
loss-of-function mutants: insufficient enzyme activity for terminal
differentiation.
We have shown in earlier deletion cloning experiments in bacteria that
the 10-kDa membrane anchorage fragment dictates the catalytic state of
the TGase 1 enzyme (26, 34). There are at least two possible ways how
this could occur. One is the modulation of substrate specificity
controlling the type of substrate that may approach the active site.
Although no direct structural data are yet available, we propose a
second possibility where at least parts of the membrane anchorage
domain fold back onto the body of the TGase 1 enzyme so as to partially
obstruct access to its active site. This model is reminiscent of what
is observed to occur for the fXIIIa activation peptide, albeit
intra-molecular instead of inter-molecular (Fig. 6). Our hypothesis is
based on two findings. In the first, in vitro overlay
experiments on the binding of different post-translationally modified
10-kDa domains showed that S-myristoyl and
S-palmitoyl modifications of the cysteine-rich cluster
(47CCGCCSC52) are required for interactions
with the 67-kDa and 67/33-kDa components to form the highly active
67/33/10-kDa complex form of the TGase 1 enzyme (27). Second, in this
study, we have recognized regions of high sequence homology of the
membrane anchorage domain of TGase 1 (residues 97-102, and perhaps
residues 55-60) with the region encompassing residues 20-25 of the
fXIIIa activation sequence that is responsible for the inhibition of
the fXIIIa enzyme. The contact points of this interaction involve
specifically the catalytic domain residue Arg252 (fXIIIa)
and Arg314 (TGase 1). Together, these data indicate the
existence of rather specific interactions between the membrane
anchorage and amino-terminal
-sandwich/catalytic core domains of the
TGase 1 enzyme. Thus in the R314L mutant, the substitution of
Arg314 and failure to make key interactions (Figs. 6 and 9)
partially releases the inhibitory effect of the membrane anchorage
domain, resulting in a molecule of increased specific activity. Given the structural context around Arg314 in our TGase 1 model
(Fig. 9), we furthermore conclude that
this molecular event also increases the stability of the protein and interferes with proteolytic activation. Mutation of Arg314
into Leu is likely to affect Arg92 directly preceding the
97NAAGDG102 hexapeptide motif and, as a
consequence, alter the accessibility to the cleavage site.

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Fig. 9.
Stereo images illustrating the effect of the
R314L mutation on the interaction between Arg314 and the
97NAAGDG102 hexapeptide motif. Detailed
view of the predicted interaction between the
97NAAGDG102 hexapeptide following the 10-kDa
membrane anchorage domain and the body of the TGase 1 enzyme.
Asp305 and Arg314 take the role of fXIIIa
Asp243 and Arg252 in linking the fragment to
the catalytic core domain. Mutation of R314 into Leu (L314,
shown in green) will disrupt the chain of electrostatic and
hydrogen bond interactions between the catalytic domain and functional
backbone groups around Asn97. As a result, we predict that
local structural changes occur affecting the conformation of residues
preceding Asn97, including also Arg92, which is
the site of proteolytic cleavage. For illustrative purposes a tentative
position for Arg92 has been included in the figure.
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In the case of the S41Y mutant form, a similar scenario has to be
assumed, although in this case our model does not allow a direct
correlation with structural information. However, we note the presence
of an Arg-Arg dipeptide (35RR36) immediately
upstream Ser41 that could in principle assume a function
similar to fXIIIa Arg11/Arg12, which contact
acidic residues near the active site thus blocking enzymatic activity
(39). Conformational alterations caused by the presence of the
substituted tyrosyl side chain could prevent the 10-kDa fragment to
exhibit its full inhibitory effect and may also explain why the S41Y
mutant form was less efficiently bound to membranes (Fig. 4).
Our data confirm the key role of the 10-kDa membrane anchorage domain
for TGase 1 activity and highlight the sensitivity of the enzyme toward
alterations affecting this region. Any mutation that disturbs the
proper conformation of the 10-kDa membrane anchorage could therefore
have drastic consequences for enzymatic activity or substrate
specificity. More detailed structural information on this domain
therefore seems warranted.
Altogether, the present studies demonstrate that loss of TGase 1 enzyme
activity, which results in LI disease, can arise either from mutations
that affect directly the correct folding of the protein or which
instead affect the subsequent postsynthetic processing of the protein
into its highly active functional forms. Accordingly, it is to be
expected that LI disease can be caused by deficient TGase 1 activity
due to adverse effects on other processing events, such as
N-myristoylation, membrane anchorage due to failed lipid S-myristoylation, or S-palmitoylation,
phosphorylation, etc. Furthermore, it is conceivable that mutations
that affect directly those enzyme systems responsible for these
post-synthetic modifications should result in a TGase 1 enzyme of
greatly reduced specific activity or potential for proteolytic
processing, with consequences of LI disease. In this regard, it is
noteworthy that cases of CRI have been identified that do not involve
mutations in the TGM1 gene (11-13). Further work must be
done now to explore whether mutations in these ancillary gene systems,
directly or indirectly affecting the TGase 1 enzyme, are the cause of
CRI or LI disease.
During the preparation of this manuscript, another paper was published
describing several new mutations of the TGM1 gene in LI, and
structural modeling was performed to explain the diminished TGase 1 activity of the mutant forms (46). Many of the data and conclusions
conform to our analyses described here. Two common mutant forms were
analyzed. As in the present study, the mutant form S41Y was
demonstrated to be less efficiently bound to membranes and to have a
higher activity than the wild type (when assayed following transfection
into keratinocytes derived from LI patients with no background TGase 1 activity). However, that study did not identify the observation
discovered here that under differentiating conditions, the S41Y mutant
form is likely to be disease-causing since it could not be processed
into a highly active functional enzyme. Our study offers a possible
explanation for this in the association of the membrane anchorage
region of TGase 1 to the
-sandwich domain. Similarly, that study
described a mutant form R314C, which possessed low specific activity,
presumably due to protein misfolding. In the present work, we show that
the Leu substitution instead leads to an excessively stable protein
that cannot be processed. Thus, biochemical analyses as performed in this study, coupled with activity and structural analyses, have provided more profound information about the properties of the TGase 1 enzyme system.