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INTRODUCTION |
The cell envelope (CE)1
is a highly insoluble structure assembled just inside the plasma
membrane of stratified squamous epithelia and is essential for
effective barrier function. To form the CE, specialized keratinocyte
proteins are expressed and subsequently made insoluble by cross-linking
by both disulfide bonds and isopeptide bonds formed by
transglutaminases (TGases) (1-5).
Emerging data suggest that the protein composition of CEs varies widely
between epithelia and even different body sites of epithelia such as
the epidermis (6, 7). However, involucrin seems to be a ubiquitous
component of most if not all CEs. Indeed, several types of data imply
that it is one of the first proteins to be cross-linked to initiate CE
assembly. First, expression studies have revealed that involucrin
deposition at the cell periphery precedes all other suspected or
confirmed CE protein constituents (8-15). Second, shadowing and
scanning transmission electron microscopy suggest that a monomolecular
layer of involucrin is overlayered on the cytoplasmic side by other CE
structural proteins (16). Third, extant models of CE structure based on
biochemical characterization and protein sequencing indicate that
involucrin becomes cross-linked to several cell peripheral proteins
including desmoplakin, envoplakin, keratin intermediate filaments, as
well as other CE proteins including members of the small proline-rich
family, cystatin
and loricrin (6, 17, 18). Fourth, recent data have
shown that involucrin is a major target for the covalent attachment of
ceramide lipids from the exterior surface of the CE, which could only
occur if involucrin was deposited in the intimate vicinity of the
keratinocyte membrane at an early time (19). Human involucrin contains
150 glutamine and 45 lysine residues (20), and it appears that
mammalian involucrins have undergone extensive expansion of various
glutamine-rich repeating motifs during evolution perhaps to increase
the sites suitable or available for TGase-mediated cross-linking (21). However, sequencing studies of this laboratory have shown that only a
limited number of these residues are used for cross-linking in
vivo (17). Moreover, whereas involucrin appears to be a good substrate for TGases in in vitro reactions (11, 22), extant data have provided no information on which of these enzyme(s) are
responsible for cross-linking in vivo.
TGases are Ca2+-dependent enzymes that catalyze
an acyl transfer reaction between the
-carboxamide group of
protein-bound glutamine and the
-amino group of lysine residues. Of
the seven known human TGases, four (TGases 1, 2, 3, and X)
are expressed in terminally differentiating epithelia such as the
epidermis (23, 24), but to date only limited data are available on
their substrate specificities and relative contributions in CE
assembly. Of these, the TGase 2 enzyme is thought to play only a minor
role, and the properties of the newly discovered TGase X
enzyme await characterization. The TGase 1 and 3 enzymes are essential
for the cooperative cross-linking of such substrates as loricrin (25),
trichohyalin (26), and small proline-rich proteins 1 (27) and 2 (28).
The TGase 3 enzyme is soluble and requires proteolytic activation
before it can function (29). The TGase 1 enzyme was first discovered in keratinocytes and is usually anchored to membranes by way of acyl N-myristoyl and S-myristoyl or
S-palmitoyl adducts near the amino terminus of the protein
(30-32). However, virtually all studies with the TGase 1 enzyme to
date have involved assays with poorly defined keratinocyte particulate
fractions (8-11) or solution assays conducted in the absence of
membranes (24-26, 30, 32).
In the present study, we have used synthetic lipid vesicles (SLV) of
composition similar to those of eukaryote plasma membranes in order to
explore how membranes affect TGase reaction and residue specificity.
First, we show that only the TGase 1 enzyme can associate with SLV.
Second, we show that of several proven CE structural proteins available
to us, only involucrin associates with SLV and in a
Ca2+-dependent manner. When both TGase 1 and
involucrin are attached to membranes, there is remarkable specificity
of glutamine residue usage for cross-linking. These data have important
implications for CE assembly and ichthyosiform diseases caused by
enzyme or substrate abnormalities.
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MATERIALS AND METHODS |
Production of Recombinant TGase 1 and 3 Enzymes--
Recombinant
full-length human TGase 1 and TGase 3 enzymes were expressed in
Sf9 cells by the BaculoGold system using the pVL1392 plasmid
vector (PharMingen, San Diego, CA) as described previously (33). TGase
1 was recovered in the particulate fraction after sonication in lysis
buffer (33). In some experiments this crude particulate fraction was
used as the source of enzyme activity in amounts standardized to
incorporate 0.7 pmol/min of [14C]putrescine into
succinylated casein. In most experiments, it was solubilized from
membranes by sonication in lysis buffer with 4% Triton X-100 or in
some cases with 1 M NH2OH-HCl (32). TGase 3 was
recovered in the cytosolic fraction after lysis by sonication. These
solutions were clarified by centrifugation and the enzymes subsequently
purified by fast protein liquid chromatography on MonoQ Sepharose as
before (33). Active fractions were brought to 1 M
Na2SO4 and rechromatographed on a 1-ml Resource
Phe hydrophobic interaction column (Amersham Pharmacia Biotech) using a
gradient from 1 M Na2SO4, 20 mM Tris-Cl (pH 8.0) to 20 mM Tris-Cl (pH 8.0) in 30 min at a 1 ml/min flow rate. The TGase 1 and 3 enzymes were >98% pure by SDS-polyacrylamide gel electrophoresis, and could be
stably stored as a suspension in 1.5 M
Na2SO4 for some weeks at 4 °C. Amounts were
determined by amino acid analysis following acid hydrolysis. TGase 3 was activated with 0.1 units/100 µg dispase for 15 min at 23 °C
and purified from the protease on a MonoQ column as above.
Expression and Purification of Recombinant Human
Involucrin--
A full-length cDNA clone of human involucrin was
obtained by polymerase chain reaction from human chromosomal DNA.
Polymerase chain reaction primers used were
(+)-GTAGCTTCTCATATGTCCCAGCAAC and (
)-CCCTTGTATGAGACGATCTGAG. These
were designed to create an NdeI restriction site to be
compatible with the pET expression system (Novagen, Madison, WI). The
polymerase chain reaction product was cloned into the pCR2.1 plasmid
using the TA Cloning kit (Invitrogen, Carlsbad, CA) and verified by DNA
sequencing. Following subcloning into the pET11a vector and
transfection into the BL21(DE3)pLysS strain of Escherichia
coli (Novagen), protein expression was induced by 1 mM
isopropyl-1-thio-
-D-galactopyranoside for 3 h. Cell
mass was pelleted and lysed by freeze-thawing. Particulate matter was removed by centrifugation, and involucrin protein was enriched by heat
precipitation as described (22). It was purified to >97% (by
SDS-polyacrylamide gel electrophoresis) by anion exchange chromatography on a HiTrap Q column (Amersham Pharmacia Biotech) using
20 mM Tris-HCl (pH 8.0) and gradient elution with the same buffer containing 1 M NaCl. By circular dichroism, it
possessed an estimated
-helix content of 68%. As this is similar to
native involucrin isolated from keratinocytes (12), it is likely that the recombinant protein had assumed its native configuration.
Preparation of Synthetic Lipid Vesicles (SLV)--
Mixtures of
dipalmitoyl-phosphatidylcholine, cholesterol,
dipalmitoyl-phosphatidylserine (PS), and other lipids where indicated (all from Sigma) were made in chloroform/methanol (95:5). In all cases,
the mixtures contained 30% cholesterol. When the amounts of PS or
other individual components were varied, phosphatidylcholine was added
to make the mixture to 100%. Mixtures were made in 0.5 ml and
contained 10 µmol of total lipids. The solvent was flushed away under
a stream of N2, dried further under high vacuum for 4 h, and then resuspended by vortexing in 0.5 ml of a buffer containing 50 mM Tris-Cl (pH 8.0), 100 mM NaCl, 3 mM NaN3, 5 mM dithiothreitol, and
200 mM sucrose. The mixture was sonicated on ice five times each for 1 min using a Branson 250 sonifier with micro probe tip, and
allowed to stand at 23 °C to facilitate assembly of SLV. After 1 h the SLV were diluted with 0.5 ml of the above buffer without sucrose, and 200-µl aliquots were centrifuged at 100,000 × g for 30 min in a Beckman Airfuge using the A-10 rotor. The
top 175 µl was removed, and the pellet was resuspended in another 150 µl of sucrose-free buffer. The final stock concentration was 11 µmol/ml.
Assaying Membrane Association of TGase 1, TGase 2, TGase 3, Involucrin, Loricrin, SPR1, SPR2, and Succinylated Casein--
Liver
TGase 2 enzyme was obtained from Sigma. The recombinant human CE
proteins loricrin (25), SPR1 (27), and SPR2 (28) were expressed and
purified as described previously. Succinylated casein was a generous
gift of Dr. Soo-Youl Kim.
All binding assays were done at 23 °C in a final volume of 200 µl
by mixing SLV with protein amounts empirically found to exceed at least
2-fold the binding capacities of the SLV as follows: for TGase 1, 20 µg (0.2 nmol) were mixed with 0.1 µmol of SLV lipids; for
involucrin, 1.2 nmol were mixed with 1 µmol of SLV lipids; for all
other proteins, 50 µg were used with 1 µmol of SLV lipids. In all
cases, the buffer contained 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM dithiothreitol, 3 mM
NaN3, and other additives where noted. Some mixtures also
contained 1 mM CaCl2 or 2 mM EDTA.
After mixing, the samples were centrifuged for 45 min at 100,000 × g, and the protein content from 50 µl of supernatant was determined by amino acid analysis after acid hydrolysis. Data were
usually not corrected for loss of SLV lipids, as in control experiments, cumulated losses were <4% as assayed by
[14C]phosphatidylcholine tracer, and thus neglected from
the calculations.
Preparation of Free Ca2+ Concentrations--
The
Ca2+ concentrations in the micromolar range were set by
buffering free Ca2+ with chelators. The desired
Ca2+/chelator ratios were calculated by the WinMaxC 1.7 computer program (34) and were made by adding the required amounts of 1 M CaCl2 to 0.2 M stock solutions of
the following chelators at pH 8.0. Final chelator concentrations in the
samples were 10 mM in all cases. Sodium citrate was the
chelator for the 20-500 µM Ca2+ range,
sodium nitrilotriacetate for the 1-20 µM
Ca2+ range, and sodium
1,2-bis(aminophenoxy)ethane-N,N,N'',N'-tetraacetate for the
0.1-1 µM Ca2+ range. The actual
concentration values were not significantly different from the
theoretical calculations (p > 0.2, n = 5) in the 2-100 µM range as assayed by arsenazo III dye
spectrophotometry (35).
Identification of [14C]Putrescine-labeled
Involucrin Fragments and Measurement of the Reaction Rate--
Three
sets of experiments were performed to determine reactive Gln residues
in involucrin by TGase 1 and labeling with
[14C]putrescine as follows: TGase 1 bound to SLV (2 µmol of lipid) containing 15% PS; the TGase 1 enzyme present in
crude Sf9 cell particulate fractions; or solubilized TGase 1 in
reaction buffer. In each case, the amount of TGase 1 activity was
standardized to 0.7 pmol/min using succinylated casein and
[14C]putrescine, as described previously (36), and
corresponded to about 0.9 pmol. This was set far below saturating
amounts on SLV in order to ensure that all enzyme was bound to the SLV.
All reactions were done in 250 µl and contained 1.2 nmol of
involucrin, 1 mM CaCl2, 20 mM
putrescine with 100 nCi of [14C]putrescine (NEN Life
Science Products, 110 Ci/mmol).
After 4 h incubation at 37 °C, the reactions were stopped by
addition of EDTA to 10 mM and 100 µl 20% SDS and
vortexed. This mixture was precipitated and washed three times with
acetone/triethylamine/acetic acid (90:5:5) (37) to remove the SDS and
the lipids. After repeated washings with acetone, the pellet was
redissolved in a buffer of 50 mM Tris-Cl (pH 7.5) and
digested for 16 h at 37 °C with 2% (by weight) of modified
trypsin (Boehringer Mannheim). Aliquots of 20 µg were resolved on a
250 × 4.6 mm Beckman Ultrasphere C18 HPLC column. Separated peaks
were analyzed for radioactivity, and peaks containing activity were
attached to a solid support for sequencing as before (38). As the
peptide number 40 eluted by 50% acetonitrile (see Fig. 9) was too long
for direct sequencing, it was subjected to limited proteolysis by
dispase and was sequenced from four peptides isolated by HPLC
chromatography as above.
The Gln residues reacted by putrescine by TGase 1 were identified by
standard protein sequencing analyses. The PTH-derivative of the
-glutamylputrescine eluted as a novel peak at 13.55 min in the HPLC
separation step of the Porton Instruments LF 3000 protein sequencer.
This was confirmed by measurement of the [14C]putrescine
label. This always corresponded to those cycles where a Gln residue was
also present.
Determination of Kinetic Parameters of Putrescine Incorporation
by TGase 1--
Values were measured at 37 °C using 0.9 pmol of
TGase 1, 20 mM putrescine with 100 nCi of
[14C]putrescine, 5 mM CaCl2, SLV
containing a total of 2 µmol total lipid, five concentrations of
substrate proteins (0.2, 0.5, 1, 2, and 5 µM for
SLV-bound TGase 1 with involucrin or 5, 10, 20, 50, and 100 µM for solubilized TGase 1 and soluble substrates) as
described (25). The calculated KM values pertain to
the protein substrates; Vmax and
kcat data are those for putrescine incorporation. The molar mass of succinylated casein was taken to be 25 kDa. The reaction rates were quantified by measuring the incorporated
radioactivity as before (35). Kinetic constants were obtained as before
(35) using the curve-fitting and regression analysis with Sigmaplot 4.0 software. All data points represent the mean of three measurements,
each performed in triplicate. The size of SLV (>85% below 100 nm, as
determined by size-exclusion on Sepharose CL-4B chromatography) was not
significantly altered during the binding and labeling reactions.
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RESULTS |
The present experiments were designed to explore the properties
and function of the TGase 1 enzyme on a lipid surface using SLV that
mimic the plasma membranes of eukaryotic cells. In preliminary experiments we made the serendipitous discovery that of several known
CE substrates, involucrin also binds efficiently to such membranes
under the conditions typically used for TGase assays. This observation
has important implications for CE assembly, and the details are
examined systematically in this paper.
Of Several Known CE Proteins Only Involucrin Attaches to SLV
Containing Physiological Concentrations of PS--
By using SLV
formulations initially taken from established methods for assaying
protein kinase C activation (39) that mimic the cytoplasmic surface of
plasma membranes of eukaryotic cells, we found that involucrin was
readily adsorbed to the SLV. By using saturating amounts of involucrin
(0.6 nmol of protein/µmol of lipid) and 1 mM
Ca2+ (typical for TGase assays), the soluble involucrin
content of the inter-SLV buffer was measured after pelleting of the SLV
by ultracentrifugation. Increasing the PS content of SLV from 0 to 30%
in 1% increments increased binding of involucrin sigmoidally between 4 and 15 mol % PS (Fig. 1A) and
could be fitted with r = 0.93 to a log
(y/100
y) = 3.82 log [PS%]
0.904
Hill's equation, where y is the ratio of SLV-bound
involucrin to the maximal binding. With increasing concentrations of
involucrin, SLV containing 15-30% PS could be saturated by 0.43 ± 0.02 nmol of involucrin/µmol of lipid. This corresponds to about
one involucrin molecule/500 nm2 of surface (40). Since an
involucrin molecule is 46 × 1.5 nm (70 nm2 (12), this
means that a remarkably high value of about 15% of the SLV surface can
be decorated by involucrin. The adsorption of involucrin to the SLV was
specific to PS since substitutions by phosphatidic acid,
phosphatidylglycerol, or phosphatidylinositol did not enhance
binding (Fig. 1B).

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Fig. 1.
The attachment of involucrin to SLV.
A, sucrose-loaded synthetic lipid vesicles (SLV)
containing a total of 2 µmol of lipid were formulated with 0-30 mol
% PS. 1.2 nmol of recombinant purified human involucrin was added to
the SLV in the presence of 1 mM Ca2+ at
23 °C. The binding was assessed by measurement of protein
concentrations in the supernatants before and after pelleting the SLV
by ultracentrifugation. The Hill coefficient of the fitted sigmoidal
regression curve is 3.82. Points represent the mean ± S.D. of
three independent measurements. B, anionic phospholipids
other than PS do not increase involucrin binding to SLV containing 8%
PS; binding of involucrin to SLV containing 8% PS plus 0-20%
phosphatidic acid (closed circles), phosphatidylglycerol
(open circles), or phosphatidylinositol (closed
inverted triangles). Points represent the mean ± S.D. of
three independent measurements.
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We also examined whether a variety of other known CE substrate proteins
could bind to SLV. The recombinant proteins available to us included
loricrin and the small proline-rich proteins 1 and 2. None of these
became attached to SLV of any composition tested (Fig.
2). Similarly, succinylated casein, a
commonly used artificial substrate for amine incorporation by TGases,
did not associate with SLV.

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Fig. 2.
Of the several proven in vivo
substrates of the TGase 1 enzyme, only involucrin binds to
SLV. Binding of excess amounts of recombinant human involucrin,
loricrin, SPR1, SPR2, or succinylated casein to SLV formulated from 0 or 15% PS in the presence or absence of free Ca2+ was
performed as in Fig. 1. Error bars represent the mean ± S.D. of three independent measurements. Values except for those of
involucrin with 15% PS and 1 mM EDTA do not represent
significant binding (p > 0.1).
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Ca2+ Ion Dependence of Binding Involucrin to
SLV--
Next, we determined the optimal free Ca2+
concentration required for binding of saturating amounts of involucrin
to SLV containing 15 mol % PS (0.6 nmol/µmol lipid) (Fig.
3). Maximal binding occurred with >20
µM Ca2+, and half-maximal binding was
estimated at 4.2 ± 0.7 µM, but binding was first
detected at 1 µM. This Ca2+-induced binding
was completely reversible by excess of the chelators EGTA or EDTA.
Magnesium and monovalent ions did not support binding of involucrin to
SLV (not shown).

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Fig. 3.
Effect of the free Ca2+
concentration on involucrin binding to SLV. Involucrin binding was
assayed as in Fig. 1 in the presence of 0-1000 µM
CaCl2. Half-maximal binding was calculated at 4.2 ± 0.7 µM. Binding was reversible in the presence of EDTA.
Points represent the mean ± S.D. of three independent
measurements.
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Solubilized Recombinant TGase 1 Binds Spontaneously to SLV--
We
have shown previously that the recombinant TGase 1 enzyme expressed in
the baculovirus system is constitutively N-myristoylated and
S-myristoylated or S-palmitoylated on its
amino-terminal 10-kDa portion and can be found mostly in the
particulate fraction of the insect cell homogenates (33). As for the
native enzyme expressed in epidermal keratinocytes, the recombinant
TGase 1 can be solubilized from the membranes by extraction with the
detergent Triton X-100, and the lipid adducts on the enzyme are
retained. Alternatively, the TGase 1 may be solubilized by use of 1 M NH2OH-HCl which hydrolyzes the
S-acyl adducts off the enzyme (32). When purified from the NH2OH-HCl method, no TGase 1 enzyme bound to the SLV, as
indicated by amino acid analysis of pelleted SLV mixtures and TGase
assays of the resulting supernatants (data not shown). However, when the TGase 1 enzyme purified from Triton X-100 extracts was mixed to
SLV, they spontaneously reassociated, as indicated by the disappearance of detectable enzyme activity from the supernatants of pelleted SLV
mixtures. Using SLV composed of 15% PS, the saturating amount was
0.9 nmol of TGase 1/µmol of lipid (Fig.
4). The binding of recombinant TGase 1 protein to SLV was not influenced by Ca2+ ions or EGTA or
to SLV prepared with varying formulations of ingredients including
anionic (20% PS), neutral (only phosphatidylcholine and cholesterol),
or cationic (5% stearylamine) lipids (data not shown). Thus the extent
of constitutive myristate and palmitate modifications of the
recombinant TGase 1 protein by baculovirus is sufficient to permit
spontaneous anchorage onto lipid bilayers per se. In
contrast, neither the cytosolic TGase 2 nor activated TGase 3 enzymes
associated with SLV of any composition tested (Fig. 4).

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Fig. 4.
Purified recombinant TGase 1 expressed in
baculovirus spontaneously associates to SLV but TGases 2 and 3 do
not. Binding was assayed as in Fig. 2 in SLV formulated from 0 or
15% PS in the presence or absence of free Ca2+.
Error bars represent the mean ± S.D. of three
independent measurements. Binding of TGases 2 or 3 is not statistically
significant (p > 0.1).
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Effect on Kinetic Parameters of TGase 1 Reaction following
Attachment to SLV--
Kinetic parameters for
[14C]putrescine incorporation by SLV carrying 0.94 pmol
of recombinant TGase 1/µmol of lipid were determined at 37 °C
using three different protein substrates as follows: succinylated
casein, human SPR 2 (28), and human involucrin (Table
I), of which only the latter was found to
attach to the SLV (Figs. 1A and 2). Kinetic constants for
the recombinant SPR2 substrate differed only by about a 2-fold increase
in KM if the PS content of SLV was 0 or 15%. As a
similar change in KM was observed both with
putrescine and succinylated casein, we attribute this
KM increase to the reduced diffusional mobility of
the enzyme after attachment to the SLV. Interestingly, attachment of
TGase 1 to SLV drastically affected its kinetic parameters with respect
to the incorporation of putrescine into the standard succinylated
casein substrate, so that the Vmax was reduced
by about 5-fold (Table I). The probable reason for this is the reduced
access to many of the Gln residues after anchorage of the enzyme. On
the other hand, the Vmax value for the
recombinant SPR2 substrate was almost unchanged. Sequencing analyses
revealed that of its several Gln residues, only Gln-6 was reacted by
putrescine in all three of the TGase 1 enzyme formulations described in
Table I, in agreement with previous findings (28). These data seem consistent with the fact that SPR2 is a much smaller and more flexible
substrate with only one reactive Gln residue. The kinetic parameters
for involucrin as a substrate were immeasurably low when the SLV were
formulated in the absence of PS. In comparison to solubilized TGase 1 enzyme and soluble involucrin, SLV containing 15% PS yielded a
200-fold decreased kcat value and an
approximately 40-fold decreased KM. In view of the
above observations for succinylated casein, these changes might also
reflect drastic changes in the availability of Gln residues on
involucrin for reaction with putrescine.
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Table I
Apparent kinetic parameters for putrescine incorporation into SPR2,
involucrin, and succinylated casein by 0.94 pmol of membrane-bound
and solubilized TGase 1
The PS content of the SLV is shown in parentheses.
KM(app) values pertain to the protein, and
Vmax and kcat data are that of
putrescine incorporation.
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The data of Table I also demonstrate that the kinetic constants of
putrescine incorporation into involucrin by TGase 1 alter with the PS
content of the SLV. We show further in Fig.
5 that the reaction rate follows a
sigmoidal shape with a sharp increase between 6 and 11% with maximal
incorporation at 10-20% PS content. This observation is to be
expected from the maximal binding of involucrin to SLV (Fig.
1A) and thus serves as a valid control. Beyond 20% PS
content, the efficiency of the reaction declines (Fig. 5 and Table I),
possibly because of inhibition of TGase 1 by excessive charge.

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Fig. 5.
Effect of PS content of SLV on incorporation
of [14C]putrescine into involucrin by TGase 1. TGase
1 (0.9 pmol) was bound to SLV formulated with 0-30% PS, to which were
added 1.2 nmol of involucrin, 20 mM
[14C]putrescine, and 1 mM Ca2+,
and reacted for 30 min at 37 °C. Incorporation of radioactivity
shows a sigmoidal effect of PS between 3 and 12% and a decline above
25% PS. Points represent the mean ± S.D. of three independent
measurements.
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Ca2+ Requirements for TGase 1 Reaction Are Markedly
Lowered following Attachment to SLV--
To the best of our knowledge,
the optimal Ca2+ ion concentration required for the TGase 1 reaction has not been measured for any substrate; standard in
vitro solution assays usually contain 1-5 mM
Ca2+ (41-43). In the absence of SLV and solubilized
recombinant TGase 1 in a mixture containing 1.2 nmol of involucrin, we
found that the reaction followed an apparent sigmoidal curve, with
half-maximal incorporation occurring at 310 ± 80 µM
Ca2+ (Fig. 6, yellow
squares). However, when an equimolar amount of TGase 1 was bound
to SLV containing 15% PS and saturating amounts of involucrin, the
Ca2+ activator constant was estimated at 22 ± 1.3 µM Ca2+ (Fig. 6, orange
triangles). Similar values (19 ± 2.7 µM) were obtained for succinylated casein, a substrate that is not significantly bound to SLV (Fig. 2). However, when TGase 1 was attached to neutral SLV containing 0% PS, the half-maximal enzyme activity with
succinylated casein was calculated at 370 ± 110 µM
Ca2+ (data not shown). Together, these control data suggest
the changes in Ca2+ sensitivity reflect the altered local
Ca2+ micro-environment on PS containing bilayers, rather
than of enhanced enzyme affinity toward Ca2+ or its
substrates in the membrane-bound state. The local concentration of
Ca2+ ions may be much higher in the intimate proximity of
PS-containing membranes as compared with the bulk solution (44),
although a wide range of dissociation constants of PS-Ca2+
adducts have been reported (for review see Ref. 45). Estimates of the
local absolute free Ca2+ concentrations on the surface of
SLV are technically not feasible. Nevertheless, our data strongly
suggest that cross-linking of involucrin by membrane-bound TGase 1 occurs at a 10-fold lower Ca2+ concentration than that
required for the soluble enzyme and substrate. Moreover, comparison
with the data of Fig. 3 reveals that this concentration is severalfold
higher than that required for efficient involucrin binding to SLV,
which thereby suggests a likely temporal order to these processes
in vivo.

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Fig. 6.
Binding to SLV lowers free Ca2+
ion levels required for involucrin cross-linking by TGase 1. [14C]Putrescine incorporation into 1.2 nmol of involucrin
by TGase 1 was measured in the absence (yellow squares) or
presence (orange triangles) of SLV (2 µmol of lipid)
containing 15% PS at different free Ca2+ concentrations.
Activity values are given as percent of the values measured at 1 mM Ca2+. Also shown for comparison are the data
from Fig. 3 showing that involucrin binds to SLV at much lower
Ca2+ concentrations (brown circles). Points
represent the mean ± S.D. of three independent
measurements.
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Solubilized Recombinant TGase 1 Reacts with the Majority of Gln
Residues of Involucrin--
The Gln residues of involucrin used by
recombinant TGase 1 were analyzed in the absence or presence of either
natural insect cell membranes or SLV as a catalytic cofactor.
Analysis of [14C]putrescine incorporation into involucrin
was done following trypsin digestion and separation of tryptic peptides on a C18 reverse phase HPLC column. Labeled peptide peaks were dried
and identified by sequencing. The PTH-derivative of
-glutamylputrescine isopeptide formed in the TGase reaction was
identified as a distinct peak in the Porton Instruments LF 3000 gas-phase sequencer (Fig. 7).

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Fig. 7.
Identification of
-glutamylputrescine by amino acid sequencing.
This assay identifies Gln residues that have been modified by
putrescine as a result of the TGase reaction. After Edman degradation,
the PTH-derivative of -glutamylputrescine appears as a novel peak
eluting at 13.55 min in the sequencing cycles where only a Gln residue
would normally be expected. The relative amounts of this peak and that
of PTH-Gln provide an estimate of the extent of modification.
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A total of 40 tryptic peptides was reliably separable, of which 29 (Fig. 8A, arrows) were labeled by
[14C]putrescine in a reaction with solubilized
recombinant TGase 1 and involucrin. Sequencing identified 80 labeled
Gln residues Table II (part A), with a
total of about 20 mol of putrescine incorporated per mol of involucrin
(Table II, part A). Human involucrin contains 150 Gln residues (20),
which means that most were promiscuously labeled under these
experimental conditions.

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Fig. 8.
Identification of tryptic peptides of
involucrin labeled with [14C]putrescine by various forms
of TGase 1. Involucrin was reacted with 20 mM
[14C]putrescine using the following: solubilized
recombinant baculovirus TGase 1 not attached to SLV (A) and
TGase 1 bound to SLV containing 15% PS (B). Tryptic
peptides of involucrin separated by C18 reverse phase HPLC were
collected and assayed for isotope incorporation. Forty involucrin
tryptic peptides were resolved in this system. Labeled peptides are
shown by the numbered arrows. Note that in B,
additional minor peptide peaks were contributed by insect proteins, but
the same numbering system was retained for clarity.
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Table II
Glutamine residues in involucrin that serve as acyl donors by TGase 1
Tryptic peptide peaks that contained [14C]putrescine label
indicated by arrows in Fig. 9 were sequenced. The location
and amount of modified Gln residues were identified by the
appearance and quantitation of PTH- -glutamylputrescine (Fig. 8).
Underlined residues denote those seen in in vivo
cross-linking (17).
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Attachment of TGase 1 to Insect Cellular Membranes or SLV Reveals
Highly Specific Utilization of Gln Residues of Involucrin--
These
experiments were repeated using recombinant TGase 1 and involucrin
bound to the crude particulate membranes of Sf9 insect cells. In
this case, only five tryptic peptide peaks were labeled (Table II, part
B), involving five different Gln residues. Similarly, following binding
of recombinant TGase 1 and involucrin to SLV containing 15% PS (Fig.
9B), the same five labeled
tryptic peptides were recovered which encompassed the same five Gln
residues (Table II, part C). In both cases, only about 1 mol of
putrescine was incorporated per mol of involucrin of which Gln-496 was
the most brightly labeled. Interestingly, these data corroborate an
earlier study (11) in which Gln-496 was identified as the most strongly labeled residue in a reaction of involucrin with the TGase 1 enzyme associated with crude keratinocyte membranes. Four other Gln residues (Gln-107, Gln-118, Gln-122, and Gln-133) located in the evolutionarily conserved head domain or ancestral portion of involucrin were also each
slightly labeled.

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Fig. 9.
Model for the alignment of involucrin and
TGase 1 on SLV on the inner surface of the plasma membrane of
keratinocytes. Newly expressed TGase 1 (green sphere)
attaches to the membranes by way of the lipid adducts on its
amino-terminal portion. As the Ca2+ concentration at the
micro-environment of the membrane surface rises above a critical
threshold level at the advent of terminal differentiation, the
involucrin (red rod) attaches spontaneously. This binding is
fostered through ionic interactions of multiple Glu residues of
involucrin, Ca2+, and the anionic PS-rich membrane surface
(yellow). We propose that these binding processes align only
certain Gln residues of involucrin near the active site of juxtaposed
TGase 1 molecules. Cross-linking reactions are initiated as the
Ca2+ concentration rises further. The activated Gln
residues may then be transferred to other nearby substrates including
desmoplakin, envoplakin, etc. (purple spheres) to initiate
CE assembly.
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DISCUSSION |
Of several enzymes likely to be involved in cross-linking
reactions to form the CE barrier during terminal differentiation in
stratified squamous epithelia, the TGase 1 enzyme is perhaps the most
complex since it exists in multiple forms (32, 46). This enzyme is of
critical importance in skin barrier function in particular since
mutations in its gene resulting in loss of activity cause the
devastating life-threatening disease lamellar ichthyosis (47-49). Most
of the TGase 1 enzyme resides on membranes through
N-myristoyl and S-myristoyl or
S-palmitoyl linkages (30-32), although minor amounts
dissociate to reside in the cytosol (46). To date, almost all studies
of this enzyme have involved solution assays of soluble substrates with
TGase 1 enzyme stripped and purified from the cellular membranes or
soluble recombinant expressed TGase 1 enzymes. In this study, we have
systematically developed a more physiological model system to explore
the properties of this enzyme. Our data introduce a so far unstudied
catalytic cofactor, the membrane surface, in regulating the interaction
of membrane-bound TGase 1 enzyme and its substrates. In particular, we
have found that of several of its known and proven in vivo
substrates, involucrin also binds to SLV membranes of similar PS
content to those of the cytoplasmic surface of plasma membranes of
eukaryote cells (50). Our data provide evidence that the residue
specificity of the TGase 1 reaction is dependent on the attachment of
itself and involucrin to the membrane surface (Fig. 9). These
observations have important implications for the mechanism of assembly
of the CE barrier structure in stratified squamous epithelia.
Optimized involucrin cross-linking by membrane-bound TGase 1 is largely
dependent on the ingredients of the membrane and requires Ca2+ ions and PS, an inherent constituent of the
cytoplasmic face of membranes in living eukaryote cells (50). PS is
required for a number of other physiological processes, where
Ca2+-dependent binding of proteins to cell
membranes is an essential condition for enzyme activity, as exemplified
in the case of protein kinase C activation (39, 51) or blood clotting
(52). As for these two well studied processes, we found that the
activating properties of PS were not substitutable by other natural
anionic phospholipids, presumably because both the carboxyl and amino groups are required for sequestration of Ca2+ ions on the
membrane surface.
Several studies have examined total Ca2+ concentrations in
keratinocytes. Available evidence suggests there is a Ca2+
concentration gradient in the epidermis, for example, from a low level
in basal cells which gradually increases toward the granular layer
(53). In addition, Ca2+ concentrations are indirectly known
to be much higher on cell membrane surfaces (54). In keratinocytes
grown in submerged cultures in low Ca2+ medium, under which
conditions they do not embark on terminal differentiation, net
intracellular Ca2+ concentrations are about 50-100
nM (55). When cells are grown in higher Ca2+
medium (0.5-1.5 mM), net intracellular Ca2+
levels rise briefly to about 100-200 nM (55), and the
stratification and the terminal differentiation program proceeds (56).
Similarly, normalization of a Ca2+ gradient is essential
for terminal differentiation and improved barrier function in
reconstructed cultured epidermis (57). However, in none of these
studies has it been possible to measure the micro-environmental concentration at or near the membrane surface.
Typically, involucrin is expressed in mid-late spinous layers in the
epidermis (or comparable levels in other stratified squamous epithelia)
and is expressed early in cultured keratinocytes as elevated
environmental Ca2+ levels initiate terminal differentiation
(58). The TGase 1 enzyme is expressed to a minor extent in basal
keratinocytes, but its major expression program approximately coincides
with that of involucrin in differentiating keratinocytes (59). Our new
data from our model SLV system demonstrate that involucrin begins to
associate onto SLV above 1 µM (Fig. 3). These
observations favor the view that involucrin binds to the plasma
membranes shortly after its expression. Furthermore, our data
demonstrate that the cross-linking of involucrin does not begin until
the net Ca2+ concentration rises about 10-fold higher than
that required for involucrin binding (Fig. 6). Thus, we propose that
the involucrin substrate and TGase 1 enzyme remain in close
juxtaposition on cellular membranes until local Ca2+
concentrations rise above a threshold level. Accordingly, together with
available in vivo data, our new results offer the
possibility that the Ca2+ gradient not only orchestrates
the expression of differentiation-specific genes (5, 9, 56) but also
creates the environment required for the initiation of CE barrier
formation by juxtaposed attachment of involucrin and TGase 1 to
membranes for their subsequent cross-linking together (Fig. 9).
We show here that cross-linking of involucrin by TGase 1 in solution
in vitro is an efficient process (Table I) involving the
utilization of more than half of the total Gln residues of involucrin
(Fig. 8, Table II). Published data from this laboratory have identified
27 Gln residues that are used for cross-linking in vivo
(17), 23 of which were used in the present in vitro experiments (Table II). This suggests that these 80 Gln residues are
the most available for reaction on involucrin. Thus it is unlikely that
the utilization of multiple Gln residues was due to degradation or
denaturation of involucrin (11, 22). Conversely, these data also allow
the speculation that involucrin may be cross-linked in vivo
by soluble TGases as well, including perhaps the minor cytosolic forms
of TGase 1.
The most striking observation in the present study is the specificity
of Gln usage for cross-linking of bound involucrin and TGase 1; more
than 50% of TGase 1 reaction involved the single Gln-496. Moreover,
this specificity was observed for both crude insect membranes, as well
as SLV of several confections, providing the PS content exceeded 5%.
Interestingly, this residue was identified as the most reactive in an
earlier in vitro study (11). This earlier experiment
utilized the TGase 1 activity of a crude keratinocyte membrane
fraction, solubilized involucrin and 5 mM Ca2+.
Our present data demonstrate that the added involucrin would have
immediately attached to the membranes. As earlier reports showed that
shorter CNBr or tryptic peptides of involucrin did not afford such
specificity (11, 22), together, the previous and our present data
indicate that the stereochemistry of binding of intact involucrin and
subsequent cross-linking by bound TGase 1 are critical determinants of
this specificity.
The kinetic data of Table I shed light on the high degree of
specificity of Gln utilization. We propose that the reason for the
lowered kcat and KM values of
membrane-bound TGase 1 using bound involucrin as substrate is due to
restriction of available Gln residues for reaction. Furthermore, the
maximal reaction velocity must be limited by the following: (i) the
quantity of involucrin molecules attached to a unit of membrane
surface; (ii) the lateral diffusion rate of enzyme and substrate along the membrane surface; and (iii) the rate of exchange between soluble and membrane-attached involucrin. An alternative hypothesis that association with the SLV membranes might change the conformation and
thus specificity of TGase 1 remains to be tested experimentally, but in
control experiments documented in Table I, we found no change in
substrate specificity of the SPR2 substrate. Furthermore, in the insect
membrane or SLV reactions only five Gln residues were used to insert
about 1 mol of putrescine/mol of involucrin, of which Gln-496 was the
most labeled. In contrast 80 Gln residues were labeled in the soluble
reaction to insert about 20 mol/mol of putrescine. Thus the overall
kinetic efficiency value for Gln-496 was at least 2-fold higher than
that of the average Gln residue in a solution reaction. Therefore,
these five residues, and Gln-496 in particular, must be particularly
favorably aligned with respect to the active site of the neighboring
bound TGase 1 enzyme. It seems obvious that the active site of
membrane-bound TGase 1 must be located at a certain distance from the
plane of the membrane, and thus the five utilizable Gln residues in
involucrin can only be those that are at a compatible distance (Fig.
9). Clearly, further studies on the three-dimensional structures of
involucrin and TGase 1 are warranted.
We have documented that involucrin is cross-linked in vivo
to a variety of structural proteins, including in particular
desmoplakin at the site of desmosomes, envoplakin, and perhaps
periplakin located primarily on plasma membranes between desmosomes, as
well as to itself. Furthermore, the predominant cross-linking site with
desmoplakin was through Gln-496 of involucrin (17). Together, these and
the present data offer a tantalizing snapshot of the earliest stages of
CE assembly. It appears that involucrin and TGase 1 associate with the
plasma membrane shortly after expression. As the localized
Ca2+ concentration rises, the TGase 1 enzyme activates
Gln-496 which is favored for transfer to Lys acceptor residues on
desmoplakin. Thus we propose that the desmosome is an important site
for the initiation of CE assembly. Furthermore, these observations have important implications for the disease lamellar ichthyosis caused by
lack of a functional TGase 1 enzyme (33, 47-49). If the initiation of
CE assembly as envisaged above cannot occur, we should expect profound
problems with formation of an effective barrier, as it is clear that
the TGase 2 and 3 enzymes also expressed in terminally differentiating
keratinocytes which do not bind to membranes (Fig. 4) cannot compensate
for this critical initial step.