(Received for publication, October 26, 1994; and in revised form, June 5, 1995)
From the
The transglutaminase 1 (TGase 1) enzyme is required for the
formation of a cornified cell envelope in epidermal keratinocytes. We
show here that in addition to its membrane-anchored form, soluble forms
of it are also important in keratinocytes. Proliferating cells contain
soluble full-length enzyme of 106 kDa, but terminally differentiating
cells contain a soluble 67-kDa form often complexed with a 33-kDa
protein as well. The amino terminus of the 67 kDa form is residue 93 of
the TGase 1 protein, corresponding to the site of proteolytic
activation of the factor XIIIa TGase. The amino terminus of the 33-kDa
protein is residue 573, corresponding to the site of a second
proteolytic cleavage site of factor XIIIa, and of the site for
proteolytic activation of the TGase 3 enzyme. The specific activity of
the 67/33-kDa soluble complex is twice that of the soluble 67-kDa form
and 10 times that of full-length TGase 1. The half-lives of the 67/33-
and 106-kDa forms are about 7 or 20 h, respectively. Thus the TGase 1
enzyme is complex, since it exists in keratinocytes as multiple soluble
forms, either intact or proteolytically processed at conserved sites,
and which have varying specific activities and likely functions. Of the six known active transglutaminases (TGases) In addition, a truncated
recombinant form of 467 amino acid residues, which retained half of the
activity of the full-length TGase 1 expressed form or native enzyme
from cultured cells(21) , has been used to make a TGase 1
polyclonal antibody. This antibody decorates the entire epidermis,
including the basal layers and epidermal derivative organs such as the
hair follicle(22, 23) . In contrast, a widely used
commercially available TGase 1 monoclonal antibody (B.C1) decorates
only the granular layer of the
epidermis(20, 24, 25, 26) . By
Western blotting methods, the monoclonal antibody recognizes a band of
about 90 kDa(20, 26) , thought to be the size of the
full-length TGase 1 enzyme in cultured epithelial cells and epidermal
tissue extracts, but the major proteins recognized and
immunoprecipitated by it have molecular masses of 10-20 kDa,
which we have recently demonstrated are the SPR1 and SPR2 proteins also
expressed in the epidermis(22) . However, our new antibody
recognizes a major band of 106 kDa, which is apparently the true
full-length size of the TGase 1 enzyme in
keratinocytes(21, 22) . This 15% increase in size may
be due to postsynthetic modifications of a basic core protein of 92
kDa(7, 8, 27) . In addition to the band of
106 kDa, our antibody recognized several other minor bands of lower
molecular weight(21, 22) , also reported
earlier(2) , that may be due to degradation of the TGase 1
protein or cross-reactivity with other TGase proteins of the
keratinocytes. In the process of a systematic analysis of these
possibilities by use of immunoprecipitation and Western blotting
experiments, we have found to our surprise that the TGase 1 system in
cultured epidermal keratinocytes and foreskin epidermal cells is far
more complicated than heretofore described. It has been thought that
most of the ``soluble'' (that is, cytosolic) TGase activity
in cultured epidermal cells is due to the TGase 2
enzyme(2, 16, 18, 19, 28) ,
whereas most of the TGase 1 activity is anchored to
membranes(2, 18, 19) . We describe here that
most of the soluble TGase activity in cultured keratinocytes is in fact
due to soluble full-length or smaller more active forms of the TGase 1
enzyme, generated by proteolytic processing of the full-length protein
at specific sequence sites comparable with the sites of activation of
other TGases.
Freshly excised foreskins were cut open and cultured for 4
h in suspension organ culture (5 ml/tissue) in Dulbecco's
modified Eagle's medium in the absence or presence of 100 µCi
each of [ Total In some experiments, aliquots of
the total cell lysates or cytosolic fractions were boiled in
polyacrylamide gel loading buffer containing 2% SDS and 2%
2-mercaptoethanol, and proteins were resolved on 10% linear or
10-20% gradient gels. Following transfer to PVDF membranes, bands
were identified by Western blotting with specific TGase antibodies and
developed with the Bio-Rad reagent(16) .
In most experiments, the absorbed
antigens and primary antibodies were harvested by boiling the washed
beads in 50 µl of SDS sample buffer containing 10%
2-mercaptoethanol, resolved by electrophoresis on 10% linear or
10-20% gradient polyacrylamide gels, dried, and autoradiographed.
Gels were exposed to x-ray film for 1-15 days. In some
autoradiograms, selected bands were quantitated by scanning in a
computing densitometer with ImageQuant software version 3.0 (Molecular
Dynamics). Standard protein markers (Life Technologies, Inc.) were
used. In some experiments, the absorbed active TGase proteins were
recovered from the washed beads with 100 µl of elution buffer
(Pierce). After 30 s, the suspension was neutralized with 25 µl of
1 M Tris acetate buffer (pH 7.5) and then pelleted to remove
the beads. The time of exposure to the low pH (
TGase
enzyme forms were also recovered from the cytosolic fraction of 3-day
post-confluent NHEK cells that had been grown in high Ca The specificities of the TGase antibodies used
was also examined in double immunoprecipitation reactions. In this
case, the three
Figure 2:
Double immunoprecipitation reactions show
that the TGase antibodies are very specific. A,
Figure 4:
The smaller 67- and 33-kDa forms of the
TGase 1 protein are present in foreskin epidermal keratinocytes.
Foreskin epidermal cells labeled with
[
Figure 1:
Immunoprecipitation reactions of the
cytosolic fractions of
However, in order to ascertain that these
bands are not due to cross-reactivity of our polyclonal TGase 1
antibody with other proteins or TGases, a series of control experiments
was performed. First, we purified active
Figure 7:
Fractionation of TGase forms from foreskin
epidermal basal (A) and suprabasal (B) cells.
Cytosolic fraction of each cell population was chromatographed on Mono
Q FPLC as in Fig. 5.
Figure 5:
Fractionation of TGases by Mono Q FPLC.
Chromatography of the cytosolic fractions of: unlabeled post-confluent
NHEK cells grown 3 days in high Ca
These controls show that the three
antibodies are highly specific, since they display only trace amounts
of cross-reactivity, in confirmation of our earlier
data(16, 22, 34) . Thus the prominent 67- and
33-kDa species and numerous other minor species seen in the TGase 1
immunoprecipitation reactions are likely to be due to smaller forms of
the full-length TGase 1 protein and not due to cross-reactivity with
the other epidermal TGases or proteins. In a third set of control
experiments, we examined whether or not this multiplicity of bands was
due to degradation of the TGase 1 protein during isolation, a problem
which has complicated earlier studies with this
enzyme(2, 20) . Cell lysates of 3-day post-confluent
NHEK cells grown in high Ca
Figure 3:
The multiple TGase 1 species are not due
to degradation during isolation. Cell lysates from post-confluent NHEK
cells grown in high Ca
Together, these experiments suggest that the TGase 1
system in cultured NHEK cells consists of the expected full-length
106-kDa form, as well as major 67/33-kDa forms, and minor forms of
other size.
Comparisons of the peaks of Fig. 5, A and B, indicate that the various TGase forms and mixtures have
widely different activities. More quantitative information was
determined by first measuring the amount of active TGase protein in
each peak of Fig. 5A by titration with iodoacetamide (21) and then calculation of specific activities, which are
(nmol of putrescine/h/pmol of protein): peak 1, 0.20; peak 2, 0.02;
peak 3, 0.08; peak 4, 0.45; and peak 5, 0.04. Thus when the 67- and
33-kDa portions are mixed together, their specific activity is more
than 10-fold greater than the full-length 106-kDa enzyme, but the
67-kDa form alone has a 5-fold increased specific activity over the
full-length enzyme. In addition, the specific activity of the 67-kDa
form is similar to that of the equivalent sized bacterially expressed
truncated form (construct 4,
Figure 6:
Sequence alignments of the TGase 1, factor
XIIIa, and TGase 3 enzymes reveal conserved cleavage activation sites
in the TGase 1 system. The three sequences are aligned for maximal
homology(8) ; gaps denote comparative sequence deletions. The
sequences determined at the amino termini of the 106-, 67-, and 33-kDa
species are highlighted. The large arrowheads denote
the identified amino termini of the 106 (residue 3)-,
These sequence
data can explain why the novel 33-kDa band is seen on Coomassie-stained
gels (not shown) and autoradiograms of immunoprecipitated In addition, the data indicate that 67-
and 33-kDa bands can be co-precipitated and co-eluted from the Mono Q
FPLC column. This means that although the TGase 1 protein chain has
been cleaved at specific sites, the two portions remain complexed
together by secondary bonding interactions. This is reminiscent of what
occurs for the 50- and 27-kDa portions of the proteolytically activated
TGase 3 enzyme(16, 17) . Together, the data suggest
that in the cytosolic fraction of cultured NHEK cells or foreskin
epidermal cells, the
However, together
these data indicate that the processed soluble forms of the TGase 1
system are major contributors to total TGase activity in both basal and
suprabasal keratinocytes.
With this
background, we wanted to know whether it was possible to activate by
limited proteolysis in vitro the soluble full-length 106-kDa
TGase 1 protein obtained by immunoprecipitation from the cytosolic
fraction of
Figure 8:
Dispase activation of the full-length
106-kDa TGase 1 form. A,
In an
identical reaction using unlabeled protein, the 30-min dispase
digestion resulted in a To further explore this in vitro dispase activation
process, we used several bacterially expressed recombinant TGase 1
constructs described previously (21) that possessed much higher
specific activities than the full-length protein. Fig. 9shows
that constructs 1 (full length), 2 (
Figure 9:
Dispase activation of bacterially
expressed truncated forms of the TGase 1 protein. The constructs 1
(full-length 92-kDa protein), 2 (
Figure 10:
The soluble 67/33-kDa processed forms
have shorter half-lives than the soluble full-length 106-kDa form of
TGase 1. A, autoradiogram of SDS gels of TGase 1 proteins
immunoprecipitated from cytosolic extracts of 2-day post-confluent
cells grown in high Ca
Moreover, the data show that the specific activity
of the major 67-kDa form is about 5-fold higher, and the 67/33-kDa
complex is Published
data have suggested that the bulk of the ``keratinocyte''
TGase 1 activity in cells is membrane-associated (1, 2, 6, 27; for
review, see (18) and (19) ), and indeed, this fraction
has been used widely for in vitro assays with this enzyme.
Other studies have shown that the membrane associated enzyme can be
released in vitro by proteolysis with trypsin to produce an
How do these different forms arise in
keratinocytes? In vivo, it is possible that the soluble TGase
1 forms reported here arise because not all enzyme is esterified by
fatty acids and anchored to membranes, leaving soluble full-length
protein, some of which is later processed by intracellular
proteases(6, 27, 56) . An alternative or
concurrent possibility is that proteolytic processing occurs while the
full-length TGase 1 is still anchored to membranes by proteases which
themselves may be regulated during terminal differentiation, remain
held together by secondary bonding forces, and then are later released.
If so, this raises the possibility that the membrane anchored enzyme
also may exist in a multiple forms. More work will be required to test
these ideas.
By way of comparison, the
TGase 1 enzyme has a longer amino-terminal sequence containing the
known site(s) of fatty acyl esterification to permit membrane
anchorage. This sequence apparently masks the active site pocket only
partly, because the full-length molecule is quite active, although its
activity can be increased up to 20-fold if 60-90 residues of
these sequences are removed(21) . The beginning of the 67-kDa
species discovered here corresponds to position 93, about 15 residues
prior to the beginning of the predicted In conclusion, the present data
reveal that much of the TGase activity in foreskin epidermal or
cultured epidermal keratinocytes is due to soluble full-length or more
highly active proteolytically processed forms of the TGase 1 system.
This means that there are several likely enzymatically active forms of
the TGase 1 enzyme in keratinocytes and perhaps other stratified
squamous epithelial cells. These new findings lead to the possibility
that they may have different substrate specificities (21) and
thus functions in the assembly of the cornified cell envelope.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)in humans, three are expressed during terminal
differentiation in stratified squamous epithelia such as the epidermis.
These are the membrane-associated TGase 1 of about 92
kDa(1, 2, 3, 4, 5, 6, 7, 8) ,
the ubiquitous soluble tissue type TGase 2 of 80 kDa (9, 10, 11, 12) , and the soluble
pro-enzyme TGase 3 of 77
kDa(13, 14, 15, 16, 17) .
These enzymes are thought to be responsible at least in part for the
assembly of a cornified cell envelope, which provides a vitally
important barrier function for the tissue(18, 19) .
However, the mechanism of assembly of this structure and the substrate
preferences, if any, of these three TGases in cornified cell envelope
formation remains to be resolved. Such studies are complicated by the
fact that the TGase 1 enzyme is perhaps the most difficult to work with
because of its lability during isolation and
purification(2, 20, 21) . However, we have
recently demonstrated that a recombinant TGase 1 enzyme can be
expressed in bacteria, which has an activity comparable with that
isolated from cultured keratinocytes(21) . Furthermore, by
deletion cloning, removal of the first 36-98 residues results in
large increases in specific activity as well as changes in reactivities
toward and kinetic efficiencies with a number of potential cornified
cell envelope substrates, but removal of up to 240 residues from the
carboxyl-terminal end has little affect on activity(21) . Thus
an important question arises as to whether smaller highly active forms
of the TGase 1 system exist in cells.
Keratinocyte Protein Extracts
Normal human
foreskin epidermal keratinocytes (NHEK) (Clonetics Corp., San Diego,
CA) were seeded at a density of 5.10 cells/cm
in 15-cm dishes and grown in low Ca
KGM medium
(0.05 mM CaCl
) as recommended by the manufacturer.
In some cases, at confluence (about 3 days) the medium CaCl
concentration was raised to 0.6 mM (high
Ca
), previously determined to be optimal for
induction of terminal differentiation in human epidermal keratinocytes
in cell culture(29) . In other high Ca
experiments, the calcium ionophore A23187 (Calbiochem) (30) was also added to cultures at confluence (25 µg/ml,
final concentration). In most experiments, the cultures were
metabolically labeled with a mixture of
[
S]cysteine and
[
S]methionine (1 µCi each/ml of medium)
(Amersham Corp.). This was added either (i) 4 h before planned
harvesting of cells, or (ii) in pulse-chase experiments, for 4 h in 2
day post-confluent cultures grown in the presence of high
Ca
, followed by replacement with unlabeled medium. As
required, the cells were harvested by scraping and sonicated in a
buffer containing 0.1 M Tris acetate, 0.15 M NaCl, 1
mM EDTA (pH 7.5) (TBS) (4
10
cells/ml) in
the presence (or absence in the case of one control set of experiments)
of a mixture of protease inhibitors leupeptin (1 mM),
4-(2-aminoethyl)-benzenesulfonyl fluoride (0.2 mM), calpain
inhibitor (10 µM), and aprotinin (0.1 unit/ml) (Boehringer
Mannhiem). The lysate was clarified by centrifugation at 10,000
g for 20 min at 4 °C to obtain the cytosolic (soluble)
fraction.
S]cysteine and
[
S]methionine. Following thorough washing in
phosphate-buffered saline, the tissues were floated on trypsin (Difco)
overnight at 4 °C to separate the epidermis, from which a total
epidermal cell suspension was recovered by standard procedures. These
cells were then plated on plastic for 4 h exactly as described
previously(31) , during which time essentially only the basal
cells attached(32) . Both the attached and unattached
suprabasal cells committed to terminal differentiation were then
harvested, sonicated in the TBS buffer, and pelleted to recover the
cytosolic fraction as above.
S-labeled foreskin
tissues (epidermis + dermis) were used to prepare human actin
exactly as described(33) .
Expression in Bacteria of Full-length and Truncated Forms
of TGase 1
Six recombinant deletion constructs of the human
TGase 1 enzyme that produced particularly stable active enzymes were
prepared exactly as described previously (21) and were
constructs 1 (full length, 92 kDa), 2 (N37), 3 (
N52), 4
(
N61), 5 (
N97), and 20 (
N108
C575).
[
S]Cysteine-labeled construct 1 was routinely
used as a marker for autoradiography of SDS gels. These recombinant
proteins, as well as the native full-length 106-kDa enzyme (prepared
from cultured NHEK cells as described below), were substrates for
proteolysis using 0.01 unit/ml of dispase (Boehringer Mannhiem) (about
1:20 protease to TGase ratio).
Immunoprecipitation of Three TGase Activities from Cell
Lysates
The three specific affinity-purified antibodies used
were: our new polyclonal anti-human TGase 1 made in goats(22) ,
polyclonal anti-guinea pig liver TGase 2 made in rabbits which
cross-reacts with the human enzyme(34) , and polyclonal
anti-guinea pig skin TGase 3 made in rabbits which cross-reacts with
the human enzyme (16) . Each of the three antibodies used in
this study was serially diluted 10 to
10
in TBS to explore optimal precipitation of enzyme
activity. In control experiments, we found that essentially complete
immunoprecipitation of the respective
S-labeled antigens
and activity occurred with a 1:50 dilution of each
antibody(21, 22) . Cell lysate fractions of NHEK cells
or separated foreskin epidermal cells (200 µl) were incubated with
the antibodies for 1 h at 4 °C by gentle shaking. Protein
A-conjugated agarose beads (ImmunoPure Plus, Pierce) were
pre-equilibrated in TBS buffer, and resuspended as a 1:1 slurry in TBS,
of which 20 µl was then added to the cytosolic primary antibody
mixtures. Following incubation for 1 h at 4 °C in a rotary shaker,
the conjugated beads of each reaction were collected by centrifugation
at 10,000
g for 2 min and washed twice with TBS to
remove unabsorbed proteins.
2.8) elution buffer
was strictly limited to
30 s. Control experiments showed that (i)
>95% of the
S-labeled TGase protein antigens were
eluted within 30 s, and (ii) the half-life of TGase 1 activity in the
low pH buffer is about 4 min.
Separation and Characterization of TGase Activities in
Foreskin Epidermal or NHEK Cell Cytosolic Fractions
The TGase 1,
2, and 3 enzymes were recovered from cytosolic fractions of unlabeled
or S-labeled foreskin epidermal ``suprabasal''
cells (for the TGase 3 proenzyme) or ``basal'' cells (for the
soluble TGase 1 and TGase 2 enzymes). Samples were chromatographed by
FPLC on a 0.5
5 cm mono-Q FPLC column equilibrated in a buffer
of 50 mM Tris acetate (pH 7.5) containing 1 mM EDTA
using 60 ml of a 0-0.5 M NaCl linear gradient, and
collected into 0.5-ml fractions, essentially as described(16) .
Peaks of TGase activities were ascertained by assays of every second
fraction. The TGase 3 eluted in the column wash, and the TGase 1 and 2
enzymes eluted at about 0.2 or 0.3 M NaCl, as
expected(2, 16) .
S-Labeled TGases from
immunoprecipitation reactions of cytosolic fractions with either the
TGase 1 (22) or TGase 2 (34) antibody were resolved
similarly and detected by counting every second fraction. In this case,
the eluted
S-labeled TGases were neutralized with 1 M Tris acetate (pH 7.8) and diluted to 1 ml before loading.
and in the presence of the calcium ionophore. The
S-labeled cytosolic fraction was immunoprecipitated with
antibodies, eluted, neutralized, and the products chromatographed as
described above and counted. An alternative method to recover the
full-length 106-kDa TGase 1 enzyme was to fractionate the unlabeled
cytosolic fraction from 1-day post-confluent NHEK cells grown in low
Ca
.
S-labeled TGases purified from foreskin
epidermal cells as described above were used for a second round of
immunoprecipitations with various combinations of antibodies (see Fig. 2A). Alternatively, TGase 1 or TGase 2 antigens of
3-day post-confluent NHEK cells grown in high Ca
were
first harvested from separate affinity columns, eluted, neutralized,
and concentrated, exactly as described
before(21, 22) .
S-labeled full-length 106-kDa TGase 1, 80-kDa TGase 2, and
77-kDa TGase 3 enzymes were purified from foreskin epidermal cell
cytosolic extracts by immunoprecipitation as described under
``Materials and Methods'' (and see Fig. 4,
5A, and 7) (lanes 1, 5, and 9,
respectively). Equal amounts of label of each were then used for
reprecipitation by the TGase 1 (lanes 2, 6, and 10),
TGase 2 (lanes 3, 7, and 11), or TGase 3 antibodies (lanes 4, 8, and 12). Autoradiograms of the dried SDS
gels were exposed for 15 days. B, the
S-labeled
cytosolic fraction of 3-day post-confluent NHEK cells grown in high
Ca
was chromatographed on separate TGase 1 or TGase 2
antibody affinity columns (lanes 1 and 5,
respectively). Equal amounts of label were then used for
immunoprecipitation by the TGase 1 (lanes 2 and 6),
TGase 2 (lanes 3 and 7), or TGase 3 (lanes 4 and 8) antibodies. These autoradiograms were exposed for
7 days. C, control of
S-labeled bacterially
expressed full-length (92 kDa) TGase 1. Sizes of bands (left)
are based on molecular mass markers (right). Note that the
TGase 2 protein is partially degraded, some low molecular mass portions
of which are differentially precipitated by the TGase 1 and 3
antibodies.
S]methionine/cysteine were fractionated as
described under ``Materials and Methods'' into basal and
suprabasal populations. Aliquots of cytosolic fractions were
immunoprecipitated with either the TGase 1, TGase 2, or TGase 3
antibodies. Lanes 1, 4, and 7, total epidermal cell
population; lanes 2, 5, and 8, basal cell fraction; lanes 3, 6, and 9, suprabasal cell fraction. The
immunoprecipitated products were resolved on 10-20% gradient SDS
gels, dried, and autoradiographed for 11 days. Loadings were adjusted
for equal amounts of protein. The sizes of the major protein components
are shown.
Measurement of TGase Specific Activities
Standard
TGase assays were performed by measurement of the incorporation of
[H]putrescine (Amersham) into succinylated
casein(21) . Protein assays were performed colorimetrically
(Bio-Rad)(35) . Samples of unlabeled active TGase species from
the Mono Q FPLC experiments were used for titrations with 5 mM
[
C]iodoacetamide to measure the amount of active
TGase protein(13, 21) . Subsequent immunoprecipitation
of the inactivated [
C]methylcarboxamide
cysteine-TGase(s) with the polyclonal TGase 1, 2, or 3 antibodies
enabled measurement of radioactivity incorporated, from which the
amount of each TGase protein and its specific activity was then
calculated(21) . While it was possible in the present work to
purify
S-labeled TGases, direct specific activity
measurements were hampered by the overlap of energies of
decay of
the [
S]methionine/cysteine and
[
H]putrescine isotopes. Specific activities were
not measured on TGase proteins previously exposed to the low pH
antibody elution buffer of either ImmunoPure beads or affinity columns.
Microsequencing
The products of some
immunoprecipitation reactions using the TGase 1 antibody from the
cytosolic fractions of NHEK cells, as described above, were resolved on
a 10% polyacrylamide gel run in Tricine buffers and transferred to PVDF
membranes. The bands containing 2-10 pmol of the 106-, 72
(minor)-, 67-, 65 (minor)-, and 33-kDa proteins were excised, placed in
a LF3500 gas-liquid phase sequencer (Porton), and run for 15 Edman
degradation cycles. Released phenylthiohydantoin-derivatized amino
acids were resolved and quantitated by on-line analytical high
performance liquid chromatography (Beckmam Instruments, using System
Gold software). The 106-kDa band initially did not give a sequence.
Another sample of this band on PVDF membrane was boiled in 5.7 N HCl at 106 °C in vacuo for 2 h to hydrolyze off the
probable NH-terminal blocking adduct. The acid solution was
dried, redissolved in 5 µl of 50% aqueous acetonitrile, and
covalently attached to a PVDF solid support (Sequelon-AA, Millipore)
for sequencing.
Complexity of Soluble TGase 1 Species in the Cytosolic
Fraction of Cultured NHEK Extracts
The purpose of the present
experiments was to better characterize the properties of the TGase 1
system than has heretofore been possible. Our earlier work has
documented significant differences in the published expression
properties of the TGase 1 enzyme system using our new polyclonal
antibody (21, 22) as compared with use of a commercial
monoclonal antibody (B.C1)(24, 25, 26) . In
addition, Western blots of cytosolic fractions from NHEK cells using
the polyclonal antibody recognized a major band of TGase 1 protein of
106 kDa, as well as minor bands between 70 and 90 kDa and below 50 kDa (21, 22) . However, because these bands were
relatively weak, in the present work we have metabolically labeled NHEK
cells or human foreskin epidermis with
[S]methionine and
[
S]cysteine and immunoprecipitated the TGase
proteins in order to better visualize them by autoradiography. The
amount of
S label incorporated during 4 h in cell culture
was in the range of 0.5-3
10
dpm/10
cells. When cultured for several days post-confluence in low
Ca
medium, under which conditions the cells do not
differentiate to a significant extent(29) , most of the TGase 1
immunoprecipitable protein was a major band of 106 kDa, as expected, as
well as minor bands of about 67 and 33 kDa, and other very minor
species of 70-95 and 45-65 kDa (Fig. 1A).
When confluent cultures were changed to high Ca
medium (not shown, but see Fig. 2B, lane 1), and
in the presence of the calcium ionophore A23187 (Fig. 1B), the 67- and 33-kDa forms were major products
of the immunoprecipitation reaction, which increased in time as the
cells differentiated.
S-labeled cultured NHEK cells
reveals the presence of multiple forms of TGase 1. NHEK cells were
plated and grown in: low Ca
medium (A) or in
high Ca
medium (B) in the presence of
calcium ionophore. Cells were harvested at the indicated days post
confluence immediately after a 4-h pulse with
[
S]methionine/cysteine. TGase 1
antibody-immunoprecipitated products of the cytosolic fractions were
resolved on 10-20% SDS gels and autoradiographed for 7 days.
Loadings were adjusted for equal amounts of cytosolic protein (measured
before immunoprecipitation). Lane C, control of
S-labeled bacterially expressed full-length (92 kDa) TGase
1. Sizes of TGase 1 antibody immunoprecipitated bands are as shown (center), based on standard molecular mass markers (left and right).
S-labeled TGase
1, 2, and 3 enzymes from labeled foreskin epidermal cells as described (16) (see Fig. 4and Fig. 7) and used them for
double immunoprecipitation reactions (Fig. 2A). In the
case of our polyclonal TGase 1 antibody, a second immunoprecipitation
reaction was able to reprecipitate 98% of the
S-labeled
106-kDa band from its first precipitation reaction (Fig. 2A,
lane 2), but the TGase 2 antibody could only precipitate 2% (lane 3), and the TGase 3 antibody, <1% (lane 4).
Likewise, the TGase 2 antibody used could reprecipitate 97% of its
first reaction of 80 kDa (lane 7), but the TGase 1 and TGase 3
antibodies could precipitate only 1% or <1% (lanes 6 and 8, respectively). The TGase 3 antibody displayed a similar
degree of specificity: <1% of its immunoprecipitation product of
about 77 kDa could be reprecipitated by the antibodies for TGases 1 and
2 (lanes 10 and 11, respectively). Two internal
controls were done: (i) the second antibody (ImmunoPure beads) used as
the primary antibody bound <2% of the
S label in each
case, and (ii) each of these three antibodies could precipitate <1%
of
S-labeled actin either in the first or second
precipitation reactions (data not shown). In a second series of control
experiments, the TGase 1 and 2 antibodies were used harvest antigens
from separate affinity columns (21, 22) using the
cytosolic fractions of
S-labeled confluent NHEK cells
cultured in the high Ca
for 3 days. Then in
immunoreprecipitation reactions, the TGase 1 antibody reprecipitated
>95% of its first reaction of the 106, 67, and 33 kDa bands (Fig. 2B, compare lane 2 with lane
1), but the TGase 2 and 3 antibodies reprecipitated <1% and
about 2% (lanes 3 and 4, respectively). Likewise, the TGase 2 antibody
reprecipitated most of its first reaction, but <2% could be
reprecipitated by the TGase 1 antibody (compare lanes 6 and 8 with lane 7).
and with the
calcium ionophore (A). B, same as A, but
S-labeled cells were first immunoprecipitated with the
TGase 1 antibody. C, autoradiograms of SDS gels of samples
from peaks of Fig. 5B and exposed for 2 days. D, same as B but immunoprecipitated with the TGase 2
antibody.
were prepared and
incubated in the presence or absence of a set of protease inhibitors
for up to 2 h. The data of Fig. 3show, however, that the
absence of the protease inhibitors neither significantly altered the
net TGase activity nor the pattern of bands recognized on Western
blots. Note in Fig. 3that the band of 33 kDa was not seen by
the antibody.
medium for 3 days were
prepared and incubated at 23 C for up to 2 h in the presence or absence
of a commercial mixture of protease inhibitors. A, assayed
TGase activity. B, Western blots of 10% polyacrylamide gels.
Neither the total enzymic activity nor the multiple bands recognized by
the antibody changed significantly, indicating little if any random
proteolysis during the extraction
procedures.
The Smaller Soluble 67/33-kDa Forms of the TGase 1 System
Are Present in Foreskin Epidermal Cells
We wanted to know
whether the smaller forms of the TGase 1 system seen in NHEK cells
grown in submerged cultures were of broader physiological significance
and are seen in epidermis as well. Foreskin epidermal cells labeled
with [S]methionine/cysteine were fractionated
into populations of mostly basal cells (those that could attach to
plastic in <4 h(32) ) and mostly suprabasal cells committed
to terminal differentiation (those which did not attach in 4 h). The
two cytosolic fractions were then separately immunoprecipitated with
the specific TGase antibodies. Fig. 4shows that the TGase 1
enzyme of basal cells is almost entirely full length (lane 2),
whereas suprabasal cells contained both the full-length and 67/33-kDa
forms (lane 3). The TGase 2 enzyme was mostly restricted to
the basal cell population (lane 5). The TGase 3 pro-enzyme was
mostly restricted to the suprabasal population (lane 9), but
in this case, a small amount of the protein also appeared as the
activated form of
50- and 27-kDa bands(16) . Accordingly,
these data support the in vitro cultured NHEK data and
moreover, show that the appearance of the soluble 67/33-kDa forms of
the TGase 1 system correlates with the commitment to terminal
differentiation.
The Soluble 67- and 67/33-kDa Forms of TGase 1 Have Much
Higher Specific Activities than the Full-length 106-kDa
Enzyme
The appearance of smaller soluble forms of the TGase 1
enzyme system in epidermal cells is reminiscent of our earlier studies
with bacterially expressed truncated recombinant forms of this
enzyme(21) . One notable discovery in those studies was that
deletion of the first 37-97 residues resulted in forms of much
higher specific activity. We next set out to determine the specific
activities of the soluble TGase 1 forms seen in foreskin epidermal or
cultured NHEK cells. Two sets of experiments were done. First, the
cytosolic fraction of unlabeled NHEK cells cultured in high
Ca and in the presence of the calcium ionophore for 3
days were fractionated on a Mono Q FPLC column as described under
``Materials and Methods,'' from which five peaks of TGase
activity were recovered (Fig. 5A). Second, a similar
but
S-labeled cytosolic fraction was first
immunoprecipitated with either our TGase 1 antibody (Fig. 5B) or the TGase 2 antibody (Fig. 5D). From the elution profiles obtained, it is
clear that peaks 1-4 of activity of Fig. 5A are due to the TGase 1 proteins, and peak 5 of Fig. 5A is TGase 2 protein. When the aliquots of the
peaks of Fig. 5B were resolved on SDS gels and
autoradiographed (Fig. 5C), they contained the 67-kDa
proteins only (peak 1), a mixture of 106- and 67-kDa proteins (peak 2), mostly the full-length 106-kDa protein (peak
3), or a mixture of 67- and 33-kDa proteins (peak 4). The
minor peak of
S-labeled protein eluted with about 0.4 M NaCl in Fig. 5B contains mostly the 33-kDa
protein from TGase 1 (Fig. 5C, lane 6) and was inactive (Fig. 5A). The 106-kDa protein could also be isolated
from the unlabeled cytosolic fraction of 1-day post-confluent NHEK cell
cultures grown in low Ca
, immunoprecipitated by our
TGase 1 antibody, and eluted at the position of peak 5 (not shown).
N61) of TGase 1(21) . On the
other hand, when mixed with the full-length 106-kDa form, the specific
activity of the 67-kDa form was diminished.
Amino Acid Microsequencing Reveals Specific Cleavage
Sites in the TGase 1 Protein
Amino acid sequencing was used in
order to further characterize the nature of the TGase 1 forms. Aliquots
of the peaks of S-labeled TGase 1 proteins of Fig. 5C were used for sequencing. These samples were
chosen, because most of the antibody molecules had been resolved away
from the desired bands in order to obtain ``clean''
sequencing data. Following resolution on 4-12% SDS gels in
Tricine buffers (Novex) and transfer to PVDF membranes, the major bands
of 106, 67, and 33 kDa and minor bands of about 70 and 65 kDa (see Fig. 5B) were excised and then subjected to amino acid
microsequencing. A sample containing about 7 pmol of the 106-kDa band
did not yield a sequence, suggesting that its amino terminus was
blocked, as has been found for three other TGase
proteins(16, 27, 36, 37) .
Accordingly, another sample was subjected to partial acid hydrolysis to
cleave off a likely NH
-terminal adduct, and then a sequence
for 15 cycles was obtained beginning from residue position 3 of the
known full-length sequence of TGase 1 protein (7) (Fig. 6). Thus the 106-kDa form does indeed
represent the full-length form of the TGase 1 enzyme. Perhaps during
the partial acid hydrolysis the first two amino acids were lost. The
major 67-kDa band (about 12 pmol) also gave a clean sequence for >15
cycles beginning at residue position 93. Likewise, the other minor
bands of
72 and 65 kDa (1-2 pmol each) yielded useful
sequences beginning at residue positions 83 and 89, respectively.
Sequence alignments of several TGase proteins (Fig. 6) reveal
that the amino terminus of the major 67-kDa band is exactly equivalent
to the site at which the blood-clotting TGase (the a subunit of factor
XIII) is proteolytically activated(38, 39) . The other
minor bands of
70 and 65 kDa begin 10 or 5 residues shifted prior
to this site. The amino terminus of the 33-kDa band resides at residue
position 573, which coincides with the position of a known thrombin
cleavage site of factor XIIIa(39) , and one residue prior to
the site at which the pro-enzyme TGase 3 is proteolytically cleaved for
activation (16, 17) (Fig. 6).
67 (residue
93)-, and 33-kDa (residue 573) forms. The smaller arrowheads denote the amino termini of the minor
72- and
65-kDa
forms. The closed arrows mark the sites of activation by
proteolytic cleavage of factor XIIIa (residue 37) and a second thrombin
cleavage site of factor XIIIa (residue 514). The open arrow marks the position of proteolytic activation of the TGase 3
pro-enzyme system (residue 472).
S-labeled cells, but not Western blotted (Fig. 3B) gels. The polyclonal antibody used in this
work was elicited against a severely truncated active form of
bacterially expressed TGase 1 protein, missing the carboxyl-terminal
241 residues (from residue position 575)(21, 22) .
Thus, because only 2 residues of this region were present on the
immunogen, the antibody is unable to recognize the 33-kDa inactive
fragment beginning at residue position 573. The remaining 10 kDa of
TGase 1 protein, located at the amino terminus, were probably not
associated with the active enzyme, since Coomassie-stained gels of
immunoprecipiated protein did not reveal smaller peptide species (data
not shown). However, the minor TGase 1 bands of 70-90 kDa that
could be identified by the antibody (Fig. 1-4) may
represent species that retain varying portions of the amino-terminal 10
kDa (first 92 residues).
67-kDa species can exist alone, complexed
with the full-length 106-kDa protein or complexed with the 33-kDa
species.
The Soluble TGase 1 Species Are Major Components of the
Total TGase Activity in Differentiating Cultured NHEK or Epidermal Cell
Cytosolic Extracts
The experimental approach of Fig. 5A allowed an estimate of the amounts of the
soluble TGase 1, 2, and 3 activities present in keratinocytes (Table 1). First, the total cell lysates and cytosolic (soluble)
fractions of foreskin basal and suprabasal cells and confluent cultured
NHEK cells maintained in low Ca, or transferred to
high Ca
media, were assayed in order to estimate the
relative amounts of activity in the insoluble (probably
membrane-associated) or soluble fractions. In differentiating cells,
about half of total cellular TGase activity was soluble, compared with
about 75% in proliferating cells (Table 1). When each of these
soluble fractions was resolved by FPLC chromatography (Fig. 7, A and B, for foreskin epidermal basal and suprabasal
cells, respectively; other curves not shown), we found that the various
forms of TGase 1 were major components of the total soluble TGase
activity, contributing about 35% in proliferating cells or up to about
90% in differentiating cells. Of these forms, the 67/33-kDa complex was
always a prominent form. TGase 2 was the major enzyme activity in
proliferating cells, but only a minor component in differentiating
cells (e.g.Fig. 7, A and B,
respectively). However, estimates of the amounts of the TGase 3 enzyme
were complicated (i) because the amounts were very low in cultured
cells as described previously(17) ; (ii) by the presence of
both the full-length (low specific activity) pro-enzyme and
proteolytically activated (high specific activity) enzyme (see Fig. 4, lane 9), which could not be resolved by the
Mono Q FPLC column, since they co-eluted with the column
wash(16) ; and (iii) because different preparations of
suprabasal foreskin keratinocytes presumably contained varying amounts
of basal cells and/or activated TGase 3 enzyme.
The Soluble 106-kDa Full-length Form of TGase 1 Can Be
Activated in Vitro at Conserved Sites by Proteolysis with
Dispase
Controlled activation of enzymes by limited proteolysis
is well documented in many physiological processes, such as the blood
coagulation cascade, the complement activation system, fibrinolysis,
kinin activation, apoptosis, and terminal
differentiation(40, 41, 42, 43, 44) .
Proteases are also important in the activation of the factor XIII
(a subunit) (3, 45, 46) , TGase
3(16) , ``TGase B'' from rat chondrosarcoma cells (47, 48) , and now for TGase 1 as well (present work
and see Refs. 49 and 50). The activation usually occurs as a result of
cleavage at specific site(s); in the cases of factor XIIIa or TGase 1
(present work), this is located toward the amino terminus; in the cases
of TGase B, TGase 3, and TGase 1 (present work), a site is located
toward the carboxyl terminus. In addition, cleavage of TGase 1 near its
amino terminus is required for its release from membranes(6) .
Thus there are two separate known activation sites in different domains
toward each end. Our new sequencing data, together with the existing
data for factor XIIIa and TGase 3, indicate that these sites have been
conserved. Furthermore, the site toward the carboxyl terminus shared by
TGases 1 and 3 coincides with a second site of cleavage of factor
XIIIa, which results in its deactivation(38) .
S-labeled confluent NHEK cells grown in low
Ca
medium. A variety of proteases was used to assess
their potential for activation. Whereas enzymes trypsin and thrombin
resulted in rapid degradation of the protein and loss of activity,
limited proteolysis with dispase resulted in specific cleavage patterns
reminiscent of in vivo processed TGase 1. By autoradiography,
major but somewhat broad bands at
85, 70, 33, and
10 kDa were
generated within 30 min of digestion, in addition to the uncleaved
full-length form (Fig. 8A). Following transfer to PDVF
membranes, these bands were cut out and used for microsequencing. The
70- and 33-kDa bands (2-3 pmol) each yielded multiple sequences,
indicating cleavage polymorphism, but a predominant sequence could be
read for 7-9 Edman degradation cycles, beginning from residue
positions 83 or 573, respectively. Thus the dispase had cleaved the
protein at the same sites as seen in vivo (Fig. 6). The
106- and
10-kDa species did not possess free amino termini,
suggesting they may have originated from the beginning of the protein.
If so, this means that the
10-kDa species contains the membrane
anchorage sequences, as described previously(6) .
S-labeled 106-kDa
protein (27 µg (
5
10
dpm) in 100 µl
volume) isolated from confluent NHEK cells grown in low Ca
and fractionated as in Fig. 5A was digested with
dispase for up to 30 min. Equal aliquots were resolved on a
10-20% gradient SDS gel, dried, and autoradiographed for 13 days. B, a similar unlabeled sample was used to measure total TGase
activity before and after 30-min dispase digestion. C, a
similar unlabeled sample was digested with dispase for 30 min,
chromatographed on the Mono Q FPLC column, and fractions were assayed
for total TGase activity. Sequencing analyses indicate that the protein
eluted in fraction 36 contains the
67-kDa active form, and the
protein eluted in fraction 74 contains the
67/33-kDa complex, as
characterized in Fig. 5.
100% increase in total enzyme activity (Fig. 8B). These products were also resolved on the
Mono Q FPLC column, from which four peaks of activity were recovered (Fig. 8C), that have similar elution properties to
those seen in Fig. 5A. However, insufficient protein
was available for [
C]iodoacetamide titrations
for specific activity estimations of these peaks, but peak 4, which in
comparison with Fig. 5A is likely to contain the
67/33-kDa complex, accounted for most of the activity. Together, these
chromatographic and sequencing data suggest that highly active forms
can be generated in vitro by limited proteolysis of the
full-length TGase 1 with dispase, by cleavage at specific conserved
sites.
N37), 3 (
N52), and 4
(
N61) were further increased by about 100%, as for the native NHEK
full-length form (Fig. 8B). Recombinant constructs
having further deletions from the amino terminus (construct 5,
N97), or from both the amino- and carboxyl termini (construct
20,
N108
C575), did not result in significant net activations (Fig. 9).
N37), 3 (
N52), 4 (
N61),
5 (
N97), and 20 (
N108
C575) were purified as described
previously (21) . Aliquots containing approximately 5 µg of
protein were digested with dispase (total volume of 40 µl) for 30
min and assayed for calculation of specific activities(21) .
Note that the
100% activation afforded by dispase occurs only when
the first 61 residues of the protein are
present.
The Soluble 67/33-kDa Forms of TGase 1 Have Shorter
Half-lives than the Soluble Full-length 106-kDa Form
Analyses of
mutations in both the factor XIIIa (51, 52, 53, 54) and TGM 1 (55) genes that result in premature chain terminations have
shown that the truncated proteins are either not detectable due to mRNA
instability or, if translated, are very unstable. To assess whether the
soluble processed forms of the TGase 1 system have significant
half-lives and thus likely to be functional in keratinocytes,
pulse-chase experiments were done using 2-day post-confluent cultures
grown in high Ca medium. Following a 4-h pulse with
S-labeled methionine/cysteine, the cells were chased with
unlabeled medium for 2 more days. By quantitation of the autoradiograms
of Fig. 10A, estimates of the half-lives of the three
major TGase 1 protein forms were possible (Fig. 10B)
and are:
20 h for the full-length 106 kDa protein and
6.5-7 h for both the 67- and 33-kDa forms. Thus although the
proteolytically processed 67/33-kDa forms have significantly shorter
half-lives than the full-length soluble form, these times are
substantial in comparison with the keratinocyte cell cycle and much
larger than for the mutant forms of TGases seen in genetic diseases.
medium, labeled for 4 h, and
chased for a further 2 days. B, the autoradiograms of three
replicate experiments were quantitated to estimate the half-lives of
the major TGase 1 processed forms.
The Soluble TGase 1 Enzyme and Its Proteolytically
Processed Forms Are Major Components in Terminally Differentiating
Foreskin and Cultured Epidermal Keratinocytes
Our present data
indicate that the TGase 1 enzyme in keratinocytes is far more complex
than heretofore appreciated. Most soluble, that is cytosolic, TGase
activity in keratinocytes was thought to be due to the ubiquitous TGase
2 activity (2, 16, 18, 19, 28) . We
show here that the most likely reason for this earlier view is that the
major active 67/33-kDa forms of TGase 1 elute from ion exchange
chromatography media very close to the TGase 2 activity (Fig. 5A). Using improved FPLC techniques, our new data
show that TGase 2 is the principal enzyme of basal epidermal cells (Fig. 8A) or proliferating cultured keratinocytes,
wherein it constitutes about 60% of the total TGase 1 activity (Table 1). However, the TGase 2 enzyme is in fact only a minor
contributor to the soluble TGase activity in keratinocytes committed to
or induced to terminally differentiate, most is attributable to the
TGase 1 system instead (Fig. 8B; Table 1). More
significantly, we show here for the first time that only part of the
soluble TGase 1 activity is the full-length 106-kDa protein. In basal
foreskin epidermal cells or cultured keratinocytes grown under
proliferating conditions, it exists primarily as the full-length form,
as described previously and generally understood in the
literature(2, 18, 19, 20, 21, 22) .
However, in suprabasal foreskin epidermal cells committed to terminal
differentiation, or keratinocytes induced to terminally differentiate
in culture when grown under high Ca conditions and
permeabilized with respect to Ca
, the TGase 1 system
also consists of several proteolytically processed forms of
significantly higher specific activities. In fact, the most prominent
67- and 67/33-kDa forms of TGase 1 can account for 80-90% of the
total soluble (cytosolic) TGase activity or 40-50% of the total
cellular TGase activity in differentiating cells (Table 1).
Presumably, the membrane-associated forms of TGase 1 account for the
remainder(1, 2, 6, 20) . Thus,
soluble full-length and processed forms of the TGase 1 system are
likely to contribute in a major way to the function of this enzyme in
both basal (proliferating) and differentiating keratinocytes.
The Soluble TGase 1 Forms Are Proteolytically Processed
at Conserved Sites Leading to Increased Specific Activities
Our
amino acid sequencing data (Fig. 6) show that the smaller
soluble forms of the TGase 1 system are not due to degradation, but are
the products of very specific proteolysis events. Two major sites of
cleavage were identified, corresponding either exactly or very closely
to the sites at which other members of the TGase family are also
cleaved for activation or inactivation. In addition, the autoradiograms
showed numerous quantitatively minor species of 70-90 or
50-60 kDa that may represent other proteolytically processed
forms. The two major cleavage sites occur near residue positions 90 and
573, generating an active separable 67-kDa species, an active stable
separable 67/33-kDa complex that remains held together by secondary
bonding interactions, and an inactive 33-kDa fragment containing most
or all of the carboxyl-terminal sequences. Soluble fragment(s)
containing the amino-terminal 10 kDa of the protein, which bear
the membrane anchorage sequences and are
inactive(6, 27) , were not found in keratinocyte
extracts in the present work. In addition to the 67/33-kDa complex,
some active 106/67-kDa complex was also stably separable by FPLC
chromatography.
10-fold higher, than the full-length 106-kDa form.
However, the specific activity of the 106/67-kDa complex is reduced.
Thus the keratinocytes contain an array of active TGase 1 forms of
widely differing specific activities. Based on the in vitro substrate properties of our earlier bacterially expressed
truncated forms of the TGase 1 enzyme (21) , it is tempting to
speculate that these multiple forms in keratinocytes could have
differing functions and/or substrate specificities. However, further
work will be needed to explore this possibility in detail.
80-kDa active species(6) . Although no sequencing was
performed, it is possible this species is very similar to the 67-kDa in vivo form, and the
70-kDa in vitro dispase
species, described here.
Structural Implications for the Role of TGase 1 Activity
in Epidermal Cells
The recently solved three-dimensional
structure of the factor XIIIa TGase provides further insights into the
present data(57) . Previous studies have shown substantial
degrees of homology in amino acid sequences, predicted secondary
structures, and thus likely three-dimensional structures, of a central
450 amino acid residue ``core'' in the family of
TGases(8, 17) . Most of the variations occur in
sequences predicted to form exposed protein turns, which may explain
the high degrees of antibody specificities described here. The
conserved TGase core consists largely of the -sandwich and active
site domains that presumably define the TGase activity(57) .
The family members differ primarily in additional sequences on their
amino- and carboxyl termini that are thought to define their substrate
preferences(18, 19) . In the case of the solved
structure of the factor XIIIa enzyme, a 37-residue leader sequence at
the amino terminus masks the active site pocket and is removed on
activation of the enzyme. At the end of the active site domain, barrel
1 and barrel 2 domains extend to the carboxyl terminus. Proteolytic
cleavage at the active site-barrel 1 domain interface results in
deactivation of this enzyme (39) .
-sandwich domain. The
beginning of the 33-kDa species described here corresponds precisely to
the junction of the active site and barrel 1 domains of factor XIIIa.
However, cleavage of TGase 1 at this point results in
100% further
activation instead ( Fig. 8and Fig. 9), indicating that
there are marked differences in the three-dimensional structure of this
portion of TGase 1 in comparison with factor XIIIa. In addition, it
would seem that the amino-terminal sequences somehow affect the
specific activity of the TGase 1 enzyme following this cleavage at
residue position 573, because retention of residues 1-61 allow
the further dispase activation, but when residues 1-92 are
deleted, no further dispase activation was observed (Fig. 9). In
the TGase 3 pro-enzyme, a hinge of about 12 residues immediately
following the active site domain is cleaved to effect an
50-fold
activation(16, 17) , again indicating an important
structural dissimilarity with factor XIIIa, but suggesting some
structural similarity to TGase 1(8) . In the cases of both
TGase 1 (present work) and TGase
3(13, 16, 34, 36) , the
-sandwich/active site domain portions (67 or 50 kDa, respectively)
are active enzymes, but these activities are significantly increased if
the respective 33- or 27-kDa portions remain complexed (present work; (16) and (17) ).
We thank Drs. John Folk, Edit Tarcsa, Tonja Kartasova,
and Shyh-Ing Jang for stimulating discussions.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.