©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Highly Active Soluble Processed Forms of the Transglutaminase 1 Enzyme in Epidermal Keratinocytes (*)

(Received for publication, October 26, 1994; and in revised form, June 5, 1995)

Soo-Youl Kim (1)(§) Soo-Il Chung (2)(¶) Peter M. Steinert (1)(**)

From the  (1)Skin Biology Branch, NIAMS and the (2)Laboratory of Cellular Development and Oncology, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892-2755

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

Of the six known active transglutaminases (TGases)()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.

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.


MATERIALS AND METHODS

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.

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 [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.

Total S-labeled foreskin tissues (epidermis + dermis) were used to prepare human actin exactly as described(33) .

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) .

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 (N108C575). [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.

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 (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.

TGase enzyme forms were also recovered from the cytosolic fraction of 3-day post-confluent NHEK cells that had been grown in high Ca 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.

The specificities of the TGase antibodies used was also examined in double immunoprecipitation reactions. In this case, the three 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) .


Figure 2: Double immunoprecipitation reactions show that the TGase antibodies are very specific. A, 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.




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 [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.


RESULTS

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.


Figure 1: Immunoprecipitation reactions of the cytosolic fractions of 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).



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 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).


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 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.



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 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.


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 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.



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.

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).

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, 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).


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)-, 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).



These sequence data can explain why the novel 33-kDa band is seen on Coomassie-stained gels (not shown) and autoradiograms of immunoprecipitated 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).

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 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.



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.

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) .

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 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) .


Figure 8: Dispase activation of the full-length 106-kDa TGase 1 form. A, 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.



In an identical reaction using unlabeled protein, the 30-min dispase digestion resulted in a 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.

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 (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, N108C575), did not result in significant net activations (Fig. 9).


Figure 9: Dispase activation of bacterially expressed truncated forms of the TGase 1 protein. The constructs 1 (full-length 92-kDa protein), 2 (N37), 3 (N52), 4 (N61), 5 (N97), and 20 (N108C575) 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.


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 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.




DISCUSSION

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.

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 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.

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 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.

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.

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) .

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 -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) ).

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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Pacific Corp., Kyunggi-Gi Do 449-900, Korea.

Present address: Korea Green Cross Corp., Kyunggi-Gi Do 449-900, Korea.

**
To whom correspondence and reprint requests should be addressed: Bldg. 6, Rm. 425, NIH, Bethesda, MD 20892-2755. Tel.: 301-496-1578; Fax: 301-402-2886; pemast{at}helix.nih.gov

The abbreviations used are: TGase(s), transglutaminase(s); NHEK, normal human epidermal keratinocytes; PVDF, polyvinylidene difluoride; TBS, Tris-buffered saline; TGase 1, suggested new nomenclature for the membrane-associated enzyme (K); TGase 2, suggested new nomenclature for the tissue enzyme (C); TGase 3, suggested new nomenclature for pro-enzyme (E); FPLC, fast protein liquid chromatography; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.


ACKNOWLEDGEMENTS

We thank Drs. John Folk, Edit Tarcsa, Tonja Kartasova, and Shyh-Ing Jang for stimulating discussions.


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