We developed a method using a single set of
degenerate oligonucleotide primers for amplification of the conserved
active site of transglutaminases by reverse transcription-polymerase
chain reaction (RT-PCR) and identification of the PCR products by
cleavage with diagnostic restriction enzymes. We demonstrate
amplification of tissue transglutaminase (TGC),
keratinocyte transglutaminase (TGK), prostate
transglutaminase (TGP), the a-subunit of factor XIII, and
band 4.2 protein from different human cells or tissues. Analysis of
normal human keratinocytes revealed expression of a transglutaminase
different from the expected and characterized transglutaminase gene
products. A full-length cDNA for the novel transglutaminase
(TGX) was obtained by anchored PCR. The deduced amino acid
sequence encoded a protein with 720 amino acids and a molecular mass of
~81 kDa. A comparison of TGX to the other members of the
gene family revealed that the domain structure and the residues
required for enzymatic activity and Ca2+ binding are
conserved and showed an overall sequence identity of about 35%. Two
transcripts with an apparent size of 2.2 and 2.8 kilobases were
detected with a specific probe for TGX on Northern blots of
human foreskin keratinocyte mRNA, indicating the presence of
alternatively spliced mRNAs. cDNA sequencing revealed a shorter TGX transcript lacking the sequence homologous to that
encoded by exon III of other transglutaminase genes. TGX
expression increased severalfold when keratinocyte cultures were
induced to differentiate by suspension or growth to postconfluency,
suggesting that TGX contributes to the formation of the
cornified envelope.
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INTRODUCTION |
Transglutaminases (EC 2.3.2.13) represent a family of enzymes
capable of stabilizing protein assemblies by
-glutamyl-
-lysine cross-links. Enzymes of this family catalyze a
Ca2+-dependent transfer reaction between the
-carboxamide group of a peptide-bound glutamine residue and various
primary amines, most commonly the
-amino group of lysine residues
(1, 2). Six different transglutaminase gene products have been
characterized in vertebrates thus far by determination of their primary
structure (3). In addition to the diversity on the genetic level, these enzymes have been shown to undergo a number of different
posttranslational modifications such as phosphorylation, fatty
acylation, and proteolytic cleavage, regulating their enzymatic
activity and subcellular localization (for review, see Refs. 3, 4, 5,
6). The individual transglutaminase gene products have specialized in the cross-linking of particular proteins or tissue structures, e.g. factor XIIIa stabilizes the fibrin clot in hemostasis
and prostate transglutaminase
(TGP)1 is
involved in semen coagulation (for review see Refs. 2 and 3), or have
even adopted additional functions such as tissue transglutaminase
(TGC) in GTP-binding in receptor signaling (7, 8) or band
4.2 protein as a structural component of the cytoskeleton (9).
Three transglutaminases have been shown to be expressed in different
stages of epidermal differentiation (for review, see Refs. 3, 10). Two
of those, keratinocyte (TGK) and epidermal (TGE) transglutaminase, are associated with terminal
differentiation events of keratinocytes (4, 11) and cross-link
structural proteins forming the cornified cell envelope (12, 13). The third enzyme, TGC (14), is expressed in skin primarily in
the basal cell layer (11, 15) and plays a role in stabilization of the
dermo-epidermal junction (16-19). The importance of proper cross-linking of the cornified envelope is exemplified by the pathology
seen in patients suffering from one form of the skin diseases referred
to as congenital ichthyosis that has been linked to mutations in the
TGK gene (20, 21).
The expression of more than one type of transglutaminase in a
particular cell type, e.g. keratinocytes and chondrocytes,
and the presence of the same gene product in different cellular
compartments raises questions about the nature of the enzyme that is
involved in a particular biological process, e.g. formation
of the skin cornified envelope (4, 11) or maturation of cartilage (16, 22, 23). Sensitive and specific assays are needed to detect the
transglutaminase gene products that potentially contribute to
biological events. To address this issue, we have developed an assay
based on PCR amplification using degenerate primers specific for the
transglutaminase gene family. Analysis of human cells and tissue
revealed, besides five of the known gene products including TGC, band 4.2 protein, the a-subunit of factor XIII,
TGK and TGP, a novel transglutaminase gene
product, TGX. In the present study, we describe the
full-length cDNA sequence and deduced amino acid sequence of two
splice variants of this novel human gene.
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MATERIALS AND METHODS |
Reagents--
Oligonucleotides and restriction enzymes were from
Oligos Etc. Inc. (Wilsonville, OR) and Promega Corp. (Madison, WI),
respectively. Reagents for cell culture were from Life Technologies,
Inc.
Cells--
Human keratinocytes were isolated from neonatal human
foreskin as described previously (24). Primary keratinocyte cultures were established on mitomycin C-treated mouse Swiss 3T3 fibroblast feeder layers in 3 parts Ham's F12 plus 1 part Dulbecco's modified Eagle's medium containing 2.5% fetal bovine serum, 0.4 µg/ml
hydrocortisone, 8.4 ng/ml cholera toxin, 5 µg/ml insulin, 24 µg/ml
adenine, 10 ng/ml epidermal growth factor (EGF; R&D Systems,
Minneapolis, MN), and antibiotics (100 µg/ml streptomycin and 100 units/ml penicillin). To induce differentiation, cells were harvested
by trypsinization and cultured for the indicated time in suspension in
the same medium supplemented with 1.68% methylcellulose (4,000 centipoises; Fisher Scientific Corp.) (25). For experiments analyzing
the effect of cell density and growth factors on differentiation, cells
were grown for one passage on a feeder layer in the absence of EGF.
Subsequently, cells were grown for 24 h in the absence of a feeder
layer before supplementing the medium with 0.5 nM EGF, 0.5 nM keratinocyte growth factor (KGF; Promega), or 10 µl of
0.1% bovine serum albumin/ml of medium for the indicated time (25).
Human dermal fibroblasts, TJ6F, were established from trypsinized
foreskin tissue, and human osteosarcoma cell line MG-63 (CRL 1427) and
human fibrosarcoma cell line HT1080 (CCL 121) were purchased from the
American Type Culture Collection (ATCC, Rockville, MD) and cultured in
Dulbecco's modified Eagle's medium containing 10% fetal bovine serum
and antibiotics. Human erythroleukemia cell line HEL was kindly
provided by Dr. Mortimer Poncz, Philadelphia, PA, cultured in
suspension in RPMI 1640 medium containing 12% fetal bovine serum, 1 mM pyruvate, and antibiotics, and induced to differentiate
with 1.25% dimethyl sulfoxide for 2 days (26). Human platelets were
collected as described (27), and a contamination with leukocytes or red
blood cells was ruled out by phase contrast microscopy.
PCR Amplification of Transglutaminases with Degenerate
Primers--
Poly(A)+ RNA was prepared from about
106 cells or 10 µg total RNA by oligo(dT)-cellulose
column chromatography using the Micro-Fast Track Kit (Invitrogen, San
Diego, CA) and recovered in 20 µl of 10 mM Tris/HCl, pH
7.5. The poly(A)+ RNA (5.0 µl) was reverse transcribed
into DNA in a total volume of 20 µl using the cDNA Cycle Kit
(Invitrogen) with either 1.0 µl of random primers (1 µg/µl) or
oligo(dT) primer (0.2 µg/µl). No difference in the amount or nature
of the PCR product was observed when the reverse transcription was done
with random or oligo(dT) primers. cDNA from human prostate
carcinoma tissue was kindly provided by Dr. Erik J. Dubbink, Rotterdam,
The Netherlands (28).
PCRs were carried out with 2.5 units of Taq DNA polymerase
(Fisher Scientific) and 25% of the reverse transcriptase reaction mixture (5.0 µl) in 100 µl of 10 mM Tris/HCl, pH 8.3, 50 mM KCl containing 2 mM MgCl2,
0.2 mM dNTPs and 50 pmol of the transglutaminase-specific degenerate oligonucleotide primers D1 and D2 (see Table I). The PCR
cycles were 45 s at 94 °C (denaturation), 2 min at 55 °C
(annealing), and 3 min at 72 °C (elongation). A total of 37 cycles
were made, with the first cycle containing an extended denaturation
period (6 min) during which the polymerase was added (hot start) and the last cycle containing an extended elongation period (10 min).
The 230-bp PCR products were purified by agarose gel electrophoresis,
recovered with the Wizard PCR Preps DNA Purification System (Promega),
and cloned by taking advantage of the 3
A-overhangs generated by
Taq DNA polymerase using the Original TA-Cloning Kit
(Invitrogen). Plasmid DNA was prepared with the Wizard Minipreps DNA
Purification System (Promega) and sequencing performed by the dideoxy
chain termination method using the Sequenase Version 2.0 Kit (U. S.
Biochemical Corp.).
Cloning of TGX by Anchored PCR--
Double-stranded
cDNA was prepared from poly(A)+ RNA (prepared as above)
of cultured normal human keratinocytes with the Copy Kit (Invitrogen)
using the oligo(dT)-NotI oligonucleotide (see Fig. 2) to
prime first strand synthesis. TGX-related sequences were
amplified by anchored PCR in both directions as outlined in Fig. 2
using TGX-specific oligonucleotides and additional
degenerate primers (see Table II) or the oligo(dT)-NotI
oligonucleotide for the 3
-end. The PCRs were performed under the
conditions described above. Nested PCRs were done by replacing the
cDNA with 1.0 µl from the first PCR reaction. Since degenerate
primers to conserved sequences upstream of primer D4 did not yield PCR
products, the cDNA was purified from nucleotides using the GlassMax
DNA Isolation Kit (Life Technologies, Inc.) and tailed in the presence
of 200 µM dCTP with 10 units of terminal deoxynucleotidyl
transferase (Promega) for 30 min at 37 °C (29) to anchor the PCR at
the 5
-end. The PCR reaction was anchored by performing a total of 5 cyles of one-sided PCR at a lower annealing temperature (37 °C) with
the abridged anchor primer (Life Technologies, Inc.; see Fig. 2) only
and was followed by transfer of 25% of the reaction at 94 °C to a
new tube containing abridged anchor primer and TGX-specific primer S6 (Table II) and by amplification as above. Nested PCR reactions were done with the universal amplification primer (Life Technologies, Inc.) and internal TGX-specific primers
(Table II) as indicated in Fig. 2.
The PCR products were gel-purified using the Geneclean II Kit (BIO 101 Inc., Vista, CA) and cloned as above. Both strands were sequenced from
both directions, with additional internal primers where required, using
the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit
(Perkin-Elmer Corp.) and the automated sequencing facility at the
Biotechnology Center at the University of Wisconsin.
Northern Blotting--
3 µg of poly(A)+ RNA from
human foreskin keratinocytes was separated in a 1.2% agarose gel
containing formaldehyde and transferred to a Zeta-probe membrane
(Bio-Rad). The gel was calibrated using the 0.24-9.5-kb RNA ladder
(Life Technologies, Inc.). For preparation of the probes, an ~700-bp
DNA fragment encoding the 3
-end of TGX, TGC,
band 4.2, or TGK was prepared by restriction with
PstI and AccI, StuI and
Bsu36I, XhoI, or XcmI and
XhoI, respectively. cDNAs encoding human
TGC, band 4.2 protein, and TGK were kindly provided by Drs. Peter J. A. Davies, Houston, TX (14), Carl M. Cohen, Boston, MA (30), and Robert H. Rice, Davis, CA (31), respectively. 32P-labeled probes were prepared using random
prime labeling (Multiprime DNA labeling system; Amersham Int.,
Amersham, UK). The membrane was hybridized with the probe at 42 °C
overnight, washed with a final stringency of 0.1 × SSC, 1% SDS
at 65 °C for 30 min, and exposed to x-ray film (Kodak, Rochester,
NY) for the indicated time period.
Amplification of TGX from Different
Cells--
cDNA was prepared as described above and a 225-bp
fragment of TGX was amplified from 1.0 µl of cDNA
with specfic primers S4 and S9 (Table II) using the PCR conditions
described above except for annealing at 60 °C.
 |
RESULTS AND DISCUSSION |
Design of PCR for Amplification of Transglutaminase Gene
Products--
To analyze the expression of transglutaminases when
starting material is limited, we undertook an effort to design primers capable of specifically amplifying transglutaminase sequences by PCR.
Alignment and comparison of the different known transglutaminase gene
products on the nucleotide level revealed several conserved regions,
particularly in the catalytic core domain (Table
I; see also Fig. 7), that could serve as
targets for primers. A single set of degenerate oligonucleotide primers
(Table I) that amplify by PCR a 230-bp DNA fragment encoding the highly
conserved active site region of transglutaminases (Fig.
1) was identified by screening of
oligonucleotides based on different conserved regions in PCR reactions
using plasmid DNA of different transglutaminases. The primers are based
on the sequence YGQCWVFAGV (see Fig. 7, aa 274-283 in
TGX), which includes the active site cysteine residue, and WM_RPDLP_G (aa 342-351) (Table I). Initial attempts with shorter oligonucleotides (18 bp) designed after the conserved sequences LFNPWC
(see Fig. 7, aa 138-143 in TGX), QCWVFA (aa 276-281), and WNFHVW (aa 333-338) were unsuccessful. Also, degenerate
oligonucleotides based on the sequence WQ_LDATPQE (see Fig. 7, aa
355-364 in TGX) and F_LLFNPWC (aa 135-143) did not yield
PCR products.
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Table I
Design of degenerate primers for amplification of members of the
transglutaminase gene family by PCR
Only human sequence is available for factor XIIIa. h = human,
m = mouse, r = rat, I = inosine.
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Fig. 1.
Amplification of transglutaminases from
different human cell lines or tissues. A 230-bp fragment
corresponding to the active site of transglutaminases was amplified
with degenerate primers D1 and D2 (Table I) by RT-PCR from MG-63
osteosarcoma cells (lane A2), HEL erythroleukemia cells
(lane B1), platelets (lane C1), keratinocytes
(lane D1), and prostate carcinoma tissue (lane
E1). Cleavage of the PCR products with restriction enzymes revealed the type of transglutaminase expressed: ScaI,
TGC; BstEII, band 4.2 protein; EcoRI,
factor XIII a-subunit; Bsp1286I, TGK; and
Tth111I, TGP. In osteosarcoma cells,
ScaI (lane A3), Bsp1286I (lane
A4), and ScaI + Bsp1286I (lane
A5) reveal TGC and TGK; in erythroleukemia
cells, ScaI (lane B2), BstEII
(lane B3), and ScaI + BstEII
(lane B4) reveal TGC and band 4.2 protein; in
platelets, EcoRI (lane C2), ScaI
(lane C3), and EcoRI + ScaI
(lane C4) reveal the a-subunit of factor XIII and
TGC; in keratinocytes, Bsp1286I (lane
D2) reveals TGK; and in prostate carcinoma tissue,
Tth111I (lane E2), ScaI (lane
E3), Bsp1286I (lane E4), and
EcoRI (lane E5) reveal TGP,
TGC, TGK, and the a-subunit of factor XIII. DNA fragments were analyzed by electrophoresis in 1% agarose gels calibrated with the 1-kb DNA ladder (lane A1; Life
Technologies, Inc.).
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Identification of Transglutaminase Gene Products by Restriction
Analysis--
A facile method to identify the nature of the PCR
products amplified with the degenerate primers is restriction analysis. Restriction sites conserved among species for a particular
transglutaminase gene but not present in PCR products derived from
other members of the gene family allow identification. Based on a
comparison of the sequence information of transglutaminases for man,
the following restriction enzymes gave cleavage patterns diagnostic of
the six different human gene products: ScaI for
TGC, BclI and NcoI (AvaI)
for TGE, BstEII for band 4.2 protein,
EcoRI for factor XIII a-subunit, Bsp1286I and
NcoI for TGK, and Tth111I for
TGP (Fig. 1). This selection of restriction enzymes would
also work with known rat or mouse sequences.
Amplification of Transglutaminase Gene Products from Human Cells or
Tissue--
We selected different human cells or tissues that are
known to express a distinct transglutaminase gene product to test
whether we could amplify all gene products of the transglutaminase
family. Sequence information for all six characterized genes is only
available in man.
TGC is expressed in many cell types and tissues in the
vertebrate body (14, 17, 22, 32), and we selected primary dermal fibroblasts (33) and two tumor cell lines, fibrosarcoma HT1080 and
osteosarcoma MG-63 (34), for our analysis. In fibroblasts and HT1080
fibrosarcoma cells, only TGC was detectable (results not
shown), wheras MG-63 osteosarcoma cells expressed TGC and TGK (Fig. 1A). Band 4.2 protein is a membrane
cytoskeleton component expressed at a high level in erythroid cells
(30, 35). For this reason, a human erythroleukemia cell line (HEL) was
tested. Erythrocytes are also known to express significant amounts of TGC (2, 36). We detected both TGC and band 4.2 protein in HEL cells (Fig. 1B). Platelets were chosen for
amplification of the a-subunit of factor XIII because they are the
major source for factor XIII a-subunit in plasma (37, 38, 39) and have been shown to contain mRNA even though they are devoid of a nucleus (27). The amplification showed that the a-subunit of factor XIII is the
predominant transcript in platelets, but TGC was also detected (Fig. 1C). TGK and TGE
contribute to the formation of the cornified envelope in skin in
distinct steps of keratinocyte differentiation (4, 11, 20, 40).
Therefore, primary keratinocyte cultures that were induced to
differentiate by culture in suspension were analyzed. TGK
was detected in adherent cells (Fig. 1D) as well as in
nonadherent cells (result not shown). We were unable to detect
TGE after culture in suspension for up to 24 h. The inability to detect TGE may be due to the fact that the
sequence of TGE differs more from the consensus used to
design the primers than other transglutaminase sequences (Table I). On
the other hand, the expression of TGE in human epidermis
has been found to be very low and not detectable in cultured human
keratinocytes (11). TGP is an androgen-regulated protein
involved in semen coagulation, and its expression is restricted to
prostate (28, 41, 42). Since no human cell line with known
TGP expression was available, human prostate tissue was
tested. TGP was the major transcript deteced in prostate
carcinoma tissue, but several other transglutaminases, TGC,
the a-subunit of factor XIII, and TGK were present as well,
which is to be expected in a vascularized tissue sample that is
composed of many different cell types.
To confirm the identity of the PCR products, the 230-bp DNA fragments
were cloned using the A-overhangs produced by Taq DNA polymerase and sequenced. To facilitate cloning of rare PCR products, portions of the DNA were cleaved by a restriction enzyme that degrades
a known PCR product, and the remainder was cloned as above. Clones
containing sequences of a predicted type of transglutaminase were
obtained in all cases, demonstrating that the assay is reliable. Keratinocytes contained a minor amount of PCR products different from
TGK, which we were unable to identify by restriction
analysis (see Fig. 1D, lane 2). Cloning and
analysis of the clones derived from these products revealed that
TGC was expressed in adherent keratinocytes, as has been
suggested previously (Refs. 11 and 15; see also Fig. 4). Unexpectedly,
we also found a transcript for a transglutaminase different from the
previously characterized human transglutaminase genes. We designate
this novel transglutaminase in the following as TGX since
its function is at present unknown.
Cloning of TGX from Human Keratinocytes by Anchored PCR
and Its Deduced Amino Acid Sequence--
To obtain further sequence
information on TGX, oligo(dT)-primed double-stranded
cDNA was prepared from poly(A)+ RNA from primary
keratinocytes isolated from human foreskin. The strategy of the
anchored PCR is summarized in Fig. 2, and the sequence of the oligonucleotide primers is given in Table II. To exclude sequence mutations
introduced by Taq DNA polymerase, all DNA fragments were
amplified at least twice in independent reactions, and the sequences of
several cloned PCR products were determined and compared. Briefly,
sequences of the 3
-end of TGX were amplified by
consecutive PCR reactions using degenerate primer D1 and
TGX-specific primers S1 and S2 together with degenerate primer D3, which is derived from the conserved amino acid sequence YKYPEGS_EER (Fig. 7, aa 443-453 in TGX). The residual
3
-sequence was amplified by sequential PCR reactions using
TGX-specific primers S1, S4, and S5 in combination with the
oligo(dT)-NotI primer used for cDNA priming. Sequences
5
of the active site were amplified in consecutive PCR reactions using
degenerate primer D2 and TGX-specific primer S3 together
with degenerate oligonucleotide D4, which is based on an upstream
cluster of conserved amino acids, i.e. LD_E_ER_EYV (Fig. 7,
aa 150-160 in TGX). Attempts to amplify sequences upstream of primer D4 with additional degenerate oligonucleotides failed. To
obtain more information on the 5
-end of TGX, we used a
5
-rapid amplification of cDNA ends approach (43). A poly(dC) tail
was added to the cDNA using terminal deoxynucleotidyl transferase to anchor the PCR reaction with an oligo(dG) primer (abridged anchor
primer). The reaction was anchored with the abridged anchor primer at
low annealing temperature, and a first round of amplification was
performed with abridged anchor primer and TGX-specific
primer S6. Subsequent reactions with nested primers, universal
amplification primer and TGX-specific primers S7 and S8,
yielded TGX-related PCR products (Fig. 2). However,
heterogeneity of the sequence upstream of primer D4 was encountered,
causing considerable difficulties in obtaining 5
-sequence. Three
different sequences have been obtained 5
of the sequence EDAVY (Fig.
7, aa 145-149 in TGX), two of which have been fully
characterized and are described in the following. The third version
deviates at the same nucleotide position but yields a stop codon in the
reading frame 63 nucleotides 5
of the presumptive splice point,
indicating that the third variant arose from failure of proper splicing
out of an intron. An understanding of the significance of this product
consequently awaits information on the gene structure.

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Fig. 2.
PCR strategy for amplification of cDNA
sequences of TGX. The top line represents
the cDNA for TGX with the start and stop codon
indicated. Brackets indicate the alternatively spliced sequence. Below is an outline of the PCR strategy, showing
the consecutive PCR reactions performed with nested oligonucleotide primers to obtain PCR products visible in ethidium bromide-stained agarose gels. The length of the final PCR products is given on the
right. The sequences of the oligonucleotide primers are
given in Tables I and II. The oligo(dT)-NotI unidirectional
primer (Invitrogen), 5 -AACCCGGCTCGAGCGGCCGCT(18), was used
as the 3 -anchoring primer. The abridged anchor primer (Life
Technologies, Inc.), 5 -GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG, was used
as the 5 -anchoring primer. In this case, the subsequently used primer
for nested PCR was a shortened oligonucleotide, universal amplification
primer (Life Technologies, Inc.) consisting of the first 20 nucleotides of the abridged anchor primer.
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Table II
Sequences of oligonucleotide primers used for PCR of TGX
Primers are numbered and were used for amplification of
TGX-specific sequences as indicated in Fig. 2. "D"
indicates degenerate primer; "S" indicates TGX-specific
primer. Forward primers (sense) are labeled "f"; reverse primers
(antisense) are labeled "r". The sequence position of the primers
is based on the sequence given in Fig. 3A. The following
abbreviations are used for degenerate positions in oligonucleotides:
M = A, C; R = A, G; S = C, G; W = A, T; Y = C,
T; I = inosine.
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The obtained sequence information consists of 1958 nucleotides
containing an open reading frame of 1914 bp for the short form of
TGX and of 2204 nucleotides with an open reading frame of
2160 bp for the long form of TGX, respectively (Fig.
3, A and
B). The probable initiation codon is present in the sequence
ACCATGG that conforms to the consensus identified by Kozak
(44) as a signal for efficient translation in higher eukaryotes. No
polyadenylation signal (AATAAA) was recognized in the short
3
-untranslated region following the termination codon (TAA),
indicating that it might be incomplete. However, repeated synthesis of
double-stranded cDNA and PCR with different primers under various
conditions did not yield additional 3
-sequence. All isolated cDNAs
end within 9-34 nucleotides downstream of the pentanucleotide ATAAA at
position 1922, i.e. at position 1935, 1938, 1939, 1942, 1943, and 1958. This pentanucleotide has been shown to function as a
polyadenylation signal in other genes (45) and might be functional in
TGX, giving rise to a very short 3
-untranslated region.
The deduced protein for the short form of TGX consists of
638 amino acids and has a calculated molecular mass of 71,915 Da and an
isoelectric point of 5.9. The deduced protein for the long form of
TGX consists of 720 amino acids and has a calculated
molecular mass of 80,764 Da and an isoelectric point of 6.0.

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Fig. 3.
Nucleotide sequence and deduced amino acid
sequence of human TGX. The full-length sequence of the
short version of TGX is shown (A) with dots
marking the position of the 82 amino acid insert (B) in the
long version. The initiation and termination codons are
underlined.
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Expression of the Novel TGX and Other Transglutaminase
Genes in Human Keratinocytes--
cDNA probes spanning the
sequence that encodes the two C-terminal barrel domains of different
human transglutaminases were used to detect the novel TGX
and other transglutaminase gene products known to be expressed in
keratinocytes on a Northern blot of human foreskin mRNA (Fig.
4). mRNAs of the expected sizes were
detected for TGC, 3.7 kb, and TGK, 2.7 kb (14,
40). Two different mRNAs with sizes of about 2.2 and 2.8 kb were
detected for TGX, indicating that alternative processing of
the transcript for TGX occurs. A previously described
approximately 2.4-kb band detected with a degenerate oligonucleotide on
a Northern blot of human foreskin that was assumed to be band 4.2 protein, based on its size (11), is likely to be identical to the
smaller transcript of TGX. This is further supported by the
fact that we were unable to detect transcripts of band 4.2 protein with
a specific probe (results not shown). The probes used displayed no
significant cross-hybridization as indicated by the distinct migration
of the detected mRNAs for the different gene products in the gel.
The relative abundance of the transcripts for
TGX:TGK:TGC is about 3:80:1. This
corresponds well with the results from the PCR amplification of
transglutaminases using the degenerate primers D1 and D2 (see Fig.
1D).

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Fig. 4.
Size of transcripts of TGX in
human keratinocytes. Northern blot containing 3 µg of poly
(A)+ RNA of adherent keratinocytes probed consecutively
with a ~700-bp fragment comprising the two C-terminal -barrel
domains of TGX (lane 1), TGK
(lane 2), and TGC (lane 3). The blot
was exposed for 3 days (TGX, lane 1), 4 h
(TGK, lane 2), and 4 days (TGC, lane 3). The migration position of RNA size markers is
indicated on the left.
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The cDNA sequence of the short form of TGX is identical
to the sequence of the long form with the exception that it lacks the
sequence encoded by exon III in other transglutaminase genes (Table
III). The sizes of the mRNAs of
TGX are larger than expected from sequencing data. This is
most likely due to the presence of additional 5
or 3
non-coding
sequences. The smaller, more abundant mRNA might result from
alternative splicing of the sequence encoded by exon III. Alternatively
spliced mRNAs have been described for TGC (46, 47),
band 4.2 protein (9, 48, 49), and TGP.2 No common
pattern for alternative splicing is evident from the current data, and
different exons are apparently alternatively processed in the different
gene products. However, a band 4.2 isoform lacking exon III has been
found in endothelial cells (9), and a putative TGP isoform
lacks part of exon III.2
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Table III
Comparison of splice donor and acceptor sites used in different
transglutaminases
The splice donor and acceptor sites for the short and long form of
TGX are based on the cDNA sequences and are represented in
alignment with known splice sites in other transglutaminase genes.
Residues consistent with the splice site consensus sequence (MAG/GTRAG
and YAG/G) are underlined.
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To analyze the expression of TGX in relation to terminal
differentiation of keratinocytes, normal human keratinocytes were induced to differentiate by culture in suspension in a semi-solid methylcellulose medium. TGX was amplified by RT-PCR from an
identical amount of RNA using TGX-specific primers (Fig.
5). Even though TGX was
present in adherent cells, it appeared to be induced in differentiating
cells (Fig. 5, lanes 7 and 8). To corroborate this result, the expression of TGX was analyzed by
semi-quantitative PCR in preconfluent and postconfluent keratinocyte
cultures in the presence or absence of either EGF or KGF (Fig.
6). EGF is well known to support
keratinocyte growth while KGF has recently been shown to attenuate
differentiation specifically in postconfluent cultures (25). A
severalfold increase in TGX expression was associated with
cell density-induced differentiation (Fig. 6B, compare
lanes 1-3 with 4-6). Both, EGF- and
KGF-treated keratinocytes exhibited decreased levels of TGX
expression relative to the control in preconfluent cultures (Fig.
6B, compare lanes 4-6). In postconfluent keratinocyte cultures, TGX expression is not significantly
altered by EGF or KGF (Fig. 6B, lanes 1-3).
However, amplification of transglutaminases with the degenerate
oligonucleotides revealed a pattern of expression that is consistent
with the pattern of transglutaminase activity measured in these
cultures (results not shown; see Ref. 25) and is likely to reflect
largely the expression of TGK that is the predominant type
of enzyme expressed (see Fig. 1D).

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Fig. 5.
Amplification of the novel transglutaminase
TGX from different human cell lines. A 225-bp fragment
of TGX was amplified by RT-PCR using specific primers S4
and S9 (Table II) from dermal fibroblasts (lane 2), HT1080
fibrosarcoma cells (lane 3), MG-63 osteosarcoma cells
(lane 4), platelets (lane 5), HEL erythroleukemia cells (lane 6), adherent (lane 7) and
non-adherent (lane 8) keratinocytes, and a fetal human skin
cDNA library (lane 9; 18 weeks gestation, Invitrogen).
Normal human keratinocytes were analyzed either prior to (lane
7) or after culture in suspension for 4 h (lane
8). PCR products were analyzed by electrophoresis in 1% agarose
gels calibrated with the 1-kb DNA ladder (lane 1; Life
Technologies, Inc.).
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Fig. 6.
Expression of TGX in
differentiating keratinocytes. Normal human keratinocytes were
treated with bovine serum albumin (lanes 1 and
4), 0.5 nM EGF (lanes 2 and
5), or 0.5 nM KGF (lanes 3 and
6) in standard medium for 3 days (preconfluent, lanes
4-6) or 10 days (postconfluent, lanes 1-3).
Panel A shows amplification of transglutaminases by RT-PCR
with degenerate primers D1 and D2; panel B shows
amplification of TGX with specific primers S4 and S9.
Amplification of glyceraldehyde 3-phosphate dehydrogenase with a
control primer set (600-bp fragment; Stratagene) shows that equal
amounts of message are present in the different samples (panel
C). All primer sets span intron-exon boundaries, thereby ensuring
that the PCR products are derived from mRNA. PCR products were
analyzed in 1% agarose gels.
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Structural Features of TGX--
A comparison of
TGX with the previously characterized human
transglutaminases reveals that the structural requirements for transglutaminase activity and Ca2+ binding are conserved
(Fig. 7). The overall sequence identity between TGX and TGC, TGE, band 4.2 protein, the a-subunit of factor XIII, TGK, or
TGP is 40.1, 42.3, 31.6, 32.7, 34.9, and 31.0%, respectively. A closer comparison shows that TGX is more
closely related to the evolutionary lineage including TGC,
TGE, and band 4.2 protein (see Ref. 3) than the other
transglutaminases (Table IV). The
catalytic mechanism of transglutaminases has been solved based on
biochemical data available for several transglutaminases (for review,
see Refs. 1 and 2) and the x-ray crystallographic structure of the
factor XIII a-subunit dimer (50). The reaction center is formed by the
core domain and involves hydrogen-bonding of the active site Cys to a
His and Asp residue to form a catalytic triad reminiscent of the
Cys-His-Asn triad found in the papain family of cysteine proteases
(51). The residues comprising the catalytic triad are conserved in
TGX (Cys277, His336,
Asp359) (Fig. 7), and the core domain shows a high level of
conservation as indicated by a sequence identity of about 50% between
TGX and the other transglutaminases (Table IV). A Tyr
residue in barrel 1 domain of the a-subunit of factor XIII is
hydrogen-bonded to the active site Cys residue, and it has been
suggested that the glutamine substrate attacks from the direction of
this bond to initiate the reaction based on analogy to the cysteine
proteases (52). In TGX, the Tyr residue has been replaced
by His549 (Fig. 7), which is expected to be a conservative
change. Another set of hydrogen bonds in the a-subunit of factor XIII
involving residues His342-Glu434 and
Asp343-Arg11
(located in the activation
peptide of the second subunit in the dimer), which have been suggested
to guide the lysine substrate to the active site (50), are not
conserved in that form in TGX (Fig. 7). Crystallization
experiments with factor XIIIa indicated that four residues are involved
in binding of a Ca2+ ion, including the main chain carbonyl
of Ala457 and the side chain carboxyl groups of
Asp438, Glu485, and Glu490 (52).
All three acidic residues are conserved in TGX (Fig. 7). A
unique insertion of about 30 amino acids is present between the
catalytic core domain and the C-terminal barrel domains in TGX (Fig. 7). A smaller insertion of about 10 amino acids
was found in TGE, and TGE has been shown to
require activation by a conformational change occurring upon
proteolytic cleavage in this flexible connecting loop (11). Cleavage
between these domains has also been observed in TGK and in
the a-subunit of factor XIII (Fig. 7). While the cleaved form of
TGK is highly active (4), contradictory results have been
reported with regard to the activity of factor XIIIa that has been
cleaved by thrombin at this site (38, 53). Proteolytic activation of
transglutaminases, probably by a member of the calpain family, seems to
be a common feature for the enzymes involved in epidermal
differentiation (4), and the extended flexible hinge region between the
core domain and the C-terminal barrel domains in
TGX should be prone to proteolytic attack.

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Fig. 7.
Comparison of the amino acid sequence of
human TGX with the sequences of the other members of the
transglutaminase family: TGC, TGE, band 4.2 protein, factor XIII a-subunit, TGK and
TGP. The sequences are arranged to reflect the
transglutaminase domain structure based on the crystal structure of
factor XIII a-subunit (50, 52): N-terminal propeptide domain
(d1), -sandwich domain (d2), catalytic core
domain (d3), and -barrel domains 1 (d4) and 2 (d5) (from top to bottom). Human
sequences are shown with positions of known amino acid variation
between species denoted as small letters (for TGC (14),
TGE (11), and band 4.2 protein (30, 54), human and mouse
sequences were considered; for TGK (31, 40) and
TGP (28, 41, 42), human and rat sequences were considered).
Dashes indicate gaps inserted for optimal sequence alignment, underlined residues represent amino acids
conserved in at least four gene products. Asterisks and
open circles at the bottom of the aligned
sequences indicate positions that are occupied by identical or
chemically similar (57) amino acids in all transglutaminases. The
active site Cys residue is shown in red, and the His and Asp
residues of the catalytic triad are in pink. Additional
residues involved in substrate interaction are shown in light
blue. Residues involved in Ca2+-binding are shown in
dark blue, and protease cleavage sites in factor XIII
a-subunit (Arg37 and Lys513), TGE
(Ser469), and TGK (Arg90 and
Arg570) are marked in green. The alternatively
spliced sequence in TGX and the known splice junctions for
exons II/III and III/IV in the other transglutaminase gene products are
marked with arrowheads.
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Table IV
Similarity of TGX to the other transglutaminase gene
products in the individual domains
The domain structure is based on the X-ray crystallographic structure
of the factor XIII a-subunit dimer (50, 52) and inferred on the other
gene products based on the sequence alignment shown in Fig. 7. The
numbers reflect percent sequence identity.
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Based on the similarity of TGX to the other active members
of the transglutaminase protein family, it is tempting to speculate that the characterized cDNA is encoding an active transglutaminase. This is further supported by the fact that in band 4.2 protein, which
is the only member of this protein family without catalytic activity,
the residues directly involved in the cataytic process are not
conserved (Fig. 7). The induction of TGX in differentiating keratinocytes further suggests that it might play a role in the formation of the cornified envelope. However, expression of
TGX is not restricted to keratinocytes (Fig. 5), and
further work is required to substantiate and extend the present
findings.
Conclusions--
Using the degenerate oligonucleotides, we have
been able to amplify 5 out of 6 previously characterized
transglutaminases and the novel transglutaminase TGX. We
have not been able to detect TGE which is likely due to its
very restricted expression in the late stages of keratinocyte
differentiation, particularly in hair follicles (11). Consistent with
our observation, Kim et al. (11) reported that expression of
TGE was not detectable in human keratinocyte cultures.
Besides the expected type of transglutaminase, which turned out to be
the predominant type of transglutaminase in the analyzed cell types, we
detected other, apparently less abundantly expressed transglutaminases
(Fig. 1, A and C). The abundance of the PCR
product for a particular type of transglutaminase correlated with its
message level detected in Northern blotting (compare Fig. 1D
and Fig. 4), and the sum of the PCR products for all transglutaminases
(Fig. 6) correlated with the measured transglutaminase activity (see
Ref. 25), at least on a semi-quantitative basis. These results suggest
that the described degenerate oligonucleotides provide an excellent
tool for identifying the types of transglutaminase expressed in a
particular cell type and for cloning of new members of this growing
gene family. The homology between vertebrate and invertebrate
transglutaminases is similar to the different human transglutaminases
compared with each other (3), indicating that these primers may work in
a wide range of different species.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF035960 and AF035961