Isolation of a cDNA Encoding a Novel Member of the Transglutaminase Gene Family from Human Keratinocytes
DETECTION AND IDENTIFICATION OF TRANSGLUTAMINASE GENE PRODUCTS BASED ON REVERSE TRANSCRIPTION-POLYMERASE CHAIN REACTION WITH DEGENERATE PRIMERS*

Daniel AeschlimannDagger §, Mary Kay KoellerDagger , B. Lynn Allen-Hoffmannpar , and Deane F. Mosher§

From the Dagger  Division of Orthopedic Surgery and the Departments of § Medicine and par  Pathology, University of Wisconsin, Madison, Wisconsin 53792

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

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.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

Transglutaminases (EC 2.3.2.13) represent a family of enzymes capable of stabilizing protein assemblies by gamma -glutamyl-epsilon -lysine cross-links. Enzymes of this family catalyze a Ca2+-dependent transfer reaction between the gamma -carboxamide group of a peptide-bound glutamine residue and various primary amines, most commonly the epsilon -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.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

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
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Abstract
Introduction
Materials & Methods
Results & Discussion
References

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.).

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.

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.

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 beta -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.

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.

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.

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), beta -sandwich domain (d2), catalytic core domain (d3), and beta -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.

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.

    ACKNOWLEDGEMENTS

We are grateful to Pascale Aeschlimann for excellent technical assistance.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant HL21644 and by fellowships from the European Molecular Biology Organization (Long Term Fellowship ALTF 97-1994) and the Swiss National Science Foundation (Stipendium für fortgeschrittene Forscher 823A-046620, 1996) (to D. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF035960 and AF035961

To whom correspondence should be addressed: Division of Orthopedic Surgery, University of Wisconsin, F4/312 Clinical Science Center, 600 Highland Ave., Madison, WI 53792. Tel.: 608-263-4530/608-263-7742; Fax: 608-263-0454.

1 The abbreviations used are: TGP, prostate transglutaminase; TGC, tissue transglutaminase, transglutaminase type II; TGE, epidermal transglutaminase, transglutaminase type III; TGK, keratinocyte transglutaminase, transglutaminase type I; EGF, epidermal growth factor; KGF, keratinocyte growth factor; RT, reverse transcription; PCR, polymerase chain reaction; bp, base pair(s); aa, amino acid(s); kb, kilobase(s).

2 Thelen, K., Zippelius, A., Oberneder, R., Rietmueller, G., and Pantel, K., GenBankTM/EBI Data Bank accession number U79008.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References

  1. Folk, J. E., and Finlayson, J. S. (1977) Adv. Protein Chem. 31, 1-133[Medline] [Order article via Infotrieve]
  2. Lorand, L., and Conrad, S. M. (1984) Mol. Cell. Biochem. 58, 9-35[Medline] [Order article via Infotrieve]
  3. Aeschlimann, D., and Paulsson, M. (1994) Thromb. Haemostasis 71, 402-415[Medline] [Order article via Infotrieve]
  4. Kim, S. Y., Chung, S.-I., and Steinert, P. M. (1995) J. Biol. Chem. 270, 18026-18035[Abstract/Free Full Text]
  5. Esposito, C., Pucci, P., Amoresano, A., Marino, G., Cozzolino, A., and Porta, P. (1996) J. Biol. Chem. 271, 27416-27423[Abstract/Free Full Text]
  6. Steinert, P. M., Kim, S.-Y., Chung, S.-I., and Marekov, L. N. (1996) J. Biol. Chem. 271, 26242-26250[Abstract/Free Full Text]
  7. Nakaoka, H., Perez, D. M., Baek, K. J., Das, T., Husain, A., Misono, K., Im, M.-J., Graham, R. B. (1994) Science 264, 1593-1596[Medline] [Order article via Infotrieve]
  8. Feng, J. F., Rhee, S. G., and Im, M. J. (1996) J. Biol. Chem. 271, 16451-16454[Abstract/Free Full Text]
  9. Cohen, C. M., Dotimas, E., and Korsgren, C. (1993) Semin. Hematol. 30, 119-137[Medline] [Order article via Infotrieve]
  10. Reichert, U., Michel, S., and Schmidt, R. (1993) in Molecular Biology of the Skin (Darmon, M., and Blumberg, M., eds), pp. 107-150, Academic Press, Inc., San Diego, CA
  11. Kim, I. G., Gorman, J. J., Park, S. C., Chung, S. I., Steinert, P. M. (1993) J. Biol. Chem. 268, 12682-12690[Abstract/Free Full Text]
  12. Steinert, P. M., and Marekov, L. N. (1995) J. Biol. Chem. 270, 17702-17711[Abstract/Free Full Text]
  13. Rice, R. H., and Green, H. (1977) Cell 11, 417-422[Medline] [Order article via Infotrieve]
  14. Gentile, V., Saydak, M., Chiocca, E. A., Akande, O., Birckbichler, P. J., Lee, K. N., Stein, J. P., Davies, P. J. A. (1991) J. Biol. Chem. 266, 478-483[Abstract/Free Full Text]
  15. Lichti, U., Ben, T., and Yuspa, S. H. (1985) J. Biol. Chem. 260, 1422-1426[Abstract]
  16. Aeschlimann, D., Kaupp, O., and Paulsson, M. (1995) J. Cell Biol. 129, 881-892[Abstract]
  17. Aeschlimann, D., and Paulsson, M. (1991) J. Biol. Chem. 266, 15308-15317[Abstract/Free Full Text]
  18. Raghunath, M., Höpfner, B., Aeschlimann, D., Lüthi, U., Meuli, M., Altermatt, S., Gobet, R., Bruckner-Tuderman, L., and Steinmann, B. (1996) J. Clin. Invest. 98, 1174-1184[Abstract/Free Full Text]
  19. Martinez, J., Chalupowicz, D. G., Roush, R. K., Sheth, A., Barsigian, C. (1994) Biochemistry 33, 2538-2545[Medline] [Order article via Infotrieve]
  20. Huber, M., Rettler, I., Bernasconi, K., Frenk, E., Lavrijsen, S. P. M., Ponec, M., Bon, M. A., Lautenschlager, S., Schorderet, D. F., Hohl, D. (1995) Science 267, 525-528[Medline] [Order article via Infotrieve]
  21. Russell, L. J., DiGiovanna, J. J., Rogers, G. R., Steinert, P. M., Hashem, N., Compton, J. G., Bale, S. K. (1995) Nat. Genet. 9, 279-283[Medline] [Order article via Infotrieve]
  22. Aeschlimann, D., Wetterwald, A., Fleisch, H., and Paulsson, M. (1993) J. Cell Biol. 120, 1461-1470[Abstract]
  23. Nurminskaya, M., and Linsenmayer, T. F. (1996) Dev. Dyn. 206, 260-271[CrossRef][Medline] [Order article via Infotrieve]
  24. Allen-Hoffmann, B. L., and Rheinwald, J. G. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 7802-7806[Abstract]
  25. Hines, M. D., and Allen-Hoffmann, B. L. (1996) J. Biol. Chem. 271, 6245-6251[Abstract/Free Full Text]
  26. Martin, P., and Papayannopoulou, T. (1982) Science 216, 1233-1235[Medline] [Order article via Infotrieve]
  27. Sottile, J., Mosher, D. F., Fullenweider, J., and George, J. N. (1989) Thromb. Haemostasis 62, 1100-1102[Medline] [Order article via Infotrieve]
  28. Dubbink, H. J., Verkaik, N. S., Faber, P. W., Trapman, J., Schröder, F. H., Romijn, J. C. (1996) Biochem. J. 315, 901-908[Medline] [Order article via Infotrieve]
  29. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (eds) (1994) Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York
  30. Korsgren, C., Lawler, J., Lambert, S., Speicher, D., and Cohen, C. M. (1990) Biochemistry 87, 613-617
  31. Philipps, M. A., Stewart, B. E., Qin, Q., Chakravarty, R., Floyd, E. E., Jetten, A. M., Rice, R. H. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 9333-9337[Abstract]
  32. Thomazy, V., and Fesus, L. (1989) Cell Tissue Res. 255, 215-224[Medline] [Order article via Infotrieve]
  33. Upchurch, H. F., Conway, E., Patterson, M. K., Jr., Birckbichler, P. J., Maxwell, M. D. (1987) In Vitro Cell. Dev. Biol. 23, 795-800[Medline] [Order article via Infotrieve]
  34. Schenker, T., and Trueb, B. (1996) Apoptosis 1, 126-130
  35. Risinger, M. A., Dotimas, E. M., and Cohen, C. M. (1992) J. Biol. Chem. 267, 5680-5685[Abstract/Free Full Text]
  36. Weraarchakul-Boonmark, N., Jeong, J. M., Murthy, S. N. P., Engel, J. D., Lorand, L. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 9804-9808[Abstract]
  37. Grundmann, U., Amann, E., Zettlmeissel, G., and Kuepper, H. A. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 8024-8028[Abstract]
  38. Takahashi, N., Takahashi, Y., and Putnam, F. W. (1986) Proc. Natl. Acad. Sci U. S. A. 83, 8019-8023[Abstract]
  39. Poon, M.-C., Russell, J. A., Low, S., Sinclair, G. D., Jones, A. R., Blahey, W., Ruether, B. A., Hoar, D. I. (1989) J. Clin. Invest. 84, 787-792[Medline] [Order article via Infotrieve]
  40. Kim, H. C., Idler, W. W., Kim, I. G., Han, J. H., Chung, S. I., Steinert, P. M. (1991) J. Biol. Chem. 266, 536-539[Abstract/Free Full Text]
  41. Ho, K. C., Quarmby, V. E., French, F. S., Wilson, E. M. (1992) J. Biol. Chem. 267, 12660-12667[Abstract/Free Full Text]
  42. Grant, F. J., Taylor, D. A., Sheppard, P. O., Mathewes, S. L., Lint, W., Vanaja, E., Bishop, P. D., O'Hara, P. J. (1994) Biochem. Biophys. Res. Commun. 203, 1117-1123[CrossRef][Medline] [Order article via Infotrieve]
  43. Frohman, M. A., Dush, M. K., and Martin, G. R. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8998-9002[Abstract]
  44. Kozak, M. (1986) Cell 44, 283-292[Medline] [Order article via Infotrieve]
  45. Berget, S. M. (1984) Nature 309, 179-182[Medline] [Order article via Infotrieve]
  46. Fraij, B. M., Birckbichler, P. J., Patterson, M. K., Jr., Lee, K. N., Gonzales, R. A. (1992) J. Biol. Chem. 267, 22616-22623[Abstract/Free Full Text]
  47. Monsonego, A., Shani, Y., Friedmann, I., Paas, Y., Eizenberg, O., and Schwartz, M. (1997) J. Biol. Chem. 272, 3724-3732[Abstract/Free Full Text]
  48. Korsgren, C., and Cohen, C. M. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 4840-4844[Abstract]
  49. Sung, L. A., Chien, S., Fan, Y.-S., Lin, C. C., Lambert, K., Zhu, L., Lam, J. S., Chang, L.-S. (1992) Blood 79, 2763-2770[Abstract]
  50. Yee, V. C., Pedersen, L. C., LeTrong, I., Bishop, P. D., Stenkamp, R. E., Teller, D. C. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7296-7300[Abstract]
  51. Pedersen, L. C., Yee, V. C., Bishop, P. D., LeTrong, I., Teller, R. C., Stenkamp, R. E. (1994) Protein Sci. 3, 1131-1135[Abstract/Free Full Text]
  52. Yee, V. C., LeTrong, I., Bishop, P. D., Pedersen, L. C., Stenkamp, R. E., Teller, D. C. (1996) Semin. Thromb. Haemostasis 22, 377-384[Medline] [Order article via Infotrieve]
  53. Greenberg, C. S., Enghild, J. J., Mary, A., Dobson, J. V., Achyuthan, K. E. (1988) Biochem. J. 256, 1013-1019[Medline] [Order article via Infotrieve]
  54. Rybicki, A. C., Schwartz, R. S., Qiu, J. J., Gilman, J. G. (1994) Mamm. Genome 5, 438-445[Medline] [Order article via Infotrieve]
  55. Lu, S., Saydak, M., Gentile, V., Stein, J. P., Davies, P. J. A. (1995) J. Biol. Chem. 270, 9748-9756[Abstract/Free Full Text]
  56. Kim, I. G., Lee, S. C., Lee, J. H., Yang, J. M., Chung, S. I., Steinert, P. (1994) J. Invest. Dermatol. 103, 137-142[Abstract]
  57. Gribskov, M., and Burgess, R. R. (1986) Nucleic Acids Res. 14, 6745-6763[Abstract]


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