Evolution of Transglutaminase Genes: Identification of aTransglutaminase Gene Cluster on Human Chromosome 15q15STRUCTURE OF THE GENE ENCODING TRANSGLUTAMINASE X AND A NOVEL GENE FAMILY MEMBER,TRANSGLUTAMINASE Z*

Received for publication, March 21, 2001, and in revised form, June 1, 2001Published, JBC Papers in Press, June 4, 2001, DOI 10.1074/jbc.M102553200

Pascale Grenard‡, Mary Kay Bates§, and Daniel Aeschlimann‡§¶

From the ‡Connective Tissue Biology Laboratories, School of Biosciences, Cardiff University, Cardiff CF10 3US, UnitedKingdom and the §Division of Orthopedic Surgery, University of Wisconsin, Madison, Wisconsin 53792

We isolated and characterized the gene encoding hu-man transglutaminase (TG)X (TGM5) and mapped it tothe 15q15.2 region of chromosome 15 by fluorescence insitu hybridization. The gene consists of 13 exons sepa-rated by 12 introns and spans about 35 kilobases. Fur-ther sequence analysis and mapping showed that thislocus contained three transglutaminase genes arrangedin tandem: EPB42 (band 4.2 protein), TGM5, and a novelgene (TGM7). A full-length cDNA for the novel transglu-taminase (TGZ) was obtained by anchored polymerasechain reaction. The deduced amino acid sequence en-coded a protein with 710 amino acids and a molecularmass of 80 kDa. Northern blotting showed that the threegenes are differentially expressed in human tissues.Band 4.2 protein expression was associated with hema-topoiesis, whereas TGX and TGZ showed widespread ex-pression in different tissues. Interestingly, the chromo-somal segment containing the human TGM5, TGM7, andEPB42 genes and the segment containing the genes en-coding TGC,TGE, and another novel gene (TGM6) onchromosome 20q11 are in mouse all found on distal chro-mosome 2 as determined by radiation hybrid mapping.This finding suggests that in evolution these six genesarose from local duplication of a single gene and subse-quent redistribution to two distinct chromosomes in thehuman genome.

Transglutaminases (EC 2.3.2.13) are a family of structurallyand functionally related enzymes that stabilize protein assem-blies through the formation of intra- or intermolecular N�(�-glutamyl)lysine bonds. Enzymes of this family catalyze a Ca2�-dependent transferase reaction between the �-carboxamidegroup of a peptide-bound glutamine residue and various pri-mary amines, most commonly the �-amino group of lysine res-idues (1, 2). Seven different transglutaminase gene productshave previously been characterized in man (3, 4) and found to

have specialized in the cross-linking of proteins in differentbiological processes. Functions include fibrin clot stabilizationin hemostasis, semen coagulation, formation of cornified enve-lopes in keratinization, and stabilization of extracellular ma-trix structures (3, 5). The essential role of transglutaminases inthese processes is witnessed by the serious impairment ofwound healing and keratinization, which are associated withtransglutaminase deficiencies (6, 7). Besides playing a struc-tural role, transglutaminase cross-linking has been shown re-cently to have a profound effect on cells by regulating thebiological activity of signaling molecules such as transforminggrowth factor-�, interleukin-2, and midkine as well as by mod-ulating cell-matrix interactions (5). Transglutaminases canpromote cell-matrix interaction through both cross-linking ofcell surface-associated fibronectin (8, 9) and noncovalent bind-ing to integrins and fibronectin (10). The latter finding furthersupports a function of transglutaminases as structural proteinsin addition to their enzymatic role and is consistent with theloss of catalytic activity of one member of the gene family, band4.2 protein. Even though each type of transglutaminase has itsown typical tissue distribution, the individual enzymes arepresent in a number of different tissues and often in combina-tion with other transglutaminases. The recently reported un-expected absence of a distinct developmental phenotype inTGC

1 null mice (11) may relate to co-expression with othertransglutaminases and indicates redundancy in this proteinfamily for the first time.

All transglutaminase enzymes are encoded by a family ofclosely related genes. Alignment of the gene products reveals ahigh degree of sequence similarity, and all family membersexhibit a similar gene organization with remarkable conserva-tion of intron distribution and intron splice types. Comparisonof the structure of the individual genes shows that they may bedivided into two subclasses (3, 12), wherein the genes encodingTGC (13), TGE (14), band 4.2 protein (15), and TGP (16) contain13 exons, and the genes encoding factor XIII a-subunit (17) andTGK (12, 18, 19) contain 15 exons (for an explanation of nomen-clature see Table I). Exon IX of the former group is separatedinto two exons (X and XI) in the TGM1 and F13A1 genes, andthe nonhomologous N-terminal extensions of factor XIII a-sub-unit and TGK are comprised by an additional exon. Phyloge-netic analysis also indicated that an early gene duplicationevent gave rise to two different lineages, one comprising TGC,TGE, and band 4.2 protein and the other comprising factor XIII

* This work was supported by 1996 Swiss National Science Founda-tion Fellowship 823A-046620 for advanced researchers (to D. A.) andArthritis Research Campaign of the UK Grant A0560. The costs ofpublication of this article were defrayed in part by the payment of pagecharges. This 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 submittedto the GenBankTM/EBI Data Bank with accession number(s)AF206502–AF206512 (genomic sequences) and AF363393 (cDNAsequence).

¶ To whom correspondence should be addressed: Connective TissueBiology Laboratories, School of Biosciences, Cardiff University, Muse-ums Ave., Cardiff CF10 3US, UK. Tel.: 44-29-20876196/20875168; Fax:44-29-20874594; E-mail: AeschlimannDP@Cardiff.ac.uk.

1 The abbreviations used are: TG, transglutaminase (see Table I); kb,kilobase pair(s); BAC, bacterial artificial chromosome; PCR, polymer-ase chain reaction; bp, base pair(s); RACE, rapid amplification of cDNAends; nt, nucleotide(s); LOD, log of the ratio of odds.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 35, Issue of August 31, pp. 33066–33078, 2001© 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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a-subunit and TGK (12). Considering the similarity in genestructure, protein primary structure, and three-dimensionalfolding (20–22) as well as catalytic mechanism (23), transglu-taminase genes seem derived from a common ancestral gene,which itself is related to cysteine proteases (24). Transglutami-nase genes were thought to be scattered in the human genome,because the genes encoding TGK and factor XIII a-subunit havebeen mapped to chromosomes 14 (18) and 6 (25), respectively,whereas TGC and TGE have been mapped to chromosome 20(14, 26), band 4.2 protein to chromosome 15 (27, 28), and TGP

to chromosome 3 (29, 30).We recently have isolated a cDNA encoding a novel member

of the transglutaminase gene family, TGX, from human fore-skin keratinocytes (4). Two related transcripts with apparentsizes of 2.2 and 2.8 kb were obtained that encoded proteins of638 and 720 amino acids with molecular masses of 72 and 81kDa, respectively. We now have characterized the structure ofthe gene encoding TGX and shown that the two previouslyisolated gene products result from alternative splicing of exonIII. We have mapped the gene to the 15q15.2 region of chromo-some 15. Analysis of its flanking sequences revealed the pres-ence of a cluster of three transglutaminase genes within �100kb, including the gene encoding band 4.2 protein and a noveltransglutaminase gene (TGM7). We have further characterizedthe new transglutaminase, TGZ, encoded by the TGM7 gene bydetermining its primary structure and tissue distribution. Ourfindings provide new insights into the evolution of the trans-glutaminase gene family.

MATERIALS AND METHODS

Reagents—Oligonucleotides were from Oligos Etc., Inc. (Wilsonville,OR) or Life Technologies, Inc., and restriction enzymes were fromPromega (Madison, WI). Image clones with GenBankTM accession num-bers AI018564, AI024635, and AW511368 were obtained from GenomeSystems, Inc. (St. Louis, MO). Reagents for cell culture were from LifeTechnologies, Inc.

Genomic Library Screening—A human BAC library established in anF-factor-based vector, pBeloBAC 11, and maintained in Escherichia coliDH10B (31) was screened by PCR (Genome Systems). A 147-bp DNAfragment unique to TGX (4) was amplified from 100 ng of genomic DNAin 100 �l of 10 mM Tris/HCl, pH 8.3, 50 mM KCl containing 2 mM MgCl2,0.2 mM dNTPs using 2.5 units of Taq DNA polymerase (Fisher), 50 pmolof upstream primer P1 (5�-CCACATGTTGCAGAAGCTGAAGGCTA-GAAGC), and downstream primer P2 (5�-CCACATGTCCACATCACT-GGGTCGAAGGGAAGG). PCR cycles (Robocycler, Stratagene, La Jolla,CA) were 45 s at 94 °C (denaturation), 2 min at 60 °C (annealing), and3 min at 72 °C (elongation) for a total of 37 cycles, with the first cyclecontaining an extended denaturation period (6 min), during which theDNA polymerase was added (hot start), and the last cycle containing anextended elongation period (10 min). Two positive clones were identi-fied, BAC-33(P5) and BAC-228(P20) (Genome Systems), and their iden-tity was verified by Southern blotting. BAC plasmid DNA was preparedusing a standard alkaline lysis protocol (32) with the modificationsrecommended by Genome Systems. 2 �g of BAC plasmid DNA wasrestricted with BamHI, EcoRI, and SpeI and probed with a 32P-labeled�500-bp NcoI/BspHI and �600-bp BspHI/NdeI cDNA fragment of TGX,respectively, as described below.

Amplification of TGM5 Intron Sequences—PCRs were carried out

with 2.5 units of Taq DNA polymerase (Fisher) and 100–200 ng ofplasmid DNA from BAC clones in 100 �l of 10 mM Tris/HCl, pH 8.3, 50mM KCl containing 2 mM MgCl2, 0.2 mM dNTPs, and 50 pmol of thedesired oligonucleotide primers. PCR cycles (Robocycler) were 45 s at94 °C (denaturation), 1 min at 60 °C (annealing), and 5 min at 72 °C(elongation) for a total of 32 cycles. The following oligonucleotides wereused as upstream and downstream primers, respectively, in the individ-ual reactions: intron 2, 5�-GGACCACCTGCTTGTTCGCCGGGG and5�-AGGGGCTGGGGCTGTGATGGCGTG; intron 3, 5�-ACCTCTTGAA-AATCCACATCGACTCCT and 5�-CAGTTCTTGCTGCCTTGGTAGAT-GAAGCC; intron 4, 5�-GACAGTGAACCCCAGAGGCAGGAG and 5�-TCTGTGGCTGGGTCAGTCTGGAAGTGCA (P3); intron 5, 5�-GCCTG-CACTTCCAGACTGACCCAGCCACA and 5�-TCCAGTTTCCATTGAG-CACCCCA; intron 6, 5�-TGCTGGGTCTTTGCTGCCGTCATGTGC and5�-TCCTTCTTCTTATTCCCCAAAATCCTGCC; intron 7, 5�-TAGATG-AGTATTATGACAACACAGGCAGG and 5�-GCGTCCAGCACCTGCC-AGCCTCC; intron 8, 5�-TGAGTGCTGGATGGCCCGGAAGG and 5�-CCCGCTCGTCACTCTGGATGCTC; intron 9, 5�-TTCACCAGGACAC-GAGTTCTGTTGGCA and P2 (see above); intron 10, P1 and 5�-TCAG-GACTGCTTTTCTCTTCACCC; intron 11, 5�-ACCCCTGCAAAATCTC-CTATTCCC and 5�-AATATCACCTGTATGGAGAGTGGCTGG; and in-tron 12, 5�-TTGAGGACTGTGTGCTGACTGTGG and 5�-AATGATGCT-TGCTTGGTGTTGGGG.

A partial sequence of intron 1 was obtained initially from the directsequencing of DNA from BAC-228(P20). The complete intron 1 se-quence was subsequently amplified from human genomic DNA(Promega) using the oligonucleotides 5�-CCAGCTGTTCTGTGGGGAG-CATCC (forward) and 5�-GGTTTTCTGCCTCTGTCCAAGTCC(reverse) and 5�-AGGGGAAGTAGACACTCTGGCAC (forward) and 5�-AGCTCTGGAGGTCTGTGAGGGC (reverse). PCRs were carried outwith 1.25 units of Pfu Turbo DNA polymerase (Stratagene) and 260 ngof genomic DNA in a total of 100 �l of supplied reaction buffersupplemented with 0.2 mM dNTPs, 2 �l of Me2SO, and 50 pmol ofprimers. A total of 37 cycles were carried out in the Robocycler withannealing at 68 °C for 1 min, extension at 72 °C for 2 min, and denat-uration at 94 °C for 45 s.

DNA Preparation and Sequencing—Plasmid DNA from BAC cloneswas further purified for direct sequencing by digestion with 200 �g/mlRNase A (Sigma) for 1 h at 37 °C and by subsequent microdialysis usingSpectra/Por 2 membranes (Spectrum Medical Industries, Inc., LaguanaHills, CA). PCR products were gel-purified using the QIAquick gelextraction kit (Qiagen, Chatsworth, CA) for sequencing. Cycle sequenc-ing was performed by the dideoxy chain termination method using theCyclist Exo�Pfu DNA sequencing kit (Stratagene) and precast 6% poly-acrylamide gels with the CastAway sequencing system (Stratagene) orusing the dRhodamine Terminator Cycle Sequencing Ready reaction kit(Applied Biosystems, Warrington, UK) and an ABI 310 automatedsequencer.

Rapid Amplification of 5�-mRNA Ends—A modified RACE protocol(4) was used to determine the transcription start site and obtain addi-tional sequence information of exon I of TGX. Briefly, double-strandedcDNA was prepared from poly(A)� RNA of cultured normal humankeratinocytes (prepared as described previously (4)) with the Copy kit(Invitrogen, San Diego, CA). The cDNA was purified from nucleotidesusing the GlassMax DNA isolation kit (Life Technologies, Inc.) andtailed in the presence of 200 �M dCTP with 10 units of terminaldeoxynucleotidyl transferase (Promega) for 30 min at 37 °C to anchorthe PCR at the 5� end. The PCR was anchored by performing a total of5 cycles of one-sided PCR at a lower annealing temperature (37 °C) withthe abridged anchor primer (Life Technologies, Inc.) only. After thetransfer of 25% of this reaction at 94 °C to a new tube containingabridged anchor primer and TGX-specific primer P3 (see above), the

TABLE ITransglutaminase terminology

Gene product Alternative designations Genename

Factor XIII a-subunit Factor XIII a and plasma transglutaminase F13A1TGK Keratinocyte transglutaminase and transglutaminase type 1 TGM1TGC Tissue transglutaminase, transglutaminase type 2, and G�h TGM2TGE Epidermal transglutaminase and transglutaminase type 3 TGM3TGP Prostate transglutaminase, transglutaminase type 4, and dorsal prostate protein 1 TGM4TGX Transglutaminase type 5 TGM5TGY Transglutaminase type 6 TGM6TGZ Transglutaminase type 7 TGM7band 4.2 Erythrocyte protein band 4.2 EPB42

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first round of amplification was carried out for a total of 37 cycles underthe conditions described above except for annealing at 55 °C. NestedPCR was done with the universal amplification primer (Life Technolo-gies, Inc.) and TGX-specific primer P4 (5�-TGAAGTACAGGGTGAGGT-TGAAGG) as described above (annealing at 60 °C) using 1 �l of the firstround PCR.

Primer Extension Analysis—Oligonucleotide P5, 5�-CATGGTAGCT-GCCTCCGGTTCCTG, containing a 5�-infrared label (IRD 800) waspurchased from MWG Biotech (Ebesberg, Germany). Primer P5(5.3pmol) was hybridized to 1 �g of poly(A)� RNA from primary kera-tinocytes (4), and reverse transcription was performed with 200 units ofSuperscript II RNase H� reverse transcriptase (Life Technologies, Inc.)in a total volume of 20 �l for 90 min at 42 °C according to manufacturerinstructions. The enzyme was heat-inactivated, and the primer exten-sion products were extracted with phenol chloroform, precipitated withethanol, and then analyzed on a 4.5% denaturing polyacrylamide geladjacent to dideoxynucleotide chain termination sequencing reactions(Thermo Sequenase cycle sequencing kit, Amersham Pharmacia Bio-tech) derived from a double-stranded genomic DNA fragment using thesame primer.

Southern Blotting—18 �g of human genomic DNA was digested withBamHI, EcoRI, and HindIII restriction enzymes, separated in a 0.8%agarose gel, and transferred to a Zeta-probe membrane (Bio-Rad). Thegel was calibrated using the Lambda DNA/HindIII markers (Promega).32P-labeled probes were prepared by random prime labeling using theMultiprime DNA labeling system (Amersham Pharmacia Biotech), andPCR products corresponding to intron 2 and exon X were used as DNAtemplates. Probes were hybridized to the blot overnight at 65 °C in 500mM NaH2PO4, pH 7.5, containing 1 mM EDTA and 7% SDS. The mem-brane was washed at 65 °C to a final stringency of 40 mM NaH2PO4, pH7.5, 1 mM EDTA, and 1% SDS, and the result was developed by expo-sure of the membrane to BioMax MR film (Eastman Kodak Co.).

Chromosomal Localization—Human peripheral blood lymphocyteswere used to prepare metaphase chromosome spreads (33). Briefly, cellswere cultured in PB-Max karyotyping medium (Life Technologies, Inc.)for 72 h and synchronized by culture in the presence of 10�7 M amethop-terin (Fluka, Buchs, Switzerland) for another 24 h. Cells were releasedfrom the mitotic block by extensive washing and subsequent culture inthe above medium containing 10�5 M thymidine for 5 h. The cells weresubsequently arrested in metaphase by the addition of colcemid to afinal concentration of 0.1 �g/ml (Life Technologies, Inc.). Harvestedcells were incubated in 0.075 M KCl for 25 min at 37 °C and fixed inmethanol/acetic acid (3:1) solution, and chromosome spreads were pre-pared by dropping the cells onto the glass slides. After air drying,chromosomes were treated with 100 �g/ml RNase A in 2� standardsaline citrate for 1 h at 37 °C, denatured in 70% (v/v) formamide in 2�standard saline citrate for 3 min at 75 °C, and dehydrated in a gradedethanol series. DNA probes were prepared by random prime labeling ofplasmid DNA of BAC-33(P5) and BAC-228(P20) with fluorescein-con-jugated dUTP using the Prime-It Fluor fluorescence labeling kit (Strat-agene). The probes were denatured at 75 °C for 10 min in hybridizationbuffer consisting of 50% formamide (v/v) and 10% dextran sulfate (w/v)in 4� standard saline citrate and prehybridized at 42 °C for 20 min to0.2 �g/ml human competitor DNA (Stratagene) to block repetitive DNAsequences. The probes were subsequently hybridized to the chromo-some spreads at 37 °C overnight, followed by washing to a final strin-gency of 0.1� standard saline citrate at 60 °C. Spreads were mounted inphosphate-buffered glycerol containing 200 ng/ml propidium iodide tocounterstain chromosomes. Slides were examined by epifluorescencemicroscopy using an �100 objective, and the images were captured witha DC-330 charge-coupled device camera (DAGE-MTI, Inc., MichiganCity, IN) using an LG-3 frame grabber board (Scion Corp., Frederick,MD) in a Macintosh 8500 work station and a modified version of theNIH Image 1.6 software (Scion Corp.). Images representing fluoresceinlabeling and propidium iodide staining of the same field were superim-posed using Adobe PhotoShop 3.0 (Adobe Systems, Inc., MountainView, CA) to map the gene to a chromosomal region.

Genomic Organization of the EBP42, TGM5, and TGM7 Genes—PCRamplification of band 4.2 sequences was performed as described for thegenomic library screening but using 100 ng of BAC plasmid DNA andthe oligonucleotides 5�-GTTCTAGGCTTCTCTAGTTGGCAGG (for-ward) and 5�-CGCTGGCTTGGCTCACCCTGTCCC (reverse) for 5�-UTR/exon I or 5�-CCTGAAAACACCATGTGTGCCAAG (forward) and5�-GTTCAGGGGCTACCACGGTGACGC (reverse) for exon XIII. South-ern blots of BAC plasmid DNA restricted with BamHI, EcoRI, and SpeIwere probed with the respective 32P-labeled fragments of band 4.2protein as described and compared with those probed for TGX. Longrange genomic PCR in combination with direct sequencing from the

BAC clones was used to isolate the sequence between the EPB42 andTGM5 genes. PCRs were performed with Pfu Turbo DNA polymerase asdescribed above but using 5% Me2SO and the primers 5�-GATTCCAC-TGTGTCCCATCCAGA (forward) and 5�-GTCACCTCTCAACAGGACA-AGGG (reverse) and 5�-GGGCCATTACCCTAGTCTCTTATTG (for-ward) and 5�-TAATAAAGTGTGACCAGCCTTCCTAG (reverse). A totalof 36 PCR cycles were carried out with a combined annealing extensionstep at 68 °C for 10 min and denaturation at 94 °C for 45 s. The locationof the TGM7 gene in respect to the TGM5-EPB42 gene cluster wasconfirmed in a similar manner using oligonucleotides 5�-TGGGCAAG-GCGCTGAGAGTCCATG (forward, P7) and 5�-GTTTACCTGTCTGCC-TCTACGCTG (reverse) and the Taq-Plus Long PCR system (Strata-gene) according to manufacturer instructions.

Cloning of TGZ by Anchored PCR—PC-3 cells (European Collection ofCell Cultures, Salisbury, UK), established from a human prostate ad-enocarcinoma, were cultured in Coon’s modified Ham’s F-12 medium(Sigma) containing 10% (v/v) heat-inactivated fetal bovine serum, 100units/ml penicillin, and 100 �g/ml streptomycin. Total RNA was iso-lated using Tri Reagent™ (Sigma) according to manufacturer instruc-tions. When needed, poly(A)� RNA was prepared using the Micro-FastTrack 2.0 kit (Invitrogen). cDNA was synthesized from 2 �g of totalRNA or 500 ng of poly(A)� RNA by reverse transcription for 1 h at 42 °Cusing 200 units of Superscript II RNase H� reverse transcriptase and0.025 �g/�l oligo(dT)15 primer in a 20-�l reaction mixture containing0.5 mM dNTPs and 10 mM dithiothreitol in first strand buffer. For thecloning of TGZ, we used a series of degenerate and gene-specific oligo-nucleotides to isolate overlapping DNA fragments, essentially followingour previously described strategy (4). Isolated and sequenced TGZ frag-ments in a 5�33� direction were obtained with the following primers:5�-CAACCTTGCGGCTTGAGTCTGTCG (forward, P8) and 5�-CAGCA-GCTCTGACGGCTTGGGTC (reverse, P9); P8 and 5�-CATACACCACG-TCGTTCCGCTG (reverse, P10), with nested reactions 5�-ATCACCTT-TGTGGCTGAGACCG (forward) and 5�-CAAGGTTAAAAAGTAGGAT-GAAAGTTC (reverse) and 5�-CACAGTGTGACTTACCCGCTG (forward,P11) and P10; P11 and 5�-CGATGGTCAAGTTCCTATCCAGTTG (re-verse, P12), with nested reaction 5�-CTTAAAGAACCCGGCCAAAGA-CTG (forward) and P12; 5�-TGTTGTTTCCAATTTCCGTTCCGC (for-ward) and 5�-TCTGGCACCCTCTGGATACGCAG (reverse); 5�-CTTAG-GGATCAGCCAGCGCAGC (forward, P13) and 5�-GGGTGACATGGAC-TCTCAGCG (reverse, P14), with nested reactions P13 and 5�-TATCT-TTTAGGACCAGCATGGACCTC (reverse) and 5�-GCGGATGAACCTG-GACTTTGG (forward) and P14; and 5�-TGGGCAAGGCGCTGAGAGT-CCATG (forward) and 5�-AGGACAGAGGTGGAGCCAAGACGACATA-GCC (reverse). PCRs were performed with 2 �l of the reverse tran-scription reaction using 1.25 units of AmpliTaq Gold DNA polymerase(Applied Biosystems) and the respective buffer supplemented with 2mM MgCl2, 0.2 mM dNTPs, and 25 pmol of each primer in a total volumeof 50 �l. 40 PCR cycles were carried out in a GeneAmp 9600 thermal-cycler (Applied Biosystems), each cycle consisting of denaturation at95 °C for 45 s, annealing at 60 °C for 1 min, and extension at 72 °C for1 min, with the first cycle containing an extended denaturation period(10 min) for the activation of the polymerase and the last cycle con-taining an extended elongation period (10 min). The 5� end of the cDNAwas isolated by 5�-RACE as described above with the exception of usingthe gene-specific oligonucleotides P9, 5�-TGAAGCTCAGCCGGAGGTA-GAAG, and 5�-GACAGACTCAAGCCGCAAGGTTG. Amplified productswere analyzed on 1% agarose gels, extracted using the QIAquick gelextraction kit, and sequenced as described.

Northern Hybridization—A human RNA master blot containingpoly(A)� mRNA of 50 different tissues was obtained from CLONTECH.32P-labeled probes were prepared by random prime labeling of DNAfragments of the different transglutaminase gene products using theMultiprime DNA labeling system. DNA fragments of 500–700 bp com-prising the 3� end of TGX, TGZ, and band 4.2 protein were generated byrestriction with PstI and AccI, NcoI and NotI (exons XII and XIII), andXhoI, respectively. The cDNA encoding human band 4.2 protein (34)was provided kindly by Dr. Carl M. Cohen (Boston, MA). Hybridizationwas performed under the conditions recommended by the manufac-turer. The labeled membrane was exposed to BioMax MR film, and thefilms were developed after 15–24 h for first exposure and 3–5 days forsecond exposure.

Amplification of TGZ from Different Tissues—cDNA from various celllines and human tissue was prepared as described previously (4). Apanel of cDNAs from human tissue (multiple tissue cDNA panel I) wasobtained from CLONTECH. A 365- or 287-bp fragment of TGZ wasamplified by PCR using the oligonucleotides 5�-TGGGCAAGGCGCT-GAGAGTCCATG (forward) and 5�-GCTGGAGGGCGGGTCTCAGG-GAGC (reverse) or 5�-AGGACAGAGGTGGAGCCAAGACGACATAGCC

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(reverse), respectively, with an annealing temperature of 60 °C.Mapping of Transglutaminase Genes in Mouse Genome—The 100

radiation hybrid clones of the T31 mouse/hamster radiation hybridpanel (35) (Research Genetics, Huntsville, AL) were screened by PCR.A 139-bp fragment of the tgm5 gene was amplified with primers 5�-TGAGGACTGTGTGCTGACCTTG (forward) and 5�-TCCTGTGTCTG-GCCTAGGG (reverse), a 149-bp fragment of the epb42 gene with prim-ers 5�-CAGGAGGAGTAAGGGGAATTGG (forward) and 5�-TGCAGGC-TACTGGAATCCACG (reverse), a 400-bp fragment of tgm7 with prim-ers 5�-GGGAGTGGCCTCATCAATGG (forward) and 5�-CCTTGACCT-CACTGCTGCTGA (reverse), a �600-bp fragment of tgm3 with primers5�-TCGGTGGCAGCCTCAAGATTG (forward) and 5�-AGACATCAATG-GGCAGCATGG (reverse), and 655- and 232-bp fragments of tgm2 withprimers 5�-TTGGGGAGCTGGAGAGCAAC (forward) and 5�-ATCCAG-GACTCCACCCAGCA (reverse) and primers 5�-(GCGGCCGCTAGT)C-CACATTGCAGGGCTCCTGAC (forward) and 5�-GCTAGCCTGTGCT-CACCATGAGG (reverse), respectively. PCRs were carried out in aGeneAmp 9600 thermalcycler with 0.035 units/�l AmpliTaq Goldpolymerase in standard reaction buffer containing 2 mM MgCl2, 0.2 mM

dNTPs, 0.4 �M of each primer, and 2.5 ng/�l genomic DNA in a totalreaction volume of 25 �l. PCR conditions were: polymerase activationfor 10 min at 95 °C, annealing at 60 °C for 45 s, extension at 72 °C for1 min, and denaturation at 94 °C for 30 s for 35 cycles with a finalextension of 3.5 min at 72 °C. PCRs were analyzed by agarose gelelectrophoresis using 1 or 1.5% gels. The hybrid cell panel was analyzedat least twice in each case to exclude PCR-related errors. The data weresubmitted to the Jackson Laboratory radiation hybrid data base foranalysis and mapped relative to known genomic markers(www.jax.org/resources/documents/cmdata/rhmap).

RESULTS

Structure of the TGM5 Gene

Isolation of Genomic Clones—A unique insertion of �30amino acids between the catalytic core domain and �-barreldomain 1 found in TGX served as a template to design specificprimers for screening of a human genomic library. The charac-terization of several genes of the transglutaminase gene familyshowed that the positions of the introns have been highlyconserved (12, 13–15, 17–19, 36), and a comparison of the TGX

sequence to the sequences of the other transglutaminases in-dicated that this unique sequence is present within an exon,exon X (see Fig. 7, amino acids 460–503 in TGX). A PCR fromhuman genomic DNA using oligonucleotides P1 and P2, whichmatch sequences at either end of this unique segment, yieldeda DNA fragment of the expected size that was confirmed to bethe correct product by sequencing (results not shown). Screen-ing of a human genomic DNA BAC library by PCR using theseoligonucleotides revealed two positive clones, BAC-33(P5) andBAC-228(P20), which were subsequently shown by Southernblotting with different cDNA probes to contain sequences span-ning at least exon II to exon X of the TGM5 gene (results notshown). Restriction analysis further indicated that each of theBAC clones contained substantially more than 50 kb of humangenomic DNA.

Gene Structure—The similarity in the gene structure of the

different transglutaminase genes prompted us to approach thecharacterization of introns by PCR amplification using oligo-nucleotide primers corresponding to the flanking exon se-quences at the presumptive exon/intron boundaries. All intron/exon boundaries were sequenced from the PCR productsobtained in at least two independent PCRs, where applicablefrom both BAC clones, to exclude mutations introduced by TaqDNA polymerase, and the results were compared. When se-quences of PCR products comprising adjacent introns had nooverlap, the intervening sequence (exon sequence) was deter-mined by direct sequencing from isolated BAC plasmid DNA toconfirm the absence of additional introns. Similarly, the 3�-untranslated region was obtained by stepwise extension of theknown sequence using direct sequencing of BAC plasmid DNA.Both BAC clones terminated short of exon I, and all attempts atisolating clones spanning exon I by the screening of BAC,P1-derived artificial chromosome, and P1 libraries with acDNA probe or by PCR failed. Exon I and intron 1 sequenceswere finally derived by nested PCR from human genomic DNAusing conditions optimized for long range genomic PCR.

We established that the TGM5 gene is comprised of �35 kbof genomic DNA and contains 13 exons and 12 introns (Fig. 1).All intron/exon splice sites conformed to the known GT/AGdonor/acceptor site rule and essentially to the consensus se-quence proposed by Mount (37) (Table II). A sequence homolo-gous to the branch point consensus CTGAC (38) was found24–44 nt upstream of the 3� splice site in introns 1, 3–6, and9–12. The size of the introns varied considerably, ranging from106 bp to more than 11 kb (Fig. 1, Table III). The sequencesobtained from the two different BAC clones matched with theexception of a deletion spanning the sequence from introns 6 to8 found in BAC-33(P5) (Fig. 1).

Allelic Variants—During the course of this work, we alsoresequenced the entire coding sequence of TGX and found threepoint mutations as compared with the previously reportedcDNA sequence (4). One of the nt exchanges is silent, and theother two result in an amino acid exchange (Table IV). The firsttwo mutations were found in both BAC clones, and the thirdwas present only in BAC-228(P20) because of the deletion inthe other BAC clone. These differences might be sequencepolymorphisms in the human gene pool, because there was noambiguity of the cDNA-derived sequence in this position deter-mined from multiple independently amplified PCR products(4). However, the fact that a serine and an alanine residue arechanged into a proline and a glycine residue that constitutesthe conserved amino acid in these positions in the transglu-taminase protein family (see Fig. 7, amino acids 67 and 352 inTGX) suggested that these may have been PCR-related muta-tions in the cDNA sequence. To clarify this issue, we haveprepared cDNA from human foreskin keratinocytes from dif-ferent individuals, amplified full-length cDNA with high fidel-

FIG. 1. Genomic organization of the human TGM5 gene. The human TGM5 gene is represented with the exons numbered I-XIII andindicated by solid boxes separated by the introns 1–12. The sizes of introns and exons are given in bp. The 5�- and 3�-untranslated regions in exonI and XIII, respectively, are represented by hatched boxes with functional elements defining the transcript indicated. Additional sequence elementsfound in the TGM5 gene are indicated as follows: Alu, Alu 7SL-derived retroposon; STS, sequence-tagged site (see Footnote 3). Below the genomicmap, a representation of the sequences present in the individual BAC clones is depicted.

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ity DNA polymerase, and sequenced the respective portions ofthe cloned cDNAs. The data confirmed that allelic variantsexist with differences in these positions (Table IV).

Alternative Splicing—Isolation and sequencing of cDNAs en-coding TGX and Northern blotting with TGX cDNA probesrevealed expression of at least two differentially spliced mRNAtranscripts for TGX in human keratinocytes (4). Solving thegene structure confirmed the short form of TGX to be the resultof alternative splicing of exon III as predicted. A third isolatedcDNA that differed also at the exon III/exon IV splice junctionturned out to be the result of incomplete or absent splicing ofintron 3, because the sequence upstream of exon IV in thecDNA matched with the 3� sequence of intron 3. Exon 3 encodes

part of the N-terminal �-barrel domain of TGX, and the absenceof the sequence encoded by exon 3 is expected to result in majorstructural changes in at least this domain of the protein. Nev-ertheless, the expression of TGX in 293 cells using the full-length cDNA resulted in synthesis of two polypeptides with amolecular weight consistent with that of the predicted productsfrom the alternatively spliced transcripts (results not shown).

5�-Untranslated Region—Initially, 5�-RACE was used to de-termine the 5� end of TGX cDNAs. Transcripts starting at 77,96, and 157 nt upstream of the initiator ATG were isolated inaddition to the previously described shorter transcript (Fig. 2B,arrowheads). All of these transcripts were recovered repeatedlyin independent experiments. Finally, primer extension experi-ments located the major transcription initiation site used inkeratinocytes 157 nt upstream of the translation start codon(Fig. 2A). The proximal promoter region was analyzed for po-tential binding sites of transcription factors using MatInspec-tor (Genomatix, Munich Germany) and GCG (Genetics Com-puter Group, Inc., Madison, Wisconsin) software packages. Noclassical TATA-box sequence was found, but a number of otherpotential transcription factor binding sites could be identified(Fig. 2B), suggesting that the TGX promoter is a TATA-lesspromoter. Interaction of CCAAT/enhancer-binding protein(may bind to CAAT box), nuclear factor I (NF1), and upstreamstimulatory factor (USF) to form a core proximal promoter hasbeen demonstrated in a number of TATA-less genes. c-Myb isfound in TATA-less proximal promoters of genes involved inhematopoiesis and often interacts with Ets factors (39), andthese sites may be operative in the expression of TGX in hema-topoietic cells, e.g. HEL cells. AP1, Ets, and SP1 elements aretypically found in keratinocyte-specific genes (40, 41) and maybe involved in transcriptional regulation in keratinocytes. Sev-eral AP1 sites are present within 2.5 kb of upstream sequenceand could interact with the proximal AP1 factor for activation.SP1 sites are positioned properly upstream of the start pointsof the shorter transcripts, raising the possibility that thesecould also be functional in transcription initiation, although toa lesser degree.

3�-Untranslated Region—The last exon, exon XIII, containeda consensus polyadenylation signal AATAAA �600 bp down-stream of the termination codon (Fig. 3). This is in good agree-

TABLE IISplice donor and acceptor sequences in the human TGM5 gene

Residues consistent with the splice site consensus sequence (MAG/GTRAG and YAG/G) are underlined.

Intronnumber Donor sequence Acceptor sequence

M A Q G L E V A1 GCTACCATGGCCCAAGgtagggaaagcccctgtggccactggagtt ttttgtctaaccctggctgccccattgcagGGCTAGAAGTGGCC

F V V E T G P L P D2 TTCGTGGTTGAAACTGgtaagaaccccagctggctcacaggggctg tggagggcctcagctctacttccctcctagGACCGCTGCCAGAC

N P W C P E D A V Y3 AATCCCTGGTGCCCAGgtaaggctgggtgcccaggcggtgcctcct tgcttcgtgccctcccactctggttcctagAGGATGCTGTCTAC

W N Y G Q F E D K I4 TGGAACTATGGACAGgtgagtctcagccctgcttatggcccatcc tgccttccctctctgcctctccccccgaagTTTGAAGACAAAATC

V V C A M I N S N D5 GTGGTGTGTGCCATGgtgaggtccctggcgtgcccggggaggagg ctcacacttctctatatggcttctcttcagATCAACAGCAATGAT

A V M C T V M R C L6 GCCGTCATGTGCACAGgtaggaggtagaaaggacctcacaaaaagg acaggtgatttttttgtgccctttttgcagTGATGAGGTGTCTG

K D T I W N F H V W7 AAGGATACTATCTGgtgagaaacaacctctcaacctatttctag caacgtctcccttggctctgtttgatacagGAACTTCCATGTCTGG

Q E M S N G V Y C C8 CAGGAGATGAGCAACGgtgaggctctccagaagaaaggcaggcccc gcccaccgaggctcccctgttctccttcagGCGTCTACTGCTGT

Y K Y E E G S L Q E9 TACAAGTATGAAGAAGgttagtaagcaagccagccctactcagagc cagctggtgctgtgctctccccaacttcagGATCCCTCCAGGAG

L S P K E A K T Y P10 CTCTCTCCTAAAGAAGgtacgcatgtgcacagtttgtgtacgcaga tctcaccccatccttgtgttctttctttagCAAAGACCTACCCC

S I T I N V L G A A11 AGCATCACGATTAATgtaggcaggagtcctgcaaatggcttgtgg taattctccttcccctcctggtctgtttagGTTCTAGGAGCAGCC

Q Q K V F L G V L K12 CAGCAGAAAGTCTTgtaagtgctgcaagtgctcagccttctcct ttttctgacatgctccattctctgttgcagCCTTGGAGTCCTCAAA

TABLE IIIIntron sizes and splice types in the human TGM5 gene

The sizes of introns are estimated to be within �100 bp unlessindicated to be sequenced entirely.

Intronnumber

Splicetype Size Method

1 1 6,300 bp PCR2 1 102 bp Sequencing3 1 3,300 bp PCR4 0 2,900 bp PCR5 0 600 bp PCR6 1 �11,800 bp PCR and Restriction Analysis7 2 1,600 bp PCR8 1 106 bp Sequencing9 1 2,900 bp PCR

10 1 545 bp Sequencing11 0 1100 bp PCR12 2 209 bp Sequencing

TABLE IVApparent polymorphisms in the cDNA and genomic DNA sequences

for TGx

The positions with nucleotide and amino acid variations are under-lined.

Residue cDNA (4) Gene cDNAa

67 S TCA P CCA P CCA220 Y TAC Y TAT Y TAC352 A GCA G GGA A GCA

a Additional sequence variant isolated in this work.

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ment with the size of the mRNA (2.8 kb) encoding full-lengthTGX expressed in human keratinocytes as detected by North-ern blotting (4) considering the length of the coding sequence(2160 bp). A CAYTG signal that binds to U4 small nuclear RNA(42), which is identical for 4 of 5 nt, is present in tandem inthree copies 7 nt downstream of the polyadenylation signal. Aclose match (YCTGTTYY) of another consensus sequence YGT-GTTYY that is found in many eukaryotic transcripts and pro-vides a signal for efficient 3� processing (43) is present 46 ntdownstream of the polyadenylation signal. However, we have

reported previously that all cDNAs isolated by reverse tran-scription PCR with an oligo(dT) oligonucleotide from humankeratinocytes ended within 9–34 nt downstream of the pen-tanucleotide ATAAA at position 2317 (Fig. 3) (4). It has beenshown that this pentanucleotide functions as a polyadenylationsignal in other genes (42), and it is apparently functional inTGX. Indeed, the most frequently found poly(A) addition sites(indicated in Fig. 3) showed cleavage at a C(A) boundary, whichis the preferred sequence for cleavage/polyadenylation in eu-karyotic genes (44). However, no CAYTG or YGTGTTYY signalwas found immediately downstream of the ATAAA signal se-quence. A possible explanation for these findings is that mosttranscripts use the AATAAA signal for polyadenylation, assuggested by the Northern blotting data, and occasionally theATAAA signal functions as a polyadenylation signal and theseshorter transcripts are selectively enhanced by PCR amplifica-tion because of the smaller size of the PCR product.

The TGM5 Gene Is Part of a Cluster of TransglutaminaseGenes

Chromosomal Localization of the TGM5 Gene—To addressthe genomic organization of the TGM5 gene or genes in thehuman genome, we performed Southern blot analysis of humangenomic DNA cut with BamHI, EcoRI, and HindIII restrictionenzymes using probes derived from intron 2 as well as from thesequence encoded by exon X that is unique to TGX. Bands of4.5, 6.0, and 10.5 kb and 4.3, 9.3, and 2.6 kb were revealed withthe respective probes. The simple pattern of restriction frag-ments hybridizing with the probes indicated that the haploidhuman genome contains only one TGM5 gene.

The TGM5 gene was subsequently localized to chromosome15 by fluorescent in situ hybridization on human metaphasechromosome spreads using genomic DNA derived from eitherBAC clone as a probe (Fig. 4A). A comparison of the probesignal to the 4,6-diamino-2-phenyl indole (DAPI) banding pat-tern localized the TGM5 gene to the 15q15 region. The local-ization was subsequently refined by determining the distanceof the fluorescent signal to the centromere as well as to eitherend of the chromosome on 13 copies of chromosome 15 andexpressing it as a fractional distance of the total length of thechromosome. These measurements placed the TGM5 gene closeto the center of the 15q15 region, i.e. to the 15q15.2 locus(Fig. 4B).

TGM5 and EPB42 Genes Are Arranged in Tandem—TheEPB42 gene (Table I) has been assigned previously to the15q15 locus on chromosome 15 (27, 28). This raised the possi-bility that the EPB42 gene is in close proximity to the TGM5gene. Indeed, PCR with specific primers for sequences derivedfrom the 5� and 3� ends of the EPB42 gene yielded products ofappropriate size from both, BAC-33(P5) and BAC-228(P20)

FIG. 2. Structure of the 5�-untranslated region of the humanTGM5 gene and mapping of the transcriptional start site. A,primer extension analysis of poly(A)� RNA isolated from primary hu-man keratinocyte prior to (lane 1) or after (lane 2) culture in suspensionfor 12 h. Extension products were separated on denaturing polyacryl-amide gel alongside a Sanger dideoxynucleotide sequencing reaction ofthe appropriate genomic DNA fragment primed with the same oligonu-cleotide. The transcriptional start site is indicated by the arrow. B, thenucleotide sequence of the proximal 5� region of the TGM5 gene. 5� endsof mRNA from primary keratinocytes mapped by RACE are indicatedby arrowheads. The major transcription start site identified by primerextension is highlighted with an asterisk (labeled �1). Consensus se-quences for putative regulatory elements are underlined.

FIG. 3. Structure of the 3�-untranslated region of the humanTGM5 gene. The 3�-flanking sequence is shown with sequences homol-ogous to known consensus sequences for 3� processing of transcripts(AATAAA, CAYTG, and YGTGTTYY) underlined. The terminationpoints of cDNAs isolated from human keratinocytes (4) by 3�-RACE areindicated by arrowheads. A pair of inverted long repeat sequences ishighlighted in italics.

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(data not shown), and sequencing confirmed the identity ofthe PCR products. Southern blotting of BAC plasmid DNAwith cDNA probes comprising the 5� or 3� end of the EPB42gene and subsequent comparison of the pattern of labeledrestriction fragments with that of the TGM5 gene allowed usto map this locus in more detail. The EPB42 and TGM5 genesare arranged in the same orientation, being spaced apart by�11 kb (Fig. 4C).

Identification of a Novel Transglutaminase Gene,TGM7—We developed a method for the detection and identifi-cation of transglutaminase gene products based on reversetranscription PCR of the conserved active site sequence withdegenerate primers, and using this method we discovered thegene product of the TGM5 gene (4). Using this same method,we have now identified another new transglutaminase geneproduct in human prostate carcinoma tissue that we desig-nated TGZ. The full-length cDNA for this new gene product wasobtained by anchored PCR as described below. A comparison ofthe TGZ cDNA sequence to the sequences in the GenbankTM

data base revealed a match with two draft sequences of humanBAC clones (AC009852 (assigned to chromosome 15) andAC009825 (assigned to chromosome 8)). These DNA contingen-cies also gave a perfect match with our TGM5 gene sequences,suggesting that the TGM7 gene is located in close proximity tothe TGM5 and EPB42 genes. Although the human BAC clones(Fig. 1) terminate within the TGM5 gene and do not containsequences of the TGZ gene product, we have isolated a mouse

BAC clone that contains all three genes (data not shown). Tofurther confirm the organization of the TGM7 gene in thehuman genome, we used long range genomic PCR with differ-ent combinations of primers designed from the flanking se-quences of the TGM5-EPB42 gene and the TGZ cDNA sequenceto explore whether the TGM7 gene was present in close prox-imity to the other two transglutaminase genes. This placed theTGM7 gene �9 kb upstream of the TGM5 gene and demon-strated that the genes are arranged in tandem fashion (Fig.4C). The sequence between the TGM7 and TGM5 genes wasfurther found to contain a pseudogene of the mitochondrialATPase D subunit, the relevance of which is unclear.

Determination of the cDNA and Amino Acid Sequence ofthe TGM7 Gene Product

Based on the initial PCR amplification of a TGZ cDNA frag-ment from prostate carcinoma tissue, we analyzed prostate-derived cell lines for expression of TGZ by PCR. A full-lengthcDNA sequence for TGZ was obtained by anchored PCR usingcDNA prepared from the human prostate carcinoma cell linePC-3, essentially following the strategy described previously(4). 5�-RACE was used to determine the 5� end of the cDNA.The isolated 3�-end sequence was confirmed by sequencing ofmatching expressed sequence tag clones in the GenbankTM

data base (accession numbers AI018564, AI024635, andAW511368). The obtained sequence information consists of

FIG. 4. Chromosomal localization of the human TGM5 gene by fluorescence in situ hybridization. A, a representative picture offluorescein-labeled genomic DNA of BAC-228(P20) (green fluorescence, arrows) hybridized to metaphase spreads of human chromosomes stainedwith propidium iodide (red fluorescence). B, an ideogram of banded chromosome 15 showing the localization of the fluorescent signal on 13chromosomes. C, a schematic map of the respective locus showing the organization of the genes encoding TGX (TGM5), band 4.2 protein (EPB42),and TGZ (TGM7) as well as other genes (D-type cyclin-interacting protein 1 (DIP1), ? (EST, GenbankTM accession numbers AA457639 andAA457640), and mitochondrial ATPase subunit D pseudogene (Mito ATPase L1 Sub D)), an L1 repetitive element, and genetic markers (forL926G10 see Footnote 3).

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2318 nt (Fig. 5) containing an open reading frame of 2130 nt.The probable initiation codon is present in the sequenceGAGATGG, which presents only limited homology to the con-sensus identified by Kozak (45), which acts as a signal forefficient transcription in eukaryotes. However, the critical pu-rine in position �3 as well as the G in position �4 are con-served. The signal for polyadenylation (AATAAA) is located158 nt downstream of the termination codon (TGA). The de-duced protein consists of 710 amino acids and has a calculatedmolecular mass of 80,065 Da and an isolelectric point of 6.6.

During the course of the TGz cDNA sequence determination,a number of aberrantly spliced gene products were isolated.These lacked exon VI or part of exon IX (5� end) or retained thewhole or part of intron 11. These products are unlikely to be ofphysiological significance but may point out that the splicing ofcertain introns in this gene is a difficult and inefficient process.Interestingly, in the variant lacking part of exon IX, splicingoccurred from the donor site of exon VIII to an acceptor site inthe middle of exon IX, which corresponds to the exact positionwhere an additional intron is present in the TGM1 and F13A1genes (Fig. 7).

Gene Products of the Transglutaminase Gene Cluster AreDifferentially Expressed in Tissues

We have shown previously that TGX is expressed in a num-ber of different cell types (4). To obtain a more complete pictureof the expression pattern of TGX and the novel gene product, weperformed a dot-blot Northern blot analysis of more than 50adult and fetal human tissues. Probes comprising sequences of

the �-barrel domains were employed, the specificity of whichhas been documented previously (4). Band 4.2 protein wasexpressed at high level in bone marrow and fetal spleen andfetal liver, consistent with its role in hematopoietic cells, andvirtually undetectable in all other tissues (Fig. 6). In contrast,TGX and TGZ showed widespread expression at low level. Thehighest levels of TGX and TGZ mRNA were present in thefemale reproductive system (uterus, placenta, ovaries, andmammary gland) and fetal tissues, and in testis and lung,respectively (Fig. 6). Reverse transcription PCR analysis onhuman cell lines and tissues confirmed these results and dem-onstrated that TGZ is expressed in osteosarcoma cells (MG-63),dermal fibroblasts (TJ6F, HCA2), erythroleukemia cells (HEL),primary keratinocytes, mammary epithelium carcinoma cells(MCF7), HELA cells, testis, skin, brain, heart, kidney, lung,pancreas, placenta, skeletal muscle, fetal liver, prostate, andprostate carcinoma tissue (data not shown). These results dem-onstrate that all three gene products of this gene cluster aredifferentially expressed in tissues.

Evolution of Transglutaminase Genes

Mapping of Mouse Genes—To further analyze the relation-ship between the most closely homologous genes of the trans-glutaminase gene family (TGM7, TGM5, and EPB42 on humanchromosome 15q15.2 and TGM2 and TGM3 on chromosome20q11/12), we mapped the respective mouse genes using radi-ation hybrid mapping. All genes mapped to the distal part ofmouse chromosome 2. The genes for tgm7, tgm5, and epb42showed a best-fit location for the segment defined by D2Mit104

FIG. 5. Nucleotide sequence and de-duced amino acid sequence of humanTGZ. The initiation and terminationcodons as well as the polyadenylation sig-nal (AATAAA) are underlined. The activesite Cys residue is shown in red, and theHis and Asp residues of the catalytic triadare shown in purple. Additional residuesthought to be involved in substrate inter-action are shown in green, and residuesinvolved in Ca2� binding are depicted inblue.

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proximal and D2Mit396 distal, with a highest LOD of 14.5,16.6, and 15.5 to the anchor marker D2Mit395 (66.9 cM) and anLOD of �20 to D2Ertd616e (69.0 cM). This is in good agree-ment with the assigned locus of epb42 67.5 cM distal from thecentromere (46). The tgm3 gene showed a best-fit location forthe segment defined by D2Mit447 proximal and D2Mit258distal, with a highest LOD of 14.8 and 12.2 to D2Mit258 (78.0cM) and D2Mit338 (73.9 cM), respectively. The tgm2 geneshowed a best-fit location for the segment defined by D2Mit139proximal (86.0 cM) and D2Mit225 distal (91.0 cM), with ahighest LOD of 17.0 to the anchor marker D2Mit287, consist-ent with its assigned locus 89.0 cM from the centromere (47).

DISCUSSION

In this study, we have shown that human TGX is the productof an �35-kb gene located on chromosome 15q15.2 and contain-ing 13 exons and 12 introns. The intron splice sites conform tothe consensus for splice junctions in eukaryotes (37). The tran-scription initiation site is localized 157 nt upstream of theinitiator methionine, and the likely polyadenylation site islocalized �600 nt downstream of the stop codon, which isconsistent the previously reported transcript size of about 2.8kb (4). The short form of TGX, which we have identified inkeratinocytes (4), is the product of alternative splicing of exonIII. The TGM5 gene is part of a cluster of three transglutami-nase genes arranged in tandem, with a novel transglutaminasegene proximal to the TGM5 gene and the gene encoding eryth-rocyte band 4.2 protein distal. We have used the designation

TGM7 for the novel gene and TGZ for its gene product, which isin line with using sequential numbers and letters for genes andproteins, respectively, in the order of discovery.

Structural and Functional Implications for the Novel Trans-glutaminase Gene Product Based on Comparative Sequence Anal-ysis—We have determined the full-length cDNA sequence of thenovel transglutaminase gene product, TGZ. A comparison of TGZ

to the previously characterized human transglutaminases re-veals that the structural requirements for transglutaminase ac-tivity and Ca2� binding are conserved. The structure of severaltransglutaminases has been solved by x-ray crystallography andshows a high degree of similarity (20–22). The reaction center isformed by the core domain and involves hydrogen bonding of theactive site Cys to a His and Asp residue to form a catalytic triadreminiscent of the Cys-His-Asn triad found in the papain familyof cysteine proteases (23). The residues comprising the catalytictriad are conserved in TGZ (Cys279, His338, and Asp361) (Figs. 5and 7), and the core domain shows a high level of conservation asindicated by a sequence identity of �50% as compared with theother transglutaminases. A Tyr residue in the barrel 1 domain ofthe a-subunit of factor XIII is hydrogen-bonded to the active siteCys residue, and it has been suggested that the glutamine sub-strate attacks from the direction of this bond to initiate thereaction based on analogy to cysteine proteases (24). It has fur-ther been proposed that this hydrogen bond prevents the forma-tion of a disulfide bond between the active site residue and aneighboring Cys, which would result in enzyme inactivation (22).

FIG. 6. Expression of TGX, band 4.2 protein, and TGZ in different fetal and mature human tissues. Human tissue Northern dot blotnormalized for average expression of nine different housekeeping genes (CLONTECH) probed with a fragment corresponding to the C-terminal�-barrel domains of TGX (A), TGZ (B), and band 4.2 (C). A diagram showing the type of poly (A)� RNA dotted onto the membrane is shown in D.

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In TGZ, the Tyr residue has been replaced by a His residue(His538), similar to TGX (Fig. 7). This is expected to be a conserv-ative change, which is supported by our data demonstrating thatrecombinant TGX produced in 293 cells has transglutaminaseactivity.2 The Trp residue (Trp279 in factor XIII), which isthought to stabilize the oxyanion intermediate generated in theproposed reaction mechanism (23), is conserved in TGZ (Trp243).All these residues involved in the catalytic process are conservedin the different transglutaminase gene products with the excep-tion of band 4.2 protein, which is the only member of this genefamily without catalytic activity (Fig. 7). Crystallization experi-ments with factor XIIIa further indicated that four residues areinvolved in the binding of a Ca2� ion, including the main chaincarbonyl of Ala457 and the side chain carboxyl groups of Asp438,Glu485, and Glu490 (24). All three acidic residues are conserved inTGZ (Asp403, Glu450, and Glu455). Based on the preservation ofcritical residues for enzyme function and domain folding and theextensive overall similarity of TGZ to the other members of thetransglutaminase family with catalytic activity, it is likely thatthe characterized cDNA encodes an active transglutaminase.

Organization of Transglutaminase Genes in the Human andMouse Genome—Having established the chromosomal localiza-tion of the TGM5 and other human transglutaminase genes, itis of interest to compare it with the equivalent chromosomalregions in the genomes of other species and in particular of themouse, for which many genetic aberrations have been mapped.

This may provide insights into the evolution of a gene family aswell as help in the identification of candidate genes for humanpathologies.

In this study, we have mapped the gene encoding TGX tochromosome 15q15 by fluorescent in situ hybridization. Band4.2 protein has been mapped previously to this chromosomalregion (27, 28) and subsequently has been assigned to 15q15.2by expression mapping of the LGMD2A locus on chromosome15 (48). A short sequence encompassing the left arm of one ofthe yeast artificial chromosome clones (926G10)3 used for ex-pression mapping matched with the sequence of intron 12 ofthe TGM5 gene, which is consistent with our data placing thetwo genes encoding TGX and band 4.2 protein in direct appo-sition. In mouse, the syntenic region is found on chromosome 2,2F1–F3 (48, 49), and our radiation hybrid data place all threegenes, tgm7, tgm5, and epb42, in this segment of chromosome2. In addition, we have isolated a mouse BAC clone and shownthat it contains all three genes. Even though the exact posi-tioning awaits further sequence data, this suggests that allthree mouse genes are in close proximity, presumably arrangedin a similar fashion to their human counterparts. Our mappingdata are consistent with the previously determined location ofthe epb42 gene by interspecific backcross analysis (46). Database analysis identified two mutations that are associated with

2 P. Grenard, and D. Aeschlimann, manuscript in preparation.

3 CEPH-Genethon yeast artificial chromosome 926_g_10 left arm:Fougerousse, F. (GenBankTM/EBI Data Bank accession numberX75198).

FIG. 7. Comparison of the structure of the different human transglutaminase genes. An alignment of the eight characterized humangene products (TGZ, this study; TGX (4); band 4.2 (34); TGE (54); TGC (55); factor XIII a-subunit (56, 57); TGK (58, 59); and TGP (30, 60)) is shown,with dashes indicating gaps inserted for optimal sequence alignment and underlined residues representing amino acids conserved in at least fourgene products. The sequences are arranged to reflect the transglutaminase domain structure based on the crystal structure of factor XIII a-subunit(20): N-terminal propeptide domain (d1), �-sandwich domain (d2), catalytic core domain (d3), and �-barrel domains 1 (d4) and 2 (d5) (from top tobottom). Known intron splice sites are marked by arrowheads (12–19). The aberrant splice donor site within exon IX of TGZ is marked by a blacksquare.

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this locus, ro (rough) and pa (pallid). In fact, it had beensuggested that pa might be a mutation in band 4.2 protein (46),but more recently it has been shown that pa/pa mice expressnormal band 4.2 by cDNA analysis and that the two loci seg-regate in an interspecific cross (50). The phenotype associatedwith neither of these mutations seems to be a match with

expected tissue impairments of a transglutaminase deficiency.Similarly the pathologies of known congenital human diseasesassociated with the respective locus are not consistent with atransglutaminase deficiency.

Interestingly, the syntenic region of human chromosome20q11 is found on distal mouse chromosome 2 adjacent to the

FIG. 8. Phylogenetic tree of thetransglutaminase gene family andgenomic organization of the genes inman and mouse. Phylogenetic treesbased on the amino acid sequence homol-ogy of the gene products have been con-structed for the individual domains aswell as for the entire gene products usingthe neighbor-joining method (61) of thePHYLIP or Treecon 1.3b (van de Peer,University of Antwerp, Belgium) softwarepackages. Sequences were aligned usingClustalX and corrected by hand as needed(nonhomologous N- and C-terminal exten-sions were excluded) to maximize homol-ogy as shown in Fig. 7 except includingsequences from different species as avail-able. A representative tree based on full-length sequences is shown in A (Treecon,distance estimation according to Tanjaand Nej). Bootstrap values are given inpercentage and the scale bar reflects 0.05substitutions per site. In B, a hypotheticalpedigree for the gene family is given thatis consistent with the data on the se-quence relationship of the individual geneproducts (A) as well as with the data ongene structure and genomic organization(C).

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locus of the identified transglutaminase gene cluster. Thegenes encoding TGC and TGE, which are more closely related tothe TGM5, TGM7, and EPB42 genes than the other transglu-taminase genes based on amino acid sequence comparison andsimilarity in gene structure (Fig. 7), have been mapped tohuman chromosome 20q11 (14, 26). We recently have clonedTGY, which is the product of the TGM6 gene that neighbors theTGM3 gene.4 We have now identified the chromosomal locationof the mouse tgm2 and tgm3 genes by radiation hybrid map-ping and identified positions �20 and 5 cM distal to the tgm7/tgm5/epb42 gene cluster, respectively. This places a total of sixtransglutaminase genes on distal mouse chromosome 2 andraises questions about their evolutionary and present-dayrelationship.

Evolution of Transglutaminase Genes—To analyze the evo-lutionary1 relationship between the transglutaminase genes inmore detail, we calculated the amino acid similarity essentiallybased on the sequence alignment shown in Fig. 7 and calcu-lated evolutionary distances using different algorithms. Allalgorithms predicted a close relationship between factor XIIIa-subunit and TGK (lineage 1), and TGX, TGZ, TGE, TGY, band4.2 protein, and TGC (lineage 2), respectively (Fig. 8). Thegenomic organization and gene structure also support the closerelationship of the latter six genes and corroborate the place-ment of these in a separate phylogenetic branch. The exactrelationship of TGP to these two lineages is less certain, but itis likely to have branched off from lineage 2 approximately atthe same time as factor XIII a-subunit and TGK diverged. Onlya single transglutaminase gene has been identified in genomesof invertebrate species, and it is thus likely that the separationof transglutaminases into four branches is likely to have oc-curred after divergence of invertebrates from proto-verte-brates. In fact, a similar relationship between invertebratesand vertebrates has been found for many orthologous genes,and it has been proposed recently that octaploidy in earlyvertebrates may have resulted in two successive genome dupli-cations in early vertebrates (52, 53). Our phylogenetic analysisis consistent with this model. One branch of lineage 2 hassubsequently undergone multiple duplications locally to gener-ate the six genes that are clustered on mouse chromosome 2.Despite the close relationship of neighboring genes within theclusters, one possible scenario is that a single transglutami-nase gene initially locally duplicated and was followed by aduplication of a larger segment of the chromosomal region,giving rise to the organization of the genes seen in mouse. Inhumans these chromosomal regions were apparently redistrib-uted to two different chromosomes. Our analysis further indi-cates that orthologues of these six genes are likely to exist in allhigher vertebrate species.

Functional Redundancy in Transglutaminase Gene Family—Despite differential expression in tissues and apparently dis-tinct promoter organization (5, 51), the expression patterns ofTGC, TGZ, and TGX and to a more limited degree TGE overlap.We have preliminary data showing that in TGC null mice (11),the expression of several other transglutaminases (includingTGX and TGE) is up-regulated.5 Based on the apparent lack ofa phenotype in response to TGC gene ablation in mice, the closesimilarity of these gene products, and their overlapping expres-sion, we hypothesize that these gene products have overlappingfunctions.

In conclusion, the identification of several new transgluta-minases that are closely related to TGC could explain some ofthe contradictory data and large number of proposed functions

in the literature regarding this enzyme and should stimulatethe re-evaluation of these experiments in the light of thesefindings.

Acknowledgments—We are grateful to Dr. Lorraine Meisner for pro-viding expertise for the interpretation of the chromosomal fluorescencein situ hybridization experiments. We are also grateful to Drs. DennisHeisey and Andy Weightman for help with the construction of thephylogenetic trees. We thank Pascale Aeschlimann for excellent tech-nical assistance and Andrea Schmick for help with the preparation ofthe art work.

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Pascale Grenard, Mary Kay Bates and Daniel AeschlimannTRANSGLUTAMINASE Z

TRANSGLUTAMINASE X AND A NOVEL GENE FAMILY MEMBER, ENCODINGCluster on Human Chromosome 15q15: STRUCTURE OF THE GENE

Evolution of Transglutaminase Genes: Identification of a Transglutaminase Gene

doi: 10.1074/jbc.M102553200 originally published online June 4, 20012001, 276:33066-33078.J. Biol. Chem. 

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