Proc. Nail. Acad. Sci. USAVol. 89, pp. 9804-9808, October 1992Biochemistry

Cloning and expression of chicken erythrocyte transglutaminase(protein-glutamine -glutamyltransferase/mRNA expresdon/erythroid development)

N. WERAARCHAKUL-BOONMARK, J.-M. JEONG, S. N. P. MURTHY, J. D. ENGEL, AND L. LORANDDepartment of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208-3500

Contributed by L. Lorand, July 29, 1992

ABSTRACT We report the sequences of cDNAs encodingchicken erythrocyte transglutaminase (EC 2.3.2.13). The com-plete mRNA consists of 3345/3349 nucleotides and predicts asingle open reading frame. Nine peptide sequences derivedfrom partial digests of the isolated protein agreed with thecorresponding translation of the open reading frame. Approx-imately 60% identities between the avian protein and threerelated mammalian enzymes were found. Chicken erythrocytetransglutaminase mRNA is most abundant in red blood cellsand kidney, and it accumulates during erythroid cell differen-tiation.

Cytosolic transglutaminases (protein-glutamine y-glutamyl-transferases, EC 2.3.2.13) were first described by Waelschand collaborators in the 1950s (1) and were assumed to beprimarily involved in the metabolism of amines (such ashistamine) by conjugation to proteins (2). These Ca2+-dependent thiol enzymes are widely distributed in verte-brates and are also present in a number of invertebrates [e.g.,sea urchin egg, Homarus hemocyte, Limulus amoebocyte(3), Homarus muscle (4), and marine sponge cells (5)], someof which served as a rich source for their purification. Theintracellular function of transglutaminases is now thought tobe the posttranslational crosslinking of proteins, triggeredperhaps by a substantial increase in free Ca2+ (approaching0.1 mM), the appearance of an activating metabolite, or theremoval ofan inhibiting substance (6). Significant amounts ofN6-(-y-glutamyl)lysine, the product of the transglutaminase-mediated crosslinking of proteins, were identified in a varietyof cellular structures [e.g., in membrane skeletal polymers inhuman red blood cells (7, 8), in the cornified envelope ofhuman keratinocytes (9), in polymers from human lens cat-aracts (10), and in apoptotic bodies of degenerated liver cells(11)]. Membrane-bound variants of intracellular transgluta-minases have been identified in keratinocytes (12). Secretedforms of transglutaminases participate in the clotting ofseminal vesicle secretory proteins of the prostatic fluid ofrodents (13-16) and in the clotting of Homarus and Limulusblood (3, 17, 18). One of the subunits (designated A) of thefactor XIII zymogen (fibrin-stabilizing factor) circulating inhuman plasma also belongs to this class ofgene products (19,20). Transglutaminase was shown to become activated in seaurchin eggs soon after fertilization (21, 22) and in A431epidermal carcinoma cells exposed to epidermal growthfactor (23), and it is expressed in induced murine erythro-leukemia cells well before the appearance ofhemoglobin (24).

This paper deals with the identification, isolation, andsequence analysis ofcDNAs encoding chicken red blood celltransglutaminase* and shows that the mRNA for this proteinis present only in very low amounts in embryonic erythroidcells or in retrovirally transformed erythroid progenitor cellsbut increases significantly during erythroid cell maturation.

MATERIALS AND METHODSPurification and Peptide Sequencing of Chicken Red Blood

Cell Transglutaminase. Erythrocyte transglutaminase waspurified from chicken blood (collected in heparin; Pel-FreezBiologicals) to apparent SDS/PAGE homogeneity by DavidSchilling, using slight modifications of the procedure de-scribed for human red cells (25). To obtain partial peptidesequences, purified protein was digested with 100:1 weightratios ofeither Staphylococcus aureus V8 protease, endopro-teinase Lys-C (Boehringer Mannheim), L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin(Worthington), or 7-amino-1-chloro-3-tosylamido-2-hep-tanone ("Na-p-tosyl-L-lysine chloromethyl ketone," TLCK)-treated a-chymotrypsin (Sigma). Some of the peptides werepurified by reverse-phase (C3 column) HPLC. Other frag-ments were separated by SDS/PAGE (26) and were elec-troblotted onto poly(vinylidene difluoride) transfer mem-branes (27) prior to sequencing (Applied Biosystems model177A; Northwestern University Biotechnology Facility).

Screening ofcDNA Libraries. Two types ofcDNA librarieswere used to isolate the chicken erythrocyte transglutamin-ase cDNA clones. The BV4 cDNA library in Agtll wasderived from poly(A)+ RNA isolated from a pool of 11-day-old chicken embryos (28), whereas the B21 cDNA library inAZAPII vector was derived from poly(A)+ RNA isolatedfrom erythroid cells of 13- to 14-day-old B21 chicken embryos(29). Recombinant cDNAs were identified by immunoscreen-ing (30) of the BV4 library with a rabbit antiserum raisedagainst purified chicken erythrocyte transglutaminase. Aftertreatment with dithiothreitol, the protein was subjected toSDS/PAGE, and rabbits were injected with the gel slicecorresponding to a band of Mr 78,000 (using Freund's com-plete adjuvant for initial injection and Freund's incompleteadjuvant for subsequent injections). The antiserum recog-nized chicken erythrocyte transglutaminase preferentially inboth the native and the denatured form; it crossreacted withhuman red cell transglutaminase and with guinea pig livertransglutaminase but did not recognize human factor XIIIsubunit a or human erythrocyte membrane protein 4.2.Positive clones were plaque-purified, and phage DNA wasisolated (31). The BV4 library was then screened succes-sively by plaque hybridization using as probes either theEcoRI-Sac I [297 nucleotides (nt)] cDNA fragment of clone27c (Fig. 1), the 54 nt of DNAsynI (5'-GAATTTGGGGT-TGATGTCCAGCATCTCGAGGCAGATGGCCAAGATC-TCATCTTC-3') complementary to nt 1034-1087 in the com-piled cDNA sequence (Fig. 2), or the EcoRI-Kpn I fragment(374 nt) from clone NW1. The B21 library was screened usingthe synthetic 21-base oligonucleotide TG1 (5'-GTCTCCAG-CACCAGCTCTTCG-3', complementary to nt 484-504 in thecDNA sequence; Fig. 2) and the EcoRI-Kpn I fragment (374nt) as probes. The positive clones derived from hybridizationscreening were amplified by PCR using an internal cDNA

Abbreviations: aa, amino acid(s); nt, nucleotide(s).*The sequence reported in this paper has been deposited in theGenBank data base (accession no. L02270).

9804

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Biochemistry: Weraarchakul-Boonmark et al. Proc. Natl. Acad. Sci. USA 89 (1992) 9805

Ball Sad Ball

EcoRI

== 3 Ckrbctg

EcoRI-Sacl (297 nt)

- 27 C4- 4- 4- 4-

- DNAsynl 154 nt)D2040

4- 4,-

.

EcoRI-Kpnl (374 nt)

4--.. TG1 (21 nt)

1----b 0

.*---- TG6 121 nt)

NW1

NW3

FIG. 1. Restriction map and sequencing strategy for the chickenred blood cell transglutaminase (Ckrbctg) cDNA clones. The shadedbox represents the coding region, and the unshaded boxes show the5' and 3' untranslated regions. Originally, clone 27c was isolated byimmunological screening (30) of a Agtll cDNA library prepared from11-day-old chicken embryos (BV4; ref. 29) using an antiserum raisedin rabbits against purified chicken red blood cell transglutaminase.D2040 was isolated by using an a-32P random-labeled EcoRI-Sac I

(297 bases) cDNA fragment of 27c as probe; NW1 was isolated usinga y-32P end-labeled oligonucleotide, DNAsynI (54 nt), as probe. NW3was isolated from a AZAPII cDNA library (B21; ref. 29) preparedfrom erythroid cells of 13- to 14-day-old chicken embryos by usingan a-32P random-labeled EcoRI-Kpn I (374 nt) cDNA fragment ofNW1 and a y-32P end-labeled oligonucleotide, TG1 (21 nt), as probes.Solid arrows indicate the extent and direction ofDNA sequencing foreach strand of the individual cDNA clones; dotted arrows indicatethe relative position and orientation of synthetic oligonucleotides.Restriction sites employed for subcloning and DNA sequencing areindicated. kb, Kilobase.

sequence and a primer within the A vector. Clones thatcontained the longest 5' inserts were then plaque-purified andfurther characterized.DNA Sequencing. All independent cDNA clones were

subcloned into pGEM-3Z(+) or pGEM-7Zf(+) (Promega) orpBluescript SK(+) (Stratagene). Plasmid DNA was se-quenced (32) using 2'-deoxyadenosine 5'-[a[35S]thio]triphos-phate and Sequenase (United States Biochemical). Occasion-ally the Taq polymerase sequencing system (Promega) wasemployed. For three clones (D2040, NW1, and NW3), nu-cleotide sequence determinations were derived from bothDNA strands by using a combination of random deletionsubcloning and synthetic oligonucleotide priming. Conve-niently located restriction sites (Fig. 1) were used to preparemost subclones. Additional subclones were prepared usingexonuclease III (33). Three other clones (27c, 30, and 31a)were sequenced for their full length, but only on one DNAstrand. The nucleotide sequences were assembled and ana-lyzed with the IBI/Pustell sequence-analysis software.

Northern Blot Analysis. Samples of poly(A)+ RNA (2 ,ug)from a variety of chicken tissues and cell lines were dena-tured, electrophoresed in a 1.3% agarose gel containing 0.2Mformaldehyde (34), and transferred to a nitrocellulose mem-brane (GeneScreenPlus, DuPont). The blot was hybridizedwith a 32P random-labeled EcoRI-Sac I cDNA fragment (297nt) from clone 27c and washed as described (35). The blot wasstripped and rehybridized to a mixture of cDNA probesencoding the chicken ,B-actin and erythrocyte band 3 proteinsin order to determine the integrity, size, and relative amountsof the mRNA samples. The band 3 hybridization signal wasused to check the degree of erythroid cell contamination ofthe various nonerythroid chicken tissues.Primer Extension Analysis. The 21-mer TG6 (5'-GAACTG-

GTACAGCTTCAGCAC-3'), complementary to nt 72-92 of

1 CGGCAACGGTGGAGCGC18 TACTGTGAGACGCAGGCTAAGGAGAATGCCTACGATCTGGAGGCCAACCTGGCTGTGCTGAAGCTGTACCAGTTC93 AACCCCNCCTTCTTCCAGACCACAGTGACGGCGCCAGATCCTGCTGAAGGCCCTCACCAACCTCGCCCCACACTGA

168 CTTCAGCCTTTGCAAGTGCATGATCGACCAGGCCACCAGCAGGACGGCCATCCGCCAGATCCTGTACCTGGGGGA243 GCTGCTGGAGACGTGCCACTTCCAGTCCTTCTGGCAAGCTCTGGATGAGAACATGGAGCTGTTGGATGGGATTGC319 TGGTTTTGAGGACTCTGTCCGAAAATTCATCTGCCACGTGGTGGGTATCACATACCAACACATCGACCGATGGCT393 GCTGGCTGAGATGCTGGGGGACCTCTCAGAGGCACAGCTGAAGGTGTGGATGAGCAAAT ATG GCT GGA CGG

M G P 3

464 GGA CCG GAC GGG ACC ATG GCC GMA GAG GTG GTG C7G GAG ACG TGC GAG CTG CAG GCC

G P G T M A E E L V L E T C L C 22

521 GAG CGC SAT GGC CGC GAG CAC CGG ACG GAG GAG A7G GIG AGC GAG GAG CTG GTG GTG

E N G E R T E E M G L V V 41

578 CGG CGC GGG GAG CCC TTC ACC ATC ACC CTG AAC TiC GCC GGA CGG GGC TAG GAG GAG

R R4 G P F T I T L N F A G R G Y4 E E 60635 GGG G7G GAG AMA CTC GCC TTC GAC GTG GAG ACG GGG CCC TGT CCC GTT GAG ACG TCA

G V KI L A F D V E C P V E T 79

692 GGC ACC AGG TCG CAC TTC ACC T75 ACC GAG TGC CCC GAG GAG GGG ACC TGG AGT GCA

G T R F T L T C P 7 A 98

749 GTG TTG GAG GAG GAG SAT GCC ACG CTC TCT GTA TCG CTC T14C TCC CCC AGC ATT

V L 0 0 G A T L C V L C P 117

806 GGC CGC CTG CCC CCC TAC CCC CTG ACG CTG GAG GCC TCC ACG GGG TAC CAG GGC TCCA R V G R Y L T L E A S T G Q G 136

863 AGC TTG GAG GTC GGG GAG TTG GTT CTG CTC TTC AAT GCC TGG GAG CCA GAG SAT GGA

F L G F V L L F N A W H P E A 155

92G GTG TAG CTG AAG GMAGA GAG GASA CGG CGT GAG TAG GTG CTG TGG GAG GAG GGG CTCV Y L K E E V L L 174

977 ATC TAG ATG GGC TCC ASS SAG TAG ATA AGC TCC AGA CCC TGC AAC GAG TTT

R I P W6 N F G Q F 193

:1034 GMA GAT SAG ATG TTG 14CC ATG TGC GTG GAG ATG TGT GAG ATC AAC CCC MAA TTC CTC

E L A C L E L N P K F L 202

1091 CCC GAG CGA AAC CTG SAC TGC TCC CGT CGC MAT GAG CCT TAT ATT CGC ASS G75

R D N L C S R R N P V G R V 231

1148 GTG AIG GCC ATG GTC AAC 7CC AAT GASGSACGAG CCCCTCGGGTCCTGCT CCC

V A V N C N V L L R W6 250

1205 SAG AAT GAG TAT CAG SAT CGC ATG ACC CCA ATC GCA TIC ATC CCC AGC GTG SAC ATEN H E G P M A W6 G S V 269

1262 AAG AGS TGG AGG ASS TTG GGC TGG GAG GTC AAG4A~::C4?>9l288

1319 TTC OCT ICC ITS GCG TGC ACT GTC ATC CCC 7CC CTG SIC GTG CCC...A'GC' "CIT "GT'G" GTCF A A V A C V C L V P R V V 3147

1376 ACG AAC TAG AAC TCT CCC GAG GAG ACC AACGGSC AAC CTG GTG ATC SAC CCC TAG CTGY N A H T N L V Y L 326

1433 ASG GAC ACG CCC ATC SAC SAGCGIG rGC 7CC AC GSAC ATS ATC TGG AAC TTC GAG TGCE R R M W N F H C 345

1490 TGC GTC CAG TIC ATG AGG GIG GCT SAG CTG CGA CGT SCA TACGSAT GGG TIC CAA

V C R P L A P G Y 364

1.547 GCG CTG GAG CCC AGO CCCCAGAGCASAM ASC CAA STT TAG TIC TCC CCG GCCCA L P P K V Y C C P A 383

1604 CC? OTT MSG GCC ATC AAG CAG CCC GAG CGT CGA GTG GAG TACGSAC ATC CCC TIC GTC

P V K A K L V Y I P F V 402

1661 GCG GAC CTC MAT CCT GACGCTG GTG TAG TOO ATC GTT CGA ACT GACGGST SAG MSGF A V N A D V V W V K 421

1718 MCG AAG ASC ACC GAG 7CC TEA GTG GTG SGCG AAGAC ATC AGC ACC AAC ACC CTC GCCK K H V V K N T K V 440

1775 AGG GAG AGC CGC GAG GAG ATC ACC GAG ACC TAG AAG TAT CCC SAG TCT SAG MCG

R H P K 459

1832 GAO CGA SAG GTG TTC AGC MSG GCG GAG GACGSAC AAG AGC 7CC CTC COT SAG CGA GAAV F K A H K1 L 478

1889 CAC CTC GAG ATG GIG ATC CTG 7CC CAC OCT CCC AAC MGC CCC 7CC SAG TTC

L H M R K L G A N N F 497

1946 SAC GTC TTC GCT 7TC ATC AGC AAC SAG ACG SAG AAC SAGC GT CAG 7CC CGTV F A F I N K R C L R 516

2003 CGC TCC GCC CCC 6CC 0CC 6CC TAG MAC SOC GAS GTC 14CC CCGC AGA TCC CCC TTC MAGL C A R A Y N V P C F K 535

2060 GAG CTG CGT AAC CTC 6CC CTC CAC CCC GAG ATG GMA CGA 6CC GTC CCC TTG CG1C ATC

L L L L P H 0 V P L R 554

2117 CTC TAT SAGCAGA TAG CCC AAC CTG 6CC GAG SAC MAC ATG ATC CTG GTG GCT

L E Y P N L Q D N M K V V 573

2174 CTC CTG ACT GAS TAT GAO ACT GAG 6CC GTC GTG GCC ATC G67 GTC TAG ATC

L L Y D V V A R V Y I 592

2231 CGA AAC CCA GAS ATC ATC AGG ATT GGG SAC CCA A7GCAGA SAC CCC AAG CTGN P K R L P 0 E R K L 611

2298 GTC GCA SAC ATC CTG GTO AAC CCA CGT GCA SAGCGCA CTC MAC AAC 7CC TTCV A I R L V N P L A P L N C F 630

2345 CTC GTC CMA GGC GCC GGC CTC ACT GAS GAS ASS ATC SAC SAGCGTT GAS SAT CCTV V A L R L P 649

2402 CTC SAC CCC CMA GCA GAG 0CC AAG TTC CGG ATG SAC TTC CTG CCC CCC GAG ICA

V P A K F R F V P R Q 666

2459 CGTCGAG MSG CGT ATG SAC GAS 6CC GAG CTG 6CC GGA GTG MCG ICC TAG

L H K L V F K L V K Y 687

2516 COG AAC GTC ATC ATC GCA CCC CTG CCC AAG TGA GGCCCCCCCGAGCCCCCACCCTGCTCCAGCCR N V A P L P K 697

2580 CTGGGCM7OGCTGCGAAACAAAGCCATAAGCCTTAGCCCMACCTGCACCMSCCGCATCCSACCCCGCACCTCCG2655 ACTAOCCCACTG4CCACCACAGCCCCTCTCCATCACTGCCACTCCCAGCCGGCCGGGGGACAGTGACMACTGTGGTAC2730 TGCAGAC9TGCCACCGGCTGAGCCATGCCTTC7CCTCCTCTCCCTCCCCATGGACCTCGACGCAIGCTCCAGCTCGO2805 GGCCCTC CTGCAGCASGCCAGCAGAMCCGTCOGTCATTTCTGCAGCTCTAAACACGCCGOCCCCGACGCAGAGCAC2680 9TAACGCAMACATAGAMAGACAATCTTCTCCTGCACCCGATGGCGCAGCAGCAGCACTCATGCCTGCCAG2955 TTAAAATGCTCTGAATGCAATTTCCTAGAGAAMCATCGTATACTGTGAGCACGMAGCTGTTTATATGCTATATA3030 CACATATAIGATAMATCTATTTATAGCTCTATMAATACATACTGCCGAGAACCCTGCTGCATAGGTMSGCAGGTT3105 67771GCTTTMTAACTGTTCCTGTCACCGCAGAGATCCTGCCCTGAGGAATGCGACATTGCCTTGATGCGAATTA3180 ACTATGCAAGCACGT ACCTCTATTTTTCACTTTTTAMAGCAAAAAMAAAAAAAAAAAA~k

FIG. 2. Nucleotide and deduced amino acid sequence of chicken

red blood cell transglutaminase cDNA. Sequences of the overlappingcDNA clones (Fig. 1) were determined by the dideoxynucleotide

technique (32). Nucleotide residues are shown in the 5' to 3'

orientation beginning at the 5' end of recombinant NW3 (Fig. 1). The

sequence reveals a single open reading frame of 2094 nt [698 amino

acids(a) flanked by 451 nt at the 5' end and by 700 nt at the 3' end.

The stop codon is indicated by an open circle. The consensus

polyadenylation signal AATAAA is located 26 nt 5' to the poly(A)tail. The pentapeptide sequence containing the active-site Cys is

shaded. Matching amino acid sequences of peptides isolated from

partial proteolytic digests of purified chicken red blood cell trans-

glutaminase (Materials and Methods) are underlined. A peptideisolated from the endoproteinase Lys-C digest of the protein (PN-

LHGPEILDVP), and three other peptides obtained in very low

yields, did not match the cDNA-derived sequence.

the chicken transglutaminase cDNA, was 32P-end-labeled bypolynucleotide kinase. RNA from MSB-1 lymphoid cells (36)or adult chicken (definitive) reticulocytes was hybridized to

EcoRI Kpnl Xhol

5.1 1 1 1

I kb

BamHl

I I I

9806 Biochemistry: Weraarchakul-Boonmark et al.

this oligonucleotide, and cDNA was synthesized by avianmyeloblastosis virus reverse transcriptase (37). To accu-rately assess the position of the mRNA cap site (the 5' end ofthe primary transcript), the primer extension products werecoelectrophoresed in aDNA sequencing gel directly adjacentto a "ladder" produced by dideoxy sequencing of clone NW3with the same primer.

RESULTS AND DISCUSSIONIsolation of Chicken Transglutaminase cDNA Clones. Im-

munoscreening with a rabbit antiserum raised against chickenerythrocyte transglutaminase allowed the initial identifica-tion of 20 clones in the BV4 cDNA library, and these wereplaque-purified. Three clones (27c, 30, and 31a) harboring thelargest fusion proteins (on Western blots) were further ana-lyzed and found to have overlapping nucleotide sequences(Fig. 1). Clone 27c contained an insert of 1704 nt, whereasclones 30 and 31a contained overlapping inserts of 270 and250 nt (data not shown). The 1704 nt of 27c represented asingle open reading frame followed by a stop codon and anuntranslated sequence of 678 nt at the 3' end. This clone wasconfirmed to be an authentic cDNA segment encodingchicken erythroid transglutaminase by matching to peptidesequences derived from a partial digest of the purified pro-tein.ADNA fragment (297 nt, EcoRP-Sac I; Fig. 1) correspond-

ing to the 5' end of clone 27c was then used to rescreen thesame library, resulting in the identification of four morepositive clones. One of these (D2040) contained 3446 nt withan open reading frame from nt 1219 to nt 2746 (Fig. 1). Thisclone encoded 509 aa, including the conserved pentapeptidearound the active-site Cys (38) for transglutaminase (aa283-287 in Fig. 2) as well as all the 3' sequence encodedwithin 27c. Eight peptide fragments from the purified enzyme[aa 189-199 (chymotrypsin digest), 250-270 (trypsin digest),391-409 (S. aureus V8 protease digest), 414-424 (chymo-trypsin digest), 438-456 (endoproteinase Lys-C digest), 465-480 (chymotrypsin digest), and 472-491 and 658-669 (en-doproteinase Lys-C digest) as indicated in Fig. 2] matchedwith segments of the conceptually translated nucleotidesequence. A sequence of 1218 nt at the 5' end of this clone(Fig. 1; dashed line on the map) was subsequently found torepresent an unspliced intron of 647 nt, as well as additional5' cDNA coding sequence of 571 nt.

After screening of the same library with a 54-base oligo-nucleotide (DNAsynl, Fig. 1), two additional clones wereobtained of which the longer (NW1) was partially sequenced(a total of 1250 nt; of these, 1244 nt matched the 5' end ofD2040, corresponding to aa 13-426 of Fig. 2). Unlike D2040,the recombinant NW1 contained no intron, but in conceptualtranslation NW1 was found to be only 6 nt longer than D2040.An EcoRI-Kpn I fragment (374 nt) of clone NW1 and

oligonucleotide TG1 (see Materials and Methods) were nextused as probes to screen a different (B21) cDNA library (29).Sixty independent recombinants were isolated, and 10 ofthese were further characterized by PCR. Three clones werefound to contain longer inserts than NW1; the longest one,NW3 (484 nt longer than NW1) was subcloned, and the 5' endwas sequenced (a total of 856 nt; of these, 372 nt matched the5' end of NW1, corresponding to aa 11-134 of Fig. 2). NW3contained the Met initiation codon preceded by an untrans-lated region of 451 nt.

Nucleotide Sequence of cDNA and the Deduced Amino AcidSequence. The nucleotide sequence of the cDNAs coding forchicken erythrocyte transglutaminase was constructed fromoverlapping sequences of cDNA clones D2040, NW1, andNW3 (Fig. 2). The compiled sequence contained 3245 nt witha single open reading frame beginning with an ATG initiationcodon (nt 452-454) and ending with a TGA stop codon (nt

2546-2548), predicting a coding sequence of 698 aa. Analysisof NW1 and NW3 provided further results matching onepeptide sequence (aa 17-30) isolated from the V8 proteasedigests of purified chicken erythrocyte transglutaminase.Two ATG triplets were found, at nt 452-454 and 479-481.

According to Kozak's rule (39), either of these could serve asan initiation codon; the latter exactly fits the consensus(ACCATGG) as the translation initiating site, while theformer is divergent (AATATGG). However, the location ofa purine residue at position -2 has been shown to have a<2-fold effect on translational efficiency (39). The ATG at nt452-454 might be suggested to be the initiation site becauseit would represent the first Met codon following severalin-frame termination codons. The translated sequence, end-ing in TGA (nt 2546-2548), was followed by 697 nt of the 3'noncoding region, including a 3' poly(A) sequence. Theputative polyadenylylation signal AATAAA was found 26 nt5' to the poly(A) tail.To locate the mRNA initiation site, primer extension was

carried out using total RNA isolated from chicken reticulo-cytes and a synthetic DNA oligonucleotide (TG6) comple-mentary to nt 72-92 of the chicken erythroid transglutamin-ase cDNA clone (Fig. 1). Two discrete products, 192 and 196nt, were obtained in the reverse transcriptase reaction (Fig.3). Therefore, the cap site is located 100-104 nt 5' to thecloned cDNA sequence, indicating a total length of 3345 and3349 nt for the mRNA.The deduced amino acid sequence of chicken erythrocyte

transglutaminase corresponds to a protein of 697 aa com-posed of 324 nonpolar amino acids (Ala36Gly56Ile3OLeu58-Met18Phe23Pro35Trp12Val56), 179 polar amino acids(Asn29Cys21Gln3oSer4lThr34Tyr24), 108 acidic amino acids(Asp43Glu65), and 86 basic amino acids (Arg^4His14Lys28)with a calculated pI of 4.7 and a molecular weight of 78,621.This value agrees well with the molecular weight of 78,000estimated by SDS/PAGE ofthe purified protein (40). The Glyresidue following Met at the initiation site is thought torepresent the N terminus of the protein, perhaps in anacetylated form. In eukaryotic proteins, removal of the first

nt 12 3 4 5 6

192 tit -

179

126

65

4

-

an-

in-Im

=n

FIG. 3. Primer extensionanalysis of chicken erythrocytetransglutaminase mRNA. Anoligonucleotide primer (TG6; 5'-GAACTGGTACAGCTTCAG-CAC-3', Fig. 1) complementaryto nt 72-92 of the cDNA se-quence (Fig. 2) was end-labeledusing polynucleotide kinase. To-tal RNA from the MSB-1 T-cellline (lane 1) or from adultchicken red blood cells (lane 2)was hybridized with this oligo-nucleotide, and cDNA was syn-thesized using avian myeloblas-tosis virus reverse transcriptase(37). The resulting productswere fractionated by electropho-resis in a 6% polyacrylamide/urea sequencing gel in parallelwith restriction fragment sizemarkers. Lanes 3-6 (G, A, T,and C, respectively) show a par-allel sequencing ladder of cloneNW3 for the same TG6 primer.

Proc. Natl. Acad. Sci. USA 89 (1992)

Proc. Natl. Acad. Sci. USA 89 (1992) 9807

Ckrbctg~~~~~~~WGG..DTMAE-liTpLQCR---R E2RTEZMGSQ QLWV-RRQW2F- TI LNF--AG R8GYEEGVDXKL AFDVET-GPCP VET0SGT.RS8F TLTDCE GT 95

Gpigltg-----MA EkDLI'LERC-DL 0LkVN---R DMRADhLCRE RL.V.LRPO8QPF' WLTLHF--EG I9GYEAGV.DTOL TFNAVT.GPDP' SEEAGT84AR SLSSAVEGGT 86

Humentg NYI2ASV82SL T 8'S WTGPAP SQEACIThAP Pt0&GAV E? 86

Mumactg -----MA FLLLRGL E0QANG---R DHHT-ADLCQE Kt7V.811 RLTLYF~--EG I6GYQASVDSL T8'GAVTG PD S8ZAG9KA~r SLSDNVEEGS 86

Hemnp4.2 -----MG QAI.GIKSGDF QAARMHN---E Z.HHTKALSSR RLFVI8-GP9. 01 YFRAPV RAFLPALKKV ALTAQTGEQP SKINRTQOATF PIrSSLGDRKW 88

Hfx~iia PRGVNLQEFL NVTSVHLFkE RWDTNK---V DHHTDKYENN KLIVHG SF YVQIDT S RPYDPRRDLF RVEYVI1GRYP QENxrtyipv PIVSELQSQK 129

Ratketg NAAGDGTIRE GML-VVNGVDL LCSRS0QNRR E1HHTDEFEYD EELIT PAGQP 81L--TLHumketg NAAlGDGTIRE GMLVVNGVDL LSSRSDQNRA E88FTDEYEYD ELIVRRGO0PF HMLL-L--LS RTYESS-DRI1 TLELLIGNNP: EVGKGTHVII PVGK-GGSGG 192

Ckrbctg MSAVLQQQDG ATLCVSLCSP SIARGRYARL :TLEASTGY- -QGS.SFHL-- -GDFVLLF NAWHPE-DA-V LKEEDERREY VLSQQGLI.. YM GSRDYITSTP 187

Gpigltg 86Sa.SAVDOQD STVSLL-LSTP ADAPIGLYL SI&LF -W..Ssr L---GHFILLY U4PRCPAOGA.VX MDSDQERQEY VGEOOGEI.FYQ t; AKFIDNGIP 178

Humnentg MTA77VVDQOD CLSLQLTT.P ANAPIGLY~ SLER$GY-- -QGS$9.VL --GHFILLF M.MI'GV DSE9QY V8X'IQW !YF.Q G F$AHFXMIP 178Mumnactg W SSVLDQQD AN"PIGiLY St AAS GV8 -GGNS9'VL 8ACADDV-Y.- L0SSEStI8RR V-1,T (XW O$VKFI5K5VPHemnp4.2 WSA11"VVEERD0A QSWTIS TTP` ADAVIGHMS LLQVSGRK(--0--LL-L--S--QFTLLF _8W~4AF LXNYAPE

Hfxiiia WGAX(IVMRED RSVRLSIQSS PKCIVGEKFR''M YVAVW¶DPYGV LRTSRNPE----TDTYILF. 8PWCEDDAVY LONEKEREEY V.L"NDIGVIFY GEVNDIKTRS 224

Ratketg WKAOVTKTNG HNGETLRVHTS PNAIItEFQF 1TVRTRS--- EAGEFO18F 0PRNE0YIL?. NPWCPM~3IV''Y' VDHE WROEYX VLNESCRIYY. GTEAQIGERT 295

Hureketg !MAQVVKASG QNL-NLRVHTS PNAIIGKFQF TVRTQS--- DAGEv-t2PF 2.PRNEIYIL'F N'PWCPE''IVY VDHFDWRkQSY VLNESGR 1Y''Y G'TEAQIGERT 287

Ckr-bctg WN8'GfZDET' LAIC.LEMWDI NPKP'LI9RQNL DCSRRNDPV/0 IGRVV.SAMVNGE8 GFkRM02IPHYE DG MSPMAWIGSV DI-LKIMWRRLG Y8(G.QCW

GpiglIg W-NFGG8 'E0XI XD[LMLWDT NP8(F XNAGQ 'DCSMRSRP.VY VG88WVS MVM OND-OOGV*DRL. D. YSDG VS0PMSWIGSV 1D0LR18WDYr CORViIYGOGH 277

Humnentg MMFGO8'QDG0- LDICLILLDV NPKF-LKNAGR 'DCSRASSPV.Y VGRVGSGMVN CND2-DQGVIL GRWMONNY.GDG '~SPFMSWlGS.V DILRRH8PNHO CQRVYGOG 277

Mumactg WWMFGGMQDGI LOT(LMLL4M NPKFLENRSR DCSR.SSPI Y VWWMVVSDMVN CN]2-DQGVLEL GRWI2NNYGDG. ISPMAH8IGSV D1)IPRWKE G- COQVyaQCW 277

D8GV'E8GDV IDLSLRLLS----KDQV E--KWSQP.Vf VARkVLGAL LH FLKEQR-VLP TPQTQATQEG ALLNKRRGSV P11 Q86LTGR GRPV-YDG;QAW.268ix...a ....F8DG. ----YH RAQ-M DLSGRGNPIK VSRVGSAMVN AKD2DE-GVLV G-S88JNIYAYG VPPSAWT~sV V1LYSE-FRGC 315

Ratketg 8INYG.QFDHGV LDAiLYI-Lb-----RRGM PYGGRGD.PVS VSRVWSAM¶JN SL'D-DNGV.LI GNMTGDY.SRG TNPSAW.VGV ETl1S G Y-SVPX.GQCM 3886

Humketg MNYGOPDHGV LDAfLYILD----- RRCM PYGGRGDP'VN VSRV.ISAMVN SLD-DNGVLI GNWSGDYSRG TNPSAWVGSV EILLSYLRTG' Y-SV-PYGQCMW 378

WAMVAGEVM AC SVPSRW1-. TY.. AHD. NL.VZGR .L.S ETG-ME-ZRR OHMYC87R G.--WW Qn PYT OG KSEG V'4Z

Gp glgtg VFAAVAG SG-EI-E1GN K-SEM1W F-H SLLGG-VVDD QAGFG-AMRG~ V% DHP ES8ITYCCGP 373

Humnentg VMA"VCMLV RCXGIPTRVV TH8YWZHbA8QH SNL-LIEYFRN CFG-EI-QGV K-SEM1INMFH '.''.S.M --YE(; WQAD YPQi RES82TYCCGP, 372

Mumactg VEFAAVAGTV RCLGIPTRVV T YMSAAIDQH SNLLIEYFRN EFdG-EL-ETN K-S CIM "W".'ME""S"WM"TA-"F' DLP-YGEO2WTQHemnp4.2 VL6AMACIV., RGLGIPARVV TTFASAQGTG GRLLIDEYYN EEG-LQ-NGE GQRGR188IFO TSTECWT(R-P ALPQG--YDG

Hfxiiia VFAGVFNTFL RCLGIPARIV TN.YFSAMDND ANLOMDIFLE EDGNVN-SKL T-KOSVMNYH CWNEAWMYRP. DLPVG.--FGG WQ.AVD.STPQE NDMCGP

Ratketg VPAGVTTTVL RCLG~LATRTV TNFNSAHDYD TSLTMID7YFT ENM4-KPLEHL N-HIDSVWNFS8 VMNDCWM14RF DLPSQ--FDG- WoVVDATP TSICGP48

Humketg VFAGVTTTVL RMCLGLATRTV TNFNSARDY.D TSLTMDIYS'D ENM-KPLEHL N-H8DSVWNFH V882DCH8.K(P DLPSG--FDG WQVVDATPQE TSSG.IFCCGP 474

Ckrbctg APV3AJ9QI LQVQYDIPF.V FAEVH-.'VVY WIVQSDGEK- KKSTHSS-W GKNIS.TKSVG RDSRW I1H8T YMMPGSENE R9VYSKAM---H---K 471

Gpigltg VVR.A13(RGH LNVK4YDAPFV MAVNDVVN WIQRQI4DGSL- RKSINHL-W GLK1ISTMSVG DEk2YrOP8GELRAFVRAN-- HLNKLA--T- 4686H~urneng VPD'V-RA- n.PG LSTKYDAWV FAEVNAGDV-VD WcDG'SV- H~S-INRsLIV- G-LKISMIV RDREZITl Y?YPEG.SSEE RE".TRAN--- H----L--N- 462

Mumactg VSVRA194EXGI) LSTK4YDAPFV FAEVNADWVD WIORDEGSV- LKMMNRSLIV.. GQKISTM.SVG RDDR8IDITH7Y YKYPEGSPEE REVE.TEAP-- 8----L--N- 462

Hemp4.2 VPVRAVKEGST VGLTPAVSDL EA-AINASCVV MWKCCEDG.TL- ELTDSNTKYV. GNN7STEG VG SDR8CLDlD..QN YXY PFG.SLQ X9F2LERVE-- K E- KM 455

Hfxiiia ASV.QAI3GHGH VCFQFDAPFV PAEVN SGLIY ITAKKDG.T9V VENVDAT-HI GKIVTRQIG GDGMMD1TDT YIFFQECQEKE, RLALETALMY G----A--k- 503

Ratket-g CSVES1LENGL VYMK%. TPPI- PAE-SDKV .QRQDDGSF- K.IVYVEEKAI OTLIVTI(AIN SNMA) ,I EI Y MHP8IGSEAZ RKIAVEKAR -- A----HGSK- 574

Humketq CSVESI'KNGL VYMKYDTPP:I EAEVVI4S'GR'V'Y' WGRQDDGSF- KIVYVEEKAI OTLOVt14AIS SNMMRED&YL tRHPEGSDAA RKAVETA-A-- A----HGSK- 566

Ckrbctg------ SSL-GEQEEg LEMRI0HL-S---EGASONG 51FFVFAFIS NDTDKEHER LRLCARTASY NGEVGPQCGF -KDL-LNLLSL QL>HMEQSVPL, 552

Gpigltq ------KEE-AQEEIIG VA98R0IV-G----QNMTMG SDFDIFA-YIT NGTAESH8ICQ LLLCARIVSY NG.VLGPVCST -NDL-LNLTL DPFSENSITPL 547

Humnentg ------KLA-EKEETG MAMRIRV-G---QSMNNG SDFDVFAHIT NNTAEEYVCR LLLCRRTVSY NGILGRECGT -KYL-LN'LTTI EPFSEKSVPL 543

Mumactg ------KLA-EKEtTG VAMR0MRV-G --Q-YEHG NDFDVFAHIG NKDTSETRECR LLLCkART-VS.Y iGVLGPiErGT -ED--INd'LYL DpySkNS'iPL 541

Hemnp4.2 EREKDNG-IR PPS-LETASP rYLLLkA--P -SSLPLR GDAQISVTLV WHSEQEEKAVQ LAIGVQAVHY NGVLAAK4-LW -RKK-LAHLTL SANLEKIITI 544

Hfxliia ------KPL-'-NTEGV MKSR.SNV-DM DFEVENAVLG Kt EKLSITFR RNSHNRYTIT AYLSAN4TFYT---VP AEF KKET-FDVTL ELSKEA58

Ratketg------ PNVYATRDSA EDVAMQVEA --QDAVM6G QDLTV.SVVLT kMRGSSRARTVK L6.LYLCVTYY TGVSGP--TF -1M.TKKEVVt; APGASDTVAM4 656

Humketg ------PNVYANRGSA EDVAM4QVEA---QDAVMG QDLMVsVmLI N.HSSSRRTVK LHLYLSVTFY TGVSGTI--F --RETKKEVEL APGASDRV.TM 648

Ckrbctg RIL'YEQGM. L.T-QDNMIX0CV ALLThEYM'D SW-AIRM3-Y 0QNPRE8(IRI LGEPMQEREL. VAEIRLVNPL AEPINNMCDFV V6IGAGLTEGO RIEELEDEVE 651

Gp-lgitg HO:LTEKY4GDY L-TESNLIKVR GLLIE-PAANS YVLAERDI-Y LENP 51 M RV LGEPKQN4R8L. IAtVSLKNPL, PVPLLGCr-FT VEFGAG-LYKDQ. KSVEMVPDPVE 646

Hurnentg CI-LYEKYRDC LTkESNL OKVR AL.LVE.PVINS YLLA-ERD.L-Y TENPYE.-iRl LGEP-XOXRKL VAEVSLQMPL PVALEGCTFT VE-GAGOIOEE' 'KTVE-IPDPV.E 642

Mumactg RILYREKSSC LtESMLIKV GLLTEPAANS YLLAERDL-Y VENPE0H7RV LGEP3ONXL. VArEVSLKNPLt SDPLYDCIFT2 VE'G'A' LOTKEQ' KSVEVSD VP 640

He.np4 .2 GLFFSNFERH8 PPENTFLRLT AMAIHSFESNL SCFAQEDI-A rCRPHLAT:KM PEKAEOYQPI. TASVSLQNSL DAPMEDCVIS 11LGRGLIHRE 'SYRF-RSVW4 642

HfX4iia LDQAGEYO4GQ LLEQASLHFF VTARIMETP6D VLAI(QKST-V LTI EI TKV RGTQVVGSDM TVTVQFTNPL KETLRNVMVH LDGPGVT--R PMARKWP'EIR 684

Ratketg PVASO4EY'OPH LVDQGAM-LL NVSGHVKESG QVLARQHTFR LRTDLSLTL LGAAVVGQEC EVQIVFKN L. PITLTNVVFR L EGSGI-QRP KVLN`VG!D-IG 753

Humketg PVAS34EYRS'.H LVDQGAM-LL NVSGHVKESG QVLAJ4QHTFR LRTPDLSLTL L-GAAVVGQEC EVQI0VFKNPIL PVTLTNVVFR LEGSG.L-QRP KILNVGD,-IG 745

Ckrbctg PQAEAO4FRME FVPRQAGLHK LMVDF-ESHL~TGVKGY.RWNVI 1AP'LPK 687

Gpigltg AGEQAI(VR:VD LLPTEVGLHK MVVNECDIM4 KAvXGTRwNVT MMPA-- 6890Hurnentg AGEEVKV8MD LWFLHMdtONK'- L'VV'N?.E''SOXML: KAVKGFRNVI-1 VGPA-- 686

Mumactg AGDLVOARVD LSPTDIG-L K LWNKQC D KSSGRHV0

Hemp4.2 PENTMCAO4FQ FT#THVGLQR LTVEVDCNMF QNLTNYKSVT VVAPEL 688

Hfxiiia RNSTVQWEEV CRPMWVSG8HIX LIASMOS$ RHV-YGELDVQ IGQRRPS 730

Ratketg GNETVTLRQT FVPVRPGPRQ LIASLDSPOL~SQVHGVIQVD VAPSSG 799

Humketg GNETVTLH42S FVPVRPGPRQ LIASLDSPQL: SQ.VHGVIQVD VASPAFG 781

Met is often accompanied by acetylation of the adjacent Ser,Ala, or Gly residue, which becomes the N terminus of themature protein (41). Our failure to directly sequence thechicken erythroid transglutaminase by Edman degradation(42) also indicates that its N terminus may be blocked. TheC terminus ofthe enzyme is designated as Lys697, because theLys codon is followed by a TGA stop codon (Fig. 2). Thededuced amino acid sequence reveals five potential Asn-linked glycosylation sites at positions 433, 493, 505, 539, and562.The avian protein displays >60o identity with three re-

lated mammalian enzymes [60.2% with guinea pig livertransglutaminase (43); 64% with human endothelial cell trans-glutaminase (44), 62.9% with mouse macrophage transgluta-minase (44); Fig. 4], which, considering the evolutionarydistance of 140 million years (49) from bird to man, is ratherstriking. The midsections of these proteins, which comprisethe conserved active center (aa 283-287) and the putativeCa2+-binding (43, 47) regions (aa 455-462 and 476-480),contain the sequences of highest identities. Interestingly, theerythrocyte transglutaminase sequence shows only 36.1%identity with subunit a of human placental factor XIII (47)and 33.6% identity with the human erythrocyte membraneprotein 4.2 (45, 46). Residues 9-20 (MAEELVLETCDL) atthe N terminus of the chicken erythrocyte transglutaminaseshowed remarkable identities with a similar stretch of resi-dues in the human endothelial (91.7%), mouse macrophage(83.3%), and guinea pig liver (75%) transglutaminases. Theseare thought to represent the class of tissue transglutaminasesthat can bind to fibronectin with high affinity (50), and it is

FIG. 4. Comparison of pro-tein sequences for several trans-glutaminases and related pro-teins. Deduced amino acid se-quences for chicken red bloodcell transglutaminase (Ckrbctg),guinea pig liver transglutamin-ase (Gpigltg; ref. 43), human en-dothelial cell transglutaminase(Humentg; ref. 44), mouse mac-rophage transglutaminase (Mu-mactg; ref. 44), human erythro-cyte membrane protein 4.2(Hemp4.2; refs. 45 and 46), hu-man placental factor XIII sub-unit a (Hfxiiia; ref. 47), rat ke-ratinocyte transglutaminase(Ratketg; ref. 48), and humankeratinocyte transglutaminase(Humketg; ref. 48) are shown.Amino acids are numbered be-ginning with the first amino acidfollowing the initiator Met resi-due. Gaps, indicated by hy-phens, were introduced into thealignment to achieve maximumhomology by using the GAP pro-gram from the Genetics Com-puter Group (GCG) sequence-analysis software package. Iden-tical amino acid residues areshaded. Sequence data for Gpig-ltg, Humentg, Mumactg,Hemp4.2, Hfxiiia, Ratketg, andHumketg were obtained fromthe GenBank/EMBL data base.

suggested that the above sequence of transglutaminase mightbe involved in this important interaction.

Expression of Chicken Erythrocyte Transglutaminase.Northern blot hybridization with the EcoRI-Sac I fragmentof clone 27c (297 nt; Fig. 1) was used to examine theexpression of chicken erythrocyte transglutaminase mRNAin various cell types. The results (Fig. 5) reveal a high degreeof tissue specificity, showing that appreciable amounts of themRNA are present only in adult chicken erythrocytes, adultkidney, and adult cardiac muscle. Expression was much lessabundant in embryonic liver, adult liver, and chicken em-bryonic fibroblasts. Expression was very low in the retrovi-rally transformed HD6 erythroid progenitor cell line, in theMSB-1 T-lymphoid cell line and in embryonic skeletal mus-cle. Actual transglutaminase enzyme activity (40) in thelysates of adult chicken erythrocytes was about 10 timesgreater than that found in HD6 cells. Also, the level ofexpression of transglutaminase mRNA in embryonic (prim-itive) reticulocytes was far less than that seen in adult(definitive) reticulocytes. These findings suggest that theexpression of transglutaminase mRNA is regulated duringerythroid cell differentiation and development at a step priorto translation.

Both N.W.-B. and J.-M.J. are considered as first authors. We areespecially grateful to Dr. M. M. Miller (Department of MolecularBiochemistry and Division of Immunology, Beckman ResearchInstitute, City of Hope Medical Center, Duarte, CA), for the B21AZAP cDNA library. It is a pleasure to thank Drs. M. W. Leonardand M. Yamamoto for their advice. This work was aided by a PublicHealth Service Research Career Award (HL03512) and a ProgramProject Grant (HL45168).

Biochemistry: Weraarchakul-Boonmark et al.

9808 Biochemistry: Weraarchakul-Boonmark et a!. Po.Nt.Aa.Si S 9(92

RBC LIVER MUSCLE

cY>

~~~~~~~~4.3kb

-2.0 kb

FIG. 5. Northern blot analysis for expression of chicken red

blood cell transglutaminase mRNA. Poly(A)+ RNA (2 l.tg) was

isolated from several chicken cell lines and tissues at different stages

of development. The RNAs, from adult (AD) chicken red blood cells

(RBC), the HD6 erythroid progenitor cell line, embryonic (EMB)

chicken red blood cells, MSB T-lymphoid cell line (T CELL),

embryonic chicken brain (BRAIN), embryonic chicken liver, adult

chicken liver, adult chicken kidney (KID), adult chicken cardiac

muscle (CARD), embryonic chicken skeletal muscle (SKEL), and

chicken embryo fibroblasts (CEF), were electrophoresed in a 1.3%

agarose gel containing formaldehyde (34) and transferred to Gene-

Screen nylon membrane (DuPont/NEN). The blot was hybridized

with a random-labeled EcoRI-Sac 1 (297 nt) cDNA fragment probe

from clone 27c (see Fig. 1). The tissue or cell line mRNAs showed

only minimal (<5%) contamination with erythrocyte-related material

(band 3), and the amounts ofmRNA applied to each lane werejudged

to be nearly identical by hybridization with a chicken P-actin cDNAprobe (data not shown). Size markers shown at right correspond to

the position of f3-actin (2 kb; ref. 51) and erythrocyte band 3 (4.3 kb;

ref. 52) mRNAs. Exposure time was 48 hr. The size of the mRNA for

transglutaminase was estimated from the Northern blot to be -3.4

kb, in good agreement with the total length of 3345/3349 nt obtained

from the cDNA analysis (Figs. 2 and 3).

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