Vittorio Gentile$, Margaret Saydak$, E. Antonio ChioccaS, Olanike AkandeS, Paul J. ..... Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman,.
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 hy The American Society for Biochemistry and Molecular Biology, Inc
Vol 266, No. 1, Issue of January 5 , pp. 478-483,1991 Printed in U.S.A.
Isolation and Characterizationof cDNA Clones to Mouse Macrophage and Human Endothelial Cell Tissue Transglutaminases* (Received for publication, April 30, 1990)
Vittorio Gentile$, Margaret Saydak$, E. Antonio ChioccaS, Olanike AkandeS, Paul J. BirckbichlerQ, Kyung N. Lees, Joseph P. SteinSV, andPeter J. A. Davies$(( From the $Departments of Pharmacology and Medicine, University of Teras Medical School, Houston, Teras 77225 and §The SamuelRoberts Noble Foundation, Ardmore, Oklahoma 73402
The deduced amino acid sequences for tissue transglutaminases from human endothelial cells and mouse macrophages have been derived from cloned cDNAs. Northern blot analysis of bothtissue transglutaminases shows a message size of approximately 3.6-3.7 kilobases. The molecular weights calculated from the deduced amino acid sequences were 77,253 for human endothelial tissue transglutaminase and 76,699 for mouse macrophage tissue transglutaminase. The deduced amino acid sequence for the human endothelial transglutaminase was confirmed by comparison with the amino acid sequence obtained by cyanogen bromide digestion of the human erythrocyte transglutaminase. The amino acid sequences of both human endothelial and mouse macrophagetissue transglutaminases were compared to other transglutaminases. A very high degree of homology was found between human endothelial, mouse macrophage, and guinea pig liver tissue transglutaminase (>80%).Moreover, human endothelial tissue transglutaminase was compared with human Factor XIIIa and a very high degree of homology(75% identity) was found in the active siteregion.
is distinct from Factor XIII. Recently a second intracellular transglutaminase has been cloned from rabbit epithelial cells and hasbeen shown to be distinct from both Factor XI11 and tissue transglutaminase (6). We have been interested in the transglutaminase activity regulatedby retinoids (7). Retinoic acid has beenshown to inducehigh levels of transglutaminase activity inmyeloid (8,9) and endothelial cells (10). To extend our knowledge of the relationship among the cellular transglutaminases, we have isolatedcDNA’s encoding tissue transglutaminase incells with high constitutive expression (human umbilical veinendothelial cells) and incells showing amarked inductionafterstimulation by retinoids(mouse macrophages). We have then compared the deduced amino acid sequences of these enzymes with guinea pig livertissue transglutaminase and transglutaminasesfrom other species. High homology was found when we compared the enzymes from human endothelial and mouse macrophage cells with guinea pig liver tissue transglutaminase. A lower degree of homology was found when we compared human endothelial and mouse macrophage amino acid sequences (32 and 36%, respectively) with rabbit tracheal epithelial cell transglutaminase. EXPERIMENTAL PROCEDURES AND RESULTS’
DISCUSSION
Transglutaminases (E.C. 2.3.2.13) are calcium-dependent enzymes that catalyze the cross-linking of proteins by promoting the formation of isopeptide bonds between proteinboundglutamineand lysineresidues. These enzymes also catalyze the conjugation of polyamines to proteins. Interest inthe possible physiological role of proteincross-linking reactions in both normal and pathological processes has led to the identification of transglutaminase activity in many cells and tissues (1). However, the relationship among the enzymes responsible for these activities has remainedlargely a matter of conjecture. The best characterized transglutaminase, plasma transglutaminase(FactorXIII)(2),hasbeen purified, cloned, and sequenced (3-4). Ikura and colleagues ( 5 ) have cloned the transglutaminase from guinea pig liver and have shown that this enzyme (tissue transglutaminase)
Cloning of Tissue Transglutaminases-We have been interested in the factors that regulate theexpression of transglutaminases incells and tissues. In order to pursue this question we have cloned tissue transglutaminase frommouse and human cells. In the case of the mouse tissue transglutaminase, screening of mouse macrophage and heart libraries resulted in the isolation of several overlapping clones. One of them (mouse clone TGHZ3) included 29 nucleotides of 5”untranslated sequence and 1775 nucleotides of codingsequence. Screening of a human endothelial cell cDNA library, on the other hand, resulted in the isolation of a cDNA clone that included the entire 5”untranslated sequence (as determined by primer extension analysis), the coding domain, and 1058 nucleotides of 3”untranslated sequence. This clone lacked a consensus polyadenylation sequence and is slightly shorter * This work was supported by Research Grants DK27078 (to P. J. than the 3,6 kilobases full-length transcript (as determined A. D.) and CA41829 (to J. P. S.) from the National Institutes of by Northern blot analysis of human endothelial cell RNA) Health. The costsof publication of this article were defrayed in part suggesting that it lacks approximately 300 base pairs of 3’by the paymentof page charges. This article must therefore be hereby untranslated sequence. marked “advertisement” in accordance with 18 U.S.C. Section 1734 We confirmed the identity of the human tissue transglutasolely to indicate this fact. The nucleotide sequence(s) reported in thispaper has been submitted minase clone by in vitro translation. The clone encoded a
to theGenBankTM/EMBLDataBank with accession number(s) M55153andM55154. 1 Current address: Dept. of Pharmacology, State Universityof New York, Syracuse, NY 13210. 11 Established Investigator for the American Heart Association. To whom the correspondence should be addressed.
Portions of thispaper(including“Experimental Procedures,” “Results,” andFigs. 1-7) are presented in miniprintat the endof this paper. Miniprint iseasily read with the aidof a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that isavailable from Waverly Press.
478
Mouse Transglutaminases and Tissue Human
479
suggested that these enzymes are likely to contain highly polypeptide that comigrated withauthentichumantissue transglutaminase and reacted witha monoclonal antibody t o conserved functional domains. The cysteineresidue a t codon the guinea pig liver transglutaminase. The apparentmolecular 277 of the human aminoacid sequence is in the active siteof the enzyme. The high degree of homology in this region of weight of the translated product, M, 85,000 (based on its mobility in sodium dodecyl sulfate-polyacrylamide gel electro- the human,mouse, and guinea pig enzymes (49 of 51 residues phoresis), is higher than its deduced molecular weight (Mr are identical between the three enzymes in this region) sug77,253) indicating that human tissue transglutaminase migests that the active site of these three enzymes has been grates anomalously on sodium dodecyl sulfate gels (17). The stringently conserved. A second functional feature of these translated product also catalyzed the calcium-dependent co- enzymes is their activation by Ca2+. Ikura etal. (5) suggested valent conjugation of putrescine to dimethylcasein indicating that theregion between codons446 and 453 showed properties that the clone encoded a catalytically active enzyme. These associated with a Ca2'-binding pocket. This region is also studies indicate that unlike Factor XI11 post-translational highly conserved in the human and mouse enzymes increasing modification of tissue transglutaminase is not a prerequisite the likelihood thatthis region isinfactthatfunctional for enzymaticactivity. calcium-bindingdomain.Recentstudies (28, 29) have reComparison of Tissue Transglutaminase Sequences-The ported that tissue transglutaminases are GTP-binding profirst tissue transglutaminase to beextensively characterized teins that show GTPaseactivityand whose cross-linking was from the guinea pig liver. This enzyme was first purified activity can be inhibited by GTP. However, sequence comby Folk and Cole in 1966 (18)and was cloned and sequenced parison between the three translgutaminases and GTP-bindby Ikura etal. in 1988 (5).We havecloned tissue transglutam- ing proteins shows no elementsof the consensus GTP-binding inases from human endothelial cells and mouse macrophages. site (30). The molecular basis for this GTP-binding activity Comparison of the deduced amino acid sequences of the three remains to be established. enzymes gives some indication of the degree of species and Comparison with Other Transglutaminases-The preceding tissue homologies of this class of enzymes. It is clear from analysis indicates that there is high homology between the this comparison that the amino acid sequences of the tissue differenttissuetransglutaminases.We were interestedin transglutaminases arehighly conserved. The overall degreeof whether this homology extended to other transglutaminases. the homology between the human and mouse enzymesis The cloning of the human endothelial tissue transglutaminase greater than84%, between human andguinea pig the homol- allows the direct sequence comparison of two human transogy is 81%.Most of the differences in thenucleotide sequences glutaminases, tissue transglutaminase and subunit a of the are attributable tosingle nucleotide changes, the majorityof human Factor XI11 (3,4). Theoverall homology between the which are silent in terms of the amino acid sequence. There two enzymes is not very high, sequence comparison at the are some substantial differences in regions of the human and amino acid level yielded a homology value of 41%. However, guinea pig enzymes.Betweennucleotides 1114-1203 and there were regions of marked homology interspersed with 1008-1097 of the human and mousenucleotidesequences, regions where the two polypeptides were completely different. respectively, the deduced amino acid sequences are identical, Fig. 9 shows the regions of tissue transglutaminase homolohowever, the insertion of a cytosine nucleotide at position gous to the subunit a of Factor XIII. There is an extended 1077 in the guinea pig sequence introduces a shift in reading zone of high homology (75%) that includes the active site of frame that results in 15 amino acid residues quite different the two proteins. In addition, the first of the three putative Deletion of a calcium-binding domains of subunit a of Factor XI11 (amino from the human or mouse sequence (Fig. a). guanosine nucleotide in the guinea pig sequence terminates acids 468-479) is highly conserved in human tissue transgluthe reading frame shift and results in re-establishment of taminase.Thisisthesame region that was foundtobe homology between the three species. A second major differextensively conservedin thecomparison of the human,mouse, ence is found in the second putative calcium-binding domain and guinea pig tissue transglutaminases. Takahashi and his of the guinea pig transglutaminase. There appears to have colleagues (4) have suggested that the first calcium-binding been the insertion of a 12-nucleotide segment in the guinea domain of the subunita of human Factor XI11 includes a loop pig enzyme that was not found in either the human mouse or enzymes (Fig. 8B). Ikura has suggested that this region may flanked by 10residues of 0 sheet structure on the aminocontribute to the calcium-binding properties of the guinea pig terminal side and 16residues of a-helix on thecarboxyl side. The same secondary structure was found when we analyzed enzyme. The human and mouseenzymes have comparable the amino acid sequence of this region of the human tissue calcium activation kinetics but lack this segment making it unlikely that this second Ca2+-binding domain contributes to transglutaminase. These similaritiessuggest the conservation of structural homology between the different transglutamithe functional propertiesof the enzyme. The kinetic properties of the tissue transglutaminaseshave nases (31). Recently, Floyd and Jetten (6) have reported the partial nucleotide sequence of a third type of transglutaminase, a human 3 1 9 EFGEIQGDKSEMIWNFH-CWESWMTRPDLQPGYEGWQA 356 transglutaminase isolatedfrom rabbit tracheal epithelial cells. A g.pig 319 ESGEIEGNKSEMIWNFHSLLGGVVDDQAGPGAWVRGVQA 351 mouse 3 1 9 EFGELETNKSEMIWNFH-CWESWMTRPDLQPGYEGWQA 356 Comparison of the aminoacid sequence of this rabbit epithe*l**IIII********* IIIIIIIIIIIIIIIII*I** lial enzyme with human tissue transglutaminase shows some human 464 KLAEKEE----TGMAMRIRVGQSMNMGSDFDVFAHITNNTAEEYVCR
506 g.pig464 KLATKEEAQEETGVMARIRVGQNMTMGSDFDIFAYITNGTAESHECQ 5 1 0 mouse 464 KLAEKEE----TGVAMRIRVGQ-YEHGNDFDVFAHIGNDTSETRECR 5 0 5 ***I*** **I*******. I I*I***1*+,*1* .I*
B
FIG. 8. Comparison between human endothelial, guinea pig liver, and mouse macrophage tissue transglutaminases. Human endothelial (human) and mouse macrophage (mouse) deduced amino acid sequences were aligned with the published sequence for guinea pig liver tissue transglutaminase (guinea pig). Symbols: asterisk (*), points of sequence homology between allthreeproteins; uertical line ( I ), points of sequence homology between two of the three proteins.
human
TGase
274
YGQCWVFAAVACTVLRCLGIPTRWTNYNSAHD
306
human F a c t o r X I I I a
311
mFAGVFNTFLRCLGIPARIVTNYFSAHD
343
human t i s s u e TGase
423
GLKISTKSVGRDEREDITHTYKYPEGSSEEREA
455
human F a c t o r X I I I a
461
GKLIVTKQIGGDGMMDITHTYKFQEGQEEERLA
493
tiSSUe
* * * * * * * * * * * * ** * * *
********
*
* **
* *
*******
**
*** *
FIG. 9. Comparison between human endothelial tissue transglutaminase and human Factor XIIIa. Theamino acid active sitecysteine sequence of thepentapeptidecontainingthe residue in underlined.
480
Transglutaminases Tissue Humanand Mouse
homology. It is interesting that the degree of homology between the regions of the rabbit epithelial transglutaminase that have beencloned and thecorresponding region of human tissue transglutaminase (32%)is comparable to thehomology between the corresponding regions of human tissue transglutaminase and Factor XI11 (36%). The similarities between epithelial and tissue transglutaminase and the a subunit of Factor XI11 suggest that these enzymes were derived from a common ancestral gene. Endothelial Cell Transglutaminase-Greenberg et al. (27) reported that cultured human and bovine endothelial cells contain high levels of transglutaminase activity and immunochemical studies, suggested that theendothelial cell enzyme cross-reacted with antibodies to tissue transglutaminase. We found that the human endothelial cell cDNA libraries contained tissue transglutaminase clones at high frequency (0.25 clones/10,000 plaques screened) and Northern blot analysis indicated that the enzyme was abundant in these cells. The striking similarity in the sequence of the endothelial cell enzyme and the enzyme from macrophages and liver confirms that this is an authentic tissue transglutaminase. Transglutaminme Expression in Mouse Tissue-There has been considerable speculation on multiple isoforms of the various transglutaminases (1). This speculation has been based on the fact that Factor XI11 has been detected as both an intracellular and an extracellular enzyme, tissue transglutaminase has been detected intracellularly, associated with membranes and possible on the outside of cells (32). In addition, multiple different molecular weights and evidence for the electrophoretic heterogeneity of the enzyme have been reported (27). These observations have suggested that alternative forms of the enzyme might be present in cells and tissues. Our studies do not support this possibility. Analysis of genomic DNA indicates that tissue transglutaminase is presentasa single copygene.' Northern gel analysis of multiple tissues have identified a single transcript of 3.6 kilobases and sequence analysis of overlapping cDNA clones has given no evidence of sequence heterogeneity in transglutaminase transcripts. Furthermore, invitro translation of the transglutaminase enzyme yields an active enzyme with electrophoretic mobility identical to that of the purified enzyme (17). Thus, all our results are compatible with the idea that tissue transglutaminase is the product of a single gene, encoded into a single transcript, and expressed in cells without significant post-translational modification. Acknowledgments-We would like to acknowledge the excellent technical assistance of Nubia Alban and Mary Sobieski and the secretarial assistance of Joan Jennings in support of these studies. J. P. Stein, unpublished observations.
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
REFERENCES Folk, J. E. (1980) Annu. Reu. Biochem. 49,517-531 Lorand, L. (1972) Ann. N. Y. Acad. Sci. 202,6-30 Ichinose, A., Hendryckson, L. E., Fujikawa, K., and Davie, E. W. (1986) Biochemistry 25, 6900-6906 Takahashi, N., Takahashi, Y., and Putman, F. W. (1986)Proc. Natl. Acad. Sci. U. S. A. 8 3 , 8018-8023 Ikura, K., Nasu, T., Yokota, H. Tsuchiya, Y., Sasaki, R., and Chiba, H. (1988) Biochemistry 2 7 , 2898-2905 Floyd, E. E., and Jetten, A. M. (1989) Mol. Cell. Biol. 9 , 48464851 Chiocca, E. A., Davies, P. J. A., and Stein, J. P. (1988) J. Biol. Chem. 263,11584-11589 Murtaugh, M. P., Arend, W. P., and Davies, P. J. A. (1984) J. Exp. Med. 159, 114-125 Moore, W. T., Jr., Murtaugh, M. P., and Davies, P. J. A. (1984) J. Biol. Chem. 2 5 9 , 12794-12802 Nara, K., Nakanishi, K., Hagiwara, H., Wakita, K., Kojima, S., and Hirose, S. (1989) J. Biol. Chem. 2 6 4 , 19308-19312 Kessler, S. W. (1975) J. Zmmunol. 1 1 5 , 1617-1624 Ausubel, F.M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (eds) (1987) Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular cloning: A Laboratorv Manual. Cold SDringHarbor Laboratorv. -. Cold Spring Harbor, NY Maxam. A.M.. and Gilbert. W. (1977) Proc. Natl. Acad. Sci. U. S. A. 74,560-564 Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Sci. U. S. A. 74,5463-5467 Wilbur, W., and Lipman, D. J. (1983) Proc. Natl. Acad. Sci. U. S. A. 8 0 , 726-730 Lee, K. N., Birckbichler, P. J., and Fesus, L. (1986) Prep. Biochem. 16,321-335 Folk, J. E., and Cole, P. W. (1966) J. Biol. Chem. 2 4 1 , 32383240 Lorand. L.. CamDbell-Wilkes. L. K.. and CooDerstein, L. (1972) "
14. 15. 16. 17. 18. 19.
,
,
I
Anal.'Biochem.*50,623-631 20. Bradford. M. M. (1976) Anal. Biochem. 72,248-254 21. O'Dowd,'B. F., Quan,'F., Willard, H. F.,. Lamhonwah, A. M., Korneluk, R. G., Lowden, J. A., Gravel, R.A., and Marhuan, D. J. (1985) Proc. Natl. Acad. Sci. U. S. A. 8 2 , 1184-1188 22. Legendre, N., and Matsudaira, P. (1988) Biotechniques 6, 153159 23. Laemmli, U. K. (1970) Nature 227,680-685 24. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76,4350-4358 25. Birckbichler, P. J., Upchurch, H. F., Patterson, M. K., Jr., and Conway, E. (1985) Hybridoma 4,179-186 26. Kozak, M. (1986) Cell 44,283-292 27. Greenberg, C. S., Achyuthan, K. E., Borowitz, M. J., and Shuman, M. A. (1987) Blood 7 0 , 702-709 28. Achyuthan, K. E., and Greenberg, C. S. (1987) J . Bid. Chem. 262,1901-1906 29. Lee, K. N., Birckbichler, P. J., and Patterson, M. K., Jr. (1989) Biochem. Biophys. Res. Commun. 1 6 2 , 1370-1375 30. Tanabe, T., Nukuda, T., Nisbikawa, Y., Sugimoto, K., Suzuki, H., Takahashi, H., Noda, M., Haga, T., Ichiyama, A., Kengawa, K., Minamino, N., Matsuo, H., and Numa, S. (1985) Nature 315,242-245 31. Gamier, J., Osguthorpe, D. J., and Robson, B. (1978) J. Mol. Biol. 120,97-120 32. Thomazy, V., and Fesus, L. (1989) Cell Tissue Res. 255,215-224
Mouse and Human Tissue Transglutaminmes SUPPLEMENTAL MATERIAL TO ISOLATION AND CHARACTERIZATION OF eDNA CLONES TO MOUSE MACROPHAGE AND HUMAN ENDOTHELIAL CELL TISSUE lRANSGLUTAMiNASES*" V. Gentile', M. Sayd&*. €.A. Chior.ca*.O. Akande'. P.I. Birrkbichierf K.N. Lee+, I.P. Stein@ and P.I.A. Davis*#
481
482
Mouse and Tissue Human
Transglutaminases
Transglutaminuses Tissue Human and Mouse 1
TGase
+
.
- ..
2
3
4
5
483 Kb 9.49 7.46
85kD
4 40 f-
43kD
TGase
2.37 1.35
0.24
A
