- Email: [email protected]
Evidence That Caspase-13 Is Not a Human but a Bovine Gene
Biochemical and Biophysical Research Communications 285, 1150 –1154 (2001) doi:10.1006/bbrc.2001.5315, available online at http://www.idealibrary.com on
Evidence That Caspase-13 Is Not a Human but a Bovine Gene Ulrich Koenig, Leopold Eckhart, and Erwin Tschachler 1 Division of Immunology, Allergy, and Infectious Diseases, Department of Dermatology, University of Vienna Medical School, Vienna, Austria
Received July 3, 2001
Caspase-13 was reported to be a member of the human caspase family of proteases (Humke, E., et al., J. Biol. Chem. 273, 15702–15707, 1998). By contrast, a recent study (Lin, X., et al., J. Biol. Chem. 275, 39920 – 39926, 2000) could not confirm caspase-13 expression in human tissues. When we searched the GenBank database we found several expressed sequence tags (ESTs) from bos taurus completely matching the published caspase-13 sequence. Reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that bovine but not human peripheral blood mononuclear cells express caspase-13. From these cells we cloned two bovine caspase-13 splice variants and found that the sequence of the larger variant was identical to the mRNA published by Humke et al. Our findings strongly suggest that the previously published caspase-13 sequence is not of human origin but represents a bovine gene. © 2001 Academic Press
Caspases are a family of cysteine-dependent aspartate-specific proteases. Several caspases are essential for the initiation and execution of apoptosis, others have a function not related to cell death, as they play critical roles in the processing of IL-1 beta and IL-18 (1). So far 14 mammalian caspases have been described, many of which exist in orthologous forms in different species. 12 human caspases, i.e., caspases-1 through -10 as well as caspases-13 and -14, have been published (2). In mouse and rat, caspases-1, -2, -3, -6, -7, -8, -9, and -11 have been described (3, 4). Additionally, caspases-12 and -14 were identified in mouse but not in rat (5). A caspase with partial homology to caspase-1 and caspase-13 was detected in cat and in dog (6). 1
To whom correspondence should be addressed at Division of Immunology, Allergy, and Infectious Diseases, Department of Dermatology, University of Vienna Medical School, Wa¨hringer Gu¨rtel 18-20, A-1090 Vienna, Austria. Fax: (43)-1-403 4922. E-mail: [email protected]. 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
Despite the increasing knowledge of apoptosis and caspases, the biology of certain members of the caspase family has not been studied in detail. One example is caspase-13, which was first described in 1998 (7). Strikingly, the only study published after the initial report yielded results that were contradictory to the former (8). In particular, no caspase-13 expression was detected by RT-PCR screening of any human tissue that had been described to be positive by Humke and colleagues, based on Northern blot analysis. Since we are in the process of characterizing the caspase expression profile of several human tissues we investigated the expression of caspase-13 more closely, and were able to demonstrate that caspase-13 does not belong to the human caspase family. MATERIALS AND METHODS Isolation of peripheral blood mononuclear cells (PBMCs) and preparation of RNA. PBMCs were isolated from bovine and human blood using Ficoll Paque (Amersham Pharmacia Biotech, Sweden) gradient centrifugation. Total RNA was prepared using the RNAzol method (Biotecx Laboratories, U.S.A.). In brief, PBMCs were lysed in RNAzol, chloroform extracted, precipitated with isopropanol and washed twice with 70% ethanol. RNA was resuspended in diethyl pyrocarbonate-treated water and incubated with DNase I (Roche, Switzerland) at 37°C for 10 min, then extracted with phenol and precipitated. cDNA synthesis. Approximately 5 g total RNA and 1 g oligo(dT) 15 primer in a total volume of 11.5 l were denatured for 10 min at 70°C and cooled to room temperature. For cDNA synthesis the following reagents were added: 6 l 5 ⫻ AMV buffer (Roche, Switzerland), 10 l dNTP mix 2.5 mM each (TaKaRa, Japan), 1 l RNasin (40 u/l) (Roche, Switzerland), 1.5 l AMV reverse transcriptase (10 u/l) (Roche, Switzerland). RNA was reverse transcribed at 42°C for 1 h. The reaction was stopped by incubation at 95°C for 3 min, the cDNA was stored at ⫺20°C. PCR, cloning, and sequencing. For PCR amplification the following reagents were combined: 1 l cDNA (diluted 1:40 in water), 4 l 10 ⫻ PCR-buffer (TaKaRa, Japan), 3.2 l dNTP mix 2.5 mM each (TaKaRa, Japan), 2 l 5⬘-primer: casp13s (5⬘-TATAAAAGCTCCTGAGGAAACT-3⬘) (5 pmol/l), 2 l 3⬘-primer casp13as (5⬘TCTGGGCTTTAACATTTGGTTT-3⬘) (5 pmol/l) and 0.4 l Taq DNA polymerase (5 u/l) (TaKaRa, Japan). The volume was adjusted to 40 l with water. Amplification was performed in a thermocycler
1150
Vol. 285, No. 5, 2001
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 1. Caspase-13 is expressed in bovine but not in human PBMCs. RNA was prepared from human and bovine PBMC. To test for the presence of contaminating DNA human (lane 1) and bovine (lane 4) RNA was subjected to PCR without prior reverse transcription. Amplification with caspase-13 specific primer pair (casp13s and casp13as) was performed on cDNAs from human (lane 2) and bovine (lane 5) PBMCs. Integrity of the respective cDNAs was tested by GAPDH PCR (lanes 3 and 6). The size of the DNA marker is indicated at the right side of the panels.
(Perkin Elmer, U.S.A.): 96°C ⫻ 3 min, 35 ⫻ (96°C ⫻ 30 s, 55°C ⫻ 30 s, 72°C ⫻ 1 min), 72°C ⫻ 7 min. The integrity of the bovine cDNA was tested with the primer pair bGAPDH(F) (5⬘-ATGGTGAAGGTCGGAGTGAACG-3⬘) and bGAPDH(R) (5⬘-TGCAGAGATGATGACCCTCTTGGC-3⬘). The integrity of the human cDNA was tested with the primer pair hGAPDH(F) (5⬘-AAGGTGAAGGTCGGAGTCAACG-3⬘) and hGAPDH(R) (5⬘-GGCAGAGATGATGACCCTTTTGGC-3⬘) under the same PCR conditions as described above. Ten microliters of the PCR-product was analyzed on a 1.5% agarose gel. Amplification of the caspase-13 transcript containing the ORF was performed with the primer pair casp13start (5⬘-CCTTCAAGGCAGTAGGAAGATACTAA-3⬘) and casp13stop (5⬘-GCTGATAAATGGTTGAATTTCCTGT-3⬘) under the same conditions as described above, except for performing elongation for 2 min at 72°C. One microliter of the PCR-product was directly ligated in pCR2.1-TOPO vector (Invitrogen, The Netherlands) according to the standard protocol. Competent E. coli (Invitrogen, The Netherlands) were transformed with 2 l ligation product. Clones were PCR-screened with the caspase-13 primer pair casp13start and casp13stop under the same conditions as described above. Positive clones were sequenced on an ABI PRISM sequencer (Applied Biosystems, U.S.A.).
RESULTS BLAST search of the human genome working draft sequence (http://genome.ucsc.edu/), which covers 90% of the human genome sequence (9) with the coding sequence of caspase-13 (GenBank Accession No. AF078533) (1) revealed no identity to the human genome sequence over stretches longer than 30 nucleotides. Similarily, a search in the human EST database (http://www.ncbi.nlm.nih.gov/blast/) yielded only human ESTs with identities of ⱕ85% to caspase-13. These ESTs belong to the human caspase-4. In contrast extended nucleotide BLAST search on non-human sequences (http://www.ncbi.nlm.nih.gov/ blast/) revealed 4 matching bovine EST entries with 99% sequence identity to different stretches of more than 200 nucleotides of the caspase-13 sequence. Three EST sequences (nucleotide sequence GenBank Accession Nos. BE485042, BG692978, BE477605) were derived from the library of pooled bovine mammary tissues, named BARC 5BOV (Sonstegard, T., Capuco, A., Van Tassell, C., Ashwell, M., Wells, K., U.S.A.). The fourth EST (nucleotide sequence GenBank Accession
No. AV618261) was derived from a library of bovine ovary fetus (Sugimoto, Y., Hirotsune S., Takasuga, A., Itoh, R., Jitohzono, A., Suzuki, H., Japan). To clarify expression of caspase-13 in cattle and in humans, we isolated peripheral blood mononuclear cells (PBMCs) from both species. RT-PCR analysis with the caspase-13 specific primer pair confirmed expression of caspase-13 in bovine but not in human PBMCs (Fig. 1). The integrity of the cDNAs was confirmed by amplification of transcripts of the housekeeping gene GAPDH (Fig. 1). In addition to RNA from bovine PBMCs, RNA prepared from bovine chondrocytes also yielded a caspase-13 RT-PCR product of identical size (not shown). Another caspase-13 primer pair flanking the entire open reading frame was used in a second PCR. The bovine cDNA yielded a PCR product of the expected length of 1207 bp (Fig. 2). In addition, a second less abundant PCR product of smaller size was detected. Cloning and sequencing of the longer PCR product revealed that the sequence of this bovine amplification product completely matched the sequence published as human caspase-13/ERICE (7). This sequence contains one continuous ORF coding for a hypothetical protein of 377 amino acids with a molecular weight (MW) of 43 kDa (Fig. 3). Clones of the shorter PCR product contained a cDNA of 1036 bp length that differed from the longer PCR product by an internal deletion of 171 nucleotides indicating that it corresponds to an alternatively spliced transcript of the same gene. It encodes a protein of 320 amino acids with a molecular weight (MW) of 37 kDa. We propose to name the two novel gene products bovine caspase-13/a and bovine caspase13/b (Fig. 3). (Nucleotide sequence GenBank Accession Nos. AF383946 and AF383947). DISCUSSION We could demonstrate, that caspase-13/ERICE can be amplified from bovine PBMCs cDNA but not from human PBMCs. Our data strongly suggest that caspase-13/ERICE was mistakenly published as a human gene as we hypothesize that most likely due to a contamination of cDNA libraries with sequences from
FIG. 2. Two species of caspase-13 transcripts can be amplified from RNA derived form bovine PBMCs. Using a primer pair spanning the entire open reading frame of caspase-13 amplification products of about 1200 nts caspase-13/a (Casp-13/a) and 1030 nts caspase-13/b (Casp-13/b) were detected. The size of the DNA marker is indicated at the right side of the panel.
1151
Vol. 285, No. 5, 2001
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 3. Nucleotide and amino acid sequences of the bovine caspase-13/a (Casp-13/a) and caspase-13/b (Casp-13/b) isoforms.
bos taurus. Based on our data, we propose to remove caspase-13 from the family of human caspases. Although we do not have a suggestion how a contamina-
tion with mammalian cDNA might have occurred, contamination of cDNA libraries with sequences from prokaryotic organisms has been reported previouls.
1152
Vol. 285, No. 5, 2001
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 3—Continued
For example the clontech kidney cDNA library (HL1123a) was reported to be contaminated with the bacterium Leuconostoc lactis (10). Also public databases are not unaffected of this problem: ESTs submitted to the European Molecular Biology Laboratory database in 1993 derived from a T lymphoblastoid cell line, were heavily contaminated by prokaryotic sequences (11). In addition to the published cDNA sequence of caspase-13/a which we found to be the predominant form expressed in bovine cells we were able to identify an additional shorter isoform of this caspase, caspase13/b. Whether this transcript is translated to a protein in the same approximated 10:1 ratio of caspase-13/a to caspase-13/b seen in RT-PCR remains to be investigated. In conclusion our data suggest that the classificatin of human caspases should be reevaluated.
Karin Macfelda for providing bovine chondrocytes, Sabine Gerlitz for technical support, and Michael Rendl for critical discussions.
REFERENCES
ACKNOWLEDGMENTS The authors thank the members of the 2nd Medical Department of the Veterinarian University of Vienna for providing bovine PBMCs, 1153
1. Zeuner, A., Eramo, A., Peschle, C., and De Maria, R. (1999) Caspase activation without death. Cell Death Differ. 6, 1075– 1080. 2. Eckhart, L., Ban, J., Fischer, H., and Tschachler, E. (2000) Caspase-14: Analysis of gene structure and mRNA expression during keratinocyte differentiation. Biochem. Biophys. Res. Commun. 277, 655– 659. 3. Van de Craen, M., Vandenabeele, P., Declercq, W., Van den Brande, I., Van Loo, G., Molemans, F., Schotte, P., Van Criekinge, W., Beyaert, R., and Fiers, W. (1997) Characterization of seven murine caspase family members. FEBS Lett. 403, 61– 69. 4. Van de Craen, M., Van Loo, G., Declercq, W., Schotte, P., Van den Brande, I., Mandruzzato, S., Van der Bruggen, P., Fiers, W., and Vandenabeele, P. (1998) Molecular cloning and identification of murine caspase-8. J. Mol. Biol. 284, 1017–1026. 5. Van de Craen, M., Van Loo, G., Pype, S., Van Criekinge, W., Van den Brande, I., Molemans, F., Fiers, W., Declercq, W., and Vandenabeele, P. (1998) Identification of a new caspase homologue: caspase-14. Cell Death Differ. 5, 838 – 846. 6. Taylor, S., Hanlon, L., McGillivray, C., Gault, E. A., Argyle, D. J., Onions, D. E., and Nicolson, L. (2000) Cloning and sequencing of
Vol. 285, No. 5, 2001
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
feline and canine ice-related cDNAs encoding hybrid caspase-1/ caspase-13-like propeptides DNA Seq. 10, 387–394. 7. Humke, E. W., Ni, J., and Dixit, V. M. (1998) ERICE, a novel FLICE-activatable caspase. J. Biol. Chem. 273, 15702–15707. 8. Lin, X. Y., Choi, M. S., and Porter, A. G. (2000) Expression analysis of the human caspase-1 subfamily reveals specific regulation of the CASP5 gene by lipopolysaccharide and interferon-␥. J. Biol. Chem. 275, 39920 –39926.
9. International human genome sequencing consortium. (2001) Initial sequencing and analysis of the human genome. Nature 409, 860 –921. 10. Dean, M., and Allikmets, R. (1995) Contamination of cDNAlibraries and expressed-sequence-tags databases. Am. J. Hum. Genet. 57, 1254 –1255. 11. Charalambos, S. (1993) Contamination of cDNA sequences in databases. Science 259, 1677–1678.
1154
Evidence That Caspase-13 Is Not a Human but a Bovine Gene Ulrich Koenig, Leopold Eckhart, and Erwin Tschachler 1 Division of Immunology, Allergy, and Infectious Diseases, Department of Dermatology, University of Vienna Medical School, Vienna, Austria
Received July 3, 2001
Caspase-13 was reported to be a member of the human caspase family of proteases (Humke, E., et al., J. Biol. Chem. 273, 15702–15707, 1998). By contrast, a recent study (Lin, X., et al., J. Biol. Chem. 275, 39920 – 39926, 2000) could not confirm caspase-13 expression in human tissues. When we searched the GenBank database we found several expressed sequence tags (ESTs) from bos taurus completely matching the published caspase-13 sequence. Reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that bovine but not human peripheral blood mononuclear cells express caspase-13. From these cells we cloned two bovine caspase-13 splice variants and found that the sequence of the larger variant was identical to the mRNA published by Humke et al. Our findings strongly suggest that the previously published caspase-13 sequence is not of human origin but represents a bovine gene. © 2001 Academic Press
Caspases are a family of cysteine-dependent aspartate-specific proteases. Several caspases are essential for the initiation and execution of apoptosis, others have a function not related to cell death, as they play critical roles in the processing of IL-1 beta and IL-18 (1). So far 14 mammalian caspases have been described, many of which exist in orthologous forms in different species. 12 human caspases, i.e., caspases-1 through -10 as well as caspases-13 and -14, have been published (2). In mouse and rat, caspases-1, -2, -3, -6, -7, -8, -9, and -11 have been described (3, 4). Additionally, caspases-12 and -14 were identified in mouse but not in rat (5). A caspase with partial homology to caspase-1 and caspase-13 was detected in cat and in dog (6). 1
To whom correspondence should be addressed at Division of Immunology, Allergy, and Infectious Diseases, Department of Dermatology, University of Vienna Medical School, Wa¨hringer Gu¨rtel 18-20, A-1090 Vienna, Austria. Fax: (43)-1-403 4922. E-mail: [email protected]. 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
Despite the increasing knowledge of apoptosis and caspases, the biology of certain members of the caspase family has not been studied in detail. One example is caspase-13, which was first described in 1998 (7). Strikingly, the only study published after the initial report yielded results that were contradictory to the former (8). In particular, no caspase-13 expression was detected by RT-PCR screening of any human tissue that had been described to be positive by Humke and colleagues, based on Northern blot analysis. Since we are in the process of characterizing the caspase expression profile of several human tissues we investigated the expression of caspase-13 more closely, and were able to demonstrate that caspase-13 does not belong to the human caspase family. MATERIALS AND METHODS Isolation of peripheral blood mononuclear cells (PBMCs) and preparation of RNA. PBMCs were isolated from bovine and human blood using Ficoll Paque (Amersham Pharmacia Biotech, Sweden) gradient centrifugation. Total RNA was prepared using the RNAzol method (Biotecx Laboratories, U.S.A.). In brief, PBMCs were lysed in RNAzol, chloroform extracted, precipitated with isopropanol and washed twice with 70% ethanol. RNA was resuspended in diethyl pyrocarbonate-treated water and incubated with DNase I (Roche, Switzerland) at 37°C for 10 min, then extracted with phenol and precipitated. cDNA synthesis. Approximately 5 g total RNA and 1 g oligo(dT) 15 primer in a total volume of 11.5 l were denatured for 10 min at 70°C and cooled to room temperature. For cDNA synthesis the following reagents were added: 6 l 5 ⫻ AMV buffer (Roche, Switzerland), 10 l dNTP mix 2.5 mM each (TaKaRa, Japan), 1 l RNasin (40 u/l) (Roche, Switzerland), 1.5 l AMV reverse transcriptase (10 u/l) (Roche, Switzerland). RNA was reverse transcribed at 42°C for 1 h. The reaction was stopped by incubation at 95°C for 3 min, the cDNA was stored at ⫺20°C. PCR, cloning, and sequencing. For PCR amplification the following reagents were combined: 1 l cDNA (diluted 1:40 in water), 4 l 10 ⫻ PCR-buffer (TaKaRa, Japan), 3.2 l dNTP mix 2.5 mM each (TaKaRa, Japan), 2 l 5⬘-primer: casp13s (5⬘-TATAAAAGCTCCTGAGGAAACT-3⬘) (5 pmol/l), 2 l 3⬘-primer casp13as (5⬘TCTGGGCTTTAACATTTGGTTT-3⬘) (5 pmol/l) and 0.4 l Taq DNA polymerase (5 u/l) (TaKaRa, Japan). The volume was adjusted to 40 l with water. Amplification was performed in a thermocycler
1150
Vol. 285, No. 5, 2001
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 1. Caspase-13 is expressed in bovine but not in human PBMCs. RNA was prepared from human and bovine PBMC. To test for the presence of contaminating DNA human (lane 1) and bovine (lane 4) RNA was subjected to PCR without prior reverse transcription. Amplification with caspase-13 specific primer pair (casp13s and casp13as) was performed on cDNAs from human (lane 2) and bovine (lane 5) PBMCs. Integrity of the respective cDNAs was tested by GAPDH PCR (lanes 3 and 6). The size of the DNA marker is indicated at the right side of the panels.
(Perkin Elmer, U.S.A.): 96°C ⫻ 3 min, 35 ⫻ (96°C ⫻ 30 s, 55°C ⫻ 30 s, 72°C ⫻ 1 min), 72°C ⫻ 7 min. The integrity of the bovine cDNA was tested with the primer pair bGAPDH(F) (5⬘-ATGGTGAAGGTCGGAGTGAACG-3⬘) and bGAPDH(R) (5⬘-TGCAGAGATGATGACCCTCTTGGC-3⬘). The integrity of the human cDNA was tested with the primer pair hGAPDH(F) (5⬘-AAGGTGAAGGTCGGAGTCAACG-3⬘) and hGAPDH(R) (5⬘-GGCAGAGATGATGACCCTTTTGGC-3⬘) under the same PCR conditions as described above. Ten microliters of the PCR-product was analyzed on a 1.5% agarose gel. Amplification of the caspase-13 transcript containing the ORF was performed with the primer pair casp13start (5⬘-CCTTCAAGGCAGTAGGAAGATACTAA-3⬘) and casp13stop (5⬘-GCTGATAAATGGTTGAATTTCCTGT-3⬘) under the same conditions as described above, except for performing elongation for 2 min at 72°C. One microliter of the PCR-product was directly ligated in pCR2.1-TOPO vector (Invitrogen, The Netherlands) according to the standard protocol. Competent E. coli (Invitrogen, The Netherlands) were transformed with 2 l ligation product. Clones were PCR-screened with the caspase-13 primer pair casp13start and casp13stop under the same conditions as described above. Positive clones were sequenced on an ABI PRISM sequencer (Applied Biosystems, U.S.A.).
RESULTS BLAST search of the human genome working draft sequence (http://genome.ucsc.edu/), which covers 90% of the human genome sequence (9) with the coding sequence of caspase-13 (GenBank Accession No. AF078533) (1) revealed no identity to the human genome sequence over stretches longer than 30 nucleotides. Similarily, a search in the human EST database (http://www.ncbi.nlm.nih.gov/blast/) yielded only human ESTs with identities of ⱕ85% to caspase-13. These ESTs belong to the human caspase-4. In contrast extended nucleotide BLAST search on non-human sequences (http://www.ncbi.nlm.nih.gov/ blast/) revealed 4 matching bovine EST entries with 99% sequence identity to different stretches of more than 200 nucleotides of the caspase-13 sequence. Three EST sequences (nucleotide sequence GenBank Accession Nos. BE485042, BG692978, BE477605) were derived from the library of pooled bovine mammary tissues, named BARC 5BOV (Sonstegard, T., Capuco, A., Van Tassell, C., Ashwell, M., Wells, K., U.S.A.). The fourth EST (nucleotide sequence GenBank Accession
No. AV618261) was derived from a library of bovine ovary fetus (Sugimoto, Y., Hirotsune S., Takasuga, A., Itoh, R., Jitohzono, A., Suzuki, H., Japan). To clarify expression of caspase-13 in cattle and in humans, we isolated peripheral blood mononuclear cells (PBMCs) from both species. RT-PCR analysis with the caspase-13 specific primer pair confirmed expression of caspase-13 in bovine but not in human PBMCs (Fig. 1). The integrity of the cDNAs was confirmed by amplification of transcripts of the housekeeping gene GAPDH (Fig. 1). In addition to RNA from bovine PBMCs, RNA prepared from bovine chondrocytes also yielded a caspase-13 RT-PCR product of identical size (not shown). Another caspase-13 primer pair flanking the entire open reading frame was used in a second PCR. The bovine cDNA yielded a PCR product of the expected length of 1207 bp (Fig. 2). In addition, a second less abundant PCR product of smaller size was detected. Cloning and sequencing of the longer PCR product revealed that the sequence of this bovine amplification product completely matched the sequence published as human caspase-13/ERICE (7). This sequence contains one continuous ORF coding for a hypothetical protein of 377 amino acids with a molecular weight (MW) of 43 kDa (Fig. 3). Clones of the shorter PCR product contained a cDNA of 1036 bp length that differed from the longer PCR product by an internal deletion of 171 nucleotides indicating that it corresponds to an alternatively spliced transcript of the same gene. It encodes a protein of 320 amino acids with a molecular weight (MW) of 37 kDa. We propose to name the two novel gene products bovine caspase-13/a and bovine caspase13/b (Fig. 3). (Nucleotide sequence GenBank Accession Nos. AF383946 and AF383947). DISCUSSION We could demonstrate, that caspase-13/ERICE can be amplified from bovine PBMCs cDNA but not from human PBMCs. Our data strongly suggest that caspase-13/ERICE was mistakenly published as a human gene as we hypothesize that most likely due to a contamination of cDNA libraries with sequences from
FIG. 2. Two species of caspase-13 transcripts can be amplified from RNA derived form bovine PBMCs. Using a primer pair spanning the entire open reading frame of caspase-13 amplification products of about 1200 nts caspase-13/a (Casp-13/a) and 1030 nts caspase-13/b (Casp-13/b) were detected. The size of the DNA marker is indicated at the right side of the panel.
1151
Vol. 285, No. 5, 2001
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 3. Nucleotide and amino acid sequences of the bovine caspase-13/a (Casp-13/a) and caspase-13/b (Casp-13/b) isoforms.
bos taurus. Based on our data, we propose to remove caspase-13 from the family of human caspases. Although we do not have a suggestion how a contamina-
tion with mammalian cDNA might have occurred, contamination of cDNA libraries with sequences from prokaryotic organisms has been reported previouls.
1152
Vol. 285, No. 5, 2001
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 3—Continued
For example the clontech kidney cDNA library (HL1123a) was reported to be contaminated with the bacterium Leuconostoc lactis (10). Also public databases are not unaffected of this problem: ESTs submitted to the European Molecular Biology Laboratory database in 1993 derived from a T lymphoblastoid cell line, were heavily contaminated by prokaryotic sequences (11). In addition to the published cDNA sequence of caspase-13/a which we found to be the predominant form expressed in bovine cells we were able to identify an additional shorter isoform of this caspase, caspase13/b. Whether this transcript is translated to a protein in the same approximated 10:1 ratio of caspase-13/a to caspase-13/b seen in RT-PCR remains to be investigated. In conclusion our data suggest that the classificatin of human caspases should be reevaluated.
Karin Macfelda for providing bovine chondrocytes, Sabine Gerlitz for technical support, and Michael Rendl for critical discussions.
REFERENCES
ACKNOWLEDGMENTS The authors thank the members of the 2nd Medical Department of the Veterinarian University of Vienna for providing bovine PBMCs, 1153
1. Zeuner, A., Eramo, A., Peschle, C., and De Maria, R. (1999) Caspase activation without death. Cell Death Differ. 6, 1075– 1080. 2. Eckhart, L., Ban, J., Fischer, H., and Tschachler, E. (2000) Caspase-14: Analysis of gene structure and mRNA expression during keratinocyte differentiation. Biochem. Biophys. Res. Commun. 277, 655– 659. 3. Van de Craen, M., Vandenabeele, P., Declercq, W., Van den Brande, I., Van Loo, G., Molemans, F., Schotte, P., Van Criekinge, W., Beyaert, R., and Fiers, W. (1997) Characterization of seven murine caspase family members. FEBS Lett. 403, 61– 69. 4. Van de Craen, M., Van Loo, G., Declercq, W., Schotte, P., Van den Brande, I., Mandruzzato, S., Van der Bruggen, P., Fiers, W., and Vandenabeele, P. (1998) Molecular cloning and identification of murine caspase-8. J. Mol. Biol. 284, 1017–1026. 5. Van de Craen, M., Van Loo, G., Pype, S., Van Criekinge, W., Van den Brande, I., Molemans, F., Fiers, W., Declercq, W., and Vandenabeele, P. (1998) Identification of a new caspase homologue: caspase-14. Cell Death Differ. 5, 838 – 846. 6. Taylor, S., Hanlon, L., McGillivray, C., Gault, E. A., Argyle, D. J., Onions, D. E., and Nicolson, L. (2000) Cloning and sequencing of
Vol. 285, No. 5, 2001
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
feline and canine ice-related cDNAs encoding hybrid caspase-1/ caspase-13-like propeptides DNA Seq. 10, 387–394. 7. Humke, E. W., Ni, J., and Dixit, V. M. (1998) ERICE, a novel FLICE-activatable caspase. J. Biol. Chem. 273, 15702–15707. 8. Lin, X. Y., Choi, M. S., and Porter, A. G. (2000) Expression analysis of the human caspase-1 subfamily reveals specific regulation of the CASP5 gene by lipopolysaccharide and interferon-␥. J. Biol. Chem. 275, 39920 –39926.
9. International human genome sequencing consortium. (2001) Initial sequencing and analysis of the human genome. Nature 409, 860 –921. 10. Dean, M., and Allikmets, R. (1995) Contamination of cDNAlibraries and expressed-sequence-tags databases. Am. J. Hum. Genet. 57, 1254 –1255. 11. Charalambos, S. (1993) Contamination of cDNA sequences in databases. Science 259, 1677–1678.
1154