The Journal of Immunology, 2006, 176: 702-703.
Copyright © 2006 by The American Association of Immunologists
The Radiation-Induced nackt (nkt) Allele Is a Loss-of-Function Mutation of the Mouse Cathepsin L Gene
Fernando Benavides*,
Carlos Perez*,
Jorge Blando*,
Jean-Louis Guénet
and
Claudio J. Conti*
* University of Texas M. D. Anderson Cancer Center Science Park-Research Division Smithville, TX 78957
Unité de Génétique des Mammifères Institut Pasteur Paris, France
We read with interest the recent article in The Journal of Immunology by Lombardi et al. (1) involving the use of nackt mutant mice (2, 3). The authors suggest the presence of a mutated cathepsin L (CTSL) protein in nackt mice that could lead to phenotypic differences with targeted mutations of the Ctsl gene (i.e., Ctsltm1Cptr/Ctsltm1Cptr) (4). Contrary to the findings by Lombardi et al. (1), our group has obtained evidence from Western blot analysis (using the same mouse CTSL affinity purified polyclonal Ab) that Ctslnkt/Ctslnkt mice do not generate any of the mature active forms of CTSL (Fig. 1). In addition, by immunohistochemistry (IHC) analysis using the same Ab, we obtained similarly intense staining of thymi from Ctsltm1Cptr/Ctsltm1Cptr and Ctsl+/Ctsl+ mice, which clearly demonstrates the low specificity of the Ab for IHC (Fig. 2). Furthermore, functional analysis by a fluorogenic assay (Calbiochem) showed similar CTSL background activity for Ctsltm1Cptr/Ctsltm1Cptr and Ctslnkt/Ctslnkt cell cultures (our manuscript in preparation). Alternatively, we could confirm the presence of a truncated unprocessed CTSL in nackt mice by Western blotting (Fig. 1). However, the suggestion that this truncated protein is responsible for an associated phenotype is highly unlikely because the nackt allele clearly behaves as a recessive loss-of-function mutation where heterozygous mice are phenotypically indistinguishable from wild-type mice, and homozygous mutants exhibit the same phenotype as Ctsl knockout mice. The minor differences reported so far on the skin phenotype (2) are presumably related to different genetic backgrounds between the nackt congenic lines and the Ctsl knockout line. If the mutated form of CTSL was responsible for phenotypic differences in nackt mice, it should function as a dominant gain-of-function mutation (e.g., a neomorph allele). Moreover, gain-of-function mutations require more restricted changes than loss-of-function mutations and are rarely produced by deletions, as is the case for the nackt allele.
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FIGURE 1. Western blot analysis of CTSL expression shows the presence of pro-CTSL, mature single-chain (SC), and double-chain (DC) (H chain) forms of CTSL in heterozygous +/Ctslnkt epidermal fibroblasts. In contrast, Ctslnkt/Ctslnkt fibroblasts, as well as Ctsltm1Cptr/Ctsltm1Cptr macrophages (a gift from Dr. A. Rudensky, University of Washington, Seattle, WA), showed the absence of processed, mature active forms of CTSL. However, a truncated (unprocessed) form of CTSL (*) is evident in Ctslnkt/Ctslnkt fibroblasts (BALB/c and DAB/2 inbred backgrounds). Western blotting was performed with polyclonal Ab AF1515 (R&D Systems), directed to aa 18–334 of a recombinant mouse CTSL.
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FIGURE 2. Immunohistochemistry of Ctsltm1Cptr/Ctsltm1Cptr (A) and Ctsl+/Ctsl+ (B) thymi (a gift from Dr. A. Rudensky) using anti-CTSL AF1515 Ab reveals strong staining in both genotypes, suggesting low specificity of the Ab in IHC (magnifications: x20). Unfortunately, the article by Lombardi et al. (1 ) did not include images of IHC that could be used for comparison.
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References
- Lombardi, G., D. Burzyn, J. Mundiñano, P. Berguer, P. Bekinschtein, H. Costa, L. F. Castillo, A. Goldman, R. Meiss, I. Piazzon, I. Nepomnaschy. 2005. Cathepsin-L influences the expression of extracellular matrix in lymphoid organs and plays a role in the regulation of thymic output and of peripheral T cell number. J. Immunol. 174: 7022-7032. [Abstract/Free Full Text]
- Benavides, F., A. Venables, H. Poetschke Klug, E. Glasscock, A. Rudensky, M. Gomez, N. Martin Palenzuela, J.-L. Guénet, E. R. Richie, C. J. Conti. 2001. The CD4 T cell-deficient mouse mutation nackt (nkt) involves a deletion in the cathepsin L (Ctsl) gene. Immunogenetics 53: 233-242. [Medline]
- Benavides, F., M. F. Starost, M. Flores, I. B. Gimenez-Conti, J.-L. Guénet, C. J. Conti. 2002. Impaired hair follicle morphogenesis and cycling with abnormal epidermal differentiation in nackt mice, a cathepsin L-deficient mutation. Am. J. Pathol. 161: 693-703. [Abstract/Free Full Text]
- Nakagawa, T., W. Roth, P. Wong, A. Nelson, A. Farr, J. Deussing, J. A. Villadangos, H. Ploegh, C. Peters, A. Y. Rudensky. 1998. Cathepsin L: critical role in Ii degradation and CD4 T cell selection in the thymus. Science 280: 450-453. [Abstract/Free Full Text]
This article has been cited by other articles:
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G. Lombardi, D. Burzyn, J. Mundinano, P. Berguer, H. Costa, A. Goldman, I. Piazzon, and I. Nepomnaschy
Response to Comment on "Cathepsin-L Influences the Expression of Extracellular Matrix in Lymphoid Organs and Plays a Role in the Regulation of Thymic Output and of Peripheral T Cell Number"
J. Immunol.,
May 1, 2006;
176(9):
5135 - 5136.
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