Western blot analysis was conducted to investigate whether the abundance of CATH protein also increases in parallel with the elevated mRNA levels in NOD mouse LGs (
Fig. 6A). CATH is produced as a proenzyme in cells (335 amino acids [aa] in humans and 333 aa in mice). Proteolytic processing of the single transcript results in the generation of active forms of human CATH containing either a minichain (98-105 aa) and a single chain (116-335 aa) or an active form containing a mini chain, a heavy chain (116-292 aa), and a light chain (293-335 aa), as demonstrated at the protein level.
36 The heavy chain and light chains are derived from further proteolytic processing of the single chain without a loss of amino acid residues. The active forms of the mouse CATH protein exhibit an identical organization, existing in two forms which were deduced by UniprotKB (
www.UniProt.org/ provided in the public domain by the UniProt Consortium, Swiss Institute of Bioinformatics, Geneva, Switzerland) according to its homology to the human ortholog at the transcriptional level. The minichain, heavy chain, and light chain of mouse CATH are annotated as 96-103, 114-290, and 291-333 aa, respectively. The major active form of CATH in Raw264.7 cells, used in our study as a positive control for CATH, appears to be the three-chain form, since the major band detected by Western blot analysis is ∼23 to 24 kDa, corresponding to the heavy chain of 177 aa (114-290 aa;
Fig. 6A, top, lane 3). Both the two-chain and the three-chain forms of CATH are present in mouse LG, as evidenced by detection of both the 23- to 24-kDa band (
Fig. 6A, top, lanes 1 and 2) and the 27- to 28-kDa band (
Fig. 6A, lane 2, top), corresponding to the heavy chain, and the single chain (220 aa), respectively. In addition, a third band at around 20 kDa was clearly detected only in NOD mouse LG lysate (
Fig. 6A, lane 1, top), which may represent a further proteolytic or truncated form of CATH. Densitometry of this Western blot quantified the intensity ratios of 27- and 28, 23- and 24-, and 20-kDa bands (NOD to BALB/c) as 14.1, 4.7, and 11.3 respectively. These results are typical of those run in multiple experiments comparing NOD and BALB/c mouse LG lysate and demonstrate the marked upregulation of CATH in the NOD mouse LG versus the BALB/c control as well as illustrate the complexity of CATH processing in different cell types.