Abstract
By using Western blot analysis, high levels of 17.5- and 20-kDa interleukin-1beta (IL-1beta) proteins were detected in the submandibular gland (SMG) of mice. Despite this fact, the amount of pro-IL-1beta protein, a precursor of IL-1beta, with a molecular size of 35 kDa in this tissue was below the detectable level, although strong expression of pro-IL-1beta mRNA was observed. A large amount of 17.5-kDa IL-1beta also appeared in the saliva of mice injected with lipopolysaccharide, suggesting that this IL-1beta is a secretory form produced by the SMG. The protein for IL-1beta-converting enzyme, a processing enzyme for pro-IL-1beta, was expressed only at a low level in the SMG as compared with its level in various epithelial tissues or lipopolysaccharide-stimulated macrophages. On the other hand, mK1, mK9, mK13, and mK22, members of the kallikrein family, were detected strongly in the SMG but not in other tissues. By incubation with mK13, but not with mK1, mK9, or mK22, the 35-kDa pro-IL-1beta was cleaved into two major products with molecular masses of 17.5 and 22 kDa, and production was inhibited by phenylmethylsulfonyl fluoride, a serine protease inhibitor, but not by IL-1beta-converting enzyme inhibitors. A peptide segment corresponding to amino acid residues 107-121 of mouse pro-IL-1beta (107WDDDDNLLVCDVPIR) was cleaved by incubation with mK13, generating two peptides, 107WDDDDNL and 114LVCDVPIR. Therefore, kallikrein mK13 would appear to hydrolyze pro-IL-1beta between its Leu113 and Leu114 residues. The results of immunohistochemistry and an autonomic therapy experiment showed that IL-1beta and kallikrein mK13 were co-localized in the secretory granules of granular convoluted tubular cells. Our present results thus suggest kallikrein mK13 is a plausible candidate for the processing enzyme for pro-IL-1beta in the SMG of mice.
Highlights
Goat polyclonal antibody raised against a carboxyl-terminal peptide of mouse IL-1, rabbit anti-IL-1converting enzyme (ICE) polyclonal antibody, and the blocking peptide, donkey anti-goat IgG (H ϩ L) conjugated with fluorescein isothiocyanate (FITC), goat anti-rabbit IgG (H ϩ L) FITC, donkey anti-goat IgG-conjugated with horseradish peroxidase, and donkey anti-rabbit IgG-horseradish peroxidase were purchased from Santa Cruz Biotechnology (Santa Cruz, CA)
A result similar to that for the submandibular gland (SMG) was obtained in the experiment in which a saliva sample from LPS-injected mice was assessed (Fig. 1A), again supporting the idea that 17.5- and 20-kDa bands would be secreted from the SMG after having been processed from their precursors
An IL-1 immunoreactive band showing a molecular mass of 35 kDa was seen in the case of lung and spleen, the level was low; but no such band was detected in the lane containing the SMG extract
Summary
IL-1␣ and IL-1 have similar but not identical multiple biological effects, which have been well characterized by in vivo and in vitro experiments [3, 4] Both forms of IL-1 are synthesized by a wide variety of tissues and affect almost all types of cells, which share a common receptor. There are many tissue kallikrein enzymes, and their genes make up a large family, the tissue kallikrein gene family [15, 16] All of these genes code for putative serine proteases and share important similarities, including the same chromosome mapping, significant homology at both the nucleotide and protein level, and similar genomic organization. We demonstrate that one of the kallikreins, mK13, known as pro-renin-converting enzyme, is able to process pro-IL-1 to form 17.5-kDa IL-1
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