Activation of Histidine Decarboxylase through Post-translational Cleavage by Caspase-9 in a Mouse Mastocytoma P-815

Kazuyuki Furuta,Kazuhisa Nakayama,Yukihiko Sugimoto,Atsushi Ichikawa,Satoshi Tanaka

doi:10.1074/jbc.m609943200

Kazuyuki Furuta, Kazuhisa Nakayama + Show 3 more

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https://doi.org/10.1074/jbc.m609943200

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Abstract

L-Histidine decarboxylase (HDC) is the rate-limiting enzyme for histamine synthesis in mammals. Although accumulating evidence has indicated the post-translational processing of HDC, it remains unknown what kinds of proteases are involved. We investigated the processing of HDC in a mouse mastocytoma, P-815, using a lentiviral expression system. HDC was expressed as a 74-kDa precursor form, which is cleaved to yield the 55- and 60-kDa forms upon treatment with butyrate. Alanine-scanning mutations revealed that two tandem aspartate residues (Asp(517)-Asp(518), Asp(550)-Asp(551)) are critical for the processing. Treatment with butyrate caused an increase in the enzyme activity of the cells expressing the wild type HDC, but not in the cells expressing the processing-incompetent mutant. An increase in histamine synthesis by butyrate was accompanied by formation of the 55- and 60-kDa form of HDC. In addition, the in vitro translated 74-kDa form of HDC was found to undergo a limited cleavage by purified human caspase-9, whereas the alanine-substituted mutants were not. Processing and enzymatic activation of HDC in P-815 cells was enhanced in the presence of a Zn(2+) chelator, TPEN. Although treatment with butyrate and TPEN drastically augmented the protease activity of caspase-3, and -9, no apoptotic cell death was observed. Both enzymatic activation and processing of HDC were completely suppressed by a pan-caspase inhibitor, partially but significantly by a specific inhibitor for caspase-9, but not by a caspase-3 inhibitor. These results suggest that, in P-815 cells, histamine synthesis is augmented through the post-translational cleavage of HDC, which is mediated by caspase-9.

Highlights

Tidine decarboxylase (HDC,2 EC 4.1.1.22) is the rate-limiting enzyme for histamine synthesis in mammals, and targeted disruption of mouse Histidine decarboxylase (HDC) gene resulted in the complete loss of de novo synthesis of histamine [8]
Post-translational Cleavage of HDC in P-815 Cells Treated with Butyrate—We previously purified HDC as a homodimer consisting of a 53-kDa subunit from P-815 cells, which were maintained in peritoneal cavity of the BDF1 mouse [11]
Because a prolonged culture of P-815 cells resulted in a decrease in the HDC level, we investigated post-translational processing of HDC expressed using a lentiviral expression in these cells

Summary

Introduction

Tidine decarboxylase (HDC, EC 4.1.1.22) is the rate-limiting enzyme for histamine synthesis in mammals, and targeted disruption of mouse HDC gene resulted in the complete loss of de novo synthesis of histamine [8]. The 74-kDa form of HDC has neither the N-terminal signal sequence nor hydrophobic membrane anchor, the in vitro translated 74-kDa HDC protein was post-translationally targeted to the microsomal fraction, which was mediated by the C-terminal 20-kDa region [17]. Fleming and Wang [18] demonstrated that rat 74-kDa form expressed in COS-7 cells is cleaved to multiple forms and that formation of the main 55-kDa form is prevented by mutations at the residues, Ser502, Lys503, and Asp504, whereas these mutations had no effects on the enzyme activity This putative processing site was not found in the primary sequence of mouse and human HDC, and no processed forms were detected in COS-7 cells expressing mouse 74-kDa HDC [17].

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