Biopharmacological study on the regulation of histamine biosynthesis

纯化的小鼠肥大细胞瘤细胞L组氨酸脱羧酶是由两个相同的53 kDa亚基组成的二聚体，其大小为74 kDa。为了阐明组胺合成的调控机制，我们对HDC酶的翻译后修饰进行了研究，并对HDC基因的5‘侧翼区进行了分析。在Sf9细胞中表达的74 kDa HDC具有较低的酶活性且存在于颗粒组分中，在体外猪胰腺弹性蛋白酶可将74 kDa HDC转化为具有较高催化活性的可溶性53 kDa HDC。对内源活性蛋白水解酶进行了检索，结果表明对苯甲双胺敏感的蛋白水解酶参与了这一过程。然后，用大鼠嗜碱性细胞系RBL-2H3研究了74 kDa和53 kDa HDC的代谢。在RBL-2H3细胞中，74 kDa酶的翻转速度很快，而53 kDa的HDC的翻转速度很慢，这表明74 kDa的HDC是由泛素蛋白酶体系统降解的。接下来，为了确定HDC的组织特异性表达区域，将HDC基因5‘侧翼区与氯霉素乙酰转移酶(CAT)基因的融合DNA导入人嗜碱性白血病KU-812-F细胞和人上皮癌HeLa细胞。CAT分析表明，HDC基因的-1003~99区含有两个正调控元件和一个负调控元件，序列分析表明在-520位有一个核因子c-Myb结合基序，KU-812-F细胞的核提取液中含有一个能与该基序结合的因子，而HeLa细胞的核提取液中则没有。这些结果表明，HDC基因5‘侧翼区至少有一个元件，包括c-Myb结合基序，负责HDC的组织特异性表达。

Purified L-histidine decarboxylase (HDC) of mouse mastocytoma cells is a dimer consisting of two identical 53 kDa subunits, whereas the size of cDNA-deduced HDC is 74 kDa. In order to clarify the regulatory mechanism of histamine synthesis, we studied on the post-translational modification of HDC enzyme and also analyzed the 5'-flanking region of the HDC gene. The expressed recombinant 74 kDa HDC in Sf9 cells had low enzyme activity and was present in the particulate fraction, and 74 kDa HDC was converted into its soluble 53 kDa HDC form with a high catalytic activity by porcine pancreas elastase in vitro. A search was made for endogenously active proteinase, and it was shown that benzamidine-sensitive proteinase was responsible for the processing. Then, metabolism of both 74 kDa and 53 kDa HDC species was investigated using rat basophilic cell line RBL-2H3. In RBL-2H3 cells, the turn-over rate of 74 kDa enzyme was very fast, while that of 53 kDa HDC was slow, and it was suggested that ubiquitinproteasome system was responsible for the degradation of 74 kDa HDC.Next, to identify the regions that regulate the tissue-specific expression of HDC,a fusion DNA with the 5'-flanking region of the HDC gene and chloramphenicol acetyltransferase (CAT) gene was transfected into human basophilic leukemia KU-812-F cells or human epithelial carcinoma HeLa cells. CAT analysis revealed that the region from -1003 to +99 of the HDC gene contained two positive and one negative regulatory elements, Sequence analysis showed a nuclear factor c-Myb binding motif at position -520, and the nuclear extract of KU-812-F cells, but not that of HeLa cells contained a factor which can bind to this motif. These results suggest that at least one element in the 5'-flanking region of the HDC gene, including c-Myb binding motif, is responsible for the tissue-specific expression of HDC.