Jansen G A, Ofman R, Denis S, Ferdinandusse S, Hogenhout E M, Jakobs C, Wanders R J
Department of Pediatrics (Emma Children's Hospital), University of Amsterdam, Academic Medical Centre, The Netherlands.
J Lipid Res. 1999 Dec;40(12):2244-54.
Phytanoyl-CoA hydroxylase (PhyH) catalyzes the conversion of phytanoyl-CoA to 2-hydroxyphytanoyl-CoA, which is the first step in the phytanic acid alpha-oxidation pathway. Recently, several studies have shown that in humans, phytanic acid alpha-oxidation is localized in peroxisomes. In rat, however, the alpha-oxidation pathway has been reported to be mitochondrial. In order to clarify this differential subcellular distribution, we have studied the rat PhyH protein. We have purified PhyH from rat liver to apparent homogeneity as judged by SDS-PAGE. Sequence analysis of two PhyH peptide fragments allowed cloning of the rat PHYH cDNA encoding a 38. 6 kDa protein. The deduced amino acid sequence revealed strong homology to human PhyH including the presence of a peroxisome targeting signal type 2 (PTS2). Heterologous expression of rat PHYH in Saccharomyces cerevisiae yielded a 38.6 kDa protein whereas the PhyH purified from rat liver had a molecular mass of 35 kDa. This indicates that PhyH is probably processed in rat by proteolytic removal of a leader sequence containing the PTS2. This type of processing has been reported in several other peroxisomal proteins that contain a PTS2. Subcellular localization studies using equilibrium density centrifugation showed that PhyH is indeed a peroxisomal protein in rat. The finding that PhyH is peroxisomal in both rat and humans provides strong evidence against the concept of a differential subcellular localization of phytanic acid alpha-oxidation in rat and human.
植烷酰辅酶A羟化酶（PhyH）催化植烷酰辅酶A转化为2-羟基植烷酰辅酶A，这是植烷酸α-氧化途径的第一步。最近，多项研究表明，在人类中，植烷酸α-氧化定位于过氧化物酶体。然而，在大鼠中，α-氧化途径据报道定位于线粒体。为了阐明这种亚细胞定位差异，我们研究了大鼠PhyH蛋白。我们从大鼠肝脏中纯化出PhyH，经SDS-PAGE判断达到了表观均一性。对两个PhyH肽段的序列分析使得编码一种38.6 kDa蛋白的大鼠PHYH cDNA得以克隆。推导的氨基酸序列显示与人类PhyH有很强的同源性，包括存在2型过氧化物酶体靶向信号（PTS2）。大鼠PHYH在酿酒酵母中的异源表达产生了一种38.6 kDa的蛋白，而从大鼠肝脏中纯化的PhyH分子量为35 kDa。这表明PhyH在大鼠中可能通过蛋白水解去除包含PTS2的前导序列而进行了加工。在其他几种含有PTS2的过氧化物酶体蛋白中也报道过这种加工类型。使用平衡密度离心法进行的亚细胞定位研究表明，PhyH在大鼠中确实是一种过氧化物酶体蛋白。PhyH在大鼠和人类中都是过氧化物酶体蛋白这一发现为反对大鼠和人类植烷酸α-氧化存在亚细胞定位差异这一概念提供了有力证据。
