Eldering J A, Grünberg J, Hahn D, Croes H J, Fransen J A, Sterchi E E
Institute of Biochemistry and Molecular Biology, and Department of Pediatrics, University of Berne, Switzerland.
Eur J Biochem. 1997 Aug 1;247(3):920-32. doi: 10.1111/j.1432-1033.1997.00920.x.
N-Benzoyl-L-tyrosyl-p-aminobenzoic acid hydrolase (PPH, human meprin), is a peptidase found in the microvillus membrane of human small intestinal epithelial cells. PPH belongs to the astacin family of zinc-metalloendopeptidases and is a protein complex composed of two glycosylated subunits, alpha and beta. The present report describes the cloning of the complete beta subunit and the remaining N2-terminal end of the alpha subunit for analysis of their primary structures in addition to the examination of their biogenesis in transfected cell cultures. The complete open reading frame of the PPH beta cDNA translates into 700 amino acid residues compared with 746 residues for the PPH alpha cDNA. The primary structure of beta and alpha subunits are 44% identical and 61% similar. As predicted from their primary structure, the two subunits of PPH have identical modular structures; starting at the N2-terminus both contain a signal peptide, a propeptide, a protease domain containing the astacin signature, a meprin A5 protein tyrosine phospatase mu (MAM) and a meprin and TRAF homology domain (MATH) domain, an epidermal growth factor(EGF)-like domain, a putative transmembrane anchor domain and a short cytosolic tail. Pulse/chase labelling and immuno-Gold electronmicroscopy of recombinant PPH beta and alpha subunits expressed in transfected Madin-Darby canine kidney (MDCK) cells show that post-translational processing and transport of the two subunits are very different. When expressed alone, the beta subunit acquired complex glycan residues, readily formed homodimers and was transported to the plasma membrane. Small amounts of PPH beta were found in the culture medium. In contrast, the cell-bound alpha subunit, when expressed alone, remained primarily in the high-mannose form, was aggregated and not expressed at the cell surface. However, the bulk of mostly endo-beta-N-acetylglucosaminidase H-resistant alpha subunit was found in the filtered culture medium. The proteolytic event that leads to the formation of this soluble transport-competent form occurs in the endoplasmic reticulum (ER). Coexpression of the alpha subunit with the beta subunit allowed the localisation of the alpha subunit to the plasma membrane. These studies indicate that assembly of the two subunits of PPH is required for the localisation of the alpha subunit to the plasma membrane. In contrast to rodent meprin, both PPH subunits are apically secreted from MDCK cells.
N-苯甲酰-L-酪氨酰-对氨基苯甲酸水解酶（PPH，人膜连蛋白）是一种在人小肠上皮细胞微绒毛膜中发现的肽酶。PPH属于锌金属内肽酶的虾红素家族，是一种由两个糖基化亚基α和β组成的蛋白质复合物。本报告描述了完整β亚基和α亚基剩余N2末端的克隆，用于分析它们的一级结构，并在转染细胞培养物中研究它们的生物合成。PPH β cDNA的完整开放阅读框翻译成700个氨基酸残基，而PPH α cDNA为746个残基。β亚基和α亚基的一级结构44%相同，61%相似。从它们的一级结构预测，PPH的两个亚基具有相同的模块化结构；从N2末端开始，两者都包含一个信号肽、一个前肽、一个含有虾红素特征的蛋白酶结构域、一个膜连蛋白A5蛋白酪氨酸磷酸酶μ（MAM）和一个膜连蛋白与肿瘤坏死因子受体相关因子同源结构域（MATH）结构域、一个表皮生长因子（EGF）样结构域、一个假定的跨膜锚定结构域和一个短的胞质尾巴。对在转染的Madin-Darby犬肾（MDCK）细胞中表达的重组PPH β和α亚基进行脉冲/追踪标记和免疫金电子显微镜观察表明，两个亚基的翻译后加工和转运非常不同。单独表达时，β亚基获得复杂的聚糖残基，容易形成同二聚体并转运到质膜。在培养基中发现少量的PPH β。相反，单独表达时，细胞结合的α亚基主要保持高甘露糖形式，聚集且不在细胞表面表达。然而，在过滤后的培养基中发现了大部分对内切β-N-乙酰葡糖胺糖苷酶H有抗性的α亚基。导致形成这种可溶的、具有转运能力形式的蛋白水解事件发生在内质网（ER）中。α亚基与β亚基共表达使α亚基能够定位到质膜。这些研究表明，PPH两个亚基的组装是α亚基定位到质膜所必需的。与啮齿动物膜连蛋白不同，PPH的两个亚基都从MDCK细胞顶端分泌。
