Hardt Birgit, Völker Christof, Mundt Susanne, Salska-Navarro Marta, Hauptmann Michaela, Bause Ernst
Institut für Physiologische Chemie, Universität Bonn, Nussallee 11, 53115 Bonn, Germany.
Biochimie. 2005 Feb;87(2):169-79. doi: 10.1016/j.biochi.2004.11.004.
The cDNA for human endo-alpha1,2-mannosidase was reconstructed using two independent EST-clones and its properties characterized. The 2837 bp cDNA construct contained a 1389 bp open reading frame (ORF) encoding for 462 amino acids and an approximately 53.6 kDa protein, respectively. Hydrophobicity analysis of this amino acid sequence, as well as proteolytic degradation studies, indicate that the enzyme is a type II protein, anchored in the membrane via a 19 amino-acid long apolar sequence close to the N-terminus. Human endo-alpha1,2-mannosidase displays a high degree of sequence identity with the catalytic domain of the homologous rat liver endo-enzyme, but differs substantially in the N-terminal peptide region, which includes the transmembrane domain. No sequence similarity exists with other processing alpha-glycosidases. Based on sequence information provided by the 2837 bp construct, the cDNA consisting of the complete 1389 bp ORF was amplified by RT-PCR using human fibroblast RNA. Incubation of E. coli lysates with this cDNA, previously modified for boost translation by codon optimization, resulted in the synthesis of an approximately 52 kDa protein which degraded [(14)C]Glc(3)-Man(9)-GlcNAc(2) efficiently, indicating that the catalytic domain of the enzyme folds correctly under cell-free conditions. Transfection of the endo-alpha1,2-mannosidase wild-type cDNA into COS 1 cells resulted in a moderate (approximately 1.5-fold) but reproducible increase of activity compared with control cells, whereas >18-fold increase in activity was measured after expression of a chimera containing green-fluorescent-protein (GFP) attached to the N-terminus of the endo-alpha1,2-mannosidase polypeptide. This, together with the observation that GFP-endo-alpha1,2-mannosidase is expressed as a Golgi-resident type II protein, points to enzyme-specific parameters directing folding and membrane anchoring, as well as Golgi-targeting, not being affected by fusion of GFP to the endo-alpha1,2-mannosidase N-terminus.
利用两个独立的EST克隆重建了人内切α1,2-甘露糖苷酶的cDNA，并对其特性进行了表征。这个2837 bp的cDNA构建体包含一个1389 bp的开放阅读框（ORF），分别编码462个氨基酸和一个约53.6 kDa的蛋白质。对该氨基酸序列进行的疏水性分析以及蛋白水解降解研究表明，该酶是一种II型蛋白，通过靠近N端的19个氨基酸长的非极性序列锚定在膜上。人内切α1,2-甘露糖苷酶与同源大鼠肝脏内切酶的催化结构域具有高度的序列同一性，但在包括跨膜结构域的N端肽区域有很大差异。与其他加工性α-糖苷酶不存在序列相似性。基于2837 bp构建体提供的序列信息，使用人成纤维细胞RNA通过RT-PCR扩增了由完整的1389 bp ORF组成的cDNA。用先前通过密码子优化进行增强翻译修饰的该cDNA孵育大肠杆菌裂解物，导致合成了一种约52 kDa的蛋白质，该蛋白质能有效降解[(14)C]Glc(3)-Man(9)-GlcNAc(2)，表明该酶的催化结构域在无细胞条件下能正确折叠。将内切α1,2-甘露糖苷酶野生型cDNA转染到COS 1细胞中，与对照细胞相比，活性有适度（约1.5倍）但可重复的增加，而在表达一种在内切α1,2-甘露糖苷酶多肽N端连接有绿色荧光蛋白（GFP）的嵌合体后，活性增加了>18倍。这一点，连同GFP-内切α1,2-甘露糖苷酶作为高尔基体驻留II型蛋白表达的观察结果，表明指导折叠、膜锚定以及高尔基体靶向的酶特异性参数不受GFP与内切α1,2-甘露糖苷酶N端融合的影响。
