Elsliger M A, Potier M
Hôpital Sainte-Justine, Département de Pédiatrie, Université de Montréal, Québec, Canada.
Proteins. 1994 Jan;18(1):81-93. doi: 10.1002/prot.340180110.
The deficiency of the lysosomal protective protein/carboxypeptidase L (CARB L) causes the lysosomal storage disorder, galactosialidosis, characterized by neuraminidase and beta-galactosidase deficiencies in patients' cells. The three enzymes form a complex inside the lysosome, and the neuraminidase and beta-galactosidase deficiencies are secondary to CARB L deficiency. Sequence similarity and common enzymological properties suggest that the protomeric tertiary structure of CARB L is conserved within a family of serine carboxypeptidases which includes the yeast carboxypeptidase Y, killer expression I gene product and several plant carboxypeptidases. We used this homology to build a model of the CARB L structure based on the recently published X-ray atomic coordinates of the wheat carboxypeptidase II (CPDW-II) which shares 32% primary structure identity with CARB L. Small insertions and deletions were accommodated into the model structure by energy minimization using the DREIDING II force field. The C alpha atomic coordinates of the final CARB L model have a RMS shift of 1.01 A compared to the corresponding conserved residues in the CPDW-II template structure. The correct orientation of the homologous catalytic triad residues Ser150, His429 and Asp392, the potential energy calculations and the distribution of hydrophobic and hydrophillic residues in the structure all support the validity of the CARB L model. Most missense mutations identified in galactosialidosis patients were located in secondary structural elements except for the Tyr211-->Asn mutation which is in a loop. The other mutant residues have their side chains deeply buried in the central beta-sheet of the model structure except for the Phe412-->Val mutation which is located in the dimer interface. The predicted effects of specific mutations on CARB L structural stability correlates well with recently published transient expression studies of mutant CARB L (Shimmoto, M. et al., J. Clin. Invest., 91:2393-2399, 1993).
溶酶体保护蛋白/羧肽酶L（CARB L）的缺乏会导致溶酶体贮积病——唾液酸贮积症，其特征是患者细胞中神经氨酸酶和β-半乳糖苷酶缺乏。这三种酶在溶酶体内形成一个复合物，神经氨酸酶和β-半乳糖苷酶的缺乏继发于CARB L的缺乏。序列相似性和共同的酶学性质表明，CARB L的原聚体三级结构在丝氨酸羧肽酶家族中是保守的，该家族包括酵母羧肽酶Y、杀伤表达I基因产物和几种植物羧肽酶。我们利用这种同源性，基于最近发表的与CARB L具有32%一级结构同一性的小麦羧肽酶II（CPDW-II）的X射线原子坐标，构建了CARB L结构模型。通过使用DREIDING II力场进行能量最小化，将小的插入和缺失纳入模型结构。最终的CARB L模型的Cα原子坐标与CPDW-II模板结构中相应的保守残基相比，RMS位移为1.01 Å。同源催化三联体残基Ser150、His429和Asp392的正确取向、势能计算以及结构中疏水和亲水残基的分布都支持CARB L模型的有效性。除了位于环中的Tyr211→Asn突变外，在唾液酸贮积症患者中鉴定出的大多数错义突变都位于二级结构元件中。除了位于二聚体界面的Phe412→Val突变外，其他突变残基的侧链都深埋在模型结构的中央β-折叠中。特定突变对CARB L结构稳定性的预测影响与最近发表的突变型CARB L的瞬时表达研究结果（Shimmoto, M.等人，《临床研究杂志》，91:2393 - 2399，1993）相关性良好。
