Recombinant Human‐Milk Bile‐Salt‐Stimulated Lipase
Recombinant Human‐Milk Bile‐Salt‐Stimulated Lipase
复制标题
重组人乳胆盐刺激脂肪酶
DOI：
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
O. Hernell
中科院分区：
文献类型：
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作者：
L. Bläckberg;M. Strömqvist;M. Edlund;K. Juneblad;L. Lundberg;L. Hansson;O. Hernell
人乳胆盐刺激的脂肪酶确保母乳喂养的婴儿有效利用乳脂。肽链的 N 端三分之二高度保守，与典型的酯酶表现出惊人的相似性。相比之下，剩余的 C 末端部分由 16 个富含脯氨酸的 O 糖基化重复序列组成，每个重复序列有 11 个残基。最近，我们可以使用重组脂肪酶变体证明，这些重复序列和单个 N 连接糖链对于催化效率都不是必需的。在本研究中，我们报告了糖基化的重要性的缺乏以及其他重要功能特性的独特重复，即胆汁盐活化、肝素结合、热稳定性、低pH下的稳定性和对蛋白水解失活的抵抗力。与天然酶相比，两种哺乳动物细胞系中产生的重组全长脂肪酶的糖基化模式略有不同，但对功能特性没有影响。此外，缺乏所有重复序列和最后一个重复序列之后的 C 末端尾部的变体表现出与纯化的天然乳酶相同的功能特征。因此，所有典型和功能上重要的特性的结构基础都存在于 N 端保守部分，尽管这些特性均不是典型酯酶所共有的。然而，我们可以证明，C 端重复是造成酶在尺寸排阻色谱中异常行为的原因，导致明显高于预期的表观分子量。
Human milk bile-salt-stimulated lipase ensures efficient utilization of milk lipid in breast-fed infants. The N-terminal two-thirds of the peptide chain is highly conserved and shows striking similarities to typical esterases. In contrast, the remaining C-terminal part consists of a unique sequence of 16 proline-rich O-glycosylated repeats of 11 residues each. Recently we could show, using recombinant lipase variants, that neither these repeats nor the single N-linked sugar chain are essential for catalytic efficiency. In the present study, we report on the lack of importance of glycosylation and the unique repeats for other important functional properties, i.e. bile-salt activation, heparin binding, heat stability, stability at low pH and resistance to proteolytic inactivation. Compared to native enzyme, recombinant full-length lipase produced in two mammalian cell lines differed slightly in glycosylation pattern with no effects on the functional properties. Moreover, a variant lacking all repeats and the C-terminal tail following the last repeat exhibited the same functional characteristics as purified native milk enzyme. Thus, the structural basis for all the typical and functionally important properties reside in the N-terminal conserved part, in spite of the fact that none of these properties are shared by typical esterases. We could however, demonstrate that the C-terminal repeats are responsible for the unusual behaviour of the enzyme in size-exclusion chromatography, resulting in a considerably higher than expected apparent molecular mass.
牛胰腺胆固醇酯酶/溶血磷脂酶的克隆。
DOI：
10.1016/0006-291x(89)91811-1
发表时间：
1989
影响因子：
3.1
作者：
Kyger,EM;Wiegand,RC;Lange,LG
通讯作者：
Lange,LG