RECOMBINANT HUMAN-MILK BILE-SALT-STIMULATED LIPASE - FUNCTIONAL-PROPERTIES ARE RETAINED IN THE ABSENCE OF GLYCOSYLATION AND THE UNIQUE PROLINE-RICH REPEATS
RECOMBINANT HUMAN-MILK BILE-SALT-STIMULATED LIPASE - FUNCTIONAL-PROPERTIES ARE RETAINED IN THE ABSENCE OF GLYCOSYLATION AND THE UNIQUE PROLINE-RICH REPEATS
复制标题
DOI：
10.1111/j.1432-1033.1995.tb20327.x
复制
发表时间：
1995-03-15
期刊：
影响因子：
--
通讯作者：
HERNELL, O
中科院分区：
文献类型：
--
作者：
BLACKBERG, L;STROMQVIST, M;HERNELL, O
人乳胆盐刺激的脂肪酶可确保母乳喂养婴儿乳脂的有效利用。多肽链的N端三分之二高度保守，与典型的酯酶有惊人的相似之处。相反，剩余的C末端部分由一个独特的序列组成，该序列由16个富含Pro的O-糖基化重复序列组成，每个重复序列11个残基。最近，我们可以利用重组脂肪酶变体证明，这些重复序列和单一的N-连接糖链对催化效率都不是必不可少的。在这项研究中，我们报告了糖基化的重要性以及对其他重要功能特性的独特重复，如胆盐激活、肝素结合、热稳定性、低pH稳定性和对蛋白水解性失活的抵抗。与天然酶相比，两种哺乳动物细胞产生的重组全长脂肪酶在糖基化模式上略有不同，对功能性质没有影响。此外，缺少所有重复序列的变体和最后一个重复序列之后的C末端尾巴显示出与纯化的天然乳酶相同的功能特性。因此，所有典型的和重要的功能性质的结构基础都存在于N末端保守的部分，尽管这些性质都不是典型的酯酶所共有的。然而，我们可以证明，C-末端重复序列是该酶在尺寸排除层析中的异常行为的原因，导致表观分子质量比预期的要高得多。
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.