Expression and purification of enzymatically active forms of the human lysyl oxidase-like protein 4
Expression and purification of enzymatically active forms of the human lysyl oxidase-like protein 4
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DOI：
10.1074/jbc.m308856200
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发表时间：
2003-12-26
影响因子：
4.8
通讯作者：
Kim, Y
中科院分区：
文献类型：
--
作者：
Kim, MS;Kim, SS;Kim, Y
赖氨酰氧化酶样蛋白 4 (LOXL4) 是赖氨酰氧化酶新兴家族的最新成员，其中几种被证明可充当铜依赖性胺氧化酶，催化细胞外基质蛋白中赖氨酸衍生的交联。除了LOX家族的特征结构域外，LOXL4还包含四个清道夫受体富含半胱氨酸的结构域，包括铜结合结构域、细胞因子受体样结构域和赖氨酰酪氨酰醌辅因子的残基。为了评估其胺氧化酶活性，我们使用大肠杆菌表达系统将 LOXL4 表达为在羧基末端附有六组氨酸残基的重组形式。重组蛋白用镍螯合亲和层析纯化，并通过逐步透析转化为酶活性形式。纯化的LOXL4蛋白对非肽基底物苯甲胺表现出0.022-0.032单位/mg的β-氨基丙腈抑制活性。这些结果表明，具有四个富含半胱氨酸的清道夫受体结构域的 LOXL4 也可能充当活性胺氧化酶。 LOXL4 的纯活性形式的可用性将极大地有助于与该胺氧化酶的底物特异性和晶体结构相关的功能研究，这将为 LOX 家族成员内的功能差异提供重要的见解。
The lysyl oxidase-like protein 4 (LOXL4) is the latest member of the emerging family of lysyl oxidases, several of which were shown to function as copper-dependent amine oxidases catalyzing lysine-derived cross-links in extracellular matrix proteins. LOXL4 contains four scavenger receptor cysteine-rich domains in addition to the characteristic domains of the LOX family, including the copper-binding domain, the cytokine receptor-like domain, and the residues of the lysyl-tyrosyl quinone cofactor. In an effort to assess its amine oxidase activity, we expressed LOXL4 as recombinant forms attached with hexa-histidine residues at the carboxyl terminus by using an Escherichia coli expression system. The recombinant proteins were purified with nickel-chelating affinity chromatography and converted into enzymatically active forms by stepwise dialysis. The purified LOXL4 proteins showed beta-aminopropionitrile-inhibitable activity of 0.022 - 0.032 units/mg toward a nonpeptidyl substrate, benzylamine. These results indicate that LOXL4, with the four scavenger receptor cysteine rich domains, may also function as an active amine oxidase. Availability of the pure and active forms of LOXL4 will be significantly helpful in functional studies related to substrate specificity and crystal structure of this amine oxidase, which should provide significant insights into functional differences within the LOX family members.