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Structure and functional studies of bovine lactoperoxidase

牛乳过氧化物酶的结构和功能研究

基本信息

批准号：
11694189
负责人：
SHIMAZAKI Kei-ichi
金额：
$ 3.14万
依托单位：
HOKKAIDO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11694189/
关键词：
lactoperoxidase heme-binding anti-microbial activity recombinant protein milk protein secondary structure 二次講造

项目摘要

The cDNA encoding bovine lactoperoxidase has been expressed in CHO cells. The recombinant lactoperoxidase was secreted as an enzymatically active single chain molecule presenting two immunoreactive forms of 88 kDa and 82 kDa, differing by their glycosylation. recombinant lactoperoxidase exhibited the characteristic absorbance spectrum with a Soret peak at 413 nm. Biochemical properties of bovine lactoperoxidase isolated from milk and recombinant bovine lactoperoxidase expressed by CHO cells were compared. The natural and recombinant lactoperoxidases showed the same conformational features as determined by CD measurements. The α-helix, β-structure and unordered structure content was found to be 17.8%, 54.2% and 28.0% for the natural lactoperoxidase and 18.6%, 50.1% and 31.3% for the recombinant lactoperoxidase, respectively. The microenvironments of aromatic amino acid residues in both lactoperoxidases seemed to be the same, although the CD spectral band due to the Soret band differed slightly. A difference in the pH-dependent spectral changes of absorbance at 413 nm was observed. From a pepsin hydrolysate of lactoperoxidase, a heme-binding peptide was isolated by reverse-phase HPLC and its amino acid sequence was examined. Engineering of recombinant lactoperoxidase into a myeloperoxidase-like molecule was attempted by substituting Gln-376 by Met, a residue known to achieve covalent binding with the heme in myeloperoxidase. However, the resulting bovine lactoperoxidase mutant failed to acquire the peculiar absorbance spectrum and the chlorinating activity of myeloperoxidase, underlining the complex nature of interactions in the heme vicinity.

牛乳过氧化物酶cDNA已在CHO细胞中表达。重组乳过氧化物酶是一种具有酶活性的单链分子，具有88 kDa和82 kDa两种免疫反应形式，其糖基化不同。重组乳过氧化物酶在413 nm处有索雷特峰。比较了从牛奶中分离的牛乳过氧化物酶和CHO细胞表达的重组牛乳过氧化物酶的生化特性。经CD测定，天然和重组乳过氧化物酶具有相同的构象特征。α-螺旋结构、β-结构和无序结构含量分别为天然乳过氧化物酶的17.8%、54.2%和28.0%，重组乳过氧化物酶的18.6%、50.1%和31.3%。两种乳酸过氧化物酶中芳香氨基酸残基的微环境似乎是相同的，尽管由于Soret带的CD光谱带略有不同。在413 nm处观察到吸光度随ph变化的光谱变化。用反相高效液相色谱法从乳酸过氧化物酶的胃蛋白酶水解产物中分离出一段血红素结合肽，并测定其氨基酸序列。通过用已知与髓过氧化物酶中的血红素实现共价结合的残基Met取代Gln-376，试图将重组乳过氧化物酶改造成髓过氧化物酶样分子。然而，由此产生的牛乳过氧化物酶突变体未能获得髓过氧化物酶特有的吸收光谱和氯化活性，强调了血红素附近相互作用的复杂性。