Eydoux Cécilia, De Caro Josiane, Ferrato Francine, Boullanger Paul, Lafont Dominique, Laugier René, Carrière Frédéric, De Caro Alain
Laboratoire d'Enzymologie Interfaciale et de Physiologie de la Lipolyse, Centre National de la Recherche Scientifique-Institut de Biologie Structurale et Microbiologie, 13402 Marseille Cedex 20, France.
J Lipid Res. 2007 Jul;48(7):1539-49. doi: 10.1194/jlr.M600486-JLR200. Epub 2007 Mar 30.
Recombinant human pancreatic lipase-related protein 2 (rHPLRP2) was produced in the protease A-deficient yeast Pichia pastoris. A major protein with a molecular mass of 50 kDa was purified from the culture medium using SP-Sepharose and Mono Q chromatography. The protein was found to be highly sensitive to the proteolytic cleavage of a peptide bond in the lid domain. The proteolytic cleavage process occurring in the lid affected both the lipase and phospholipase activities of rHPLRP2. The substrate specificity of the nonproteolyzed rHPLRP2 was investigated using pH-stat and monomolecular film techniques and various substrates (glycerides, phospholipids, and galactolipids). All of the enzyme activities were maximum at alkaline pH values and decreased in the pH 5-7 range corresponding to the physiological conditions occurring in the duodenum. rHPLRP2 was found to act preferentially on substrates forming small aggregates in solution (monoglycerides, egg phosphatidylcholine, and galactolipids) rather than on emulsified substrates such as triolein and diolein. The activity of rHPLRP2 on monogalactosyldiglyceride and digalactosyldiglyceride monomolecular films was determined and compared with that of guinea pig pancreatic lipase-related protein 2, which shows a large deletion in the lid domain. The presence of a full-length lid domain in rHPLRP2 makes it possible for enzyme activity to occur at higher surface pressures. The finding that the inhibition of nonproteolyzed rHPLRP2 by tetrahydrolipstatin and diethyl-p-nitrophenyl phosphate does not involve any bile salt requirements suggests that the rHPLRP2 lid adopts an open conformation in aqueous media.
重组人胰腺脂肪酶相关蛋白2（rHPLRP2）是在缺乏蛋白酶A的毕赤酵母中产生的。使用SP-琼脂糖凝胶和Mono Q色谱从培养基中纯化出一种分子量为50 kDa的主要蛋白质。发现该蛋白质对盖子结构域中肽键的蛋白水解切割高度敏感。盖子结构域中发生的蛋白水解切割过程影响了rHPLRP2的脂肪酶和磷脂酶活性。使用pH计和单分子膜技术以及各种底物（甘油酯、磷脂和半乳糖脂）研究了未被蛋白水解的rHPLRP2的底物特异性。所有酶活性在碱性pH值下最高，在pH 5-7范围内降低，该范围对应于十二指肠中发生的生理条件。发现rHPLRP2优先作用于在溶液中形成小聚集体的底物（甘油单酯、蛋黄卵磷脂和半乳糖脂），而不是作用于诸如三油酸甘油酯和二油酸甘油酯等乳化底物。测定了rHPLRP2对单半乳糖基二甘油酯和二半乳糖基二甘油酯单分子膜的活性，并与豚鼠胰腺脂肪酶相关蛋白2的活性进行了比较，后者在盖子结构域中有一个大的缺失。rHPLRP2中全长盖子结构域的存在使得酶活性能够在更高的表面压力下发生。四氢脂抑素和对硝基苯磷酸二乙酯对未被蛋白水解的rHPLRP2的抑制不涉及任何胆汁盐需求，这一发现表明rHPLRP2的盖子在水性介质中采用开放构象。
