Human Cathepsin G: Expression, C-Terminal Processing and Dual Specificity

人组织蛋白酶 G：表达、C 端加工和双重特异性

• 批准号: 7195586
• 负责人: David Andrew Johnson
• 金额: $ 21.02万
• 依托单位: EAST TENNESSEE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7195586
• 关键词: Academic Research Enhancement Awards; Affect; Agonist; Amino Acids; Aneurysm; Anoikis; Apoptosis; Arthritis; Azurophilic Granule; Bacteria; Bacterial Infections; Binding; Blood Platelets; C-terminal; Cardiac Myocytes; Cardiomyopathies; Cardiovascular system; Cathepsin G; Cells; Cellular biology; Chymase; Cleaved cell; Cloning; Cytoplasmic Granules; DNA; Data; Disease; Elastases; Elements; Endopeptidases; Environment; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzyme Kinetics; Enzymes; Event; Failure; Family; Fermentation; Future; Generations; Goals; Green Fluorescent Proteins; Health; Human; Human Cell Line; Hypersensitivity; Inflammation; Invaded; Kinetics; Knowledge; Laboratories; Length; Leukocyte Elastase; Liposomes; Medical Students; Membrane; Methods; Microbiological Techniques; Mucous body substance; Mutate; Mutation; Natural Immunity; Neutropenia; Pancreatic Elastase; Peptide Hydrolases; Peptides; Pichia; Plasma; Play; Predisposition; Process; Progranulocytes; Proteins; Pulmonary Emphysema; Range; Reaction; Recombinants; Research; Research Personnel; Resistance; Role; Serine Protease; Single Nucleotide Polymorphism; Site-Directed Mutagenesis; Solid; Specificity; Structure; Structure-Activity Relationship; Students; Substrate Specificity; Techniques; Testing; Thinking; Training; Trypsin; Work; Yeasts; alanylglycine; arginyllysine; career; chymotrypsin; experience; human mast cell tryptase; improved; in vivo; inhibitor/antagonist; innovation; interest; killings; mast cell; medical schools; member; mutant; neuronal cell body; neutrophil; phenylalanylleucine; protein purification; response; trafficking

DESCRIPTION (provided by applicant): Human Cathepsin G (CatG) and human neutrophil elastase (HNE) play significant innate immunity roles via neutrophil killing of bacteria. Both serine proteases are stored as active enzymes in cytoplasmic granules. They also undergo an unusual processing step that removes carboxyl terminal peptides of 10 and 19 amino acids. In the case of HNE, failure to remove this peptide results in trafficking to the membrane and neutropenia. In Aim#1 these proteases will be expressed as active recombinant enzymes via secretion from the methanolic yeast Pichia pastoris, a proven approach for the expression of similar enzymes. Both full-length and C-terminally truncated forms will be produced. In Aim#2 these recombinant enzymes will be compared with native proteases isolated from neutrophils. Likewise, full-length and C-terminally truncated enzymes will be compared with respect to kinetics, reaction with inhibitors and binding to membranes and liposomes, because membrane binding is a possible function of the C-terminal extension. Cellular trafficking of full-length and truncated forms of CatG will be compared, using similar forms of HNE as controls. Full-length forms of the enzymes are also needed for future studies to investigate the mechanism of the unusual C-terminal processing event. Although the C- terminal extension on HNE has been shown to play a role in trafficking and binding to membranes, it is unknown whether the shorter C-terminal extension on CatG has similar or different function. In Aim#3 the dual specificity CatG will be studied. Although CatG is a member of the chymotrypsin family, it has been shown to also cleave trypsin-like substrates. This dual specificity is thought to be due to Glu226 in the S1 substrate binding pocket and this residue will be mutated to other amino acids to see if the dual specificity is affected. Expected results will open the door to further studies on the function of CatG in neutrophils and mast cells, while providing presently unavailable recombinant enzymes that will aid other researchers. Recombinant HNE will allow the generation of mutants resulting from single nucleotide polymorphisms, which might contribute to other diseases such as emphysema and arthritis. This AREA project provides students with experience in DNA cloning, microbiological techniques, cell biology, fermentation methods, protein purification, structure/function studies, enzyme/inhibitor reactions and enzyme kinetics. This laboratory has a solid record in the training of undergraduates, medical students and graduate students. Knowledge to be gained through this project will improve our understanding of neutrophil function and diseases, including susceptibility to bacterial infections, inflammation and neutropenia. This Academic Research Enhancement Award will allow undergraduate and graduate students to gain important research experience in a medical school environment that will prepare them for careers in scientific research. Two human protein degrading enzymes will be produced in yeast and studied to gain additional knowledge concerning an unusual processing event. These enzymes and the cells of the body that store them are known to play critical roles in our defenses to bacterial infection and in diseases, including allergies. Consequently, the knowledge and experiences to be gained will aid in our efforts to improve health.

描述(申请人提供)：人组织蛋白酶G(CATG)和人中性粒细胞弹性蛋白酶(HNE)通过中性粒细胞杀死细菌发挥重要的先天免疫作用。这两种丝氨酸蛋白酶都以活性酶的形式储存在细胞质颗粒中。它们还经历了一个不寻常的加工步骤，去除了10和19个氨基酸的羧基末端多肽。在HNE的情况下，不能去除这种多肽会导致转运到细胞膜和中性粒细胞减少。在目标#1中，这些蛋白酶将通过甲醇酵母巴斯德毕赤酵母的分泌物表达为活性重组酶，这是一种经过验证的表达类似酶的方法。将产生全长和C末端截断的表格。在Aim#2中，这些重组酶将与从中性粒细胞中分离的天然蛋白酶进行比较。同样，全长和C端截短的酶将在动力学、与抑制剂的反应以及与膜和脂质体的结合方面进行比较，因为膜结合可能是C端延伸的功能。将使用类似形式的HNE作为对照，比较全长和截短形式的CATG的细胞转运。未来的研究还需要全长形式的酶来研究不寻常的C末端加工事件的机制。尽管HNE上的C末端延伸已被证明在运输和与膜结合方面发挥作用，但尚不清楚CATG上较短的C末端延伸是否具有相似或不同的功能。在目标3中，我们将研究CATG的双重特异性。虽然CATG是胰凝乳酶家族的成员之一，但它也被证明可以裂解类胰蛋白酶底物。这种双重特异性被认为是由于S1底物结合口袋中的Glu226，这个残基将突变为其他氨基酸，看看双重特异性是否受到影响。预期的结果将为进一步研究CATG在中性粒细胞和肥大细胞中的功能打开大门，同时提供目前无法获得的重组酶，这将有助于其他研究人员。重组hNE将允许产生单核苷酸多态导致的突变，这可能导致其他疾病，如肺气肿和关节炎。这个领域的项目为学生提供了DNA克隆、微生物学技术、细胞生物学、发酵方法、蛋白质纯化、结构/功能研究、酶/抑制物反应和酶动力学方面的经验。该实验室在本科生、医学生和研究生培养方面有着坚实的记录。通过这个项目获得的知识将提高我们对中性粒细胞功能和疾病的了解，包括对细菌感染、炎症和中性粒细胞减少症的易感性。这个学术研究促进奖将允许本科生和研究生在医学院环境中获得重要的研究经验，这将为他们在科学研究领域的职业生涯做好准备。将在酵母中生产两种人类蛋白质降解酶，并对其进行研究，以获得关于一种不寻常的加工事件的更多知识。众所周知，这些酶和储存它们的身体细胞在我们防御细菌感染和包括过敏在内的疾病中发挥着关键作用。因此，将获得的知识和经验将有助于我们努力改善健康。

项目成果

Expression of recombinant human mast cell chymase with Asn-linked glycans in glycoengineered Pichia pastoris.

重组人肥大细胞糜酶与 Asn 连接聚糖在糖工程毕赤酵母中的表达。

• DOI: 10.1016/j.pep.2014.08.005
• 发表时间: 2014
• 期刊: Protein expression and purification
• 影响因子: 1.6
• 作者: Smith,EliotT;Perry,EvanT;Sears,MeganB;Johnson,DavidA
• 通讯作者: Johnson,DavidA

High yield expression in Pichia pastoris of human neutrophil elastase fused to cytochrome B5.

• DOI: 10.1016/j.pep.2023.106255
• 发表时间: 2023-02
• 期刊: Protein expression and purification
• 影响因子: 1.6
• 作者: E. Smith;M. Kruppa;David A. Johnson;J. Van Haeften;Xingchen Chen;Darren Leahy;Jonathan Peake;Jonathan M. Harris