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. ? ? ?
| Smith, Eliot T; Perry, Evan T; Sears, Megan B et al. (2014) Expression of recombinant human mast cell chymase with Asn-linked glycans in glycoengineered Pichia pastoris. Protein Expr Purif 102:69-75 |
| Smith, Eliot T; Johnson, David A (2013) Human enteropeptidase light chain: bioengineering of recombinants and kinetic investigations of structure and function. Protein Sci 22:577-85 |
| Singh, Sanjay K; Thirumalai, Avinash; Hammond Jr, David J et al. (2012) Exposing a hidden functional site of C-reactive protein by site-directed mutagenesis. J Biol Chem 287:3550-8 |
