Coexpression of novel furin-resistant LPL variants with lipase maturation factor 1 enhances LPL secretion and activity
LPL is a secreted enzyme that hydrolyzes triglycerides from circulating lipoproteins. Individuals lacking LPL suffer from severe hypertriglyceridemia, a risk factor for acute pancreatitis. One potential treatment is to administer recombinant LPL as a protein therapeutic. However, use of LPL as a pro...
| Main Authors: | , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2018-12-01
|
| Series: | Journal of Lipid Research |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S0022227520341705 |
| _version_ | 1818420111383461888 |
|---|---|
| author | Ming Jing Wu Anna Wolska Benjamin S. Roberts Ellis M. Pearson Aspen R. Gutgsell Alan T. Remaley Saskia B. Neher |
| author_facet | Ming Jing Wu Anna Wolska Benjamin S. Roberts Ellis M. Pearson Aspen R. Gutgsell Alan T. Remaley Saskia B. Neher |
| author_sort | Ming Jing Wu |
| collection | DOAJ |
| description | LPL is a secreted enzyme that hydrolyzes triglycerides from circulating lipoproteins. Individuals lacking LPL suffer from severe hypertriglyceridemia, a risk factor for acute pancreatitis. One potential treatment is to administer recombinant LPL as a protein therapeutic. However, use of LPL as a protein therapeutic is limited because it is an unstable enzyme that is difficult to produce in large quantities. Furthermore, these considerations also limit structural and biochemical studies that are needed for large-scale drug discovery efforts. We demonstrate that the yield of purified LPL can be dramatically enhanced by coexpressing its maturation factor, LMF1, and by introducing novel mutations into the LPL sequence to render it resistant to proteolytic cleavage by furin. One of these mutations introduces a motif for addition of an N-linked glycan to the furin-recognition site. Furin-resistant LPL has previously been reported, but is not commonly used. We show that our modifications do not adversely alter LPL's enzymatic activity, stability, or in vivo function. Together, these data show that furin-resistant LPL is a useful reagent for both biochemical and biomedical studies. |
| first_indexed | 2024-12-14T12:49:16Z |
| format | Article |
| id | doaj.art-c4ed15e75e3a431ba9946ec87c0c8a82 |
| institution | Directory Open Access Journal |
| issn | 0022-2275 |
| language | English |
| last_indexed | 2024-12-14T12:49:16Z |
| publishDate | 2018-12-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Journal of Lipid Research |
| spelling | doaj.art-c4ed15e75e3a431ba9946ec87c0c8a822022-12-21T23:00:43ZengElsevierJournal of Lipid Research0022-22752018-12-01591224562465Coexpression of novel furin-resistant LPL variants with lipase maturation factor 1 enhances LPL secretion and activityMing Jing Wu0Anna Wolska1Benjamin S. Roberts2Ellis M. Pearson3Aspen R. Gutgsell4Alan T. Remaley5Saskia B. Neher6Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NCLipoprotein Metabolism Laboratory, Translational Vascular Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MDDepartment of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NCDepartment of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NCDepartment of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NCLipoprotein Metabolism Laboratory, Translational Vascular Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MDTo whom correspondence should be addressed.; Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NCLPL is a secreted enzyme that hydrolyzes triglycerides from circulating lipoproteins. Individuals lacking LPL suffer from severe hypertriglyceridemia, a risk factor for acute pancreatitis. One potential treatment is to administer recombinant LPL as a protein therapeutic. However, use of LPL as a protein therapeutic is limited because it is an unstable enzyme that is difficult to produce in large quantities. Furthermore, these considerations also limit structural and biochemical studies that are needed for large-scale drug discovery efforts. We demonstrate that the yield of purified LPL can be dramatically enhanced by coexpressing its maturation factor, LMF1, and by introducing novel mutations into the LPL sequence to render it resistant to proteolytic cleavage by furin. One of these mutations introduces a motif for addition of an N-linked glycan to the furin-recognition site. Furin-resistant LPL has previously been reported, but is not commonly used. We show that our modifications do not adversely alter LPL's enzymatic activity, stability, or in vivo function. Together, these data show that furin-resistant LPL is a useful reagent for both biochemical and biomedical studies.http://www.sciencedirect.com/science/article/pii/S0022227520341705lipoprotein lipaseprotein purificationtrafficking |
| spellingShingle | Ming Jing Wu Anna Wolska Benjamin S. Roberts Ellis M. Pearson Aspen R. Gutgsell Alan T. Remaley Saskia B. Neher Coexpression of novel furin-resistant LPL variants with lipase maturation factor 1 enhances LPL secretion and activity Journal of Lipid Research lipoprotein lipase protein purification trafficking |
| title | Coexpression of novel furin-resistant LPL variants with lipase maturation factor 1 enhances LPL secretion and activity |
| title_full | Coexpression of novel furin-resistant LPL variants with lipase maturation factor 1 enhances LPL secretion and activity |
| title_fullStr | Coexpression of novel furin-resistant LPL variants with lipase maturation factor 1 enhances LPL secretion and activity |
| title_full_unstemmed | Coexpression of novel furin-resistant LPL variants with lipase maturation factor 1 enhances LPL secretion and activity |
| title_short | Coexpression of novel furin-resistant LPL variants with lipase maturation factor 1 enhances LPL secretion and activity |
| title_sort | coexpression of novel furin resistant lpl variants with lipase maturation factor 1 enhances lpl secretion and activity |
| topic | lipoprotein lipase protein purification trafficking |
| url | http://www.sciencedirect.com/science/article/pii/S0022227520341705 |
| work_keys_str_mv | AT mingjingwu coexpressionofnovelfurinresistantlplvariantswithlipasematurationfactor1enhanceslplsecretionandactivity AT annawolska coexpressionofnovelfurinresistantlplvariantswithlipasematurationfactor1enhanceslplsecretionandactivity AT benjaminsroberts coexpressionofnovelfurinresistantlplvariantswithlipasematurationfactor1enhanceslplsecretionandactivity AT ellismpearson coexpressionofnovelfurinresistantlplvariantswithlipasematurationfactor1enhanceslplsecretionandactivity AT aspenrgutgsell coexpressionofnovelfurinresistantlplvariantswithlipasematurationfactor1enhanceslplsecretionandactivity AT alantremaley coexpressionofnovelfurinresistantlplvariantswithlipasematurationfactor1enhanceslplsecretionandactivity AT saskiabneher coexpressionofnovelfurinresistantlplvariantswithlipasematurationfactor1enhanceslplsecretionandactivity |