Characterization of recombinant β subunit of human MUC4 mucin (rMUC4β)
Abstract MUC4 is a transmembrane mucin expressed on various epithelial surfaces, including respiratory and gastrointestinal tracts, and helps in their lubrication and protection. MUC4 is also aberrantly overexpressed in various epithelial malignancies and functionally contributes to cancer developme...
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Nature Portfolio
2021-12-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-021-02860-5 |
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| author | Prakash G. Kshirsagar Mansi Gulati Wade M. Junker Abhijit Aithal Gaelle Spagnol Srustidhar Das Kavita Mallya Shailendra K. Gautam Sushil Kumar Paul Sorgen Krishan K. Pandey Surinder K. Batra Maneesh Jain |
| author_facet | Prakash G. Kshirsagar Mansi Gulati Wade M. Junker Abhijit Aithal Gaelle Spagnol Srustidhar Das Kavita Mallya Shailendra K. Gautam Sushil Kumar Paul Sorgen Krishan K. Pandey Surinder K. Batra Maneesh Jain |
| author_sort | Prakash G. Kshirsagar |
| collection | DOAJ |
| description | Abstract MUC4 is a transmembrane mucin expressed on various epithelial surfaces, including respiratory and gastrointestinal tracts, and helps in their lubrication and protection. MUC4 is also aberrantly overexpressed in various epithelial malignancies and functionally contributes to cancer development and progression. MUC4 is putatively cleaved at the GDPH site into a mucin-like α-subunit and a membrane-tethered growth factor-like β-subunit. Due to the presence of several functional domains, the characterization of MUC4β is critical for understanding MUC4 biology. We developed a method to produce and purify multi-milligram amounts of recombinant MUC4β (rMUC4β). Purified rMUC4β was characterized by Far-UV CD and I-TASSER-based protein structure prediction analyses, and its ability to interact with cellular proteins was determined by the affinity pull-down assay. Two of the three EGF-like domains exhibited typical β-fold, while the third EGF-like domain and vWD domain were predominantly random coils. We observed that rMUC4β physically interacts with Ezrin and EGFR family members. Overall, this study describes an efficient and simple strategy for the purification of biologically-active rMUC4β that can serve as a valuable reagent for a variety of biochemical and functional studies to elucidate MUC4 function and generating domain-specific antibodies and vaccines for cancer immunotherapy. |
| first_indexed | 2024-12-17T21:12:39Z |
| format | Article |
| id | doaj.art-13058b5ae1dd4ec7b5fd166b9d902f71 |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-12-17T21:12:39Z |
| publishDate | 2021-12-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-13058b5ae1dd4ec7b5fd166b9d902f712022-12-21T21:32:24ZengNature PortfolioScientific Reports2045-23222021-12-0111111310.1038/s41598-021-02860-5Characterization of recombinant β subunit of human MUC4 mucin (rMUC4β)Prakash G. Kshirsagar0Mansi Gulati1Wade M. Junker2Abhijit Aithal3Gaelle Spagnol4Srustidhar Das5Kavita Mallya6Shailendra K. Gautam7Sushil Kumar8Paul Sorgen9Krishan K. Pandey10Surinder K. Batra11Maneesh Jain12Department of Biochemistry and Molecular Biology, College of Medicine, University of Nebraska Medical CenterDepartment of Biochemistry and Molecular Biology, College of Medicine, University of Nebraska Medical CenterDepartment of Biochemistry and Molecular Biology, College of Medicine, University of Nebraska Medical CenterDepartment of Biochemistry and Molecular Biology, College of Medicine, University of Nebraska Medical CenterDepartment of Biochemistry and Molecular Biology, College of Medicine, University of Nebraska Medical CenterDepartment of Biochemistry and Molecular Biology, College of Medicine, University of Nebraska Medical CenterDepartment of Biochemistry and Molecular Biology, College of Medicine, University of Nebraska Medical CenterDepartment of Biochemistry and Molecular Biology, College of Medicine, University of Nebraska Medical CenterDepartment of Biochemistry and Molecular Biology, College of Medicine, University of Nebraska Medical CenterDepartment of Biochemistry and Molecular Biology, College of Medicine, University of Nebraska Medical CenterDepartment of Molecular Microbiology and Immunology, Saint Louis University Health Sciences CenterDepartment of Biochemistry and Molecular Biology, College of Medicine, University of Nebraska Medical CenterDepartment of Biochemistry and Molecular Biology, College of Medicine, University of Nebraska Medical CenterAbstract MUC4 is a transmembrane mucin expressed on various epithelial surfaces, including respiratory and gastrointestinal tracts, and helps in their lubrication and protection. MUC4 is also aberrantly overexpressed in various epithelial malignancies and functionally contributes to cancer development and progression. MUC4 is putatively cleaved at the GDPH site into a mucin-like α-subunit and a membrane-tethered growth factor-like β-subunit. Due to the presence of several functional domains, the characterization of MUC4β is critical for understanding MUC4 biology. We developed a method to produce and purify multi-milligram amounts of recombinant MUC4β (rMUC4β). Purified rMUC4β was characterized by Far-UV CD and I-TASSER-based protein structure prediction analyses, and its ability to interact with cellular proteins was determined by the affinity pull-down assay. Two of the three EGF-like domains exhibited typical β-fold, while the third EGF-like domain and vWD domain were predominantly random coils. We observed that rMUC4β physically interacts with Ezrin and EGFR family members. Overall, this study describes an efficient and simple strategy for the purification of biologically-active rMUC4β that can serve as a valuable reagent for a variety of biochemical and functional studies to elucidate MUC4 function and generating domain-specific antibodies and vaccines for cancer immunotherapy.https://doi.org/10.1038/s41598-021-02860-5 |
| spellingShingle | Prakash G. Kshirsagar Mansi Gulati Wade M. Junker Abhijit Aithal Gaelle Spagnol Srustidhar Das Kavita Mallya Shailendra K. Gautam Sushil Kumar Paul Sorgen Krishan K. Pandey Surinder K. Batra Maneesh Jain Characterization of recombinant β subunit of human MUC4 mucin (rMUC4β) Scientific Reports |
| title | Characterization of recombinant β subunit of human MUC4 mucin (rMUC4β) |
| title_full | Characterization of recombinant β subunit of human MUC4 mucin (rMUC4β) |
| title_fullStr | Characterization of recombinant β subunit of human MUC4 mucin (rMUC4β) |
| title_full_unstemmed | Characterization of recombinant β subunit of human MUC4 mucin (rMUC4β) |
| title_short | Characterization of recombinant β subunit of human MUC4 mucin (rMUC4β) |
| title_sort | characterization of recombinant β subunit of human muc4 mucin rmuc4β |
| url | https://doi.org/10.1038/s41598-021-02860-5 |
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