Identifying the Critical Amino Acids Within the Immunoglobulin Domain of Human Peroxidasin Necessary for Crosslinking of Basement Membranes

Undergraduate #57
Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology

Maya Gaines-Smith - Hampton University
Co-Author(s): Tanisha Maitre and Isi Ero-Tolliver, Hampton University, Hampton, VA

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Assembly and integrity of collagen IV networks is essential for the integrity of basement membranes. An integral component of collagen IV is the sulfilimine bond (S=N), between methionine-93 and hydroxylysine-211 within the NC1 domains. This bond was identified as important for stabilization of basement membrane networks. Peroxidasin has been identified as the multi-domain peroxidase responsible for catalyzing sulfilimine bond formation by using its peroxidase and n-terminus of the immunoglobulin (Ig) domain. Previously, we identified the peroxidase and immunoglobulin domain as the domains necessary for crosslinking the basement membrane. Therefore, our current work focuses on investigating and identifying the critical amino acids within the immunoglobulin domain of peroxidasin responsible for catalyzing sulfilimine bond formation. To this end, we are designing primers to introduce specific mutations into our target Ig site within peroxidasin. These mutants will later be transfected into HEK293T cells that are overlayed on uncrosslinked basement membrane generated by PFHR-9 cells. Using SDS-PAGE, western blots and commassie staining we will assess the amount of crosslinking to determine the critical amino acids of the immunoglobulin domain of peroxidsain that is necessary and sufficient for crosslinking the basement membrane. These findings will provide insight into the mechanism for crosslinking/sulfilimine bond formation within the basement membrane.

Not Submitted

Funder Acknowledgement(s): Funding provided by the National Science Foundation under cooperative agreement number HRD - 1238838

Faculty Advisor: Isi Ero-Tolliver, isi.erotolliver@hampontu.edu

Role: I am involved in the tissue culture aspect of this research, along with the design of DNA primers that will be utilized to generate the mutated peroxidasin.