Exploring and exploiting the molecular function of endosialin (CD248): a clinical biomarker in inflammation and cancer

Poyser, Callum (2014). Exploring and exploiting the molecular function of endosialin (CD248): a clinical biomarker in inflammation and cancer. University of Birmingham. M.Phil.

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Abstract

Endosialin (CD248) is a novel stromal cell surface receptor that has shown to play a role in both cancer and inflammation. Little is known about CD248’s structure, function and mechanism of action.

Deletion mutants of CD248 were generated by overlapping fusion PCR, which were then stably-transfected into MG63 cells, detected by immunofluorescence staining and flow cytometry, and analysed using a growth curve assay. A Drosophila S2 cell expression system was used to produce recombinant CD248 protein. Hanging drop crystallisation technique was used to generate protein crystals. Western blotting was used to determine the phosphorylation state of MAPK ERK1/2 in mouse osteoblasts, post-induction with platelet-derived growth factor BB (PDGF-BB).

Crystallisation screening of recombinant CD248 c-type lectin domain (CTLD) and ectodomain generated potential protein crystals for future x-ray crystallographic analysis. Growth curve analysis of CD248 cell surface-expressing MG63 cells demonstrated that the EGF repeat domain may be a key region of CD248 responsible for controlling the proliferation of MG63 cells. Primary osteoblasts from CD248-/- mice exhibited no response to stimulation with PDGF-BB, due to aberrant PDGF signal transduction in CD248-/- mice. This work provides a stepping stone to study and achieve a better understanding of CD248 structure, function and mechanism of action.

Type of Work:

Thesis (Masters by Research > M.Phil.)

Award Type:

Masters by Research > M.Phil.

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