Poster abstracts
Poster number 27 submitted by Christopher Schwebach
Identification and characterization of three categories of PLS3 mutations resulting in osteogenesis imperfecta
Christopher Schwebach (Chemistry and Biochemistry, Ohio State), Matthew Orchard (Chemistry and Biochemistry, Ohio State), Lucas Runyan (Chemistry and Biochemistry, Ohio State), Elena Kudryashova (Chemistry and Biochemistry, Ohio State)
Abstract:
Osteogenesis imperfecta (OI) results in fragile bones due to disruption of bone generation. Recently, several cases have been linked to mutations in plastin 3 (PLS3), an actin bundling protein. The role of PLS3 in bone formation is not well understood. Because of this, specific treatments for X-linked OI remain elusive. Of 24 identified mutations, five result in full-length protein with amino acid insertions (E249_A250insI-L and A253_L254insN) or substitutions (A368D, N446S, and L478P.) We purified each mutant and investigated their biochemical properties. All mutants retained the ability to bind F-actin and only L478P lost its ability to bundle actin. Both E249_A250insI-L and A368D were insensitive to Ca2+, a recognized inhibitor of PLS3 bundling activity. In contrast, A253_L254insN and N446S were more Ca2+-sensitive than wild type. Based on these findings we can classify OI causative mutations of PLS3 into four groups: bundling-incompetent (L478P), Ca2+-hyposensitive (E249_A250insI-L and A368D), Ca2+-hypersensitive (A253_L254insN and N446S), and PLS3-truncation/null. In agreement, transfection of each coding mutant into XTC fibroblasts and U2OS osteoblasts resulted in three major phenotypes. While wild type PLS3 was found in both the lamellipodia and adhesion complexes of XTC cells, Ca2+-hypersensitive and -hyposensitive mutants were localized primarily to lamellipodia or to the adhesion structures, respectively. In U2OS cells, Ca2+-hypersensitive mutants were more diffused in the cytoplasm, while Ca2+-hyposensitive mutants, in addition to strong co-localization with the adhesion complexes, induced formation of abnormal actin bundles. The bundling-incompetent L478P was defuse throughout the cytoplasm in both cell types. Future studies should be carried out to determine the effect of each of these categories on bone development.
Keywords: Plastin, Actin, Osteoporosis