Protein characterization of a candidate mechanism SNP for Crohn's disease: the macrophage stimulating protein R689C substitution.

ABSTRACT: High throughput genome wide associations studies (GWAS) are now identifying a large number of genome loci related to risk of common human disease. Each such locus presents a challenge in identifying the relevant underlying mechanism. Here we report the experimental characterization of a proposed causal single nucleotide polymorphism (SNP) in a locus related to risk of Crohn's disease and ulcerative colitis. The SNP lies in the MST1 gene encoding Macrophage Stimulating Protein (MSP), and results in an R689C amino acid substitution within the ?-chain of MSP (MSP?). MSP binding to the RON receptor tyrosine kinase activates signaling pathways involved in the inflammatory response. We have purified wild-type and mutant MSP? proteins and compared biochemical and biophysical properties that might impact the MSP/RON signaling pathway. Surface plasmon resonance (SPR) binding studies showed that MSP? R689C affinity to RON is approximately 10-fold lower than that of the wild-type MSP? and differential scanning fluorimetry (DSF) showed that the thermal stability of the mutant MSP? was slightly lower than that of wild-type MSP?, by 1.6 K. The substitution was found not to impair the specific Arg483-Val484 peptide bond cleavage by matriptase-1, required for MSP activation, and mass spectrometry of tryptic fragments of the mutated protein showed that the free thiol introduced by the R689C mutation did not form an aberrant disulfide bond. Together, the studies indicate that the missense SNP impairs MSP function by reducing its affinity to RON and perhaps through a secondary effect on in vivo concentration arising from reduced thermodynamic stability, resulting in down-regulation of the MSP/RON signaling pathway.

SUBMITTER: Gorlatova N 

PROVIDER: S-EPMC3210151 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature