title: 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 β-chain of MSP (MSPβ). MSP binding to the RON receptor tyrosine kinase activates signaling pathways involved in the inflammatory response. We have purified wild-type and mutant MSPβ proteins and compared biochemical and biophysical properties that might impact the MSP/RON signaling pathway. Surface plasmon resonance (SPR) binding studies showed that MSPβ R689C affinity to RON is approximately 10-fold lower than that of the wild-type MSPβ and differential scanning fluorimetry (DSF) showed that the thermal stability of the mutant MSPβ was slightly lower than that of wild-type MSPβ, 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.
authors.affiliation: Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, Maryland, United States of America.
authors.forename: Natalia
authors.initials: N
authors.lastname: Gorlatova
authors.forename: Kinlin
authors.initials: K
authors.lastname: Chao
authors.forename: Lipika R
authors.initials: LR
authors.lastname: Pal
authors.forename: Rawan Hanna
authors.initials: RH
authors.lastname: Araj
authors.forename: Andrey
authors.initials: A
authors.lastname: Galkin
authors.forename: Illarion
authors.initials: I
authors.lastname: Turko
authors.forename: John
authors.initials: J
authors.lastname: Moult
authors.forename: Osnat
authors.initials: O
authors.lastname: Herzberg
mesh_terms: D019943:Amino Acid Substitution; D003093:Colitis, Ulcerative; D003424:Crohn Disease / Q000235:genetics*; D015536:Down-Regulation; D017228:Hepatocyte Growth Factor / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D050505:Mutant Proteins / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide; D011485:Protein Binding / Q000235:genetics; D055550:Protein Stability; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics
chemical_list: D050505:Mutant Proteins; D011518:Proto-Oncogene Proteins; C082332:macrophage stimulating protein; D017228:Hepatocyte Growth Factor; C080835:RON protein; D020794:Receptor Protein-Tyrosine Kinases
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