title: Yersinia pestis YopM: thrombin binding and overexpression.
abstract: In previous studies, Yersinia pestis YopM has been shown through mutational analysis to be necessary for virulence in mice and found to have homology with the thrombin-binding domain of the platelet receptor GPIb alpha. In this study, YopM was purified and shown by dot blot and chemical cross-linking tests to bind to human alpha-thrombin. No cross-linked product could be detected when human prothrombin was incubated with YopM. As a functional test of thrombin binding, it was shown that native but not boiled YopM inhibits thrombin-induced aggregation of human platelets. Control tests showed that YopM did not inactivate the platelets themselves, nor was its effect a nonspecific consequence of its very acidic isoelectric point. Microsequencing of YopM revealed an intact N terminus, indicating that functional YopM is not processed at the N terminus or secreted by a mechanism involving a cleavable signal sequence. Further characterization was made of an interesting effect on yopM expression that had been noticed in a previous study. A 1.5-kb HaeIII subclone overexpressed YopM in both Y. pestis and Escherichia coli compared with a larger clone containing the 5.3-kb HindIII-F fragment. To search for a possible regulator of YopM expression, the HindIII-F fragment was sequenced, revealing several open reading frames and three large repeated sequences. Deletional analysis showed that these were not involved in regulation of yopM. The data implicated a DNA structure 5' to yopM in moderating yopM expression.
authors.affiliation: Department of Microbiology and Immunology, Chandler Medical Center, University of Kentucky, Lexington 40536-0084.
authors.forename: B S
authors.initials: BS
authors.lastname: Reisner
authors.forename: S C
authors.initials: SC
authors.lastname: Straley
mesh_terms: D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D006801:Humans; D008969:Molecular Sequence Data; D010974:Platelet Aggregation; D013917:Thrombin / Q000378:metabolism*; D015010:Yersinia pestis / Q000378:metabolism*
chemical_list: D001425:Bacterial Outer Membrane Proteins; C060772:yopM protein, Yersinia; D013917:Thrombin