Purification, characterization, and specificity of dextranase inhibitor (Dei) expressed from Streptococcus sobrinus UAB108 gene cloned in Escherichia coli.
The dextranase inhibitor gene (dei) from Streptococcus sobrinus UAB108 was previously cloned, expressed, and sequenced. Its gene product (Dei) has now been purified as a single band with apparent molecular mass of 43 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The specific activity of Dei increased 121-fold upon purification. Most Dei activity (91.2%) was located in the periplasmic fraction from recombinant Escherichia coli cells. Dei competitively inhibits dextranase (Dex). This competitive inhibition mechanism has been further shown by detection and recovery of the intermediate enzyme-inhibitor (Dex-Dei) complex by gel filtration technology using fast protein liquid chromatography. Calibration of their molecular masses indicated that native Dei exists as a tetramer, Dex exists as dimer, and the Dex-Dei complex consists of two Dex molecules with two Dei molecules. Deletion analysis indicates that the intact Dei molecule is essential for Dei activity but not for glucan binding and immune cross-reaction. Dei is a special kind of glucan-binding protein with ability to inhibit Dex with high specificity. It can inhibit endogenous Dex, which can make more branches in glucan with the cooperation of the glucosyltransferase GTF-I. This inhibition cause the accumulation of water-soluble glucan. The latter reaction product can inhibit plaque formation and adherence of the mutans group of streptococcal cells. Dei derived from S. sobrinus UAB108 can inhibit only Dex from S. sobrinus (serotypes d and g), S. downei (previously S. sobrinus, serotype h), and S. macacae (serotype h). This finding suggests that Dei is another important protein existing in some serotypes of the mutans group of streptococci which participates in sucrose metabolism through its interaction with Dex.
Sun
J W
JW
Department of Biology, Washington University, St. Louis, Missouri 63130, USA.
Wanda
S Y
SY
Curtiss
R
R
D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification; D003910:Dextranase / Q000037:antagonists & inhibitors*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D037102:Lectins; D012680:Sensitivity and Specificity; D016986:Streptococcus sobrinus / Q000737:chemistry / Q000235:genetics*; D013395:Sucrose / Q000378:metabolism
D001426:Bacterial Proteins; D002352:Carrier Proteins; D037102:Lectins; C090543:dei protein, Streptococcus sobrinus; C037937:glucan-binding proteins; D013395:Sucrose; D003910:Dextranase