Purification, characterization, and specificity of dextranase inhibitor (Dei) expressed from Streptococcus sobrinus UAB108 gene cloned in Escherichia coli

Abstract

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 the 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.The mutans group strains of streptococci are capable of converting dietary sucrose to water-soluble glucan (WSG) and water-insoluble glucan (WIG) (9,20,22,26). These glucans, particularly the latter, are the principal materials for the development of human dental plaque and subsequent caries formation (2, 22, 51, 58). There are several enzymes and proteins involved in sucrose metabolism, such as glucosyltransferases (GTFs), which include WSG synthase (GTF-S) (18,19,33,35) and WIG synthase (GTF-I) (2,15,26,29), glucan-binding protein (GBP) (34,40,42), and dextranase (Dex), responsible for the cleavage of ␣-1,6 linkages in glucan molecules (5,17,45). All of these proteins exist simultaneously in mutans group strains of streptococci. The functions and relationships among them, as well as their contributions to sucrose metabolism, have been a focus of research of mechanisms of cariogenicity by the mutans group of streptococci. In just a few years, the introduction of a molecular genetic approach to the study of glucan metabolism and related proteins in oral streptococci has contributed much information. Nevertheless, there remain many unsolved questions. Since 1982, another protein (Dex inhibitor [Dei]) in oral streptococci ...

Microbial Dextran-Hydrolyzing Enzymes: Fundamentals and Applications

“…This finding suggests that Dei exists in some serotypes of mutans group of streptococci and participates in sucrose metabolism through its interaction with dextranase (201).…”

Sugar metabolism by mutans streptococci

“…The points of cleavage have not been determined but it seems idea that GTF have a modular structure and this is supported by the fact that the Dei dextranase inhibitor also contains likely that the multiple forms result from sequential removal of C-terminal stretches of amino acids. However, the role of homologous repeat units (Sun et al 1995). Furthermore, homologous sequences are found in proteins from other the non-enzymatic C-terminal regions of these large molecules remains an enigma.…”

Glucan-binding Proteins of the Oral Streptococci

“…At high growth rates, S. mutans synthesizes predominantly water-insoluble glucan that binds Dei and suppresses its inhibitor activity. Any free Dei is bound to dextranase in enzyme-inhibitor complexes, but dextranase remains in excess (Sun et al, 1995;Shaw et al, 1997). While the pathogenic mechanisms of the MS for humans or animals are generally thought to be similar, the various numbers and types of GTFs and GBPs highlight the unique biology of each species.…”