0000000000179727
AUTHOR
Michael Moos
Cellular UDP-Glucose Deficiency Caused by a Single Point Mutation in the UDP-Glucose Pyrophosphorylase Gene
We previously isolated a mutant cell that is the only mammalian cell reported to have a persistently low level of UDP-glucose. In this work we obtained a spontaneous revertant whose UDP-glucose level lies between those found in the wild type and the mutant cell. The activity of UDP-glucose pyrophosphorylase (UDPG:PP), the enzyme that catalyzes the formation of UDP-glucose, was in the mutant 4% and in the revertant 56% of the activity found in the wild type cell. Sequence analysis of UDPG: PP cDNAs from the mutant cell showed one missense mutation, which changes amino acid residue 115 from glycine to aspartic acid. The substituted glycine is located within the largest stretch of strictly con…
Purification and evaluation of large clostridial cytotoxins that inhibit small GTPases of Rho and ras subfamilies
Publisher Summary This chapter discusses the purification and evaluation of large clostridia cytotoxins (LCTs) that inhibit small guanosine 5'-triphosphates (GTPases) of Rho and Ras subfamilies. LCTs are glycosyltransferases that inactivate GTPases of the Rho and Ras subfamilies by covalently coupling a sugar moiety (mostly glucose) to the conserved threonine residue in region switch 1 of the GTPases (T35 in Ras). This glycosylation functionally inactivates the GTPases leading to the collapse of the actin cytoskeleton and ultimately induces apoptosis of the cells. Small GTP-binding proteins are key players in the regulation of signal transducing networks of eukaryotic cells. Their regulator…
Variant toxin B and a functional toxin A produced by Clostridium difficile C34.
A particular property of Clostridium difficile strain C34 is an insertion of approximately 2 kb in the tcdA-C34 gene that does not hinder expression of a fully active TcdA-C34 molecule. Intoxication with TcdA-C34 induced an arborized appearance in eukaryotic cells (D-type cytopathic effect); intoxication with TcdB-C34 induced a spindle-like appearance of cells (S-type cytopathic effect). Inactivation of GTPases with purified toxins revealed that Rho, Rac, Cdc42, and Rap are substrates of TcdA-C34. The variant cytotoxin TcdB-C34 inactivated Rho, Rac, Cdc42, Rap, Ral, and R-Ras. Hence, this is the first ‘S-type’ cytotoxin which inactivates both Rho and R-Ras, and is coexpressed with a ‘D-type…
Impact of amino acids 22-27 of Rho-subfamily GTPases on glucosylation by the large clostridial cytotoxins TcsL-1522, TcdB-1470 and TcdB-8864
Here we report data describing some principles of the interaction between small GTP-binding proteins and large Clostridial cytotoxins (LCTs). Our investigation was based on the differential glucosylation of Rac1 versus RhoA by LCTs TcsL-1522, TcdB-1470 and TcdB-8864. Chimeric RhoA/Rac1 proteins and GTPases mutated at defined regions or single amino acids were used as substrates. Starting with chimeric Rac/Rho proteins we demonstrated that proteins containing the N-terminal 73 amino acids of Rac1 (but not those of RhoA) were efficiently glucosylated. Within this stretch, three regions differ significantly in Rac1 and RhoA. Regions containing amino acids 41-45 and 50-54 had no effect on toxin…
Clostridium difficile IStron CdISt1: Discovery of a Variant Encoding Two Complete Transposase-Like Proteins
ABSTRACT Screening a Clostridium difficile strain collection for the chimeric element Cd ISt1 , we identified two additional variants, designated Cd ISt1 -0 and Cd ISt1 -III. In in vitro assays, we could prove the self-splicing ribozyme activity of these variants. Structural comparison of all known Cd ISt1 variants led us to define four types of IStrons that we designated Cd ISt1 -0 through Cd ISt1 -III. Since Cd ISt1 -0 encodes two complete transposase-like proteins (TlpA and TlpB), we suggest that it represents the original genetic element, hypothesized before to have originated by fusion of a group I intron and an insertion sequence element.
Delineation of the catalytic domain of Clostridium difficile toxin B-10463 to an enzymatically active N-terminal 467 amino acid fragment.
Abstract In an attempt to directly approach the postulated toxic domain of Clostridium difficile 's TcdB-10463, eight subclones of different size and locations in the N-terminal third of the toxin were generated. Expression of these toxin fragments was checked in Western blots and the enzymatic activity of the expressed proteins was analyzed by glucosylating Ras related small GTP-binding proteins. Two polypeptides of 875 aa (TcdBc1–3) and 557 aa (TcdBc1-H) glucosylated their targets Rho, Rac and Cdc42 with the same activity and specificity as the holotoxin. In comparison 516 aa (TcdBc1-N) and 467 aa (TcdBc1-A) protein fragments exhibited highly reduced activity, while Tcdc1 and TcdB2–3 (aa …
A chimeric ribozyme in Clostridium difficile combines features of group I introns and insertion elements
CdlSt1, a DNA insertion of 1975 bp, was identified within tcdA-C34, the enterotoxin gene of the Clostridium difficile isolate C34. Located in the catalytic domain A1-C34, Cd/St1 combines features of two genetic elements. Within the first 434 nt structures characteristic for group I introns were found; encoding the two transposase-like proteins tlpA and tlpB nucleotides 435-1975 represent the remainder of a IS605-like insertion element. We show that the entire CdlSt1 is accurately spliced from tcdA-C34 primary transcripts and that purified TcdA-C34 toxin is of regular size and catalytic activity. A search for CdlSt1-related sequences demonstrates that the element is widespread in toxinogenic…