Secretion kinetics of endo-N-acetyl-beta-D-glucosaminidase during vegetative growth of Myxococcus xanthus.

It was recently demonstrated that endo-N-acetyl-beta-D-glucosaminidases (ENGase) acting on N-glycosylproteins are produced by myxobacteria. In this study, it was shown that the secretion of ENGase during vegetative growth of Myxococcus xanthus was cell-density-dependent. The activity produced per cell increased up to 6 x 10(8) cells/ml and stabilized thereafter (maximum level). Two of the developmental mutants used in this study (bsgA and csgA) were locked for ENGase secretion into the maximum level regardless of cell density. To explain the pattern of ENGase secretion, we postulated the presence of a molecule that induces the enzyme until it reaches a proper concentration threshold. Although the chemical structure of this cell density signal was not determined during this study, its occurrence during vegetative growth of M. xanthus was strongly suggested by the results.

Are there biological functions for bacterial endo-N-acetyl-beta-D-glucosaminidases?

The endo-N-acetyl-beta-D-glucosaminidases (ENGase) acting on the N-N'-diacetylchitobiosyl core of N-glycosylproteins are essential reagents for the investigation of the structure and the functions of glycoproteins. These enzymes were largely studied with the aim of offering more tools with new and broader substrate specificities to the community of glycobiologist. Conversely, little attention was given to their potential role in the physiology of bacteria, even though it had been shown that ENGases are important enzymes for the physiology of animal and plant cells. In this brief review, we present the main characteristics of the bacterial ENGases and confine our discussion to biological aspects of their action in bacterial systems.

An endo-N-acetyl-beta-D-glucosaminidase, acting on the di-N-acetylchitobiosyl part of N-linked glycans, is secreted during sporulation of Myxococcus xanthus.

After the demonstration that Stigmatella aurantiaca DW4 secretes an endo-N-acetyl-beta-D-glucosaminidase (ENGase), acting on the di-N-acetylchitobiosyl part of N-linked glycans (S. Bourgerie, Y. Karamanos, T. Grard, and R. Julien, J. Bacteriol. 176:6170-6174, 1994), an ENGase activity having the same substrate specificity was also found to be secreted during vegetative growth of Myxococcus xanthus DK1622. The activity decreased in mutants known to secrete less protein than the wild type (Exc +/-). During submerged development, the activity was produced in two steps: the first increase occurred during the aggregation phase, and the second one occurred much later, during spore formation. This production was lower in developmental mutants impairing cell-cell signaling, the late mutants (csg and dsg) being the most deficient. Finally, when sporulation was obtained either by starvation in liquid shake flask culture or by glycerol induction, the activity was produced exclusively by the wild-type cells during the maturation of the coat.

Organization of the bovine alpha 2-fucosyltransferase gene cluster suggests that the Sec1 gene might have been shaped through a nonautonomous L1-retrotransposition event within the same locus.

By referring to the split coding sequence of the highly conserved alpha 6-fucosyltransferase gene family (assumed to be representative of the common alpha 2 and alpha 6 fucosyltransferase gene ancestor), we have hypothesized that the monoexonic coding sequences of the present alpha 2-fucosyltransferase genes have been shaped in mammals by several events of retrotransposition and/or duplication. In order to test our hypothesis, we determined the structure of the three bovine alpha 2-fucosyltransferase genes (bfut1, bfut2, and sec1) and analyzed their characteristics compared with their human counterparts (FUT1, FUT2, and Sec1). We show that in mammals, a complex nonautonomous L1-retrotransposition event occurred within the locus of the alpha 2-fucosyltransferase ancestor gene itself. A consequence of this event was the processing in Catarrhini of a Sec1 pseudogene via several point mutations.

Three bovine alpha2-fucosyltransferase genes encode enzymes that preferentially transfer fucose on Galbeta1-3GalNAc acceptor substrates.

To investigate the synthesis of alpha2-fucosylated epitopes in the bovine species, we have characterized cDNAs from various tissues. We found three distinct alpha2-fucosyltransferase genes, named bovine fut1, fut2, and sec1 which are homologous to human FUT1, FUT2, and Sec1 genes, respectively. Their open reading frames (ORF) encode polypeptides of 360 (bovine H), 344 (bovine Se), and 368 (bovine Sec1) amino acids, respectively. These enzymes transfer fucose in alpha1,2 linkage to ganglioside GM(1)and galacto- N -biose, but not to the phenyl-beta-D-galactoside, type 1 or type 2 acceptors, suggesting that their substrate specificity is different and more restricted than the other cloned mammalian alpha2-fucosyltransferases. Southern blot analyses detected four related alpha2-fucosyltransferase sequences in the bovine genome while only three have been described in other species. The supernumerary entity seems to be related to the alpha2-fucosyltransferase activity which can also use type 1 and phenyl-beta-D-galactoside substrate acceptors. It was exclusively found in bovine intestinal tract. Our results show that, at least in one mammalian species, four alpha2-fucosyltransferases are present, three adding a fucose on alpha1,2 linkage on type 3/4 acceptor (Galbeta1-3GalNAc) and another able to transfer also fucose on phenyl-beta-D-galactoside and type 1 (Galbeta1-3GlcNAc) acceptors. The phylogenetic tree of the enzymes homologous to those encoded by the bovine fut1, fut2, and sec1 genes revealed two main families, one containing all the H-like proteins and the second containing all the Se-like and Sec1-like proteins. The Sec1-like family had a higher evolutionary rate than the Se-like family.