Congenital disorder of glycosylation Ic in patients of Indian origin.

Congenital disorder of glycosylation type Ic (CDG-Ic) is caused by mutations in ALG6, encoding an alpha 1,3-glucosyltransferase. The most frequent mutation found in this gene (C998T resulting in an A333V substitution) has until now been found only in patients of European origin. Here we describe the first occurrence of this CDG-Ic mutation in patients of Indian origin. Of three Indian patients described in this study, patient 1 was homozygous and patient 2 heterozygous for the A333V mutation. In patient 2 we also found a new mutation, IVS3+2_3insT, just 3bp away from the previously described IVS3+5G>A substitution; both mutations resulted in exon 3 skipping. We screened a panel of >350 genomic DNA samples from an ethnically diverse American population to determine the frequency of the A333V mutation. None of the samples carried this mutation, indicating the frequency of patients carrying this homozygous mutation should be <1 in 5x10(5). The discovery of the common CDG-Ic mutation A333V in an Indian population raises questions as to its ethnic origin.

The RING finger protein Siah-1 regulates the level of the transcriptional coactivator OBF-1.

The transcriptional coactivator OBF-1, which interacts with Oct-1 and Oct-2 and the octamer site DNA, has been shown to be critical for development of a normal immune response and the formation of germinal centers in secondary lymphoid organs. Here we have identified the RING finger protein Siah-1 as a protein interacting specifically with OBF-1. This interaction is mediated by the C-terminal part of Siah-1 and by residues in the N-terminus of OBF-1, partly distinct from the residues required for formation of a complex with the Oct POU domains and the DNA. Interaction between Siah-1 and OBF-1 leads to downregulation of OBF-1 protein level but not mRNA, and to a corresponding reduction in octamer site-dependent transcription activation. Inhibition of the ubiquitin-proteasome pathway in B cells leads to elevated levels of OBF-1 protein. Furthermore, in immunized mice, OBF-1 protein amounts are dramatically increased in primary activated B cells, without concomitant increase in OBF-1 mRNA. These data suggest that Siah-1 is part of a novel regulatory loop controlling the level of OBF-1 protein in B cells.

Unraveling the function of glycosyltransferases in Streptococcus thermophilus Sfi6.

Streptococcus thermophilus Sfi6 produces a texturizing exopolysaccharide (EPS) consisting of a -->3)[alpha-D-Galp-(1-->6)]-beta-D-Glcp-(1-->3)-alpha-D-GalpNAc-(1--> 3)-beta-D-Galp-(1--> repeating unit. We previously identified and analyzed a 14.5-kb gene cluster from S. thermophilus Sfi6 consisting of 13 genes responsible for its EPS production. Within this gene cluster, we found a central region of genes (epsE, epsF, epsG, and epsI) that showed similarity to glycosyltransferases. In this study, we investigated the sugar specificity of these enzymes. EpsE catalyzes the first step in the biosynthesis of the EPS repeating unit. It exhibits phosphogalactosyltransferase activity and transfers galactose onto the lipophilic carrier. The second step is fulfilled by EpsG, which transfers an alpha-N-acetylgalactosamine onto the first beta-galactoside. The activity of EpsF was determined by characterizing the EPS produced by an S. thermophilus epsF deletion mutant. This EPS consisted of the monosaccharides Gal, Glc, and GalNAc in an approximately equimolar ratio, thus suggesting that epsF codes for the branching galactosyltransferase. epsI probably codes for the beta-1,3-glucosyltransferase, since it is the only glycosyltransferase to which no gene has been assigned and it exhibits similarity to other beta-glycosyltransferases. EpsE shows the conserved features of phosphoglycosyltransferases, whereas EpsF and EpsG exhibit the primary structure of alpha-glycosyltransferases, belonging to glycosyltransferase family 4, whose members are conserved in all major phylogenetic lineages, including the Archaea and Eukaryota.

Introduction of the exopolysaccharide gene cluster from Streptococcus thermophilus Sfi6 into Lactococcus lactis MG1363: production and characterization of an altered polysaccharide.

Streptococcus thermophilus Sfi6 produces an exopolysaccharide (EPS) composed of glucose, galactose and N-acetylgalactosamine in the molar ratio of 1:2:1. The genes responsible for the EPS biosynthesis have been isolated previously and found to be clustered in a 14.5 kb region encoding 13 genes. Transfer of this gene cluster into a non-EPS-producing heterologous host, Lactococcus lactis MG1363, yielded an EPS with a similar high molecular weight, but a different structure from the EPS from the native host. The structure of the recombinant EPS was determined by means of 1H homonuclear and 1H-13C heteronuclear two-dimensional nuclear magnetic resonance (NMR) spectra and was found to be --> 3)-beta-D-Glcp-(1 --> 3)-alpha-D-Galp-(1 --> 3)-beta-D-Galp-(1 --> as opposed to --> 3)[alpha-D-Galp-(1 --> 6)]-beta-D-Glcp-(1 --> 3)-alpha-D-GalpNAc-(1 --> 3)-beta-D-Galp-(1 --> for the wild-type S. thermophilus Sfi6. Furthermore, L. lactis MG1363 (pFS101) was also lacking a UDP-N-acetylglucosamine C4-epimerase activity, which would provide UDP-GalNAc for a GalNAc incorporation into the EPS and probably caused the substitution of GalNAc by Gal in the recombinant EPS. This modification implies that (i) bacterial glycosyltransferases could potentially have multiple specificities for the donor and the acceptor sugar molecule; and (ii) the repeating unit polymerase can recognize and polymerize a repeating unit that differs in the backbone as well as in the side-chain from its native substrate.

OBF-1, a novel B cell-specific coactivator that stimulates immunoglobulin promoter activity through association with octamer-binding proteins.

Recent biochemical and genetic studies indicate that in addition to the octamer-binding proteins Oct-1 and Oct-2, other B cell components are required for lymphoid-restricted, octamer site-mediated immunoglobulin gene promoter activity. Using a genetic screen in yeast, we have isolated B cell-derived cDNAs encoding Oct-binding factor 1 (OBF-1), a novel protein that specifically associates with Oct-1 and Oct-2. Biochemical studies demonstrate that OBF-1 has no intrinsic DNA-binding activity and recognizes the POU domains of Oct-1 and Oct-2, but not those of Oct-4 and Oct-6. The OBF-1 mRNA is expressed in a highly cell-specific manner, being most abundant in B cells and essentially absent in most of the other cells or tissues tested. Furthermore, expression of OBF-1 in HeLa cells selectively stimulates the activity of a natural immunoglobulin promoter in an octamer site-dependent manner. Thus, OBF-1 has all the properties expected for a B cell-specific transcriptional coactivator protein.

Involvement of the Ets family factor PU.1 in the activation of immunoglobulin promoters.

The B cell-specific expression of immunoglobulin (Ig) genes is controlled by the concerted action of variable (V) region promoters and intronic or 3' enhancers, all of which are active in a lymphoid-specific manner. A crucial highly conserved element of the V region promoters is the octamer site -ATTTGCAT-, which can be bound by ubiquitous (Oct-1) as well as B cell-specific (Oct-2) factors. Another less conserved element found in many Ig promoters is pyrimidine-rich and has been shown to be functionally important, in particular for those Ig promoters that have only an imperfect octamer site. In this study we have analyzed the factors binding specifically to the pyrimidine-rich motif of the V kappa 19 promoter, a light chain gene promoter with an imperfect octamer site. Using nuclear extracts prepared from B cells, we detected two sets of specific complexes in electrophoretic mobility shift experiments. One complex appears to be ubiquitous but enriched in lymphoid cells and represents the binding of a potentially novel factor with an apparent molecular mass of approximately 50 kDa. The other complex was found only with extracts from pre-B or B cells as well as from a macrophage cell line and appears to be caused by the binding of PU.1, a factor of the Ets family. We show that on this Ig promoter Oct factors (Oct-1 or Oct-2) and PU.1 can bind concomitantly but without synergism. By transfection experiments in non-B cells we demonstrate that PU.1 is indeed able to activate this promoter in concert with Oct-2. Furthermore, we show that PU.1 can bind with varying affinities to the pyrimidine-rich elements of several other Ig promoters. These data suggest a more general role for PU.1 or other members of the Ets family in the activation of Ig promoters.