Carbamylated sortilin associates with cardiovascular calcification in patients with chronic kidney disease.

Sortilin, an intracellular sorting receptor, has been identified as a cardiovascular risk factor in the general population. Patients with chronic kidney disease (CKD) are highly susceptible to develop cardiovascular complications such as calcification. However, specific CKD-induced posttranslational protein modifications of sortilin and their link to cardiovascular calcification remain unknown. To investigate this, we examined two independent CKD cohorts for carbamylation of circulating sortilin and detected increased carbamylated sortilin lysine residues in the extracellular domain of sortilin with kidney function decline using targeted mass spectrometry. Structure analysis predicted altered ligand binding by carbamylated sortilin, which was verified by binding studies using surface plasmon resonance measurement, showing an increased affinity of interleukin 6 to in vitro carbamylated sortilin. Further, carbamylated sortilin increased vascular calcification in vitro and ex vivo that was accelerated by interleukin 6. Imaging by mass spectrometry of human calcified arteries revealed in situ carbamylated sortilin. In patients with CKD, sortilin carbamylation was associated with coronary artery calcification, independent of age and kidney function. Moreover, patients with carbamylated sortilin displayed significantly faster progression of coronary artery calcification than patients without sortilin carbamylation. Thus, carbamylated sortilin may be a risk factor for cardiovascular calcification and may contribute to elevated cardiovascular complications in patients with CKD.

Kaposi's sarcoma-associated herpesvirus gH/gL: glycoprotein export and interaction with cellular receptors.

The attachment, entry, and fusion of Kaposi's sarcoma-associated herpesvirus (KSHV) with target cells are mediated by complex machinery containing, among others, viral glycoprotein H (gH) and its alleged chaperone, gL. We observed that KSHV gH, in contrast to its homologues in several other herpesviruses, is transported to the cytoplasm membrane independently from gL, but not vice versa. Mutational analysis revealed that the N terminus of gH is sufficient for gL interaction. However, the entire extracellular part of gH is required for efficient gL secretion. The soluble ectodomain of gH was sufficient to interact with the surfaces of potential target cells in a heparin-dependent manner, and binding was further enhanced by coexpression of gL. Surface plasmon resonance revealed a remarkably high affinity of gH for glycosaminoglycans. Heparan sulfate (HS) proteoglycans of the syndecan family act as cellular receptors for the gH/gL complex. They promoted KSHV infection, and expression of gH/gL on target cells inhibited subsequent KSHV infection. Whereas gH alone was able to bind to HS, we observed that only the gH/gL complex adhered to heparan sulfate-negative cells at lamellipodium-like structures.

A new piece of an old jigsaw: glucose kinase is activated posttranslationally in a glucose transport-dependent manner in streptomyces coelicolor A3(2).

Members of the soil-dwelling prokaryotic genus Streptomyces are indispensable for the recycling of complex polysaccharides, and produce a wide range of natural products. Nutrient limitation is likely to be a major signal for the onset of their development, resulting in spore formation by specialized aerial hyphae. Streptomycetes grow on numerous carbon sources, which they utilize in a preferential manner. The main signaling pathway underlying this phenomenon is carbon catabolite repression, which in streptomycetes is totally dependent on the glycolytic enzyme glucose kinase (Glk). How Glk exerts this fascinating dual role (metabolic and regulatory) is still largely a mystery. We show here that while Glk is made constitutively throughout the growth of Streptomyces coelicolor A3(2), its catalytic activity is modulated in a carbon source-dependent manner: while cultures growing exponentially on glucose exhibit high Glk activity, mannitol- grown cultures show negligible activity. Glk activity was directly proportional to the amount of two Glk isoforms observed by Western blot analysis. The activity profile of GlcP, the major glucose permease, correlated very well with that of Glk. Our data are consistent with a direct interaction between Glk and GlcP, suggesting that a Glk-GlcP permease complex is required for efficient glucose transport by metabolic trapping. This is supported by the strongly reduced accumulation of glucose in glucose kinase mutants. A model to explain our data is presented.

YeeI, a novel protein involved in modulation of the activity of the glucose-phosphotransferase system in Escherichia coli K-12.

The membrane-bound protein EIICB(Glc) encoded by the ptsG gene is the major glucose transporter in Escherichia coli. This protein is part of the phosphoenolpyruvate:glucose-phosphotransferase system, a very important transport and signal transduction system in bacteria. The regulation of ptsG expression is very complex. Among others, two major regulators, the repressor Mlc and the cyclic AMP-cyclic AMP receptor protein activator complex, have been identified. Here we report identification of a novel protein, YeeI, that is involved in the regulation of ptsG by interacting with Mlc. Mutants with reduced activity of the glucose-phosphotransferase system were isolated by transposon mutagenesis. One class of mutations was located in the open reading frame yeeI at 44.1 min on the E. coli K-12 chromosome. The yeeI mutants exhibited increased generation times during growth on glucose, reduced transport of methyl-alpha-d-glucopyranoside, a substrate of EIICB(Glc), reduced induction of a ptsG-lacZ operon fusion, and reduced catabolite repression in lactose/glucose diauxic growth experiments. These observations were the result of decreased ptsG expression and a decrease in the amount of EIICB(Glc). In contrast, overexpression of yeeI resulted in higher expression of ptsG, of a ptsG-lacZ operon fusion, and of the autoregulated dgsA gene. The effect of a yeeI mutation could be suppressed by introducing a dgsA deletion, implying that the two proteins belong to the same signal transduction pathway and that Mlc is epistatic to YeeI. By measuring the surface plasmon resonance, we found that YeeI (proposed gene designation, mtfA) directly interacts with Mlc with high affinity.

GlcP constitutes the major glucose uptake system of Streptomyces coelicolor A3(2).

We provide a functional and regulatory analysis of glcP, encoding the major glucose transporter of Streptomyces coelicolor A3(2). GlcP, a member of the Major Facilitator Superfamily (MFS) of bacterial and eucaryotic sugar permeases, was found to be encoded twice at two distinct loci, glcP1 and glcP2, located in the central core and in the variable right arm of the chromosome respectively. Heterologous expression of GlcP in Escherichia coli led to the full restoration of glucose fermentation to mutants lacking glucose transport activity. Biochemical analysis revealed an affinity constant in the low-micromolar range and substrate specificity for glucose and 2-deoxyglucose. Deletion of glcP1 but not glcP2 led to a drastic reduction in growth on glucose reflected by the loss of glucose uptake. This correlated with transcriptional analyses, which showed that glcP1 transcription was strongly inducible by glucose, while glcP2 transcripts were barely detectable. In conclusion, GlcP, which is the first glucose permease from high G+C Gram-positive bacteria characterized at the molecular level, represents the major glucose uptake system in S. coelicolor A3(2) that is indispensable for the high growth rate on glucose. It is anticipated that the activity of GlcP is linked to other glucose-mediated phenomena such as carbon catabolite repression, morphogenesis and antibiotic production.

A recombinant bispecific single-chain Fv antibody against HLA class II and FcgammaRIII (CD16) triggers effective lysis of lymphoma cells.

Bispecific antibodies offer the possibility of improving effector-cell recruitment for antibody therapy. For this purpose, a recombinant bispecific single-chain Fv antibody (bsscFv), directed against FcgammaRIII (CD16) and human leucocyte antigen (HLA) class II, was constructed and tested in functional assays. RNA from the hybridomas 3G8 and F3.3, reacting with CD16 and HLA class II, respectively, was used to generate phage display libraries. From these libraries, reactive phages were isolated and the bsscFv was constructed by connecting both single-chain Fv components through a 20 amino acid flexible linker. After expression in SF21 insect cells and chromatographic purification, the bsscFv bound specifically and simultaneously to both antigens. The affinities of the anti-CD16 and the anti-HLA class II scFv components of the bsscFv were 8.6 x 10(-8) mol/l and 13.7 x 10(-8) mol/l, respectively, which was approximately sevenfold lower than the F(ab) fragments of the parental antibodies. In antibody-dependent cellular cytotoxicity experiments with human mononuclear cells as effectors, the bsscFv-mediated specific lysis of both HLA class II-positive, malignant human B-lymphoid cell lines and primary cells from patients with chronic B-cell lymphocytic leukaemia. Optimal lysis was obtained at bsscFv concentrations of approximately 400 ng/ml, similar to the concentration required for maximum lysis by the corresponding chemically linked bispecific antibody. Thus, this recombinant bsscFv-antibody is an efficient molecule for effector-cell mediated lysis of malignant human B-lymphoid cells.

Complementation of a Delta ccpA mutant of Lactobacillus casei with CcpA mutants affected in the DNA- and cofactor-binding domains.

In low-G+C Gram-positive bacteria, the regulatory protein CcpA has been shown to play a major part in the so-called carbon catabolite repression (CCR) process, as well as in the induction of basic metabolic genes, for which it is considered a global regulator. A strain of Lactobacillus casei that carried a complete deletion of ccpA has been constructed and used to test the effect of CCR on N-acetylglucosaminidase activity and growth performance of a collection of seven CcpA mutations obtained by site-directed mutagenesis. The replaced amino acids were located in the DNA- and cofactor (P-Ser-HPr)-binding domains. Mutations in the DNA-binding domain lacked CCR, as found in Bacillus megaterium. However, mutations in the cofactor-binding domain of L. casei CcpA had a different phenotype to that observed in the previous studies with B. megaterium. Two of them, S80L and T307I, displayed a significant hyper-repression, an effect never reported before for CcpA. Comparison of growth capabilities provided by the different mutants and their ability to sustain CCR demonstrated that CCR, at least on the enzymic activity tested, and the growth defect caused by the CcpA mutations are unrelated features.

In silico and transcriptional analysis of carbohydrate uptake systems of Streptomyces coelicolor A3(2).

Streptomyces coelicolor is the prototype for the investigation of antibiotic-producing and differentiating actinomycetes. As soil bacteria, streptomycetes can metabolize a wide variety of carbon sources and are hence vested with various specific permeases. Their activity and regulation substantially determine the nutritional state of the cell and, therefore, influence morphogenesis and antibiotic production. We have surveyed the genome of S. coelicolor A3(2) to provide a thorough description of the carbohydrate uptake systems. Among 81 ATP-binding cassette (ABC) permeases that are present in the genome, we found 45 to encode a putative solute binding protein, an essential feature for carbohydrate permease function. Similarity analysis allowed the prediction of putative ABC systems for transport of cellobiose and cellotriose, alpha-glucosides, lactose, maltose, maltodextrins, ribose, sugar alcohols, xylose, and beta-xylosides. A novel putative bifunctional protein composed of a substrate binding and a membrane-spanning moiety is likely to account for ribose or ribonucleoside uptake. Glucose may be incorporated by a proton-driven symporter of the major facilitator superfamily while a putative sodium-dependent permease of the solute-sodium symporter family may mediate uptake of galactose and a facilitator protein of the major intrinsic protein family may internalize glycerol. Of the predicted gene clusters, reverse transcriptase PCRs showed active gene expression in 8 of 11 systems. Together with the previously surveyed permeases of the phosphotransferase system that accounts for the uptake of fructose and N-acetylglucosamine, the genome of S. coelicolor encodes at least 53 potential carbohydrate uptake systems.

The phosphotransferase system of Streptomyces coelicolor is biased for N-acetylglucosamine metabolism.

Mutation of the crr-ptsI gene locus revealed that Streptomyces coelicolor uses the phosphotransferase system (PTS) for N-acetylglucosamine uptake. crr, ptsI, and ptsH, which encode the three general PTS phosphotransferases, are induced by N-acetylglucosamine but not by other PTS substrates. Thus, the S. coelicolor PTS is biased for N-acetylglucosamine utilization, a novel feature that distinguishes this PTS from others.

Cross communication between components of carbon catabolite repression of Lactobacillus casei and Bacillus megaterium.

In low-GC Gram-positive bacteria, carbon catabolite repression (CCR) is exerted by transcriptional regulation through a protein complex consisting of catabolite control protein CcpA and serine phosphorylated phosphocarrier protein HPr (HPr-ser-P). We investigated the interaction between these components of Lactobacillus casei and Bacillus megaterium. CcpA of L. casei could not complement a B. megaterium ccpA mutant strain, whereas it was found to be functional in Bacillus subtilis. To explore the nature of the non-complementing phenotype, we overproduced and purified CcpA and HPr of L. casei for in vitro analyses. Electrophoretic mobility shift assays revealed a failure in CCR signal transduction at the level of protein-protein interaction between L. casei CcpA and B. megateriumHPr-ser-P, while binding of CcpA to the B. megaterium target site was intact. We established a method based on surface plasmon resonance that allowed a quantitative analysis of CcpA/HPr-ser-P interactions. Calculation of the apparent dissociation constants revealed that the interaction of L. casei CcpA with B. megaterium HPr-ser-P was fivefold weaker than with its own HPr-ser-P suggesting that the reduced affinity was responsible for the non-complementing phenotype.

The MT domain of the proto-oncoprotein MLL binds to CpG-containing DNA and discriminates against methylation.

Alterations of the proto-oncogene MLL (mixed lineage leukemia) are characteristic for a high proportion of acute leukemias, especially those occurring in infants. The activation of MLL is achieved either by an internal tandem duplication of 5' MLL exons or by chromosomal translocations that create chimeric proteins with the N-terminus of MLL fused to a variety of different partner proteins. A domain of MLL with significant homology to the eukaryotic DNA methyltransferases (MT domain) has been found to be essential for the transforming potential of the oncogenic MLL derivatives. Here we demonstrate that this domain specifically recognizes DNA with unmethylated CpG sequences. In gel mobility shifts, the presence of CpG was sufficient for binding of recombinant GST-MT protein to DNA. The introduction of 5-methylCpG on one or both DNA strands precluded an efficient interaction. In surface plasmon resonance a KD of approximately 3.3 x 10(-8) M was determined for the GST-MT/DNA complex formation. Site selection experiments and DNase I footprinting confirmed CpG as the target of the MT domain. Finally, this interaction was corroborated in vivo in reporter assays utilizing the DNA-binding properties of the MT domain in a hybrid MT-VP16 transactivator construct.