The molecular weight distribution of succinoglycan produced by Sinorhizobium meliloti is influenced by specific tyrosine phosphorylation and ATPase activity of the cytoplasmic domain of the ExoP protein

It is thought that in the gram-negative soil bacterium Sinorhizobium meliloti the protein ExoP is involved in biosynthesis of the acidic exopolysaccharide succinoglycan (EPS I). The amounts and compositions of EPS I produced by mutants expressing ExoP proteins characterized by specific amino acid substitutions in the C-terminal cytoplasmic domain were analyzed. The cytoplasmic domain of the ExoP protein was shown to have ATPase activity. Mutations in the highly conserved Walker A ATP-binding motif prevented ATPase activity of the ExoP protein. Phenotypically, these mutations resulted in much lower levels of succinoglycan which consisted only of monomers of the octasaccharide repeating unit. The ExoP protein has similarities to proteins with autophosphorylating protein tyrosine kinase activity. We found that ExoP was phosphorylated on tyrosine and that site-directed mutagenesis of specific tyrosine residues in the cytoplasmic domain of ExoP resulted in an altered ratio of low-molecular-weight succinoglycan to high-molecular-weight succinoglycan.     

Low-molecular-weight succinoglycan is predominantly produced by Rhizobium meliloti strains carrying a mutated ExoP protein characterized by a periplasmic N-terminal domain and a missing C-terminal domain

The membrane topology of the Rhizobium meliloti 2011 ExoP protein involved in polymerization and export of succinoglycan was analysed by translational fusions of lacZ and phoA reporter genes to the exoP gene. Based on this analysis, the ExoP protein could be divided into an N-terminal domain mainly located in the periplasmic space and a C-terminal domain located in the cytoplasm. Whereas the C-terminal domain of ExoP is characterized by a potential nucleotide-binding motif, the N-terminal ExoP domain contains the sequence motif‘PX₂pX₄SPKX₁₁GXMXG₁′, which is also present in proteins involved in the determination of O-antigen chain length. R. meliloti strains carrying mutated exoP* genes, exclusively encoding the N-terminal ExoP domain, produced a reduced amount of succinoglycan. This reduction could be suppressed by a mutation in the regulatory gene exoR. The ratio of low-molecular-weight to high-molecular-weight succinoglycan was significantly increased in the exoP* mutant strain. In the exoP*lexoR mutant strain only low-molecular-weight succinoglycan could be detected. Based on sequence homologies and similar hydropathic profiles, the N-terminal domain of ExoP was proposed to be a member of a protein family thought to be involved in polysaccharide chain-length determination.     

Exopolysaccharide (EPS) synthesis in Bradyrhizobium japonicum : sequence, operon structure and mutational analysis of an exo gene cluster

The nucleotide sequence of a 8330-bp DNA fragment from Bradyrhizobium japonicum 110spc4 was determined. Sequence analysis revealed that six ORFs were present and the deduced amino acid sequences were homologous to enzymes involved in exopolysaccharide (EPS) biosynthesis. The genes appear to be organized into at least four different operons. One gene was found to be homologous to exoB, which encodes a UDP-galactose 4′-epimerase. Other ORFs were homologous to UDP-hexose transferases and one ORF showed similarity to Sinorhizobium (Rhizobium) meliloti ExoP, which has been suggested to be involved in EPS chain-length determination. A set of deletion and insertion mutants was constructed and the resulting B. japonicum strains were tested for their symbiotic traits. Deletion mutant ΔP22, which lacks the C-terminal part of ExoP, the UDP-hexose transferase ExoT and the N-terminal part of ExoB, shows a delayed nodulation phenotype and induces symptoms of plant defense reactions; its EPS does not contain galactose and no high molecular weight fraction is synthesized. In contrast, insertion mutant EH3, which expresses an exoP gene product that is truncated in its putative periplasmic domain, produced an EPS containing both HMW and LMW fractions. However, the interaction of EH3 with soybeans was severely perturbed. As a rule, only the initial steps of nodule formation were observed.     

Exopolysaccharide (EPS) synthesis in Bradyrhizobium japonicum : sequence, operon structure and mutational analysis of an exo gene cluster

The nucleotide sequence of a 8330-bp DNA fragment from Bradyrhizobium japonicum 110spc4 was determined. Sequence analysis revealed that six ORFs were present and the deduced amino acid sequences were homologous to enzymes involved in exopolysaccharide (EPS) biosynthesis. The genes appear to be organized into at least four different operons. One gene was found to be homologous to exoB, which encodes a UDP-galactose 4′-epimerase. Other ORFs were homologous to UDP-hexose transferases and one ORF showed similarity to Sinorhizobium (Rhizobium) meliloti ExoP, which has been suggested to be involved in EPS chain-length determination. A set of deletion and insertion mutants was constructed and the resulting B. japonicum strains were tested for their symbiotic traits. Deletion mutant ΔP22, which lacks the C-terminal part of ExoP, the UDP-hexose transferase ExoT and the N-terminal part of ExoB, shows a delayed nodulation phenotype and induces symptoms of plant defense reactions; its EPS does not contain galactose and no high molecular weight fraction is synthesized. In contrast, insertion mutant EH3, which expresses an exoP gene product that is truncated in its putative periplasmic domain, produced an EPS containing both HMW and LMW fractions. However, the interaction of EH3 with soybeans was severely perturbed. As a rule, only the initial steps of nodule formation were observed. Received: 2 January 1998 / Accepted: 24 March 1998     

The Succinyl and Acetyl Modifications of Succinoglycan Influence Susceptibility of Succinoglycan to Cleavage by the Rhizobium meliloti Glycanases ExoK and ExsH

In Rhizobium meliloti (Sinorhizobium meliloti) cultures, the endo-1,3-1,4-β-glycanases ExoK and ExsH depolymerize nascent high-molecular-weight (HMW) succinoglycan to yield low-molecular-weight (LMW) succinoglycan. We report here that the succinyl and acetyl modifications of succinoglycan influence the susceptibility of succinoglycan to cleavage by these glycanases. It was previously shown that exoH mutants, which are blocked in the succinylation of succinoglycan, exhibit a defect in the production of LMW succinoglycan. We have determined that exoZ mutants, which are blocked in the acetylation of succinoglycan, exhibit an increase in production of LMW succinoglycan. For both wild-type and exoZ mutant strains, production of LMW succinoglycan is dependent on the exoK⁺ and exsH⁺ genes, implying that the ExoK and ExsH glycanases cleave HMW succinoglycan to yield LMW succinoglycan. By supplementing cultures of glycanase-deficient strains with exogenously added ExoK or ExsH, we have demonstrated directly that the absence of the acetyl group increases the susceptibility of succinoglycan to cleavage by ExoK and ExsH, that the absence of the succinyl group decreases the susceptibility of succinoglycan to cleavage, and that the succinyl effect outweighs the acetyl effect for succinoglycan lacking both modifications. Strikingly, nonsuccinylated succinoglycan actually can be cleaved by ExoK and ExsH to yield LMW succinoglycan, but only when the glycanases are added to cultures at greater than physiologically relevant concentrations. Thus, we conclude that the molecular weight distribution of succinoglycan in R. meliloti cultures is determined by both the levels of ExoK and ExsH glycanase expression and the susceptibility of succinoglycan to cleavage.     

The ExpR/Sin quorum-sensing system controls succinoglycan production in Sinorhizobium meliloti

Sinorhizobium meliloti is a gram-negative soil bacterium capable of forming a symbiotic nitrogen-fixing relationship with its plant host, Medicago sativa. Various bacterially produced factors are essential for successful nodulation. For example, at least one of two exopolysaccharides produced by S. meliloti (succinoglycan or EPS II) is required for nodule invasion. Both of these polymers are produced in high- and low-molecular-weight (HMW and LMW, respectively) fractions; however, only the LMW forms of either succinoglycan or EPS II are active in nodule invasion. The production of LMW succinoglycan can be generated by direct synthesis or through the depolymerization of HMW products by the action of two specific endoglycanases, ExsH and ExoK. Here, we show that the ExpR/Sin quorum-sensing system in S. meliloti is involved in the regulation of genes responsible for succinoglycan biosynthesis as well as in the production of LMW succinoglycan. Therefore, quorum sensing, which has been shown to regulate the production of EPS II, also plays an important role in succinoglycan biosynthesis.     

Bacterial adaptation to cold: Conservation of a short J-domain co-chaperone and its protein partners in environmental proteobacteria

Bacterial genomes are a huge reservoir of genes encoding J-domain protein co-chaperones that recruit the molecular chaperone DnaK to assist protein substrates involved in survival, adaptation, or fitness. The atc operon of the aquatic mesophilic bacterium Shewanella oneidensis encodes the proteins AtcJ, AtcA, AtcB, and AtcC, and all of them, except AtcA, are required for growth at low temperatures. AtcJ is a short J-domain protein that interacts with DnaK, but also with AtcC through its 21 amino acid C-terminal domain. This interaction network is critical for cold growth. Here, we show that AtcJ represents a subfamily of short J-domain proteins that (i) are found in several environmental, mostly aquatic, β- or ɣ-proteobacteria and (ii) contain a conserved PX₇W motif in their C-terminal extension. Using a combination of NMR, biochemical and genetic approaches, we show that the hydrophobic nature of the tryptophan of the S. oneidensis AtcJ PX₇W motif determines the strong AtcJ–AtcC interaction essential for cold growth. The AtcJ homologues are encoded by operons containing at least the S. oneidensis atcA, atcB, and atcC homologues. These findings suggest a conserved network of DnaK and Atc proteins necessary for low-temperature growth and, given the variation in the atc operons, possibly for other biological functions.     

Rat Serum Factors Inhibiting the G1-S Transition In Hepatocytes

An attempt was made to detect the serum factors inhibiting the G₁-S transition in synchronized, baby rat hepatocytes. In untreated adult rat serum, this inhibitory activity was always linked to high molecular weight (HMW) compounds. Incubation of serum with trypsin or chymotrypsin resulted in the formation of a low molecular weight (LMW) G₁-S inhibitory factor. the same result was obtained with fractions from adult rat liver but not with kidney or spleen fractions. Separation of the LMW factor by ultrafiltration increased its specific activity by about 10³. the active period in the cell cycle of both the LMW and HMW factors was the same: the late G₁ phase. However, the activity of the LMW factor was not blocked by the Kunitz factor. an enzymatic transformation of the HMW factor might be induced by liver cell membrane-bound proteases and constitute a mechanism regulating hepatocyte proliferation.     

Function of the cytoplasmic tail of human calcitonin receptor-like receptor in complex with receptor activity-modifying protein 2

Receptor activity-modifying protein 2 (RAMP2) enables calcitonin receptor-like receptor (CRLR) to form an adrenomedullin (AM)-specific receptor. Here we investigated the function of the cytoplasmic C-terminal tail (C-tail) of human (h)CRLR by co-transfecting its C-terminal mutants into HEK-293 cells stably expressing hRAMP2. Deleting the C-tail from CRLR disrupted AM-evoked cAMP production or receptor internalization, but did not affect [¹²⁵I]AM binding. We found that CRLR residues 428-439 are required for AM-evoked cAMP production, though deleting this region had little effect on receptor internalization. Moreover, pretreatment with pertussis toxin (100 ng/mL) led to significant increases in AM-induced cAMP production via wild-type CRLR/RAMP2 complexes. This effect was canceled by deleting CRLR residues 454-457, suggesting Gi couples to this region. Flow cytometric analysis revealed that CRLR truncation mutants lacking residues in the Ser/Thr-rich region extending from Ser⁴⁴⁹ to Ser⁴⁶⁷ were unable to undergo AM-induced receptor internalization and, in contrast to the effect on wild-type CRLR, overexpression of GPCR kinases-2, -3 and -4 failed to promote internalization of CRLR mutants lacking residues 449-467. Thus, the hCRLR C-tail is crucial for AM-evoked cAMP production and internalization of the CRLR/RAMP2, while the receptor internalization is dependent on the aforementioned GPCR kinases, but not Gs coupling.     

Effects of individually silenced N-glycosylation sites and non-synonymous single-nucleotide polymorphisms on the fusogenic function of human syncytin-2

ABSTRACT

The placental syncytiotrophoblast, which is formed by the fusion of cytotrophoblast cells, is indispensable for the establishment and maintenance of normal pregnancy. The human endogenous retrovirus envelope glycoprotein syncytin-2 is the most important player in mediating trophoblast cell-cell fusion as a fusogen. We constructed expression plasmids of wild-type and 21 single-amino-acid substitution mutants of syncytin-2, including 10 N-glycosylation sites individually silenced by mutagenizing N to Q, 1 naturally occurring single-nucleotide polymorphism (SNP) N118S that introduced an N-glycosylation site, and another 10 non-synonymous SNPs located within important functional domains. We observed that syncytin-2 was highly fusogenic and that the mutants had different capacities in merging 293T cells. Of the 21 mutants, N133Q, N312Q, N443Q, C46R (in the CXXC motif) and R417H (in the heptad repeat region and immunosuppressive domain) lost their fusogenicity, whereas N332Q, N118S, T367M (in the fusion peptide), V483I (in the transmembrane domain) and T522M (in the cytoplasmic domain) enhanced the fusogenic activity. We also proved that N133, N146, N177, N220, N241, N247, N312, N332 and N443 were all glycosylated in 293T cells. A co-immunoprecipitation assay showed compromised interaction between mutants N443Q, C46R, T367M, R417H and the receptor MFSD2A, whereas N118S was associated with more receptors. We also sequenced the coding sequence of syncytin-2 in 125 severe pre-eclamptic patients and 272 normal pregnant Chinese women. Surprisingly, only 1 non-synonymous SNP T522M was found and the frequencies of heterozygous carriers were not significantly different. Taken together, our results suggest that N-glycans at residues 133, 312, 332 and 443 of syncytin-2 are required for optimal fusion induction, and that SNPs C46R, N118S, T367M, R417H, V483I and T522M can alter the fusogenic function of syncytin-2.     

The SH3 domain of HS1 protein recognizes lysine-rich polyproline motifs

HS1 is a protein involved in erythroid proliferation and apoptotic cell death, containing several structurally significant motifs including a C-terminal SH3 domain. HPK1 is a member of the Ste20-related kinase family, which contains four proline-rich sequences and is constitutively associated with HS1 in hematopoietic cells. Recombinant fusion protein GST-SH3_HS1 was expressed to assess the binding properties of 16 peptides derived from the HPK1 proline-rich regions. The binding affinities were determined by non-immobilized ligand interaction assay by circular dichroism. Our results revealed that the classical PxxPxK class II binding motif is not sufficient to induce the interaction with the GST-SH3_HS1 domain, an event dependent on the presence of additional basic residue(s) located at the C-terminus of the PxxPxK motif: Lys₋₅ in P2 peptide and Lys₋₈ in P4c peptide. Lys replacement by Arg residues decreases the ligand binding affinity. The finding that both SH3_HS1 domain and full-length HS1 protein bind to P2 peptide with similar affinity demonstrates that the whole protein sequence does not affect the interaction properties of the domain. In silico models of SH3_HS1 as a complex with P2 or P4c highlight the domain residues that interact with the recognition determinants of the peptide ligand and that cooperate in the complex stabilization.     

Mapping of functional domains of human high molecular weight and low molecular weight kininogens using murine monoclonal antibodies

Thirty-four monoclonal antibodies directed against human high molecular weight (HMW) and low molecular weight (LMW) kininogens and their derivatives were obtained, and the specificities of the antibodies were assayed by enzyme-linked immunosorbent assay (ELISA). By use of HMW kininogen, kinin-free HMW kininogen, kinin-free and fragment 1.2 (fr 1.2) free HMW kininogen, fr 1.2-light chain of HMW kininogen, LMW kininogen, kinin-free LMW kininogen, heavy chain of LMW kininogen, and light chain of LMW kininogen, the monoclonal antibodies were characterized and classified into four groups: (A) 20 monoclonal antibodies reacting with only the heavy chain, a common region of HMW and LMW kininogens; each of these monoclonal antibodies possessed the specificity to domain 1 (2 monoclonal antibodies), domain 2 (2 monoclonal antibodies), domain 3 (7 monoclonal antibodies), and both domains 2 and 3 (7 monoclonal antibodies) of the heavy chain; (B) 7 monoclonal antibodies reacting with fr 1.2, a unique histidine-rich region; (C) 5 monoclonal antibodies reacting with the light chain of HMW kininogen; (D) 2 monoclonal antibodies reacting with the light chain of LMW kininogen. Two monoclonal antibodies in the first group (group A), designated HKG H7 and H12, effectively suppressed the thiol proteinase inhibitor activity of HMW kininogen to papain and calpains and of LMW kininogen to papain, but the others did not affect it. Further, all the monoclonal antibodies which recognized the fr 1.2 or light chain of HMW kininogen (groups B and C) suppressed the clotting activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solution structure of the PX domain, a target of the SH3 domain

The phox homology (PX) domain is a novel protein module containing a conserved proline-rich motif. We have shown that the PX domain isolated from the human p47phox protein, a soluble subunit of phagocyte NADPH oxidase, binds specifically to the C-terminal SH3 domain derived from the same protein. The solution structure of p47 PX has an alpha + beta structure with a novel folding motif topology and reveals that the proline-rich motif is presented on the molecular surface for easy recognition by the SH3 domain. The proline-rich motif of p47 PX in the free state adopts a distorted left-handed polyproline type II helix conformation.     

Yield and physicochemical properties of EPS from Halomonas sp. strain TG39 identifies a role for protein and anionic residues (sulfate and phosphate) in emulsification of n‐hexadecane

In this study, we investigated the yield and physicochemical properties of the high molecular weight extracellular polymeric substance (HMW–EPS) produced by Halomonas sp. strain TG39 when grown on different types and ratios of substrates. Glucose (1% w/v) and a peptone/yeast extract ratio of 5.1 (0.6% w/v final concentration) yielded an EPS fraction (HMW‐glucose) exhibiting the highest anionic activity (20.5) and specific emulsifying activity (EI₂₄ = 100%) compared to EPS produced by cells grown on mannitol, sucrose, malt extract or no carbon source. The HMW–EPS fractions were capable of binding ≈255–464 mg of methylene blue (MB) per gram of EPS, which represents the highest reported binding of MB by a bacterial EPS. A comparative evaluation of these properties to those of commercial hydrocolloids indicated that the combined effect of protein and anionic residues of the HMW–EPS contributed to its ability to emulsify n‐hexadecane. Liquid chromatography revealed the HMW‐glucose EPS to be a heterogeneous polymer with a polydispersity index of 1.8. This work presents evidence of a correlation between the anionic nature and protein content of bacterial EPS with its emulsifying qualities, and identifies EPS produced by strain TG39 as a high MB‐binding bacterial sorbant with potential biotechnological application. Biotechnol. Bioeng. 2009;103: 207–216. © 2008 Wiley Periodicals, Inc.     

Moonlighting by different stressors: crystal structure of the chaperone species of a 2-Cys peroxiredoxin

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Highlights? The Schistosoma mansoni peroxiredoxin I (SmPrxI) is a moonlighting protein ? SmPrxI switches from LMW peroxidase to HMW chaperone due to chemical stressors ? The three-dimensional structures of both LMW and HMW SmPrxI have been solved ? SmPrxI HMW is the structure of a Prx chaperone     

Mutations in the Essential Arabinosyltransferase EmbC Lead to Alterations in Mycobacterium tuberculosis Lipoarabinomannan

The Mycobacterium tuberculosis cell wall is a complex structure essential for the viability of the organism and its interaction with the host. The glycolipid lipoarabinomannan (LAM) plays an important role in mediating host-bacteria interactions and is involved in modulation of the immune response. The arabinosyltransferase EmbC required for LAM biosynthesis is essential. We constructed recombinant strains of M. tuberculosis expressing a variety of alleles of EmbC. We demonstrated that EmbC has a functional signal peptide in M. tuberculosis. Over- or underexpression of EmbC resulted in reduced or increased sensitivity to ethambutol, respectively. The C-terminal domain of EmbC was essential for activity because truncated alleles were unable to mediate LAM production in Mycobacterium smegmatis and were unable to complement an embC deletion in M. tuberculosis. The C-terminal domain of the closely related arabinosyltransferase EmbB was unable to complement the function of the EmbC C-terminal domain. Two functional motifs were identified. The GT-C motif contains two aspartate residues essential for function in the DDX motif. The proline-rich region contains two highly conserved asparagines (Asn-638 and Asn-652). Mutation of these residues was tolerated, but loss of Asn-638 resulted in the synthesis of truncated LAM, which appeared to lack arabinose branching. All embC alleles that were incapable of complementing LAM production in M. smegmatis were not viable in M. tuberculosis, supporting the hypothesis that LAM itself is essential in M. tuberculosis.     

Cryoelectron Microscopic Examination of Human Immunodeficiency Virus Type 1 Virions with Mutations in the Cyclophilin A Binding Loop

The human immunodeficiency virus type 1 capsid protein contains a conserved P₂₁₇X₄PX₂PX₅P₂₃₁ motif. Mutation at Pro-222 decreases virion incorporation of cyclophilin A, while mutation at Pro-231 abolishes infectivity. Although viral RNA incorporation and protease cleavage of the Gag precursor were not affected by these mutations, cryoelectron microscopy revealed a loss of virion maturation in P231A particles.     

Assembly of acetylcholinesterase tetramers by peptidic motifs from the proline-rich membrane anchor, PRiMA: competition between degradation and secretion pathways of heteromeric complexes

The membrane-bound form of acetylcholinesterase (AChE) constitutes the major component of this enzyme in the mammalian brain. These molecules are hetero-oligomers, composed of four AChE catalytic subunits of type T (AChE(T)), associated with a transmembrane protein of type 1, called PRiMA (proline-rich membrane anchor). PRiMA consists of a signal peptide, an extracellular domain that contains a proline-rich motif (14 prolines with an intervening leucine, P4LP10), a transmembrane domain, and a cytoplasmic domain. Expression of AChE(T) subunits in transfected COS cells with a truncated PRiMA, without its transmembrane and cytoplasmic domains (P(stp54) mutant), produced secreted heteromeric complexes (T4-P(stp54)), instead of membrane-bound tetramers. In this study, we used a series of deletions and point mutations to analyze the interaction between the extracellular domain of PRiMA and AChE(T) subunits. We confirmed the importance of the polyproline stretches and defined a peptidic motif (RP4LP10RL), which induces the assembly and secretion of a heteromeric complex with four AChE(T) subunits, nearly as efficiently as the entire extracellular domain of PRiMA. It is noteworthy that deletion of the N-terminal segment preceding the prolines had little effect. Interestingly, short PRiMA mutants, truncated within the proline-rich motif, reduced both cellular and secreted AChE activity, suggesting that their interaction with AChE(T) subunits induces their intracellular degradation.