The adhesive and cohesive properties of a bacterial polysaccharide adhesin are modulated by a deacetylase

Bacterial exopolysaccharide synthesis is a prevalent and indispensible activity in many biological processes, including surface adhesion and biofilm formation. In Caulobacter crescentus, surface attachment and subsequent biofilm growth depend on the ability to synthesize an adhesive polar polysaccharide known as the holdfast. In this work, we show that polar polysaccharide synthesis is a conserved phenomenon among Alphaproteobacterial species closely related to C. crescentus. Among them, mutagenesis of Asticcacaulis biprosthecum showed that disruption of the hfsH gene, which encodes a putative polysaccharide deacetylase, leads to accumulation of holdfast in the culture supernatant. Examination of the hfsH deletion mutant in C. crescentus revealed that this strain synthesizes holdfast; however, like the A. biprosthecum hfsH mutant, the holdfasts are shed into the medium and have decreased adhesiveness and cohesiveness. Site‐directed mutagenesis at the predicted catalytic site of C. crescentus HfsH phenocopied the ΔhfsH mutant and abolished the esterase activity of HfsH. In contrast, overexpression of HfsH increased cell adherence without increasing holdfast synthesis. We conclude that the polysaccharide deacetylase activity of HfsH is required for the adhesive and cohesive properties of the holdfast, as well as for the anchoring of the holdfast to the cell envelope.     

The dynamic properties of neuronal chromatin are modulated by triiodothyronine

The effect of triiodothyronine (T3) on the rate of synthesis of nuclear proteins was studied during terminal differentiation of rat cortical neurons cultured in a serum-free medium. To this aim total and acid soluble nuclear proteins were analyzed by different electrophoretic techniques. Our results show that: 1) during maturation in vitro, neuronal nuclei undergo a dramatic change in the rate at which different classes of histones and high mobility group (HMG) proteins are synthesized; the synthetic activity, measured as incorporation of radioactive precursors into nuclear proteins, slows indeed down with age: especially evident is the decrease in core histones synthesis; at day 15, on the other hand, HMG 14 and 17 and ubiquitinated H2A (A24) are synthesized at a high rate, especially in T3-treated neurons; 2) neurons treated with T3 show, at any age tested, a higher level of lysine incorporation into nuclear proteins; 3) even if during the first days of culture neurons synthesize core histones more actively in the presence of T3, there is no accumulation of these proteins at later stages, as compared with untreated cells. Possible implications of these data and relationship with the chromatin rearrangement which accompanies neuronal terminal differentiation are discussed.     

Clathrin-mediated endocytosis of MUC1 is modulated by its glycosylation state

MUC1 is a mucin-like type 1 transmembrane protein associated with the apical surface of epithelial cells. In human tumors of epithelial origin MUC1 is overexpressed in an underglycosylated form with truncated O-glycans and accumulates in intracellular compartments. To understand the basis for this altered subcellular localization, we compared the synthesis and trafficking of various glycosylated forms of MUC1 in normal (Chinese hamster ovary) cells and glycosylation-defective (ldlD) cells that lack the epimerase to make UDP-Gal/GalNAc from UDP-Glc/GlcNAc. Although the MUC1 synthesized in ldlD cells was rapidly degraded, addition of GalNAc alone to the culture media resulted in stabilization and near normal surface expression of MUC1 with truncated but sialylated O-glycans. Interestingly, the initial rate of endocytosis of this underglycosylated MUC1 was stimulated by twofold compared with fully glycosylated MUC1. However, the half-lives of the two forms were not different, indicating that trafficking to lysosomes was not affected. Both the normal and stimulated internalization of MUC1 could be blocked by hypertonic media, a hallmark of clathrin-mediated endocytosis. MUC1 endocytosis was also blocked by expression of a dominant-negative mutant of dynamin-1 (K44A), and MUC1 was observed in both clathrin-coated pits and vesicles by immunoelectron microscopy of ultrathin cryosections. Our data suggest that the subcellular redistribution of MUC1 in tumor cells could be a direct result of altered endocytic trafficking induced by its aberrant glycosylation; potential models are discussed. These results also implicate a new role for O-glycans on mucin-like membrane proteins entering the endocytic pathway through clathrin-coated pits.     

Structural characterization of recombinant soluble rat neuroligin 1: mapping of secondary structure and glycosylation by mass spectrometry

Neuroligins (NLs) are a family of transmembrane proteins that function in synapse formation and/or remodeling by interacting with beta-neurexins (beta-NXs) to form heterophilic cell adhesions. The large N-terminal extracellular domain of NLs, required for beta-NX interactions, has sequence homology to the alpha/beta hydrolase fold superfamily of proteins. By peptide mapping and mass spectrometric analysis of a soluble recombinant form of NL1, several structural features of the extracellular domain have been established. Of the nine cysteine residues in NL1, eight are shown to form intramolecular disulfide bonds. Disulfide pairings of Cys 117 to Cys 153 and Cys 342 to Cys 353 are consistent with disulfide linkages that are conserved among the family of alpha/beta hydrolase proteins. The disulfide bond between Cys 172 and Cys 181 occurs within a region of the protein encoded by an alternatively spliced exon. The disulfide pairing of Cys 512 and Cys 546 in NL1 yields a structural motif unique to the NLs, since these residues are highly conserved. The potential N-glycosylation sequons in NL1 at Asn 109, Asn 303, Asn 343, and Asn 547 are shown occupied by carbohydrate. An additional consensus sequence for N-glycosylation at Asn 662 is likely occupied. Analysis of N-linked oligosaccharide content by mass matching paradigms reveals significant microheterogeneous populations of complex glycosyl moieties. In addition, O-linked glycosylation is observed in the predicted stalk region of NL1, prior to the transmembrane spanning domain. From predictions based on sequence homology of NL1 to acetylcholinesterase and the molecular features of NL1 established from mass spectrometric analysis, a novel topology model for NL three-dimensional structure has been constructed.     

Synthesis of alkyl-alpha-L-rhamnosides by water soluble alcohols enzymatic glycosylation

The synthesis of alkyl-alpha-rhamnosides by alpha-rhamnosidase was studied using rhamnose and rhamnosides, particularly the flavonoid naringin, as glycosylation agents, and water soluble alcohols as acceptors. The reaction products were analyzed by HPLC chromatography and identified by 13C y 1H NMR. The glycosylation of alcohols by reverse hydrolysis was maximum for 40% methanol, 30% ethanol, 10% propanol and 20% isopropanol. Under optimum conditions the yield of rhamnose to alkyl-alpha-rhamnoside transformation decreased from 68% for methyl-alpha-rhamnoside to 10% for isopropyl-alpha-rhamnoside. The time course of rhamnosylations produced using naringin as the donor was comparable with that of the reverse hydrolysis obtained at the same molar concentration of the donor. The flavonoids and their derivatives remaining in the solution after the glycosylation were removed by ion exchange QEAE chromatography at pH 10. These results indicate that both, reverse hydrolysis and glycosylation by naringin are acceptable procedures for the enzymatic synthesis of short chain length alkyl-alpha-L-rhamnosides.     

The nanomechanical properties of rat fibroblasts are modulated by interfering with the vimentin intermediate filament system

The contribution of the intermediate filament (IF) network to the mechanical response of cells has so far received little attention, possibly because the assembly and regulation of IFs are not as well understood as that of the actin cytoskeleton or of microtubules. The mechanical role of IFs has been mostly inferred from measurements performed on individual filaments or gels in vitro. In this study we employ atomic force microscopy (AFM) to examine the contribution of vimentin IFs to the nanomechanical properties of living cells under native conditions. To specifically target and modulate the vimentin network, Rat-2 fibroblasts were transfected with GFP-desmin variants. Cells expressing desmin variants were identified by the fluorescence microscopy extension of the AFM instrument. This allowed us to directly compare the nanomechanical response of transfected and untransfected cells at high spatial resolution by means of AFM. Depending on the variant desmin, transfectants were either softer or stiffer than untransfected fibroblasts. Expression of the non-filament forming GFP-DesL345P mutant led to a collapse of the endogenous vimentin network in the perinuclear region that was accompanied by localized stiffening. Correlative confocal microscopy indicates that the expression of desmin variants specifically targets the endogenous vimentin IF network without major rearrangements of other cytoskeletal components. By measuring functional changes caused by IF rearrangements in intact cells, we show that IFs play a crucial role in mechanical behavior not only at large deformations but also in the nanomechanical response of individual cells.     

The neutralization properties of a HIV-specific antibody are markedly altered by glycosylation events outside the antigen-binding domain

Neutralizing Abs constitute a pivotal mechanism of the adaptive immune response against HIV-1 infection. Yet, most of the Abs that appear in the circulation during HIV infection are nonneutralizing. In this study, we report a dramatic change of the neutralizing properties of a human Ab reactive with the nonneutralizing epitope termed cluster I on the HIV-1 transmembrane protein gp41 when the Ab was produced in Chinese hamster ovary (CHO)-K1 cells. Our laboratory has previously reported that the Ab F240, when produced in a hybridoma, is nonneutralizing as assessed by standard neutralization assays. The F240 IgG1 Ab expressed in CHO cells acquired a strong neutralization activity against a broad range of HIV isolates without a change in immunoreactivity. Sequencing of the F240 mRNAs produced in the parental hybridoma and CHO cells revealed identical sequences, suggesting that acquired neutralization resulted from cell-specific posttranslational modifications. We found that the Ab produced by CHO cells is glycosylated to a greater extent than the parental Ab produced by the hybridoma. Moreover, treatment with peptide N-glycosidase F abrogated F240 neutralization, in an isolate-specific manner, but not Ab b12 neutralization. Interestingly, the F240 isotype-switched variants IgG3 and IgG4, also expressed in CHO cells, exhibited identical immunoreactivity to IgG1 isotypes but had clear differences in viral neutralization. These results suggest that structural features of the Ig molecule other than the primary sequence of the variable regions play a more prominent role in HIV neutralization than anticipated.     

Transmembrane helix-helix interactions are modulated by the sequence context and by lipid bilayer properties

Folding of polytopic transmembrane proteins involves interactions of individual transmembrane helices, and multiple TM helix-helix interactions need to be controlled and aligned to result in the final TM protein structure. While defined interaction motifs, such as the GxxxG motif, might be critically involved in transmembrane helix-helix interactions, the sequence context as well as lipid bilayer properties significantly modulate the strength of a sequence specific transmembrane helix-helix interaction. Structures of 11 transmembrane helix dimers have been described today, and the influence of the sequence context as well as of the detergent and lipid environment on a sequence specific dimerization is discussed in light of the available structural information. This article is part of a Special Issue entitled: Protein Folding in Membranes.     

Effect of glycosylation on the function of a soluble, recombinant form of the transferrin receptor

Production of the soluble portion of the transferrin receptor (sTFR) by baby hamster kidney (BHK) cells is described, and the effect of glycosylation on the biological function of sTFR is evaluated for the first time. The sTFR (residues 121-760) has three N-linked glycosylation sites (Asn251, Asn317, and Asn727). Although fully glycosylated sTFR is secreted into the tissue culture medium ( approximately 40 mg/L), no nonglycosylated sTFR could be produced, suggesting that carbohydrate is critical to the folding, stability, and/or secretion of the receptor. Mutants in which glycosylation at positions 251 and 727 (N251D and N727D) is eliminated are well expressed, whereas production of the N317D mutant is poor. Analysis by electrospray ionization mass spectrometry confirms dimerization of the sTFR and the absence of the carbohydrate at the single site in each mutant. The effect of glycosylation on binding to diferric human transferrin (Fe(2) hTF), an authentic monoferric hTF with iron in the C-lobe (designated Fe(C) hTF), and a mutant (designated Mut-Fe(C) hTF that features a 30-fold slower iron release rate) was determined by surface plasmon resonance; a small ( approximately 20%) but consistent difference is noted for the binding of Fe(C) hTF and the Mut-Fe(C) hTF to the sTFR N317D mutant. The rate of iron release from Fe(C) hTF and Mut-Fe(C) hTF in complex with the sTFR and the sTFR mutants at pH 5.6 reveals that only the N317D mutant has a significant effect. The carbohydrate at position 317 lies close to a region of the TFR previously shown to interact with hTF.     

Controlled glycosylation of plant-produced recombinant proteins

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The leukocyte-endothelial cell interactions are modulated by extracellular matrix proteins.

BACKGROUND: Endothelium is supported, in normal conditions, by a basement membrane composed, among others, by collagen IV and laminin. Changes in the basement membrane composition could induce changes in endothelial cell modifying their interactions with leukocytes. METHODS AND RESULTS: Isolated polymorphonuclear cells (PMN) and peripheral blood mononuclear cells (PBMC) were added to cultured human umbilical endothelial cells (HuVEC) previously seeded on collagen IV, collagen I or gelatin. Adhesion of leukocytes to HUVEC and specific cytotoxicity were analysed. PMN adhesion and cytotoxicity were lower whereas those from PBMC were higher when HuVEC were seeded on collagen I, as compared with cells seeded on collagen IV. To analyse the mechanisms involved in these phenomena, P-selectin, ICAM-1, VCAM-1 and MCP- 1 expression were evaluated in HuVEC seeded on the different ECM components. P-selectin and mRNA expression of VCAM-1 were lower in cells seeded on collagen I. By contrast, MCP-1 expression was higher in collagen I. Collagen I-dependent effects were partially prevented when collagen I was treated with pepsin. ILK activity was lower in cells seeded on collagen I, whereas ERK 1/2 activity was enhanced. ILK overexpression reduced ERK 1/2 phosphorylation and this could promote the reduction in P-selectin and the increase in MCP-1. CONCLUSION: Collagen I decreased ILK activity and this would induce an increase in ERK 1/2 activity in HuVEC. As a consequence, the P-selectin content is diminished and, by contrast, the MCP-1 content is increased. The final effect is a lower recruitment of PMN and a higher adhesion of PBMC.     

The elastic properties of the caulobacter crescentus adhesive holdfast are dependent on oligomers of N-acetylglucosamine

The aquatic bacterium Caulobacter crescentus attaches to solid surfaces through an adhesive holdfast located at the tip of its polar stalk, a thin cylindrical extension of the cell membrane. In this paper, the elastic properties of the C. crescentus stalk and holdfast assembly were studied by using video light microscopy. In particular, the contribution of oligomers of N-acetylglucosamine (GlcNAc) to the elasticity of holdfast was examined by lysozyme digestion. C. crescentus cells attached to a surface undergo Brownian motion while confined effectively in a harmonic potential. Mathematical analysis of such motion enabled us to determine the force constant of the stalk-holdfast assembly, which quantifies its elastic properties. The measured force constant exhibits no dependence on stalk length, consistent with the theoretical estimate showing that the stalk can be treated as a rigid rod with respect to fluctuations of the attached cells. Therefore, the force constant of the stalk-holdfast assembly can be attributed to the elasticity of the holdfast. Motions of cells in a rosette were found to be correlated, consistent with the elastic characteristics of the holdfast. Atomic force microscopy analysis indicates that the height of a dried (in air) holdfast is approximately one-third of that of a wet (in water) holdfast, consistent with the gel-like nature of the holdfast. Lysozyme, which cleaves oligomers of GlcNAc, reduced the force constant to less than 10% of its original value, consistent with the polysaccharide gel-like nature of the holdfast. These results also indicate that GlcNAc polymers play an important role in the strength of the holdfast.     

Biodiversity extinction thresholds are modulated by matrix type

Biodiversity extinction thresholds are abrupt declines in biological diversity that occur with habitat loss, associated with a decline in habitat connectivity. Matrix quality should influence the location of thresholds along habitat loss gradients through its effects on connectivity; however these relationships have seldom been explored empirically. Using field data from 23 independent 1254 ha landscapes in the Brazilian Atlantic Forest, we evaluated how tropical avian biodiversity responds to native forest loss within habitat patches embedded either in homogeneous pasture matrix context (with a high proportion of cattle pastures), and heterogeneous coffee matrix context (with high abundance of sun coffee plantations). We considered taxonomic, functional, and phylogenetic diversity, and tested if matrix type and choice of diversity metric influenced the location of biodiversity thresholds along the forest cover gradient. We found that matrix type postponed the abrupt loss of taxonomic diversity, from a threshold of 35% of forest cover in homogeneous pasture matrix to 19% in heterogeneous coffee matrix. Phylogenetic diversity responded similarly, with thresholds at 30 and 24% in homogeneous‐pasture and heterogeneous‐coffee matrices, respectively, but no relationship with forest cover was detected when corrected for richness correlation. Despite the absence of a threshold for functional diversity in either matrix types, a strong decline below 20% of habitat amount was detected. Finally, below 20% native habitat loss, all diversity indices demonstrated abrupt declines, indicating that even higher‐quality matrices cannot postpone diversity loss below this critical threshold. These results highlight that taxonomic diversity is a more sensitive index of biodiversity loss in fragmented landscapes, which may be used as a benchmark to prevent subsequent functional and phylogenetic losses. Furthermore, increasing matrix quality appears an efficient conservation strategy to maintain higher biodiversity levels in fragmented landscapes over a larger range of habitat loss.     

Circadian clocks are modulated by compartmentalized oscillating translation

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Expression and analysis of the glycosylation properties of recombinant human erythropoietin expressed in Pichia pastoris

The Pichia pastoris expression system was used to produce recombinant human erythropoietin, a protein synthesized by the adult kidney and responsible for the regulation of red blood cell production. The entire recombinant human erythropoietin (rhEPO) gene was constructed using the Splicing by Overlap Extension by PCR (SOE-PCR) technique, cloned and expressed through the secretory pathway of the Pichia expression system. Recombinant erythropoietin was successfully expressed in P. pastoris. The estimated molecular mass of the expressed protein ranged from 32 kDa to 75 kDa, with the variation in size being attributed to the presence of rhEPO glycosylation analogs. A crude functional analysis of the soluble proteins showed that all of the forms were active in vivo.