Arabidopsis MAP-Kinase 3 Phosphorylates UDP-Glucose Dehydrogenase: a Key Enzyme Providing UDP-Sugar for Cell Wall Biosynthesis

The enzyme UDP-glucose dehydrogenase (UGD) competes with sucrose-phosphate synthase for the common photosynthesis product UDP-glucose. Sucrose-phosphate synthase is part of a pathway for the export of sucrose from source leaves to neighboring cells or the phloem. UGD is a central enzyme in a pathway for many nucleotide sugars used in local cell wall biosynthesis. Here, we identify a highly conserved phosphorylation site in UGD which is readily phosphorylated by MAP-kinase 3 in Arabidopsis. Phosphorylation occurs at a surface-exposed extra loop in all plant UGDs that is absent in UGDs from bacteria or animals. Phosphorylated sucrose-phosphate synthase is shifted to an inactive form which we did not measure for phosphorylated UGD. Plant UGDs have an extra loop which is phosphorylated by AtMPK3. Phosphorylation is not causing a reduction of UGD activity as found for the competitor enzymes and thus sets a preference for maintaining UDP-sugars at a constant level to prioritize cell wall biosynthesis.

     

Matrilins mediate weak cell attachment without promoting focal adhesion formation.

The matrilins form a family of non-collagenous adaptor proteins in the extracellular matrix. The extracellular ligand interactions of matrilins have been studied in some detail, while the potential interplay between matrilins and cells has been largely neglected. Except for matrilin-4, all matrilins mediate cell attachment, but only for matrilin-1 and -3 the binding is clearly dose dependent and seen already at moderate coating concentrations. Even so, much higher concentrations of matrilin-1 or -3 than of fibronectin are required for cell attachment to reach plateau values. Integrins contribute to the matrilin-mediated cell attachment, but the binding does not lead to formation of focal contacts and reorganisation of the actin cytoskeleton. Cells deficient in beta1 integrins are able to adhere, although weaker, and matrilins do not bind the soluble integrin alpha1beta1 and alpha2beta1 ectodomains. Cell surface proteoglycans may promote the attachment, as cells deficient in glycosaminoglycan biosynthesis adhere less well to matrilin-3. Even so, exogenous glycosaminoglycans are not able to compete for the attachment of HaCaT cells to matrilins.     

Heterogeneity of Collagen VI Microfibrils: STRUCTURAL ANALYSIS OF NON-COLLAGENOUS REGIONS*

Collagen VI, a collagen with uncharacteristically large N- and C-terminal non-collagenous regions, forms a distinct microfibrillar network in most connective tissues. It was long considered to consist of three genetically distinct α chains (α1, α2, and α3). Intracellularly, heterotrimeric molecules associate to form dimers and tetramers, which are then secreted and assembled to microfibrils. The identification of three novel long collagen VI α chains, α4, α5, and α6, led to the question if and how these may substitute for the long α3 chain in collagen VI assembly. Here, we studied structural features of the novel long chains and analyzed the assembly of these into tetramers and microfibrils. N- and C-terminal globular regions of collagen VI were recombinantly expressed and studied by small angle x-ray scattering (SAXS). Ab initio models of the N-terminal globular regions of the α4, α5, and α6 chains showed a C-shaped structure similar to that found for the α3 chain. Single particle EM nanostructure of the N-terminal globular region of the α4 chain confirmed the C-shaped structure revealed by SAXS. Immuno-EM of collagen VI extracted from tissue revealed that like the α3 chain the novel long chains assemble to homotetramers that are incorporated into mixed microfibrils. Moreover, SAXS models of the C-terminal globular regions of the α1, α2, α4, and α6 chains were generated. Interestingly, the α1, α2, and α4 C-terminal globular regions dimerize. These self-interactions may play a role in tetramer formation.     

A monovalent mutant of cyanovirin-N provides insight into the role of multiple interactions with gp120 for antiviral activity

Cyanovirin-N (CV-N) is a 101 amino acid cyanobacterial lectin with potent antiviral activity against HIV, mediated by high-affinity binding to branched N-linked oligomannosides on the viral surface envelope protein gp120. The protein contains two carbohydrate-binding domains, A and B, each of which binds short oligomannosides independently in vitro. The interaction to gp120 could involve either a single domain or both domains simultaneously; it is not clear which mode would elicit the antiviral activity. The model is complicated by the formation of a domain-swapped dimer form, in which part of each domain is exchanged between two monomers, which contains four functional carbohydrate-binding domains. To clarify whether multivalent interactions with gp120 are necessary for the antiviral activity, we engineered a novel mutant, P51G-m4-CVN, in which the binding site on domain A has been knocked out; in addition, a [P51G] mutation prevents the formation of domain-swapped dimers under physiological conditions. Here, we present the crystal structures at 1.8 A of the free and of the dimannose-bound forms of P51G-m4-CVN, revealing a monomeric structure in which only domain B is bound to dimannose. P51G-m4-CVN binds gp120 with an affinity almost 2 orders of magnitude lower than wt CV-N and is completely inactive against HIV. The tight binding to gp120 is recovered in the domain-swapped version of P51G-m4-CVN, prepared under extreme conditions. Our findings show that the presence of at least two oligomannoside-binding sites, either by the presence of intact domains A and B or by formation of domain-swapped dimers, is essential for activity.     

The influence of culture media on embryonic renal collecting duct cell differentiation

Summary During kidney development the embryonic ampullar collecting duct (CD) epithelium changes its function. The capability for nephron induction is lost and the epithelium develops into a heterogeneously composed epithelium consisting of principal and intercalated cells. Part of this development can be mimicked under in vitro conditions, when embryonic collecting duct epithelia are isolated from neonatal rabbit kidneys and kept under perfusion culture. The differentiation pattern is quite different when the embryonic collecting duct epithelia are cultured in standard Iscove’s modified Dulbecco’s medium as compared to medium supplemented with additional NaCl. Thus, the differentiation behavior of embryonic CD epithelia is unexpectedly sensitive. To obtain more information about how much influence the medium has on cell differentiation, we tested medium 199, basal medium Eagle, Williams’ medium E, McCoys 5A medium, and Dulbecco’s modified Eagle medium under serum-free conditions. The experiments show that in general, all of the tested media are suitable for culturing embryonic collecting duct epithelia. According to morphological criteria, there is no difference in morphological epithelial cell preservation. The immunohistochemical data reveal two groups of expressed antigens. Constitutively expressed antigens such as cytokeratin 19, P_CD 9, Na/K ATPase, and laminin are present in all cells of the epithelia independent of the culture media used. In contrast, a group of antigens detected by mab 703, mab 503, and PNA is found only in individual series. Thus, each culture medium produces epithelia with a very specific cell differentiation pattern.     

Population dynamics of Gentiana pneumonanthe and Rhynchospora fusca during wet heathland restoration

Abstract. The population dynamics and reproductive strategies of two rare wet heathland species, Gentiana pneumonanthe and Rhynchospora, were studied in experimental permanent plots in a wet heathland near Bremen, NW Germany, to assess how effective sod cutting and mowing are in promoting these species. In one experiment, small plots (0.25 m² - 4 m²) were sod-cut or mown; in the second, one large plot 30 mx 50m) was sod-cut. The development of the vegetation and the number of shoots of the two target species were recorded annually. Sod cutting lead to the highest shoot numbers of Gentiana in the long run, whilst mowing was more effective at the beginning of the experiment. Seedlings and adult shoots slowly became more and more abundant after sod cutting. In contrast, Rhynchospora soon formed closed stands after sod-cutting, first through seed germination and then through fast clonal growth of the established individuals. Moist soil or short inundations promoted the germination and seedling establishment of Gentiana, whereas drought negatively affected seedling recruitment. Long periods of inundation severely reduced the population at first, but high numbers of seedlings were found in the following growing season. For the disturbance-dependent Gentiana and Rhynchospora, the availability of gaps within the vegetation is of crucial importance. To promote existing populations, we suggest small-scale sod cuttings which create gaps without disturbing existing flowering individuals too much. For degenerated stands of wet heathland we recommend large-scale sod cutting to activate the seed bank. Additionally, seed introduction may be helpful to encourage the development of a wet heathland with characteristic floristic composition.     

Conformational gating of dimannose binding to the antiviral protein cyanovirin revealed from the crystal structure at 1.35 A resolution

Cyanovirin (CV-N) is a small lectin with potent HIV neutralization activity, which could be exploited for a mucosal defense against HIV infection. The wild-type (wt) protein binds with high affinity to mannose-rich oligosaccharides on the surface of gp120 through two quasi-symmetric sites, located in domains A and B. We recently reported on a mutant of CV-N that contained a single functional mannose-binding site, domain B, showing that multivalent binding to oligomannosides is necessary for antiviral activity. The structure of the complex with dimannose determined at 1.8 A resolution revealed a different conformation of the binding site than previously observed in the NMR structure of wt CV-N. Here, we present the 1.35 A resolution structure of the complex, which traps three different binding conformations of the site and provides experimental support for a locking and gating mechanism in the nanoscale time regime observed by molecular dynamics simulations.