Common Cell-Surface Receptors for Discoidin I and Discoidin II in Dictyostelium discoideum

Following nutrient depletion, cells of the cellular slime mould Dictyostelium discoideum become cohesive and aggregate to form multicellular complexes. Several proteins that accumulate on the cell surface during this period have been implicated in mediating aggregative-phase cell cohesion, namely contact sites A (CsA), gp 150, and two endogenous lectins (discoidin I and discoidin II). The aggregating cells also possess receptors for both discoidin I and discoidin II but these have not yet been isolated and characterised for both lectins.
In the present study we investigated the relationship between the receptors for these lectins, in particular to what extent discoidin I and discoidin II receptors are common. Radio-iodinated discoidin I and discoidin II were purified and used in binding assays for lectin receptors on the surface of aggregated (10 h stage of development) D. discoideum NC4 cells. Sugar competition of ¹²⁵I-labelled discoidin I and ¹²⁵I-labelled discoidin II binding indicated distinct but overlapping sugar specificities for these lectins when binding to their in vivo receptors. Competition of the binding of radio-iodinated lectin with either unlabelled discoidin I or unlabelled discoidin II showed that at least 50% of the cell-surface binding sites for these lectins are in common and for these receptors the binding affinity of discoidin I is 9–20 times higher than for discoidin II. Approximately 35% of discoidin II binding sites appear to be unavailable for discoidin I binding.     

Discoidin I from Dictyostelium discoideum and Interactions with Oligosaccharides: Specificity, Affinity, Crystal Structures, and Comparison with Discoidin II

Discoidin I (DiscI) and discoidin II (DiscII) are N-acetylgalactosamine (GalNAc)-binding proteins from Dictyostelium discoideum. They consist of two domains: an N-terminal discoidin domain and a C-terminal H-type lectin domain. They were cloned and expressed in high yield in recombinant form in Escherichia coli. Although both lectins bind galactose (Gal) and GalNAc, glycan array experiments performed on the recombinant proteins displayed strong differences in their specificity for oligosaccharides. DiscI and DiscII bind preferentially to Gal/GalNAcβ1-3Gal/GalNAc-containing and Gal/GalNAcβ1-4GlcNAcβ1-6Gal/GalNAc-containing glycans, respectively. The affinity of the interaction of DiscI with monosaccharides and disaccharides was evaluated using isothermal titration calorimetry experiments. The three-dimensional structures of native DiscI and its complexes with GalNAc, GalNAcβ1-3Gal, and Galβ1-3GalNAc were solved by X-ray crystallography. DiscI forms trimers with involvement of calcium at the monomer interface. The N-terminal discoidin domain presents a structural similarity to F-type lectins such as the eel agglutinin, where an amphiphilic binding pocket suggests possible carbohydrate-binding activity. In the C-terminal H-type lectin domain, the GalNAc residue establishes specific hydrogen bonds that explain the observed affinity (K_d = 3 × 10^− 4 M). The different specificities of DiscI and DiscII for oligosaccharides were rationalized from the different structures obtained by either X-ray crystallography or molecular modeling.     

Colocalization of discoidin-binding ligands with discoidin in developing Dictyostelium discoideum

The Dictyostelium discoideum lectins, discoidin I and discoidin II, and the endogenous ligands to which they bind were immunohistochemically localized in sections of this organism at successive stages of development. For these studies, an axenic strain, AX3, was grown in a macromolecule-depleted medium rather than on bacteria, which themselves contain discoidin-binding ligands. Discoidin I-binding sites (endogenous ligands) in sections of D. discoideum were concentrated in the slime coat around aggregates, whereas discoidin II-binding sites were observed in a vesicle-like distribution in prespore cells and also in spore coats. In contrast, discoidin II did not bind to the slime coat and discoidin I bound relatively poorly to prespore cells and spore coats. The distributions of the endogenous lectins themselves were the same in axenically grown cells as previously reported for cells raised on bacteria. Discoidin I was concentrated in the slime coat and around stalk cells, and discoidin II was prominent in and around prespore cells. The congruent localization of each lectin with its endogenous ligand suggests that discoidin I normally functions in association with glycoconjugates in the slime around aggregates, and discoidin II with the galactose-rich spore coat polysaccharide.     

An analysis of discoidin I binding sites in Dictyostelium discoideum (NC4).

Vegetative wild-type (strain NC4) D. discoideum cells and cells at the 10h stage of development (aggregation) were harvested in the presence of 0.5 M-galactose to remove any endogenous discoidin I already bound to the cell surface, and fixed with glutaraldehyde. Affinity-purified 125I-labelled discoidin I bound to these fixed cells in a specific manner, greater than or equal to 95% of binding being inhibited by 0.5 M-galactose. Binding of 125I-labelled discoidin I was essentially complete in 90 min at 22 degrees C. Based on specific radioactivity measurements, vegetative (0h) D. discoideum (NC4) cells bind approx. 8.4 x 10(5) discoidin I tetramers/cell and aggregated (10h) cells bind 5.1 x 10(5) discoidin I tetramers/cell, each exhibiting apparent positive co-operativity of binding with highest limiting affinity constants (Ka) of approx. 1 x 10(7) and 2 x 10(7) M-1, respectively. Klebsiella aerogenes, the food source used for growth of D. discoideum NC4 amoebae, also binds 125I-labelled discoidin I and this is greater than 99% inhibited by 0.5 M-galactose. However, at the levels of bacterial contamination present, greater than 97% of 125I-labelled discoidin I binding to D. discoideum cell preparations was to the cells themselves. Confirmation of the number of discoidin I tetramers bound per D. discoideum cell was obtained by elution of bound 125I-labelled discoidin I followed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and then quantification by scanning of stained discoidin I bands.     

Bacterial glycoconjugates are natural ligands for the carbohydrate binding site of discoidin I and influence its cellular compartmentalization

Klebsiella pneumoniae, Escherichia coli, and Bacillus subtilis, bacteria commonly eaten by Dictyostelium discoideum, contain glycoconjugates that bind discoidin I, a lectin synthesized by the slime mold as it differentiates. In cells fed bacteria that contain abundant discoidin I-binding glycoconjugates, these ligands and endogenous discoidin I accumulated in specialized structures called multilamellar bodies. In contrast, in cells fed bacteria that had been treated to thoroughly deplete them of discoidin I-binding glycoconjugates, neither endogenous discoidin I nor complementary glycoconjugates were found in the multilamellar bodies. In such cells discoidin I was located in the cytoplasm, as indicated by both immunohistochemistry with the electron microscope and immunoassay of subcellular fractions. The results indicate that a function of the carbohydrate-binding site of discoidin I is to interact with bacterial glycoconjugates, which the slime mold does not degrade. This interaction directs compartmentalization of the lectin in multilamellar bodies and its externalization from the cell in these structures.     

Purification of a high-affinity discoidin I-binding proteoglycan from axenic Dictyostelium discoideum growth medium

The axenic Dictyostelium discoideum growth medium HL-5, prepared using Difco proteose peptone No. 2, contains an extremely potent inhibitor of the binding of ¹²⁵I-labeled discoidin I to glutaraldehyde-fixed, cohesive D. discoideum cells. Axenic strain A3 D. discoideum cells bind or internalize the inhibitor during growth in HL-5 medium and subsequently shed or excrete it while differentiating in suspension. The inhibitor has been purified from Difco proteose peptone No. 2 by sequential gel filtration on Sepharose 4B and affinity adsorption using discoidin I-Sepharose. The inhibitor is heterogeneous in molecular weight (4 · 10⁵?2 · 10⁶), but is relatively homogeneous in density on CsCl density gradients. The size and activity of the inhibitor are resistant to periodate, reduction and maleylation, proteases, nucleases and heating in the absence or presence of sodium dodecyl sulfate. Mild alkali causes a partial reduction in activity and converts the higher molecular weight fraction of the inhibitor to a lower molecular weight. The purified inhibitor contains neutral hexose, hexosamine and amino acid in an approximate molar ratio of 4 : 3 : 2. These and other properties suggest that the inhibitor is an unusual proteoglycan. Certain well-characterized glycosaminoglycans are relatively potent inhibitors of discoidin I binding. The proteoglycan reported here is the most potent discoidin I-binding inhibitor ever identified.     

Guanine nucleotides modulate cell surface cAMP-binding sites in membranes from Dictyostelium discoideum

D. discoideum contains kinetically distinguishable cell surface cAMP binding sites. One class, S, is slowly dissociating and has high affinity for cAMP (K_d = 15 nM, $\text{t}\text{1}\text{2}\text{= 15 s}$). A second class is fast dissociating ($\text{t}\text{1}\text{2}$ about 1 s) and is composed of high affinity binding sites H (K_d ≈ 60 nM), and low affinity binding sites L (K_d = ≈ 450 nM) which interconvert during the binding reaction. Guanine nucleotides affect these three binding types in membranes prepared by shearing D.discoideum cells through Nucleopore filters. The affinity of S for cAMP is reduced by guanine nucleotides from 13 nM to 25 nM, and the number of S-sites is reduced about 50%. The number of fast dissociating sites is not altered by guanine nucleotides, but these sites are mainly in the low affinity state. Half-maximal effects are obtained at about 1 μM GTP, 2 μM GDP and 10 μM Gpp(NH)p(guanyl-5′-yl-imidodiphosphate); ATP and ADP are without effect up to 1 mM. These results indicate that D.discoideum cells have a functionally active guanine nucleotide binding protein involved in the transduction of extracellular cAMP signals via cell surface cAMP receptors.     

In vitro modulation of macrophage tumoricidal activity

Summary NaIO₄ treatment of mouse adherent peritoneal cells or lymphocyte-free cloned macrophages enhances their cytotoxic and tumoricidal activity. 5×10^–3 M NaIO₄ treatment of nontumoricidal BCG-activated macrophages renders them completely tumoricidal, whereas the same treatment of stimulated (peptone-normal) macrophages renders them weakly tumoricidal. Addition of LPS in nanogram quantities too low to enhance tumor cell killing by untreated peptone-normal macrophages causes NaIO₄-treated peptone-normal macrophages to be maximally tumoricidal. The activating action of NaIO₄, MAF, or LPS can be potently, but inconsistently, blocked or reversed by the reducing agent NaBH₄ or the aldehyde-reacting agent dimedone. NaIO₄ treatment of lymphocyte-free macrophage colonies does not make them cytotoxic, but NaIO₄-treated colony macrophages are cytotoxic for tumor cells when cultured in 10 ng/ml LPS (an amount of LPS inadequate to render untreated colony macrophages cytotoxic). Supernatants of NaIO₄-treated adherent peritoneal cells contain MAF activity. Thus, the NaIO₄-induced enhancement of peritoneal cell tumoricidal activity may result from both direct NaIO₄ activating effects on macrophages and indirect NaIO₄ effects through NaIO₄-induced MAF production.     

Asymmetric reduction of alkyl aryl ketones in the binding domain of bovine serum albumin

The asymmetric reduction of alkyl aryl ketones with sodium borohydride (NaBH₄) or sodium cyanoborohydride (NaBH₃CN) in the presence of bovine serum albumin (BSA) was studied. The asymmetric induction in the product alcohols was largely dependent on the BSA concentration and the pH value of the buffer solution. The maximum optical yields obtained ranged over 14–78%, varying with the alkyl and aryl substituents, using ca. $\text{1}\text{3}$ molar eq of BSA to the ketone and at pH > 9. With two exceptions, the predominant enantiomer had the R configuration in 13 ketones investigated.     

Molecular Cloning and Characterization of the cDNA for Discoidin II of Dictyostelium discoideum

By using monoclonal antibodies directed against discoidin II,we have isolated cDNA clones from axenically grown Ax-2 cells.On cDNA clone (D2) condtained a 1.2-k.b insert encoding theentire discoidin II protein, which is conposed of 257 aminoacid residuces and has a calculated molecular mass of 28,574.The amino acid sequences, determined by Edman degradation ofsix tryptic peptides of discoidin II, were identical to thosededuced from the cDNA sequences. The protein bears no resemblanceto any proteins in the data banks, except that its sequenceis 49% identical with the amino acid sequence of discoidin I.Discoidin II shares with discoidin I both a carbohydratebindingsite and an Arg-Gly-Asp (RGD) sequence, which has been foundin fibronectin in mammalian cells. With the onset of aggregation(8 h of development), a 1.3-kb discoidin II mRNA begins to accumulate.A similar pattern of regulation occurs at the protein level. ¹Present address: MRC Laboratory for Molecular Cell Biology,University College London, Gower Street, London, WC1E 6BT UnitedKingdom     

Sequence and expression of the discoidin I gene family in Dictyostelium discoideum

We have isolated recombinant phage and plasmids containing the four developmentally regulated discoidin I genes of Dictyostelium discoideum. Two of the genes are linked within 0.5 × 10³ bases with the same polarity. S¹ nuclease mapping shows that at least three members of the gene family are expressed and that the 5′ ends of the mRNAs start at equivalent sites. The genes have homologous 5′ untranslated regions and extremely divergent 3′ untranslated regions. In addition, some of the genes are flanked by homologous repeat sequences. The genes encode three different isoelectric forms of the protein. Examination of nucleotide sequences in the protein coding region shows that most nucleotide changes in the 5′ half of the gene result in amino acid substitutions while most base substitutions in the 3′ half are neutral.     

Selective down-regulation of cell surface cAMP-binding sites and cAMP-induced responses in Dictyostelium discoideum

Extracellular cAMP induces an intracellular accumulation of cAMP and cGMP levels in Dictyostelium discoideum. cAMP is detected by cell-surface receptors which are composed of a class of fast-dissociating sites ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1?2}\text{s}$) and a class of slow-dissociating sites ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 15?150}\text{s}$). Exposure of D. discoideum cells to 1 mM cAMP for 30 min induces a reduction of cAMP binding (down-regulation; Klein, C. and Juliani, M.H. (1977) Cell 10, 329–335). The number of fast-dissociating sites was reduced by 80–90% in down-regulated cells. These sites are composed of two forms with high and low affinity which interconvert during the binding reaction. In down-regulated cells this transition still occurred in the residual sites. The accumulation of cellular cAMP levels induced by a saturating stimulus decreased by 80–90%. The number of slow-dissociating sites was not significantly reduced in down-regulated cells, but their affinity decreased about 10-fold. The accumulation of cellular cGMP levels induced by a saturating stimulus was not decreased; however, about 20-fold higher cAMP concentrations were required to induce the same response. These results demonstrate that the cAMP transduction pathways to adenylate cyclase and guanylate cyclase are down-regulated differently. Furthermore, the results suggest that the fast-dissociating sites are involved in the activation of adenylate cyclase, while the slow-dissociating sites are coupled to guanylate cyclase.     

The modulation of cell surface cAMP receptors from Dictyostelium disscoideum by ammonium sulfate

Dictyostelium discoideum cells contain a heterogeneous population of cell surface cAMP receptors with components possessing different affinities (K_d between 15 and 450 nM) and different off-rates of the cAMP-receptor complex ($\text{t}\text{1}\text{2}$ between 0.7 and 150 s). The association of cAMP to the receptor and the dissociation of the cAMP-receptor complex still occur in the presence of 3.4 M ammonium sulfate. However, these processes are strongly altered. (1) Low concentrations of ammonium sulfate (≈ 50 mM) induce an approx. 2-fold increase of the number of cAMP binding sites. The same effect is induced by millimolar concentrations of CaCl₂. Ammonium sulfate and CaCl₂ are not additive, which suggests that these salts may act via the same mechanism. (2) High concentrations of ammonium sulfate (3.4 M) induce an alteration in the proportioning of the various cAMP binding sites to the components with the highest affinity. (3) High concentrations of ammonium sulfate (3.4 M) retard the dissociation of all binding sites about 3–6-fold, thus giving rise to an increase in the affinity of all cAMP-binding components.     

Electron acceptors associated with P-700 in Triton solubilized Photosystem I particles from spinach chloroplasts

Flash-induced absorption changes of Triton-solubilized Photosystem I particles from spinach were studied under reducing and/or illumination conditions that serve to alter the state of bound electron acceptors. By monitoring the decay of P-700 following each of a train of flashes, we found that P-430 or components resembling it can hold 2 equivalents of electrons transferred upon successive illuminations. This requires the presence of a good electron donor, reduced phenazine methosulfate or neutral red, otherwise the back reaction of P-700⁺ with P-430 occurs in about 30 ms. If the two P-430 sites, designated Centers A and B, are first reduced by preilluminating flashes or chemically by dithionite under anaerobic conditions, then subsequent laser flashes generate a 250 μs back reaction of P-700⁺, which we associate with a more primary electron acceptor A₂. In turn, when A₂ is reduced by background (continuous) illumination in presence of neutral red and under strongly reducing conditions, laser flashes then produce a much faster (3 μs) back reaction at wavelengths characteristic of P-700. We associate this with another more primary electron acceptor, A₁, which functions very close to P-700. The organization of these components probably corresponds to the sequence $\text{P-700-}\text{A}{}_1\text{-}\text{A}{}_2\text{-P-430[}\text{A}\text{B}\text{]}$. The relation of the optical components to acceptor species detected by EPR, by electron-spin polarization or in terms of peptide components of Photosystem I is discussed.Preliminary experiments with broken chloroplasts suggest that an analogous situation occurs there, as well.     

A cysteine-rich extracellular protein containing a PA14 domain mediates quorum sensing in Dictyostelium discoideum

Much remains to be understood about quorum-sensing factors that allow cells to sense their local density. Dictyostelium discoideum is a simple eukaryote that grows as single-celled amoebae and switches to multicellular development when food becomes limited. As the growing cells reach a high density, they begin expressing discoidin genes. The cells secrete an unknown factor, and at high cell densities the concomitant high levels of the factor induce discoidin expression. We report here the enrichment of discoidin-inducing complex (DIC), an ~400-kDa protein complex that induces discoidin expression during growth and development. Two proteins in the DIC preparation, DicA1 and DicB, were identified by sequencing proteolytic digests. DicA1 and DicB were expressed in Escherichia coli and tested for their ability to induce discoidin during growth and development. Recombinant DicB was unable to induce discoidin expression, while recombinant DicA1 was able to induce discoidin expression. This suggests that DicA1 is an active component of DIC and indicates that posttranslational modification is dispensable for activity. DicA1 mRNA is expressed in vegetative and developing cells. The mature secreted form of DicA1 has a molecular mass of 80 kDa and has a 24-amino-acid cysteine-rich repeat that is similar to repeats in Dictyostelium proteins, such as the extracellular matrix protein ecmB/PstA, the prespore cell-inducing factor PSI, and the cyclic AMP phosphodiesterase inhibitor PDI. Together, the data suggest that DicA1 is a component of a secreted quorum-sensing signal regulating discoidin gene expression during Dictyostelium growth and development.     

The Discoidin I Gene Family of Dictyostelium Discoideum Is Linked to Genes Regulating Its Expression

The discoidin I protein has been studied extensively as a marker of early development in the cellular slime mold Dictyostelium discoideum. However, like most other developmentally regulated proteins in this system, no reliable information was available on the linkage of the discoidin genes to other known genes. Analysis of the linkage of the discoidin I genes by use of restriction fragment length polymorphisms revealed that all three discoidin I genes as well as a pseudogene are located on linkage group II. This evidence is consistent with the discoidin I genes forming a gene cluster that may be under the control of a single regulatory element. The discoidin I genes are linked to three genetic loci (disA, motA, daxA) that affect the expression of the discoidin I protein. Linkage of the gene family members to regulatory loci may be important in the coordinate maintenance of the gene family and regulatory loci. A duplication affecting the entire discoidin gene family is also linked to group II; this appears to be a small tandem duplication. This duplication was mapped using a DNA polymorphism generated by insertion of the Tdd-3 mobile genetic element into a Tdd-2 element flanking the gamma gene. A probe for Tdd-2 identified a restriction fragment length polymorphism in strain AX3K that was consistent with generation by a previously proposed Tdd-3 insertion event. A putative duplication or rearrangement of a second Tdd-2 element on linkage group IV of strain AX3K was also identified. This is the first linkage information available for mobile genetic elements in D. discoideum.     

The molecular structure of human erythrocyte spectrin. Biophysical and electron microscopic studies.

Molecules of human erythrocyte spectrin have been examined by electron microscopy after low-angle shadowing. Spectrin heterodimers and tetramers were first purified and characterized by polyacrylamide gel electrophoresis and analytical ultracentrifugation under conditions which minimize proteolysis and aggregation. The heterodimers and tetramere were separated for low-angle shadowing by gel filtration in ammonium acetate buffer at physiological ionic strength, in which they showed sedimentation coefficients of 8.9 S and 12.5 S, respectively, similar to those values reported for heterodimers and tetramers in non-volatile buffers. The ammonium acetate buffer promoted the dissociation of spectrin tetramers into heterodimers under conditions in which tetramers in NaCl or KCl buffers are stable. When visualized by low-angle unidirectional and rotary shadowing, spectrin heterodimers appeared as long flexible molecules with a mean shadowed length of 97 nm. Each heterodimer, composed of the two polypeptide chains, band 1 (240,000 M_r) and band 2 (220,000 M_r), often appeared as two separate strands which lay partially separated from one another or coiled round each other in a loose double helix. The association between these polypeptides appears to be weak, except at both ends of the molecule where there are sites of strong binding. Tetramers are formed by the end-to-end association of two spectrin heterodimer molecules without measurable overlap, and have a mean shadowed length of 194 nm. This association to form tetramers probably involves head-to-head binding of the heterodimers, since the higher oligomers to be expected from a head-to-tail binding mode are not observed. The molecular shape of spectrin is quite distinct from that of myosin, to which it has often been likened.     

Uptake of alpha-aminoisobutyric acid in pig spleen lymphocytes stimulated by sodium periodate.

The effect of sodium periodate on the ability of pig spleen lymphocytes to transport the nonmetabolizable amino acid, α-aminoisobutyric acid, was studied. NaIO₄-treated cells exhibited a lowered rate of uptake of α-aminoisobutyric acid in contrast to phytohemagglutinin- and concanavalin A-treated cells. However, when periodate-treated cells were preincubated with untreated cells for 2 h, the mixed cells exhibited twofold stimulation in the uptake of α-aminoisobutyric acid as compared to untreated cells. The increased uptake of α-aminoisobutyric acid in mixed cells was due to a change in the V but not in the K_m. The observed increased uptake of α-aminoisobutyric acid in mixed cells was inhibited (24%) by ouabain, although the level of uptake in untreated and NaIO₄-treated cells was not affected. Na⁺,K⁺-ATPase activity in mixed cells, which was ouabain sensitive, was stimulated 56%. Studies also showed that there was a decrease in the fluorescence polarization (P value) of diphenyl hexatriene in mixed cells (P = 0.21) as compared to untreated cells (P = 0.24). These results demonstrate that NaIO₄ treatment induces a change in the lymphocyte cell membrane and transport of α-aminoisobutyric acid. Incubation of NaIO₄-treated cells with untreated cells is required for the stimulatory effect in the uptake of α-aminoisobutyric acid, and the stimulation appears to be due to changes in Na⁺,K⁺-ATPase activity and membrane fluidity.     

Studies on the transport mechanism of 5-methyltetrahydrofolic acid in freshly isolated hepatocytes: effect of ethanol.

The effect of ethanol on the transport of 5-methyltetrahydrofolate in freshly isolated hepatocytes in vitro resulted in about a 30% increase in accumulation of substrate. It was shown that this was not due to differences in metabolism, nor to an inhibition of efflux. Preincubation with 40 mm ethanol for 45 min resulted in a significantly increased rate of entry of 5-methyltetrahydrofolate into the cells. The stimulatory effect was specific to 5-methyltetrahydrofolate since ethanol inhibited uptake of folate and methotrexate. The increased uptake was due to metabolism of ethanol as shown by studies with pyrazole. Also, the n-alkanols, propanol through pentanol, and sorbitol but not methanol were stimulatory. Anaerobiosis and sodium azide stimulated uptake of 5-methyl-tetrahydrofolate but were inhibitory to methotrexate uptake. These data, taken together, suggest that the ethanol effect is due to increased entry of 5-CH₃-H₄PteGlu into the cells possibly as the result of an increased cellular $\text{NADH}\text{NAD}$ ratio.