ABSTRACT␤-1,4-Endoglucanases (EGases, EC 3.2.1.4) degrade polysaccharides possessing ␤-1,4-glucan backbones such as cellulose and xyloglucan and have been found among extremely variegated taxonomic groups. Although many animal species depend on cellulose as their main energy source, most omnivores and herbivores are unable to produce EGases endogenously. So far, all previously identified EGase genes involved in the digestive system of animals originate from symbiotic microorganisms. Here we report on the synthesis of EGases in the esophageal glands of the cyst nematodes Globodera rostochiensis and Heterodera glycines. From each of the nematode species, two cDNAs were characterized and hydrophobic cluster analysis revealed that the four catalytic domains belong to family 5 of the glycosyl hydrolases (EC 3.2.1, 3.2.2, and 3.2.3). These domains show 37-44% overall amino acid identity with EGases from the bacteria Erwinia chrysanthemi, Clostridium acetobutylicum, and Bacillus subtilis. One EGase with a bacterial type of cellulose-binding domain was identified for each nematode species. The leucine-rich hydrophobic core of the signal peptide and the presence of a polyadenylated 3 end precluded the EGases from being of bacterial origin. Cyst nematodes are obligatory plant parasites and the identified EGases presumably facilitate the intracellular migration through plant roots by partial cell wall degradation.

No abstract

The binding of the major water-soluble lens protein a-crystallin to the lens plasma membrane has been investigated by reassociating purified a-crystallin with a-crystallin-jepleted membranes and with phospholipid vesicles in which the lens membrane protein MP26 had been reconstituted. a-Crystallin rcassociates at high affinity (Kd = 13 x 10 ' M ) with alkali-washed lens plasma membranes but not with lens plasma membranes treated with guanidine/HCl, nor with phospholipid vesicles o r erythrocyte membranes. Binding Lo lens plasma membranes is dependent on salt, temperature and ptl and occurs in a saturable manner. Reconstitution of MP26 into phospholipid vesicles and subsequent analysis of a-crystallin binding suggests the involvement o f this transmcmbrane protein. Binding ist not influenced by prctrcatment of membranes with proteases, suggesting that the 4-kDa cytoplasmic fragment of MP26 is not necessary for a-crystallin binding. 1,abeling expcrimcnts using (trifluoromethyl)-3-(~z-[ ' I]iodophenyl)diazirine probe for intrinsic membrane proteins further showed that x-crystallin contains hydrophobic regions on its surface which might enable this protein to make contact with the lipid bilayer. Newly synthesized a-crystallin, in lens culture, is not associated with the plasma membrane, suggesting that the assembly of x-crystallin aggregates does not take place in a membrane-bound mode.Bovine 8u-crystallin is ;i structural eye lens protein which occurs a s i t n aggregatc of 600 -I000 kDa, composed of' two main types of polypeptides, aA and xB (for reviews see [I, 21).The amino acid seqirences of these 20-kDa chains are known from many vcrtcbrate species. a-Crystallin contains tnainly /i and random coil conformation and has very little a-helical content [ 3 ] . N o direct tertiary structure information about x-cryst;illin is available, but models for the quaternary structure of bovine 1-crystallin have been proposed [4].Although x-crystallin is a major component of the watcrsoluble fraction of the lens fiber cells, it has already been shown in earlier work that the insoluble protein from bovine lens is largely derived from x-crystdlin (reviewed in [S]). Part or this protein also secins to exist in close association with the fiber plasma membrane [h --81. 'The membrane-associated 2-crystallin fraction cannot be extracted with various buffers, and even 6 M urea solubilizes only part of it. 'Fhcrefore x-crystallin has sometimes been regarded a s an integral plasma mcrnbrane constituent [' ,I. Physicochemical studies have indeed indicated that x-crystallin, as compared to the other crystallins, has more hydrophobic peptide-peptide interactions. and that more of its hydrophobic amino acids are sohcnt-exposed or solvent-accessible [I 0--131. These studies also showcd that the apparent A H and A S values for the solubi1ir;ation of x-crystallin arc quite low, which is indicative of ii rather hydrophobic character [lo, 131. An intriguing feature of a-crystallin is the presence o f regions which show considerable sequence s...

In preparasitic second-stage juveniles (J(2)) of potato cyst nematode Globodera rostochiensis, six proteins with molecular masses of 30, 31a/b, 32, 39, and 49 kDa were recognized on Western blots by a monoclonal antibody (MGR48) specific for the subventral esophageal glands. All of these subventral gland proteins (svp's) focused in the basic range (pI 6.8 to 8.6) of an immobilized pH gradient. Western blotting showed that the svp's were present in preparasitic and parasitic J(2) and not in later juvenile stages and adult females. Minor svp quantities also were observed in adult males. Immunogold labeling of preparasitic J(2) showed that the svp's were localized in the rough endoplasmic reticulum and secretory granules of the subventral esophageal glands. Potato root diffusate triggered the secretion of svp's through the stylet, and 5-methoxy-N,N-dimethyltryptamine-hydrogen-oxalate had only a quantitative, additional effect. The forward flow of svp's through the metacorporal pump chamber was confirmed by the presence of svp's in the circular lumen of the esophagus (procorpus), as established by immunoelectron microscopy. Our data provide conclusive evidence that secretory proteins of the subventral glands of G. rostochiensis can be secreted through the stylet and support the hypothesis that the subventral esophageal glands play an important role in the early events of this nematode-plant interaction.

The gene encoding a protein containing a putative [6Fe-6S] prismane cluster has been cloned from Desulfovibrio vulgaris (Hildenborough) and sequenced. The gene encodes a polypeptide composed of 553 amino acids (60161 Da). The DNA-derived amino acid sequence was partly confirmed by Nterminal sequencing of the purified protein and of fragments of the protein generated by CNBr cleavage. Furthermore, the C-terminal sequence was verified by digestion with carboxypeptidases A and B. The polypeptide contains nine Cys residues. Four of these residues are gathered in a CysXaa,-Cys-Xaa7-Cys-Xaa5-Cys motif located towards the N-terminus of the protein. No relevant sequence similarity was found with other proteins, including those with high-spin Fe-S clusters (nitrogenase, hydrogenase), with one significant exception : the stretch containing the first four Cys residues spans two submotifs, Cys-Xaa2-Cys and Lys-Gly-Xaa-Cys-Gly, separated by 11 residues, that are also present in high-spin Fe-S cluster containing CO dehydrogenase. Western-blot analysis demonstrates cross-reactivity of antibodies raised against the purified protein both in Desulfovibrio strains and other sulfate-reducing bacteria. Hybridization of the cloned gene with genomic DNA of several other Desulfovibrio species indicates that homologous sequences are generally present in the genus Desulfovibrio. The biochemical characterization of the protein (purification, metal analysis, cellular localization, amino acid composition and N-terminal amino acid sequence) was further described by Pierik et al. [S]. The protein was described as a cytoplasmic, monomeric protein with a molecular mass of 52 kDa, as determined by sedimentation-equilibrium centrifugation. The iron and acid-labile-sulfur content were both estimated at approximately 6 atoms/polypeptide.In the past few years, similar very-high-spin EPR signals were found in a number of enzymes: dissimilatory sulfite two Ni [7,8] and the P cluster of the nitrogenase MoFe protein from Azatobacter vinelandii containing 24-28 Fe and two Mo [9,10]. In these enzymes, the high-spin clusters are thought LO be essential for enzyme activity. Unfortunately, these proteins all contain several iron-sulfur clusters (and/ The structure of these so-called superclusters in proteins will ultimately be given by X-ray crystallography. This requires knowledge of the primary structure of the protein containing the supercluster. In this paper, we investigate the primary structure of the prismane protein of D. vulgaris (Hildenborough). MATERIALS AND METHODS MaterialsRcstriction endonucleases, DNA polymerase I, large fragment of DNA polymerase I (Klenow fragment), Taq DNA polymerase, T4 DNA ligase, S1 nuclease, RNA markers (0.24-9.5 kb) and exonuclease 111 were purchased form Bethesda Research Laboratories. Calf intestinal phosphatase, dNTP, ddNTP and protein A were obtained from Boehringer.[ E -~~P ]~A T P (3000 Ci/mmol) and Na"'1 was purchased from Amersham. Iodo-beads iodination reagent was from Pierce. Universal M13 sequencing primer ...

Downstream of the genes for the structural alpha and beta subunits of the periplasmic Desulfovibrio vulgaris (Hildenborough) hydrogenase a DNA fragment was detected with sequence homology to these genes. This fragment was cloned in Escherichia coli and the nucleotide sequence was determined. A gene was detected on the fragment with coding capacity for a 65.8 kDa polypeptide, hyd gamma. The central part of hyd gamma has an unusually high degree of homology with the alpha subunit and the C-terminal part has similarity with the beta subunit. These results strongly suggest that the three genes for hyd gamma and the alpha and beta subunits derive from one common ancestor gene. We succeeded in the identification of the translational product of this gene in E. coli, but were unable to determine the function of hyd gamma after expression in E. coli.

No abstract

The gene encoding the prismane protein from Desulfovibrio vulgaris (Hildenborough) was inserted into broad-host-range vector pSUP104. The recombinant plasmid, pJSP104, was transferred to D. vulgaris by conjugal plasmid transfer. In the transconjugant D. vulgaris cells the prismane protein was 25-fold overproduced. The overproduced prismane protein was characterized by molecular mass, isoelectric point, iron content and spectroscopical properties. Both the iron content and the ultraviolet/ visible spectrum are identical to the wild-type protein indicating that iron incorporation in the overproduced protein is complete. EPR spectra of the dithionite-reduced form of the overproduced protein indicated that the Fe-S cluster might occur in a similar structure as found in inorganic model compounds containing a [6Fe-6S] prismane core. The as-isolated overproduced protein showed the presence of a second S = 1/2 spin system that was also detected in the corresponding prismane protein from D . desuljiuricans (ATCC 27774), but not in the protein from wild-type D . vulgaris. This additional signal was irreversibly transformed to the 'wild-type' high-spin and low-spin systems upon two reduction/re-oxidation cycles. It is shown that the EPR spectroscopy of the overproduced prismane protein is very similar to that of the D. desulfuricans enzyme and, with the exception of the second S = 1/2 spin system, to that of the prismane protein from wild-type D. vulgaris. Contrary to claims for the D. desulfuricans protein, it is shown here that all data can be fully explained assuming a single [6Fe-6S] cluster, that might be titrated into four different redox states and occurs in up to three different spin systems in the one-electron reduced state.The prismane protein from Desulfovibrio vulgaris (Hildenborough) was the first example of a protein in which a Fe-S cluster with S = 9/2 paramagnetism was discovered [l -31. Since then, high-spin ('superspin') Fe-S clusters with spin systems S > 512 have been described in a number of enzymes: dissimilatory sulfite reductase from D . vulgaris (Hildenborough) [4], CO dehydrogenase from Methanothrix soehngenii [5, 61 and the nitrogenase MoFe protein (the Pclusters in this protein) from Azotobacter vinelandii [7, 81. However, these latter enzymes all contain large numbers of iron atoms (20-30), arranged in multiple, different, clusters. This makes a study of the redox properties and the spectroscopic analysis of the high-spin clusters in these proteins rather complicated. The prismane protcin, on the other hand, was shown to contain only six irons and six acid-labile sulfide ions! molecule [9]. Therefore, this protein was used as a relatively simple model protein for the study of these high-spin Fe-S clusters, although its function is as yet unknown [lo] In the 5+ state, the cluster occurs in two magnetic forms: approximately 90% of the clusters occurs in a high-spin S = 9/2 system and 10% in a S = 1/2 spin system. In the 3 + state, the cluster occurs in a S = 1/2 spin system; the EPR spectrum of...