Two cDNAs, designated xynA and manA, encoding xylanase A (XYLA) and mannanase A (MANA), respectively, were isolated from a cDNA library derived from mRNA extracted from the anaerobic fungus, Piromyces. XYLA and MANA displayed properties typical of endo-beta 1,4-xylanases and mannanases, respectively. Neither enzyme hydrolyzed cellulosic substrates. The nucleotide sequences of xynA and manA revealed open reading frames of 1875 and 1818 base pairs, respectively, coding for proteins of M(r) 68,049 (XYLA) and 68,055 (MANA). The deduced primary structure of MANA revealed a 458-amino acid sequence that exhibited identity with Bacillus and Pseudomonas fluorescens subsp. cellulosa mannanases belonging to glycosyl hydrolase Family 26. A 40-residue reiterated sequence, which was homologous to duplicated noncatalytic domains previously observed in Neocallimastix patriciarum xylanase A and endoglucanase B, was located at the C terminus of MANA. XYLA contained two regions that exhibited sequence identity with the catalytic domains of glycosyl hydrolase Family 11 xylanases and were separated by a duplicated 40-residue sequence that exhibited strong homology to the C terminus of MANA. Analysis of truncated derivatives of MANA confirmed that the N-terminal 458-residue sequence constituted the catalytic domain, while the C-terminal domain was not essential for the retention of catalytic activity. Similar deletion analysis of XYLA showed that the C-terminal catalytic domain homologue exhibited catalytic activity, but the corresponding putative N-terminal catalytic domain did not function as a xylanase. Fusion of the reiterated noncatalytic 40-residue sequence conserved in XYLA and MANA to glutathione S-transferase, generated a hybrid protein that did not associate with cellulose, but bound to 97- and 116-kDa polypeptides that are components of the multienzyme cellulase-hemicellulase complexes of Piromyces and Neocallimastix patriciarum, respectively. The role of this domain in the assembly of the enzyme complex is discussed.

The action on tamarind seed xyloglucan of the pure, xyloglucan-specific endo-(1-->4)-beta-D-glucanase from nasturtium (Tropaeolum majus L.) cotyledons has been compared with that of a pure endo-(1-->)-beta-D-glucanase ('cellulase') of fungal origin. The fungal enzyme hydrolysed the polysaccharide almost completely to a mixture of the four xyloglucan oligosaccharides: [formula: see text] Exhaustive digestion with the nasturtium enzyme gave the same four oligosaccharides plus large amounts of higher oligosaccharides and higher-polymeric material. Five of the product oligosaccharides (D, E, F, G, H) were purified and shown to be dimers of oligosaccharides A to C. D (glc8xyl6) had the structure A-->A, H (glc8xyl6 gal4) was C-->C, whereas E (glc8xyl6gal), F (glc8xyl6gal2) and G (glc8xyl6gal3) were mixtures of structural isomers with the appropriate composition. For example, F contained B2-->B2 (30%), A-->C (30%), C-->A (20%), B2-->B1 (15%) and others (about 5%). At moderate concentration (about 3 mM) oligosaccharides D to H were not further hydrolysed by the nasturtium enzyme, but underwent transglycosylation to give oligosaccharides from the group A, B, C, plus higher oligomeric structures. At lower substrate concentrations, hydrolysis was observed. Similarly, tamarind seed xyloglucan was hydrolysed to a greater extent at lower concentrations. It is concluded that the xyloglucan-specific nasturtium-seed endo-(1-->4)-beta-D-glucanase has a powerful xyloglucan-xyloglucan endo-transglycosylase activity in addition to its known xyloglucan-specific hydrolytic action. It would be more appropriately classified as a xyloglucan endo-transglycosylase. The action and specificity of the nasturtium enzyme are discussed in the context of xyloglucan metabolism in the cell walls of seeds and in other plant tissues.

No abstract

Gram‐negative bacteria are enveloped by a system of two membranes, and they use specialized multicomponent, energy‐driven pumps to transport molecules directly across this double‐layered partition from the cell interior to the extra‐cellular environment. One component of these pumps is embedded in the outer‐membrane, and the paradigm for its structure and function is the TolC protein from Escherichia coli. A common component of a wide variety of efflux pumps, TolC and its homologues are involved in the export of chemically diverse molecules ranging from large protein toxins, such as α‐hemolysin, to small toxic compounds, such as antibiotics. TolC family members thus play important roles in conferring pathogenic bacteria with both virulence and multidrug resistance. These pumps assemble reversibly in a transient process that brings together TolC or its homologue, an inner‐membrane‐associated periplasmic component, an integral inner‐membrane translocase and the substrate itself. TolC can associate in this fashion with a variety of different partners to participate in the transport of diverse substrates. We review here the structure and function of TolC and the other components of the efflux/transport pump.

Although much effort has been expended on evaluating recombinant proteins and synthetic peptides as immunogens, they have generally proved incapable of inducing an efficient cytotoxic T-cell (CTL) response. Filamentous bacteriophage fd can display multiple copies of foreign peptides in the N-terminal region of its major coat protein pVIII, 2,700 copies of which make up the virus capsid. Here we show that fd virions displaying peptide RT2 (ILKEPVHGV), corresponding to residues 309-317 of the reverse transcriptase (RTase) of HIV-1, are able to prime a CTL response specific for this HIV-1 epitope in human cell lines. Successful priming also requires a T-helper epitope, pep23 (KDSWTVNDIQKLVGK), corresponding to residues 249-263 of HIV-1 RTase. Supplying this by displaying it on either the same or a separate bacteriophage virion led to activation of antigen-specific CD4+ T cells. Likewise, HLA-A2 transgenic mice immunized with bacteriophage virions displaying peptide RT2 were shown to mount an effective, specific anti-HIV-RT2 CTL response. This unexpected ability to elicit a designated cytolytic T-cell response, in addition to a B-cell response, has important implications for access to the class I major histocompatibility complex (MHC) loading compartment and the development of recombinant vaccines.

We have investigated the substrate subsite recognition requirement of the xyloglucan endo-transglycosylase/xyloglucan-specific endo-(1-->4)-beta-D-glucanase (NXET) from the cotyledons of nasturtium seedlings. Seed xyloglucans are composed almost entirely of the Glc4 subunits XXXG, XLXG, XXLG and XLLG, where G represents an unsubstituted glucose residue, X a xylose-substituted glucose residue and L a galactosyl-xylose-substituted glucose residue. Thus in the xyloglucan sequence shown below, the xylose (Xyl) residues at the backbone glucose (Glc) residues numbered -3, -2, +2 and +3 may be galactose-substituted, and NXET cleaves between the unsubstituted glucose at -1 and the xylose-substituted glucose at +1, which never carries a galactosyl substituent. [formula: see text] We have isolated the xyloglucan oligosaccharides XXXGXXXG and XLLGXLLG from NXET digests of tamarind seed xyloglucan, have modified them enzymatically using a pure xyloglucan oligosaccharide-specific alpha-xylosidase from nasturtium seeds to give GXXGXXXG and GLLGXLLG, and have identified and compared the products of NXET action on XXXGXXXG, GXXGXXXG, XLLGXLLG and GLLGXLLG. We have also compared the molar proportions of XXXG, XLXG, XXLG and XLLG in native tamarind and nasturtium seed xyloglucans with those in NXET digests of these polysaccharides. Using these and existing data we have demonstrated that NXET action does not require xylose-substitution at glucose residues -4, -2, +1 and +3 and that xylose substitution at +2, is a requirement. There may also be a requirement for xylose substitution at -3. We have demonstrated also that galactosyl substitution of a xylose residue at +1 prevents, and at -2 modifies, chain-cleavage. A partial model for the minimum substrate binding requirement of NXET is proposed.

Virions of filamentous bacteriophage fd are capable of displaying multiple copies of peptide epitopes and generating powerful immune responses to them. To investigate the antigen processing mechanisms in human B cell lines used as antigen presenting cells, the major coat protein (pVIII) in intact virions was fluorescently labeled, and its localization in various intracellular compartments was followed using confocal microscopy. We show that the virions were taken up and processed to yield peptides that reach both the major histocompatibility complex (MHC) class II compartment and the endoplasmic reticulum. Moreover, when exposed to bacteriophages displaying a cytotoxic T lymphocyte (CTL) epitope from the reverse transcriptase of human immunodeficiency virus type-1 (HIV-1), B cells were lysed by specific cytotoxic lymphocytes. This confirms that filamentous bacteriophage virions are capable of being taken up and processed efficiently by MHC class I and class II pathways, even in nonprofessional antigen presenting cells. These remarkable features explain, at least in part, the unexpected ability of virions displaying foreign T-cell epitopes to prime strong T-helper-dependent CTL responses. These findings have important implications for the development of peptide-based vaccines, using filamentous bacteriophage virions as scaffolds.

The α-xylosidase which is involved in the postgerminative mobilisation of xyloglucan in nasturtium seed cotyledons has now been purified to apparent homogeneity by a facile procedure involving lectin affinity chromatography. The purified enzyme, a glycoprotein, moved as a single band (apparent molecular weight 85000) on sodium dodecyl sulphate-gel electrophoresis, whilst isoelectric focusing gave a number of enzymatically active protein bands spanning the range pI = 5.0 to 7.1 (maximum activity at pI = 6.1). The enzyme did not hydrolyse the simple α-xylosides p-nitrophenyl-α-d-xylopyranoside and woprimeverose (α-d-Xyl(1→6)-d-Glc), or polymeric tamarind-seed xyloglucan. It released xylose from a complex mixture of oligosaccharides produced by exhaustive hydrolysis of tamarind seed xyloglucan using the xyloglucan-specific endo-(1→4)-β-d-glucanase from germinated nasturtium seeds (M. Edwards et al. 1986, J. Biol. Chem., 261. 9489-9494). The three xyloglucan oligosaccharides of lowest molecular size were purified from this mixture and were shown by (1)H-nuclear magnetic resonance ((1)H-NMR) and enzymatic analysis to have the structures: