Molecular cloning and expression of endo-beta-N-acetylglucosaminidase D, which acts on the core structure of complex type asparagine-linked oligosaccharides

Endo-beta-N-acetylglucosaminidase D (Endo D) produced by Streptococcus pneumoniae cleaves the di-N-acetylchitobiose structure in asparagine-linked oligosaccharides. The enzyme generally acts on complex type oligosaccharides after removal of external sugars by neuraminidase, beta-galactosidase, and beta-N-acetylglucosaminidase. We cloned the gene encoding the enzyme and expressed it as a periplasm enzyme in Escherichia coli. The first 37 amino acids in the predicted sequence are removed in the mature enzyme, yielding a protein with a molecular mass of 178 kDa. The substrate specificity of the recombinant enzyme is indistinguishable from the enzyme produced by S. pneumoniae. Endo-beta-N-acetylglucosaminidase A (Endo A) from Arthrobacter protophormiae, the molecular mass of which is 72 kDa, had 32% sequence identity to Endo D, starting from the N-terminal sides of both enzymes, although Endo A hydrolyzes high-mannose-type oligosaccharides and does not hydrolyze complex type ones. Endo D is not related to endo-beta-N-acetylglucosaminidases H, F(1), F(2), or F(3), which share common structural motifs. Therefore, there are two distinct groups of endo-beta-N-acetylglucosaminidases acting on asparagine-linked oligosaccharides. The C-terminal region of Endo D shows homology to beta-galactosidase and beta-N-acetylglucosaminidase from S. pneumoniae and has an LPXTG motif typical of surface-associated proteins of Gram-positive bacteria. It is possible that Endo D is located on the surface of the bacterium and, together with other glycosidases, is involved in virulence.     

Molecular interactions of the Gbeta binding domain of the Ste20p/PAK family of protein kinases. An isolated but fully functional Gbeta binding domain from Ste20p is only partially folded as shown by heteronuclear NMR spectroscopy

The transmission of the mating signal of the budding yeast Saccharomyces cerevisiae requires Ste20p, a member of the serine/threonine protein kinases of the Ste20p/PAK family, to link the Gbeta subunit of the heterotrimeric G protein to the mitogen-activated protein kinase cascades. The binding site of Ste20p to the Gbeta subunit was mapped to a consensus sequence of SSLphiPLI/VXphiphibeta (X for any residue; phi for A, I, L, S or T; beta for basic residues), which was shown to be a novel Gbeta binding (GBB) motif present only in the noncatalytic C-terminal domains of the Ste20p/PAK family of protein kinases (Leeuw, T., Wu, C., Schrag, J. D., Whiteway, M., Thomas, D. Y., and Leberer, E. (1998) Nature 391, 191-195; Leberer, E., Dignard, D., Thomas, D. Y., and Leeuw, T. (2000) Biol. Chem. 381, 427-431). Here, we report the results of an NMR study on two GBB motif peptides and the entire C-terminal domain derived from Ste20p. The NMR data show that the two peptide fragments are not uniquely structured in aqueous solution, but in the presence of 40% trifluoroethanol, the longer 37-residue peptide exhibited two well defined, but flexibly linked helical structure elements. Heteronuclear NMR data indicate that the fully functional 86-residue C-terminal domain of Ste20p is again unfolded in aqueous solution but has helical secondary structure preferences similar to those of the two peptide fragments. The NMR results on the two GBB peptides and the entire GBB domain all indicate that the two important binding residues, Ser(879) and Ser(880), are located at the junction between two helical segments. These experimental observations with the prototype GBB domain of a novel family of Gbeta-controlled effectors may have important implications in understanding the molecular mechanisms of the signal transduction from the heterotrimeric G protein to the mitogen-activated protein kinase cascade.     

Analysis of the subsite specificity of rat insulysin using fluorogenic peptide substrates

Recombinant rat insulysin was shown to cleave the internally quenched fluorogenic peptide 2-aminobenzyl-GGFLRKVGQ-ethylenediamine-2,4-dinitrophenol at the R-K bond, exhibiting a K(m) of 13 microm and a V(max) of 2.6 micromol min(-1) mg(-1). Derivatives of this peptide in which the P(2) leucine or the P(2)' valine were replaced with other residues were used to probe the subsite specificity of the enzyme. Varying the P(2) residue produced a 4-fold range in K(m) and a 7-fold range in k(cat). The nature of the P(2) residue had a significant effect on the site of cleavage. Leucine, isoleucine, valine, and aspartate produced cleavage at the R-K bond. Asparagine produced 36% cleavage at the N-R bond and 64% cleavage at the R-K bond, whereas with alanine or serine the A-R and S-R bonds were the major cleavage sites. With tyrosine, phenylalanine, methionine, or histidine representing the varied residue X, cleavages at F-X, X-R, and R-K were seen, whereas with tryptophan equal cleavage occurred at the F-W and W-R bonds. Variable P(2)' residues produce less of a change in both K(m) and k(cat) and have little influence on the cleavage site. Exceptions are phenylalanine, tyrosine, leucine, and isoleucine, which in addition to producing cleavage at the R-K bond, produce significant cleavage at the L-R bond. Alanine and tyrosine were unique in producing cleavage at the F-L bond. Taken together, these data suggest that insulysin specificity is directed toward the amino side of hydrophobic and basic residues and that the enzyme has an extended substrate binding site.     

Functional roles of the tyrosine within the NP(X)(n)Y motif and the cysteines in the C-terminal juxtamembrane region of the CB2 cannabinoid receptor

Sequence-dependent DNA conformations of matrix attachment regions (MARs) available in a database were calculated using the wedge model, and compared with randomly chosen genes, promoters, enhancers and transposons. The MARs had a longer bent part and higher angle/helical turn than the other regions. It is known that some MAR sequences have A-tracts that cause DNA bending, and we also found many A-tracts in examined MARs. Furthermore, non-random and clustered distribution of A-tracts shown here gave further evidence of the importance of A-tracts for MAR conformations. These results suggest that DNAs of MARs have a characteristic conformation instead of conserved sequence.     

Central beta-amyloid peptide-induced peripheral interleukin-6 responses in mice

beta-Amyloid peptides (Abetas) share with lipopolysaccharide, a potent pro-inflammatory agent, the property of stimulating glial cells or macrophages to induce various inflammatory mediators. We recently reported that central administration of lipopolysaccharide induces peripheral interleukin-6 responses via both the central and peripheral norepinephrine system. In this study, the effect of intracerebroventricular injection of various synthetic Abetas on plasma interleukin-6 levels was examined in mice. Abeta(1-42) dose-dependently increased plasma interleukin-6 levels: 'aged' Abeta(1-42) was more effective than fresh, whereas Abeta(42-1) had no effect. 'Aged' Abeta(1-42) (205 pmol/mouse i.c.v.)-induced plasma interleukin-6 peaked at 2 h post injection, which is earlier than the peak time of the Abeta(1-42)-induced brain interleukin-6, tumor necrosis factor-alpha and interleukin-1beta levels, which was 4, 4 and 24 h, respectively. Among various peripheral organs, Abeta(1-42) (205 pmol/mouse i.c.v.) significantly increased interleukin-6 mRNA expression in lymph nodes and liver. Abeta(1-42) (205 pmol/mouse i.c.v.) significantly increased norepinephrine turnover in both hypothalamus and spleen. Either central or peripheral norepinephrine depletion effectively inhibited the Abeta(1-42)-induced peripheral interleukin-6 response. Pretreatment with prazosin (alpha(1)-adrenergic antagonist), yohimbine (alpha(2)-adrenergic antagonist), and ICI-118,551 (beta(2)-adrenergic antagonist), but not with betaxolol (beta(1)-adrenergic antagonist), inhibited Abeta(1-42)-induced plasma interleukin-6 levels. These results demonstrate that centrally administered Abeta(1-42) effectively induces the systemic interleukin-6 response which is mediated, in part, by central Abeta(1-42)-induced activation of the central and the peripheral norepinephrine systems.     

Herpes simplex virus-induced keratitis: evaluation of the role of molecular mimicry in lesion pathogenesis

Viruses are suspected but usually unproven triggering factors in autoimmunity. One favored mechanism to explain the role of viruses in the genesis of autoimmunity is molecular mimicry. An immunoinflammatory blinding lesion called herpetic stromal keratitis (HSK) that follows ocular infection with herpes simplex virus (HSV) is suggested to result from a CD4(+) T-cell response to a UL6 peptide of HSV that cross-reacts with a corneal autopeptide shared with the immunoglobulin G2a(b) (IgG2a(b)) isotype. The present report reevaluates the molecular mimicry hypothesis to explain HSK pathogenesis. Our results failed to reveal cross-reactivity between the UL6 and IgG2a(b) peptides or between peptide reactive T cells and HSV antigens. More importantly, animals infected with HSV failed to develop responses that reacted with either peptide, and infection with a recombinant vaccinia UL6 vector failed to cause HSK, in spite of generating UL6 reactivity. Other lines of evidence also failed to support the molecular mimicry hypothesis, such as the failure to affect HSK severity upon tolerization of susceptible BALB/c and B-cell-deficient mice with IgG2a(b) or UL6 peptides. An additional study system revealed that HSK could be induced in mouse strains, such as the OT2 x RAG1(-/-) mice (T cell receptor transgenic recognizing OVA(323-339)) that were unable to produce CD4(+) T-cell responses to any detectable HSV antigens. Our results cast doubt on the molecular mimicry hypothesis as an explanation for the pathogenesis of HSK and indicate that if autoimmunity is involved its likely proceeds via a bystander activation mechanism.