Quantitative interactions between cryptdin-4 amino terminal variants and membranes

Paneth cells secrete alpha-defensins into the lumen from the base of small intestinal crypts, and cryptdin-4 (Crp4) is the most potent mouse alpha-defensin in vitro. Purified recombinant Crp4 and Crp4 variants with (des-Gly)-, (Gly1Val)-, (Gly1Asp)-, and (Gly1Arg)-substitutions were all bactericidal with Crp4 and (Gly1Arg)-Crp4 being slightly more active than other variants. Bactericidal activities correlated directly with permeabilization of live Escherichia coli, with equilibrium binding to E. coli membrane phospholipid bilayers and vesicles, and with induced graded fluorophore leakage from phospholipid vesicles. The Crp4 peptide N-terminus affects bactericidal activity modestly, apparently by influencing peptide binding to phospholipid bilayers and subsequent permeabilization of target cell membranes.     

Interactions of mouse Paneth cell alpha-defensins and alpha-defensin precursors with membranes. Prosegment inhibition of peptide association with biomimetic membranes

The bactericidal activity of mouse alpha-defensins (cryptdins) requires proteolytic activation of inactive precursors by matrix metalloproteinase-7 (matrilysin, EC, MMP-7(a)). To investigate mechanisms of cryptdin-4 (Crp4) peptide interactions with membrane bilayers and to determine whether MMP-7-mediated proteolysis activates the membrane disruptive activity of Crp4, associations of Crp4 and melittin with biomimetic lipid/polydiacetylene chromatic vesicles were characterized. The peptides differ in their sensitivity to vesicle lipid composition and their depth of bilayer penetration. Crp4 undergoes strong interfacial binding onto lipid bilayers with disruption of the bilayer head group region, unlike melittin, which inserts more deeply into the hydrophobic core of the bilayer. Colorimetric and tryptophan fluorescence studies showed that Crp4 insertion is favored by negatively charged phospholipids and that zwitterionic and Escherichia coli phospholipids promote stronger interfacial binding; melittin-membrane interactions were independent of either variable. In contrast to the membrane disruptive activity of Crp4, pro-Crp4 did not perturb vesicular membranes, consistent with the lack of bactericidal activity of the precursor, and incubation of Crp4 with prosegment in trans blocked Crp4 and G1W-Crp4 membrane interactions at concentrations that inhibit Crp4 bactericidal activity. CD measurements showed that Crp4 has an expected beta-sheet structure that is not evident in the pro-Crp4 CD trace or when Crp4 is incubated with prosegment, indicating that the beta-sheet signal is attenuated by proregion interactions or possibly disrupted by the prosegment. Collectively, the results suggest that the prosegment inhibits Crp4 bactericidal activity by blocking peptide-mediated perturbation of target cell membranes, a constraint that is relieved when MMP-7 cleaves the prosegment.     

Tandem mass spectrometric identification of spinach Photosystem II light-harvesting components

Light-harvesting proteins harness light energy for photosynthesis. Sequences of the Photosystem II (PS II) light harvesting proteins, Lhcb1–6, have been deduced from many plants. However, limited information is available for spinach Lhcb sequences, although a spinach PS II preparation (BBY) is commonly used as a model for plant photosynthetic oxygen evolution [DA Berthold, GT Babcock and CF Yocum (1981) FEBS Lett 134: 231–234]. In this work, we describe the use of tryptic digestion, liquid chromatography, tandem mass spectrometry, and database searching to identify light-harvesting proteins in the spinach BBY preparation. Using this approach, partial amino acid sequences were assigned to the PS II-associated light-harvesting proteins, Lhcb1–6. The identified stretches of sequence are predicted to contain intra-membranous chlorophyll ligands, extra-membranous loop regions, and lutein-binding sites. In addition, we find that at least two distinct Lhcb4 (CP29) polypeptides and two distinct Lhcb1 polypeptides are present in the BBY preparation. One of these Lhcb4 polypeptides has a subsequence that has not been reported for Lhcb4 in any other organism. This work demonstrates the utility of tandem mass spectrometry in the characterization of photosynthetic membrane proteins. This revised version was published online in June 2006 with corrections to the Cover Date.     

A Cytochemical Study of Extracellular Sheaths Associated with Rigidoporus lignosus during Wood Decay

An ultrastructural and cytochemical investigation of the development of Rigidoporus lignosus, a white-rot fungus inoculated into wood blocks, was carried out to gain better insight into the structure and role of the extracellular sheaths produced by this fungus during wood degradation. Fungal sheaths had a dense or loose fibrillar appearance and were differentiated from the fungal cell wall early after wood inoculation. Close association between extracellular fibrils and wood cell walls was observed at both early and advanced stages of wood alteration. Fungal sheaths were often seen deep in host cell walls, sometimes enclosing residual wood fragments. Specific gold probes were used to investigate the chemical nature of R. lignosus sheaths. While labeling of chitin, pectin, β-1,4- and β-1,3-glucans, β-glucosides, galactosamine, mannose, sialic acid, RNA, fucose, and fimbrial proteins over fungal sheaths did not succeed, galactose residues and laccase (a fungal phenoloxidase) were found to be present. The positive reaction of sheaths with the PATAg test indicates that polysaccharides such as β-1,6-glucans are important components. Our data suggest that extracellular sheaths produced by R. lignosus during host cell colonization play an important role in wood degradation. Transportation of lignin-degrading enzymes by extracellular fibrils indicates that alteration of plant polymers may occur within fungal sheaths. It is also proposed that R. lignosus sheaths may be involved in recognition mechanisms in fungal cell-wood surface interactions.     

Ribosomal RNA sequence phylogeny is not congruent with ascospore morphology among species in Ceratocystis sensu stricto

The genus Ceratocystis sensu stricto includes important fungal pathogens of woody and herbaceous plants. This genus is distinguished from species in Ceratocystis sensu lato by the presence of Chalara anamorphs. Ascospore shape has been used extensively in delineating Ceratocystis taxa, which show a large variety of ascospore shapes. Sequence analysis of one region of he 18S ribosomal RNA subunit and two regions of the 28S ribosomal RNA subunit showed that there was a majority of multiple substitutions at nucleotide sites and that there was a low transition/transversion ratio, T = 0.72. Both of these results suggest that these are well established, old species. Ascospore morphology, for the most part, was not congruent with the molecular phylogeny, and the use of morphological characters may be misleading in the taxonomy of these species.      

Abundant androgen regulated mRNAs in mouse submandibular gland: cell-free translation of renin precursor mRNA

Submandibular glands of male mice contain at least four abundant mRNAs that occur at low concentrations in glands of females. The male-specific mRNAs code for polypeptides of 48,000, 43,000, 29,000, and 27,000 MW. Androgenic regulation of these mRNAs is illustrated by their apparent absence in glands of castrate males and by their accumulation in glands of females treated with testosterone. Selective hybrid-arrested translation experiments also indicate reduced levels of these male-specific sequences in female gland cytoplasm. The 48,000 MW male-specific polypeptide is reduced in translation products directed by gland mRNA from C57BL10/J mice (variants deficient in salivary renin), suggesting the corresponding mRNA codes for a renin precursor. The identity of this polypeptide is confirmed by immune selection with renin-specific antibody.