A Soluble Chloroplast Protease Processes the Euglena Polyprotein Precursor to the Light Harvesting Chlorophyll a/b Binding Protein of Photosystem II

The Euglena light harvesting chlorophyll a/b binding protein of photosystem II (LHCPII) is synthesized as a polyprotein precursors composed of 8 LHCPIIs covalently joined by a decapeptide. A soluble chloroplast protease releases LHCPII from the polyprotein. The polyprotein processing peptidase has a pH optima between 8.0 and 9.0. It is inhibited by Zn2+, Cu2+, phenylmethylsulfonyl fluoride and E64 suggesting it is a novel thiol protease

Topology of Euglena chloroplast protein precursors within endoplasmic reticulum to Golgi to chloroplast transport vesicles

Euglena chloroplast protein precursors are transported as integral membrane proteins from the endoplasmic reticulum (ER) to the Golgi apparatus prior to chloroplast localization. All Euglena chloroplast protein precursors have functionally similar bipartite presequences composed of an N-terminal signal peptide domain and a stromal targeting domain containing a hydrophobic region approximately 60 amino acids from the predicted signal peptidase cleavage site. Asparagine-linked glycosylation reporters and presequence deletion constructs of the precursor to the Euglena light-harvesting chlorophyll a/b-binding protein of photosystem II (pLHCPII) were used to identify presequence regions translocated into the ER lumen and stop transfer membrane anchor domains. An asparagine-linked glycosylation site present at amino acid 148 of pLHPCII near the N terminus of mature LHCPII was not glycosylated in vitro by canine microsomes while an asparagine-linked glycosylation site inserted at amino acid 40 was. The asparagine at amino acid 148 was glycosylated upon deletion of amino acids 46-146, which contain the stromal targeting domain, indicating that the hydrophobic region within this domain functions as a stop transfer membrane anchor sequence. Protease protection assays indicated that for all constructs, mature LHCPII was not translocated across the microsomal membrane. Taken together with the structural similarity of all Euglena presequences, these results demonstrate that chloroplast precursors are anchored within ER and Golgi transport vesicles by the stromal targeting domain hydrophobic region oriented with the presequence N terminus formed by signal peptidase cleavage in the vesicle lumen and the mature protein in the cytoplasm

Human Antibodies that Recognize Novel Immunodominant Quaternary Epitopes on the HIV-1 Env Protein

<div><p>Numerous broadly neutralizing antibodies (Abs) target epitopes that are formed or enhanced during mature HIV envelope formation (<i>i</i>.<i>e</i>. quaternary epitopes). Generally, it is thought that Env epitopes that induce broadly neutralizing Abs are difficult to access and poorly immunogenic because of the characteristic oligomerization, conformational flexibility, sequence diversity and extensive glycosylation of Env protein. To enhance for isolation of quaternary epitope-targeting Abs (QtAbs), we previously used HIV virus-like particles (VLPs) to bind B cells from long-term non-progressor subjects to identify a panel of monoclonal Abs. When expressed as recombinant full-length Abs, a subset of these novel Abs exhibited the binding profiles of QtAbs, as they either failed to bind to monomeric Env protein or showed much higher affinity for Env trimers and VLPs. These QtAbs represented a significant proportion of the B-cell response identified with VLPs. The Ab genes of these clones were highly mutated, but they did not neutralize common HIV strains. We sought to further define the epitopes targeted by these QtAbs. Competition-binding and mapping studies revealed these Abs targeted four separate epitopes; they also failed to compete for binding by Abs to known major neutralizing epitopes. Detailed epitope mapping studies revealed that two of the four epitopes were located in the gp41 subunit of Env. These QtAbs bound pre-fusion forms of antigen and showed differential binding kinetics depending on whether oligomers were produced as recombinant gp140 trimers or as full-length Env incorporated into VLPs. Antigenic regions within gp41 present unexpectedly diverse structural epitopes, including these QtAb epitopes, which may be targeted by the naturally occurring Ab response to HIV infection.</p></div

Epitope competition group (CG) assignment, binding kinetics and preference of quaternary-epitope targeting Abs for binding to Env in VLPs gp140 trimers.

<p>Epitope competition group (CG) assignment, binding kinetics and preference of quaternary-epitope targeting Abs for binding to Env in VLPs gp140 trimers.</p

Disruption of mAb binding to HIV-1 gp160 by alanine replacement of amino acid residues.

<p>Disruption of mAb binding to HIV-1 gp160 by alanine replacement of amino acid residues.</p

Ferula assa-foetida L.

原著和名: アギ科名: セリ科 = Umbelliferae採集地: 千葉県 千葉市 千葉大学 (下総 千葉市 千葉大学)採集日: 1971/5/23採集者: 萩庭丈壽整理番号: JH037541国立科学博物館整理番号: TNS-VS-987541備考: DB作成協力会による補足あ

Quaternary epitope-targeting Abs variably bind gp140 trimers in a strain-dependent manner.

<p>Depending on the QtAb, variability was observed in binding BaL strain Env protein incorporated in VLPs (filled circle) versus gp140 trimers of the same strain (Clade B BaL, filled triangle) or of a different clade B strain (SF162, open triangle). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158861#pone.0158861.t001" target="_blank">Table 1</a> for corresponding binding data.</p

QtAb competition ELISA against BaL gp140 trimers revealed multiple novel quaternary epitopes.

<p>Each frame represents competitive binding against a different biotinylated competitor Ab (BCA) (using QtAbs 2C6, 8F6, 6F11, or 6F5). Unlabeled Ab to BCA ratios of 2:1 (based on EC₅₀ values) are shown. Competition was confirmed by control Ab inhibition (the unlabeled Ab was same Ab as the BCA) shown in thick black bars for each frame. Competitive inhibition of BCA binding of >40% (mean value irrespective of standard deviation), approximately twice the background % of controls (outside of the diagonally shaded areas beneath the dashed line), was considered positive. Assays were performed in duplicate and repeated twice, with consistent inter-assay results. The Abs within each dashed box represents those Abs forming a CG (A, B or C). The negative control, mAb PG9, targets a well-characterized neutralizing quaternary epitope; as an unlabeled competitor, it did not show any competition. Another unlabeled competitor, mAb G1-3E4, is a non-QtAb control Ab from the same study.</p