Molecular cloning, genomic organization and functional analysis of an anti-lipopolysaccharide factor from Chinese mitten crab Eriocheir sinensis

Anti-lipopolysaccharide factor (ALF) represents one kind of basic proteins, which binds and neutralizes LPS and exhibits strong antibacterial activity against Gram-negative R-type bacteria. The ALF gene of Chinese mitten crab Eriocheir sinensis (Milne Edwards, 1853) (denoted as EsALF) was identified from haemocytes by expressed sequence tag (EST) and PCR approaches. The full-length cDNA of EsALF consisted of 700 nucleotides with a canonical polyadenylation signal-sequence AATAAA, a polyA tail, and an open-reading frame of 363 bp encoding 120 amino acids. The high similarity of EsALF-deduced amino acid sequence shared with the ALFs from other species indicated that EsALF should be a member of ALF family. The mRNA expression of EsALF in the tissues of heart, gonad, gill, haemocytes, eyestalk and muscle was examined by Northern blot analysis and mRNA transcripts of EsALF were mainly detected in haemocytes, heart and gonad. The temporal expression of EsALF in haemocytes after Vibrio anguillarum challenge was recorded by quantitative real-time RT-PCR. The relative expression level of EsALF was up-regulated rapidly at 2 h post-injection and reached 3-fold to that in blank group. After a drastic decrease to the original level from 4 to 8h, the expression level increased again and reached 4-fold to that in the blank group at 12 h post-injection. The genomic DNA sequence of EsALF gene consists of 1174bp containing three exons and two introns. The coding sequence of the EsALF mature peptide was cloned and expressed in Escherichia coli BL21(DE3)-pLysS to further elucidate its biological functions. The purified recombinant product showed bactericidal activity against both Gram-positive (G(+)) and Gram-negative (G(-)) bacteria, which demonstrated that the rEsALF was a broad-spectrum antibacterial peptide. All these results indicated that EsALF was an acute-phase protein involved in the immune responses of Chinese mitten crab, and provided a potential therapeutic agent for disease control in aquaculture. (c) 2007 Elsevier Ltd. All rights reserved

PtdIns(3)P-bound UVRAG coordinates Golgi-ER retrograde and Atg9 transport by differential interactions with the ER tether and the beclin 1 complex.

Endoplasmic reticulum (ER)-Golgi membrane transport and autophagy are intersecting trafficking pathways that are tightly regulated and crucial for homeostasis, development and disease. Here, we identify UVRAG, a beclin-1-binding autophagic factor, as a phosphatidylinositol-3-phosphate (PtdIns(3)P)-binding protein that depends on PtdIns(3)P for its ER localization. We further show that UVRAG interacts with RINT-1, and acts as an integral component of the RINT-1-containing ER tethering complex, which couples phosphoinositide metabolism to COPI-vesicle tethering. Displacement or knockdown of UVRAG profoundly disrupted COPI cargo transfer to the ER and Golgi integrity. Intriguingly, autophagy caused the dissociation of UVRAG from the ER tether, which in turn worked in concert with the Bif-1-beclin-1-PI(3)KC3 complex to mobilize Atg9 translocation for autophagosome formation. These findings identify a regulatory mechanism that coordinates Golgi-ER retrograde and autophagy-related vesicular trafficking events through physical and functional interactions between UVRAG, phosphoinositide and their regulatory factors, thereby ensuring spatiotemporal fidelity of membrane trafficking and maintenance of organelle homeostasis

PtdIns(3)P-bound UVRAG coordinates Golgi–ER retrograde and Atg9 transport by differential interactions with the ER tether and the beclin 1 complex

ER-Golgi membrane transport and autophagy are intersecting trafficking pathways that are tightly regulated and crucial for homeostasis, development and diseases. Here, we identify UVRAG, a Beclin1-binding autophagic factor, as a PI(3)P-binding protein that depends on PI(3)P for its ER localization. We further show that UVRAG interacts with RINT-1, and acts as an integral component of the RINT-1-containing ER tethering complex, which couples phosphoinositide metabolism to COPI-vesicle tethering. Displacement or knockdown of UVRAG profoundly disrupted COPI cargo transfer to the ER and Golgi integrity. Intriguingly, autophagy caused the dissociation of UVRAG from the ER tether, which in turn worked in concert with the Bif-1-Beclin-PI(3)KC3 complex to mobilize Atg9 translocation for autophagosome formation. These findings identify a regulatory mechanism that coordinates Golgi-ER retrograde and autophagy-related vesicular trafficking events through physical and functional interactions between UVRAG, phosphoinositide, and their regulatory factors, thereby ensuring spatiotemporal fidelity of membrane trafficking and maintenance of organelle homeostasis