EHD2-mediated restriction of caveolar dynamics regulates cellular fatty acid uptake.

Eps15-homology domain containing protein 2 (EHD2) is a dynamin-related ATPase located at the neck of caveolae, but its physiological function has remained unclear. Here, we found that global genetic ablation of EHD2 in mice leads to increased lipid droplet size in fat tissue. This organismic phenotype was paralleled at the cellular level by increased fatty acid uptake via a caveolae- and CD36-dependent pathway that also involves dynamin. Concomitantly, elevated numbers of detached caveolae were found in brown and white adipose tissue lacking EHD2, and increased caveolar mobility in mouse embryonic fibroblasts. EHD2 expression itself was down-regulated in the visceral fat of two obese mouse models and obese patients. Our data suggest that EHD2 controls a cell-autonomous, caveolae-dependent fatty acid uptake pathway and imply that low EHD2 expression levels are linked to obesity.

Structural insights into the activation mechanism of dynamin-like EHD ATPases.

Eps15 (epidermal growth factor receptor pathway substrate 15)-homology domain containing proteins (EHDs) comprise a family of dynamin-related mechano-chemical ATPases involved in cellular membrane trafficking. Previous studies have revealed the structure of the EHD2 dimer, but the molecular mechanisms of membrane recruitment and assembly have remained obscure. Here, we determined the crystal structure of an amino-terminally truncated EHD4 dimer. Compared with the EHD2 structure, the helical domains are 50° rotated relative to the GTPase domain. Using electron paramagnetic spin resonance (EPR), we show that this rotation aligns the two membrane-binding regions in the helical domain toward the lipid bilayer, allowing membrane interaction. A loop rearrangement in GTPase domain creates a new interface for oligomer formation. Our results suggest that the EHD4 structure represents the active EHD conformation, whereas the EHD2 structure is autoinhibited, and reveal a complex series of domain rearrangements accompanying activation. A comparison with other peripheral membrane proteins elucidates common and specific features of this activation mechanism.

Halilectin 1 (H-1) and Halilectin 2 (H-2): two new lectins isolated from the marine sponge Haliclona caerulea.

Two new lectins named Halilectin 1 (H-1) and Halilectin 2 (H-2) were isolated from the marine sponge Haliclona caerulea using a combination of affinity chromatography on stroma fixed onto Sephadex G-25 and cation and anion exchange chromatography. H-1 is a monomeric protein with a molecular mass of 40 kDa estimated using sodium dodecyl sulfate polyacrylamide gel electrophoresis and 15 kDa estimated using a TSK gel. Conversely, H-2 is a homodimeric protein with 15 kDa monomers linked via weak interactions. H-1 more effectively agglutinates trypsinized rabbit erythrocytes, whereas H-2 more effectively agglutinates native rabbit erythrocytes. The hemagglutinating activity of H-1 could be not inhibited by any tested sugars, but H-2 was inhibited by orosomucoid and porcine stomach mucin. Neither lectin was dependent on divalent ions. H-1 was stable at basic pH range and temperatures up to 50 °C, whereas H-2 was stable at acid pH range and temperatures up to 80 °C. The H. caerulea lectins exhibited dose-dependent toxicity against Artemia nauplii. Additionally, 76% of the primary structure of H-2 was determined using tandem mass spectrometry to contain a unique amino acid sequence with no similarity to any members of the animal lectin family.

Lysine acetylation regulates the interaction between proteins and membranes.

Lysine acetylation regulates the function of soluble proteins in vivo, yet it remains largely unexplored whether lysine acetylation regulates membrane protein function. Here, we use bioinformatics, biophysical analysis of recombinant proteins, live-cell fluorescent imaging and genetic manipulation of Drosophila to explore lysine acetylation in peripheral membrane proteins. Analysis of 50 peripheral membrane proteins harboring BAR, PX, C2, or EHD membrane-binding domains reveals that lysine acetylation predominates in membrane-interaction regions. Acetylation and acetylation-mimicking mutations in three test proteins, amphiphysin, EHD2, and synaptotagmin1, strongly reduce membrane binding affinity, attenuate membrane remodeling in vitro and alter subcellular localization. This effect is likely due to the loss of positive charge, which weakens interactions with negatively charged membranes. In Drosophila, acetylation-mimicking mutations of amphiphysin cause severe disruption of T-tubule organization and yield a flightless phenotype. Our data provide mechanistic insights into how lysine acetylation regulates membrane protein function, potentially impacting a plethora of membrane-related processes.

L-Rhamnose-binding lectin from eggs of the Echinometra lucunter: Amino acid sequence and molecular modeling.

An L-rhamnose-binding lectin named ELEL was isolated from eggs of the rock boring sea urchin Echinometra lucunter by affinity chromatography on lactosyl-agarose. ELEL is a homodimer linked by a disulfide bond with subunits of 11 kDa each. The new lectin was inhibited by saccharides possessing the same configuration of hydroxyl groups at C-2 and C-4, such as L-rhamnose, melibiose, galactose and lactose. The amino acid sequence of ELEL was determined by tandem mass spectrometry. The ELEL subunit has 103 amino acids, including nine cysteine residues involved in four conserved intrachain disulfide bonds and one interchain disulfide bond. The full sequence of ELEL presents conserved motifs commonly found in rhamnose-binding lectins, including YGR, DPC and KYL. A three-dimensional model of ELEL was created, and molecular docking revealed favorable binding energies for interactions between ELEL and rhamnose, melibiose and Gb3 (Galα1-4Galß1-4Glcß1-Cer). Furthermore, ELEL was able to agglutinate Gram-positive bacterial cells, suggesting its ability to recognize pathogens.

A chromophore-containing agglutinin from Haliclona manglaris: purification and biochemical characterization.

A new chromophore-containing agglutinin (Haliclona manglaris agglutinin (HMA)) was isolated from the tropical sponge H. manglaris. HMA was purified by a combination of hydrophobic interaction chromatography and ion exchange chromatography. Native HMA is a heterotrimer formed by two ß-chains (15 kDa) and one α-chain (22 kDa). HMA is a glycoprotein and possesses three intrachain disulfide bonds. Hemagglutinating activity of HMA was stable at neutral pH and temperatures up to 60 °C. HMA was only inhibited by thyroglobulin. Mass spectrometry sequencing and Edman degradation revealed a unique amino acid sequence of about 30%. Moreover, HMA has an organic chromophore of 581 Da, and this characteristic seems to be important to its antioxidant activity. Interestingly, while HMA showed no toxicity against Artemia nauplii and was unable to agglutinate bacterial cells, it did show a high capacity to protect ß-carotene against oxidation. Thus, our findings suggest the putative involvement of HMA in the protection of the sponge against oxidation.

HGA-2, a novel galactoside-binding lectin from the sea cucumber Holothuria grisea binds to bacterial cells.

A novel lectin, HGA-2, was isolated from the sea cucumber Holothuria grisea. The protein was isolated by a single chromatographic step using a column of Guar Gum as affinity. HGA-2 showed an apparent molecular mass of 17 kDa and 34 kDa under reducing and nonreducing conditions, respectively. The hemagglutinating activity was specific for rabbit erythrocytes, showing no activity for human blood A, B and O. Its hemagglutinating activity was inhibited by carbohydrates containing galactose, with higher affinity for GalNAc and glycoprotein porcine stomach mucin (PSM). HGA-2 was stable at pH 6-10, significantly declining at pH 5 and a temperature of 40°C, with its activity being abolished at 100 °C. The HGA-2 protein was found to be Ca(2+)-dependent; it was highly toxic against Artemia nauplii and able to recognize and agglutinate cells of Escherichia coli. Amino acid sequences of tryptic peptides of HGA-2 strongly suggest that HGA-2 is a member of the C-type lectin family.

Toxicity and binding profile of lectins from the Genus canavalia on brine shrimp.

Lectins are sugar-binding proteins widely distributed in nature with many biological functions. Although many lectins have a remarkable biotechnological potential, some of them can be cytotoxic. Thus, the aim of this study was to assess the toxicity of five lectins, purified from seeds of different species of Canavalia genus. In order to determine the toxicity, assays with Artemia nauplii were performed. In addition, a fluorescence assay was carried out to evaluate the binding of lectins to Artemia nauplii. In order to verify the relationship between the structure of lectins and their cytotoxic effect, structural analysis was carried out to evaluate the volume of the carbohydrate recognition domain (CRD) of each lectin. The results showed that all lectins exhibited different toxicities and bound to a similar area in the digestive tract of Artemia nauplii. Concerning the structural analysis, differences in spatial arrangement and volume of CRD may explain the variation of the toxicity exhibited by each lectin. To this date, this is the first study that establishes a link between toxicity and structure of CRD from Diocleinae lectins.

H-3, a new lectin from the marine sponge Haliclona caerulea: purification and mass spectrometric characterization.

A new lectin from the marine sponge Haliclona caerulea (H-3) was isolated using a combination of hydrophobic interaction chromatography and ion-exchange chromatography. H-3 is a protein with three distinct bands on SDS-PAGE: 9 kDa, 16 kDa and 18 kDa. Nevertheless, on gel filtration and N-PAGE, H-3 showed a symmetrical peak and a unique band, respectively. Hemagglutinating activity of H-3 was stable at neutral pH and temperatures up to 60 °C. N-Acetylgalactosamine and porcine stomach mucin were the most potent inhibitors of H-3. Primary structure of the lectin was determined using tandem mass spectrometry, and it showed no similarity to any members of the animal lectin families. Top down fragmentation revealed some posttranslational modifications in H-3, including glycosylation. The glycan composition of H-3 was determined, and its structure was predicted. Furthermore, H-3 is a blue protein, binding to a chromophore(-597) by weak interactions, and this is the first time that the interaction between one lectin and a natural chromophore has been shown.