Generation of antibodies to an extracellular region of the transporters Glut1/Glut4 by immunization with a designed antigen.

Monoclonal antibodies are one of the fastest growing class of drugs. Nevertheless, relatively few biologics target multispanning membrane proteins because of technical challenges. To target relatively small extracellular regions of multiple membrane-spanning proteins, synthetic peptides, which are composed of amino acids corresponding to an extracellular region of a membrane protein, are often utilized in antibody discovery. However, antibodies to these peptides often do not recognize parental membrane proteins. In this study, we designed fusion proteins in which an extracellular helix of the membrane protein glucose transporter 1 (Glut1) was grafted onto the scaffold protein Adhiron. In the initial design, the grafted fragment did not form a helical conformation. Molecular dynamics simulations of full-length Glut1 suggested the importance of intramolecular interactions formed by surrounding residues in the formation of the helical conformation. A fusion protein designed to maintain such intramolecular interactions did form the desired helical conformation in the grafted region. We then immunized an alpaca with the designed fusion protein and obtained VHH (variable region of heavy-chain antibodies) using the phage display method. The binding of these VHH antibodies to the recombinant Glut1 protein was evaluated by surface plasmon resonance, and their binding to Glut1 on the cell membrane was further validated by flow cytometry. Furthermore, we also succeeded in the generation of a VHH against another integral membrane protein, glucose transporter 4 (Glut4) with the same strategy. These illustrates that our combined biochemical and computational approach can be applied to designing other novel fusion proteins for generating site-specific antibodies.

Isolation of state-dependent monoclonal antibodies against the 12-transmembrane domain glucose transporter 4 using virus-like particles.

The insulin-responsive 12-transmembrane transporter GLUT4 changes conformation between an inward-open state and an outward-open state to actively facilitate cellular glucose uptake. Because of the difficulties of generating conformational mAbs against complex and highly conserved membrane proteins, no reliable tools exist to measure GLUT4 at the cell surface, follow its trafficking, or detect the conformational state of the protein. Here we report the isolation and characterization of conformational mAbs that recognize the extracellular and intracellular domains of GLUT4, including mAbs that are specific for the inward-open and outward-open states of GLUT4. mAbs against GLUT4 were generated using virus-like particles to present this complex membrane protein in its native conformation and using a divergent host species (chicken) for immunization to overcome immune tolerance. As a result, the isolated mAbs recognize conformational epitopes on native GLUT4 in cells, with apparent affinities as high as 1 pM and with specificity for GLUT4 across the human membrane proteome. Epitope mapping using shotgun mutagenesis alanine scanning across the 509 amino acids of GLUT4 identified the binding epitopes for mAbs specific for the states of GLUT4 and allowed the comprehensive identification of the residues that functionally control the GLUT4 inward-open and outward-open states. The mAbs identified here will be valuable molecular tools for monitoring GLUT4 structure, function, and trafficking, for differentiating GLUT4 conformational states, and for the development of novel therapeutics for the treatment of diabetes.

Leishmania infantum modulates host macrophage mitochondrial metabolism by hijacking the SIRT1-AMPK axis.

Metabolic manipulation of host cells by intracellular pathogens is currently recognized to play an important role in the pathology of infection. Nevertheless, little information is available regarding mitochondrial energy metabolism in Leishmania infected macrophages. Here, we demonstrate that during L. infantum infection, macrophages switch from an early glycolytic metabolism to an oxidative phosphorylation, and this metabolic deviation requires SIRT1 and LKB1/AMPK. SIRT1 or LBK1 deficient macrophages infected with L. infantum failed to activate AMPK and up-regulate its targets such as Slc2a4 and Ppargc1a, which are essential for parasite growth. As a result, impairment of metabolic switch caused by SIRT1 or AMPK deficiency reduces parasite load in vitro and in vivo. Overall, our work demonstrates the importance of SIRT1 and AMPK energetic sensors for parasite intracellular survival and proliferation, highlighting the modulation of these proteins as potential therapeutic targets for the treatment of leishmaniasis.

Novel link between inflammation and impaired glucose transport during equine insulin resistance.

Although insulin resistance (IR) has been increasingly recognized in horses, a clear understanding of its pathophysiology is lacking. The purpose of the present study was to determine the early pathologic changes in IR horses by characterizing alterations in proteins that play key roles in innate immunological responses and inflammatory pathways, and by identifying potential links with glucose transport and insulin signaling. Visceral (VIS) and subcutaneous (SC) adipose tissue and skeletal muscle (SM) biopsies were collected from horses, which were classified as insulin-sensitive (IS) or IR based on the results of an insulin-modified frequently sampled intravenous glucose tolerance test. Protein expression of Toll-like receptor 4 (TLR-4), suppressor of cytokine signaling 3 (SOCS-3) and tumor necrosis factor alpha (TNF-α) were quantified by Western blotting in VIS and SC adipose depots and SM, as well as insulin receptor substrate 1 (IRS-1). To better characterize the potential relationship between inflammation, IR and impaired glucose transport, we correlated active cell surface glucose transporter 4 (GLUT-4) content (measured by a cell surface biotinylated assay) with individual- and tissue-specific data related to inflammation. IR was associated with a significantly increased expression of TLR-4 and SOCS-3 in SM and VIS tissue, without a significant change in SC site. We also observed a significant increase in TNF-α in VIS, but not in SC, tissue of IR vs. IS horses. There was no difference in total content or serine phosphorylation of IRS-1 for any sampling site in IR compared to IS horses. We further observed a significant positive correlation between TLR-4 content and SOCS-3, as well as a significant negative correlation between SOCS-3 content and GLUT-4 trafficking. Taken together, the data suggested a pro-inflammatory state in SM and VIS, but not SC, adipose depot during compensated IR. In addition, SOCS-3 appears to be a novel link between inflammation and dysregulated glucose metabolism and insulin sensitivity during the early pathogenesis of insulin resistance.