Antibodies elicited by yeast glycoproteins recognize HIV-1 virions and potently neutralize virions with high mannose N-glycans.

The glycan shield on the human immunodeficiency virus 1 (HIV-1) envelope (Env) glycoprotein has drawn attention as a target for HIV-1 vaccine design given that an increasing number of potent and broadly neutralizing antibodies (bNAbs) recognize epitopes entirely or partially comprised of high mannose type N-linked glycans. In an attempt to generate immunogens that target the glycan shield of HIV-1, we previously engineered a triple mutant (TM) strain of Saccharomyces cerevisiae that results in exclusive presentation of high mannose type N-glycans, and identified five TM yeast glycoproteins that support strong binding of 2G12, a bNAb that targets a cluster of high mannose glycans on the gp120 subunit of Env. Here, we further analyzed the antigenicity and immunogenicity of these proteins in inducing anti-HIV responses.

Yeast-elicited cross-reactive antibodies to HIV Env glycans efficiently neutralize virions expressing exclusively high-mannose N-linked glycans.

The HIV envelope (Env) protein uses a dense coat of glycans to mask conserved domains and evade host humoral immune responses. The broadly neutralizing antibody 2G12, which binds a specific cluster of high-mannose glycans on HIV Env, shows that the glycan shield can also serve as a target for neutralizing antibodies. We have described a triple mutant Saccharomyces cerevisiae strain that expresses high-mannose glycoproteins that bind to 2G12.

Protein-protein interactions involving congenital cataract T5P gammaC-crystallin mutant: a confocal fluorescence microscopy study.

The human lens crystallin gene CRYGC T5P is associated with Coppock-like cataract and has a phenotype of a dust-like opacity of the fetal lens nucleus and deep cortical region. Previous in vitro mutation studies indicate that the protein has changed conformation, solubility, and stability, which may make it susceptible to aggregation, as seen in cataractous lens and cell culture expression. To investigate the mechanisms leading to these events, we studied protein-protein interactions using confocal fluorescence resonance energy transfer (FRET) microscopy.

Methylglyoxal induces prostaglandin E2 production in rat mesangial cells.

The formation of methylglyoxal (MG), a reactive dicarbonyl compound, is accelerated under hyperglycemia, presumably contributing to tissue injury in diabetes. On the other hand, prostaglandin E2 (PGE2) has been implicated in glomerular hyperfiltration, a characteristic change in the early stage of diabetic nephropathy. We therefore examined whether MG was capable of inducing PGE2 production in rat mesangial cells (RMC) to address a possible mechanism by which hyperglycemia-derived dicarbonyls accelerated the development of diabetic nephropathy.

Protein-protein interactions between lens vimentin and alphaB-crystallin using FRET acceptor photobleaching.

Purpose: The R120G mutation of alphaB-crystallin is known to cause desmin-related myopathy, but the mechanisms underlying the formation of cataract are not clearly established. We hypothesize that alteration of protein-protein interaction between R120G alphaB-crystallin and lens intermediate filament proteins is one of the mechanisms of congenital cataract.

Methods: Protein-protein interactions were determined by confocal fluorescence resonance energy transfer (FRET) microscopy using green fluorescence protein (GFP) as the donor and red fluorescence protein (RFP) as the acceptor.

Confocal fluorescence microscopy study of interaction between lens MIP26/AQP0 and crystallins in living cells.

MIP26/AQP0 is the major lens fiber membrane protein and has been reported to interact with many other lens components including crystallins, lipid, and cytoskeletal proteins. Regarding crystallins, many previous reports indicate that MIP26/AQP0 interacts with either only alpha-crystallin or some specific gamma-crystallins. Considering the possibly important role of MIP26/AQP0 in the reduction of light scattering in the lenses, we have further investigated its interaction with crystallins using confocal fluorescence resonance energy transfer (FRET) microscopy.

Degradation of C-terminal truncated alpha A-crystallins by the ubiquitin-proteasome pathway.

Purpose: Calpain-mediated C-terminal cleavage of alpha A-crystallins occurs during aging and cataractogenesis. The objective of the present study was to explore the role of the ubiquitin-proteasome pathway (UPP) in degrading C-terminal truncated alpha A-crystallins.

Methods: Recombinant wild-type (wt) alpha A-crystallin and C-terminal truncated alpha A(1-168)-, alpha A(1-163)-, and alpha A(1-162)-crystallins were expressed in Escherichia coli and purified to homogeneity.

Protein-protein interactions among human lens acidic and basic beta-crystallins.

Human lens beta-crystallin contains four acidic (betaA1-->betaA4) and three basic (betaB1-->betaB3) subunits. They oligomerize in the lens, but it is uncertain which subunits are involved in the oligomerization. We used a two-hybrid system to detect protein-protein interactions systematically.

Confocal fluorescence resonance energy transfer microscopy study of protein-protein interactions of lens crystallins in living cells.

Purpose: To determine protein-protein interactions among lens crystallins in living cells.

Methods: Fluorescence resonance energy transfer (FRET) microscopy was used to visualize interactions in living cells directly. Two genes, one (alphaA-crystallin) fused with green fluorescence protein (GFP) and the other (each of the following genes: alphaB-, betaB2-, gammaC-crystallin, and R120G alphaB-crystallin mutant) fused with GFP variant red fluorescence protein (RED), were cotransfected into HeLa cells.

Glutathiolation enhances the degradation of gammaC-crystallin in lens and reticulocyte lysates, partially via the ubiquitin-proteasome pathway.

Purpose: S-glutathiolated proteins are formed in the lens during aging and cataractogenesis. The objective of this work was to explore the role of the ubiquitin-proteasome pathway in eliminating S-glutathiolated gammaC-crystallin.

Methods: Recombinant human gammaC-crystallin was mixed with various concentrations of glutathione (GSH) and diamide at 25 degrees C for 1 hour.

Fluorescence resonance energy transfer study of subunit exchange in human lens crystallins and congenital cataract crystallin mutants.

Lens alpha-crystallin is an oligomeric protein with a molecular mass of 500-1000 kDa and a polydispersed assembly. It consists of two types of subunits, alphaA and alphaB, each with a molecular mass of 20 kDa. The subunits also form homo-oligomers in some other tissues and in vitro.

Effect of methylglyoxal modification and phosphorylation on the chaperone and anti-apoptotic properties of heat shock protein 27.

Heat shock protein 27 (Hsp27) is a stress-inducible protein in cells that functions as a molecular chaperone and also as an anti-apoptotic protein. Methylglyoxal (MGO) is a reactive dicarbonyl compound produced from cellular glycolytic intermediates that reacts non-enzymatically with proteins to form products such as argpyrimidine. We found considerable amount of Hsp27 in phosphorylated form (pHsp27) in human cataractous lenses.

A novel alphaB-crystallin mutation associated with autosomal dominant congenital lamellar cataract.

Purpose: To identify the mutation and the underlying mechanism of cataractogenesis in a five-generation autosomal dominant congenital lamellar cataract family.

Methods: Nineteen mutation hot spots associated with autosomal dominant congenital cataract have been screened by PCR-based DNA sequencing. Recombinant wild-type and mutant human alphaB-crystallin were expressed in Escherichia coli and purified to homogeneity.

Domain interaction sites of human lens betaB2-crystallin.

betaB2-crystallin, the major component of beta-crystallin, is a dimer at low concentrations but can form oligomers under physiological conditions. The interaction domains have been speculated to be the beta-sheets, each of which is formed by two or more beta-strands. betaB2-crystallin consists of 16 beta-strands, 8 in the N-terminal domain and 8 in the C-terminal domain.

Dicarbonyl stress and apoptosis of vascular cells: prevention by alphaB-crystallin.

Methylglyoxal (MGO) is an alpha-dicarbonyl compound produced from triose phosphate intermediates of glycolysis. It reacts rapidly with proteins to produce advanced glycation products. We have studied the effect of MGO modification of fibronectin on retinal capillary cell viability.

Interaction and biophysical properties of human lens Q155* betaB2-crystallin mutant.

Purpose: Missense mutations in crystallin genes have been identified in autosomal dominant congenital cataracts. A truncation in the CRYBB2 gene (Q155*) has been associated with cerulean cataract, however its effects on biophysical properties have not been reported. We sought to determine the changes in conformation and protein-protein interactions brought about by this mutation.

AlphaB-crystallin inhibits glucose-induced apoptosis in vascular endothelial cells.

Recent studies implicate hyperglycemia as a cause of vascular complications in diabetes. Our study confirmed that high concentration of glucose (30 mM) induces apoptosis in cultures of human umbilical vein endothelial cells. After 5 days of culture TUNEL positive cells in high concentration of glucose were nearly 63% higher when compared to normal concentration of glucose (5 mM).

Effect of dicarbonyl modification of fibronectin on retinal capillary pericytes.

Purpose: To determine effects of alpha-dicarbonyl modification of an extracellular matrix protein on retinal capillary pericyte attachment and viability.

Methods: Primary cultures of bovine retinal pericytes (BRPs) were seeded on either normal fibronectin (FN) or FN modified by methylglyoxal (MGO) and glyoxal (GO). Apoptosis was measured by flow cytometry along with caspase-3 activity.

Relation between serum 3-deoxyglucosone and development of diabetic microangiopathy.

Objective: 3-Deoxyglucosone (3-DG), a highly reactive intermediate of the glycation reaction, has been suggested to contribute to the development of diabetes complications. To verify this hypothesis, we assessed the relation between serum 3-DG concentrations and the severity of diabetic microangiopathy in diabetic patients.

Research Design And Methods: We conducted a high-performance liquid chromatography assay to determine the serum 3-DG concentrations of 110 diabetic patients with different degrees of severity of diabetic microangiopathy and 57 age-matched control subjects.

Methylglyoxal induces apoptosis through activation of p38 mitogen-activated protein kinase in rat mesangial cells.

Background: The formation of methylglyoxal (MG), a highly reactive dicarbonyl compound, is accelerated through several pathways, including the glycation reaction under diabetic conditions, presumably contributing to tissue injury in diabetes. On the other hand, apoptotic cell death of glomerular cells has been suggested to play a role in the development of glomerulosclerosis in various types of glomerular injuries. We therefore examined whether MG was capable of inducing apoptosis in rat mesangial cells to address the possible mechanism by which hyperglycemia-related products accelerated pathologic changes in diabetic glomerulosclerosis.