Adjuvant potential of aggregate-forming polyglutamine domains.

Aggregation may significantly affect the fate of a polypeptide, including its susceptibility to proteasome-dependent or autophagic degradation, its interaction with chaperones, etc. Since all these factors may affect the antigenicity of a polypeptide, we hypothesized that stimulating aggregation of an antigenic protein by its fusion to polyQ domain may enhance its antigenic potential. This hypothesis was tested with the weakly immunogenic model antigen GFP, which was fused to either long polyQ domain that triggers protein aggregation (103Q), or short polyQ domain that does not promote aggregation (25Q). Plasmids encoding control pGFP or soluble 25Q-GFP generated a very weak antibody response, while a significant increase in anti-GFP antibody titer was seen in groups immunized with DNA encoding aggregating 103Q-GFP. Similarly, fusion with 103Q strongly enhanced anti-GFP CTL activity, compared to fusion with 25Q. No apparent toxicity was observed after immunization with polyQ-GFP fusions. These data suggest that fusion of an antigen with expanded polyQ domains could have a significant adjuvant potential.

Prime-boost vaccination with a combination of proteosome-degradable and wild-type forms of two influenza proteins leads to augmented CTL response.

Targeting viral antigens for proteosomal degradation has previously been proposed as a means for immunogenicity augmentation. However, utilization of modified unstable antigens may be insufficient for potent T-cell cross-presentation by APCs, a mechanism that requires high levels of the antigenic protein. Therefore, we hypothesized that a recombinant vaccine utilizing a combination of proteosome-sensitive and proteosome-resistant versions of an antigen in a prime-boost regimen may provide the most efficient CTL response. To address this hypothesis, we utilized conserved proteosome-resistant influenza A virus proteins M1 and NS1. Unstable versions of these polypeptides were constructed by destroying their 3D structure via truncations or short insertions into predicted alpha-helical structures. These modified polypeptides were stabilized in the presence of the proteosome inhibitor MG132, strongly suggesting that they are degraded via a ubiquitin-proteosome pathway. Importantly, with both M1 and NS1antigens, homologous DNA vaccination with a mixture of unstable and proteosome-resistant wt forms of these proteins resulted in significantly higher CTL activity than vaccination with either wt or degradable forms. The most dramatic effect was seen with NS1, where homologous immunization with a mixture of these two forms was the only regimen that produced a notable elevation of CTL response, compared to vaccination with the wt NS1. Additionally, for M1 protein, heterologous vaccination utilizing the unstable form as prime and wild-type form as boost, demonstrated significant augmentation of the CTL response. These data indicate that combining proteosome-sensitive and proteosome-resistant forms of an antigen during vaccination is advantageous.

A Glut4-vesicle marker protein, insulin-responsive aminopeptidase, is localized in a novel vesicular compartment in PC12 cells.

Glut4-containing vesicles represent a regulated recycling compartment in insulin-sensitive fat and skeletal muscle cells, the nature and origin of which are not fully understood. In addition to Glut4 itself, these vesicles compartmentalize a number of proteins, at least one of which, insulin-responsive aminopeptidase, or IRAP, is completely colocalized with Glut4 in insulin-sensitive tissues. However, unlike Glut4, IRAP is expressed in a variety of other tissues and cell lines. Here, we explored the intracellular localization of IRAP in the rat pheochromocytoma cell line PC12. We found that this protein is present in a distinct population of slowly recycling light vesicles. By gradient centrifugations, immunoadsorption and double immunofluorescent staining, these vesicles are different from transferrin-containing endosomes, small synaptic vesicles and secretory granules and may thus represent a novel compartment in PC12 cells. Glut4-GFP chimera transiently expressed in PC12 cells is targeted to IRAP-containing vesicles indicating that cotargeting of Glut4 and IRAP is not specific for adipocytes and myocytes, but is faithful in a foreign cell type. We suggest that PC12 cells may possess a novel type of a vesicular carrier that may represent the homolog of Glut4-vesicles.

Identification and characterization of leptin-containing intracellular compartment in rat adipose cells.

The major leptin-containing membrane compartment was identified and characterized in rat adipose cells by means of equilibrium density and velocity sucrose gradient centrifugation. This compartment appears to be different from peptide-containing secretory granules present in neuronal, endocrine, and exocrine cells, as well as from insulin-sensitive GLUT-4-containing vesicles abundant in adipocytes. Exocytosis of both leptin- and GLUT-4-containing vesicles can be induced by insulin; however, only leptin secretion is responsive to serum stimulation. This latter effect is resistant to cycloheximide, suggesting that serum triggers the release of a stored pool of presynthesized leptin molecules. We conclude that regulated secretion of leptin and insulin-dependent translocation of GLUT-4 represent different pathways of membrane trafficking in rat adipose cells. NIH 3T3 cells ectopically expressing CAAT box enhancer binding protein-alpha and Swiss 3T3 cells expressing peroxisome proliferator-activated receptor-gamma undergo differentiation in vitro and acquire adipocyte morphology and insulin-responsive glucose uptake. Only the former cell line, however, is capable of leptin secretion. Thus different transcriptional mechanisms control the developmental onset of these two major and independent physiological functions in adipose cells.

Glucose transporter Glut3 is targeted to secretory vesicles in neurons and PC12 cells.

In rat brain and cultured neuroendocrine PC12 cells, Glut3 is localized at the cell surface and, also, in a distinct population of homogenous synaptic-like vesicles. Glut3-containing vesicles co-purify with "classical" synaptic vesicles, but can be separated from the latter by sucrose gradient centrifugation. Unlike classical synaptic vesicles, Glut3-containing vesicles possess a high level of aminopeptidase activity, which has been identified as aminopeptidase B. This enzyme has recently been shown to be a marker of the secretory pathway in PC12 cells (Balogh, A., Cadel, S., Foulon, T., Picart, R., Der Garabedian, A., Rousselet, A., Tougard, C., and Cohen, P. (1998) J. Cell Sci. 111, 161-169). We, therefore, conclude that Glut3 is targeted to secretory vesicles in both neurons and PC12 cells.

Two distinct populations of synaptic-like vesicles from rat brain.

In nonneuronal cells, several plasma membrane proteins such as exofacial enzymes, receptors, and ion channels recycle between their intracellular compartment(s) and the cell surface via an endosomal pathway. In neurons, however, this pathway has not been extensively characterized. In particular, it remains unclear whether or not it is related to the recycling of small synaptic vesicles, the major pathway of membrane traffic in nerve terminals. To approach this problem, we purified and studied a vesicular fraction from rat brain synaptosomes. Two distinct populations of vesicles with different buoyant densities and sedimentation coefficients were detected in this fraction by sucrose gradient centrifugation and Western blot analysis of the individual proteins. Both populations contain proteins that are markers of synaptic vesicles, namely, SV2, synaptotagmin, synaptophysin, secretory carrier membrane proteins (SCAMPs), synaptobrevin, and rab3a. A striking difference between the two populations is the presence of arginine aminopeptidase activity (a previously suggested marker for the regulated endosomal recycling pathway) exclusively in the lighter less-dense vesicles. The same two vesicular populations were also detected in the preparation of clathrin-coated vesicles isolated from whole rat brain or purified synaptosomes after removal of their clathrin coats by incubation at pH 8.5. We conclude, therefore, that both types of vesicles recycle in synaptosomes via a clathrin-mediated pathway. These data present experimental evidence for biochemical heterogeneity of synaptic-like vesicles in rat brain.

Exploring protein interactions.

Protein Targeting Protocols Edited by Robert A. Clegg. Totowa, Humana, 1998, $79.50 (xii+ 331 pages), ISBN 0-896-03450-X.

The T-tubule is a cell-surface target for insulin-regulated recycling of membrane proteins in skeletal muscle.

(1) In this study we have determined the distribution of various membrane proteins involved in insulin-activated glucose transport in T-tubules and in sarcolemma from rat skeletal muscle. Two independent experimental approaches were used to determine the presence of membrane proteins in T-tubules: (i) the purification of T-tubules free from sarcolemmal membranes by lectin agglutination, and (ii) T-tubule vesicle immunoadsorption. These methods confirmed that T-tubules from rat skeletal muscle were enriched with dihydropyridine receptors and tt28 protein and did not contain the sarcolemmal markers dystrophin or beta 1-integrin. Both types of experiments revealed an abundant content of GLUT4 glucose carriers, insulin receptors and SCAMPs (secretory carrier membrane proteins) in T-tubule membranes. (2) Acute administration in vivo of insulin caused an increased abundance of GLUT4 in T-tubules and sarcolemma. On the contrary, insulin led to a 50% reduction in insulin receptors present in T-tubules and in sarcolemma, demonstrating that insulin-induced insulin receptor internalization affects T-tubules in the muscle fibre. The alteration in the content of GLUT4 and insulin receptors in T-tubules was a consequence of insulin-induced redistribution of these proteins. SCAMPs also redistributed in muscle membranes in response to insulin. They were recruited by insulin from intracellular high-density fractions to intracellular lighter-density fractions and to the cell surface, showing a pattern of insulin-induced cellular redistribution distinct from those of GLUT4 and the insulin receptor. (3) In conclusion, the T-tubule is a cell-surface target for membrane proteins involved in recycling such as SCAMPs or for membrane proteins that acutely redistribute in response to insulin such as GLUT4 or insulin receptors.

Immunological analysis of GLUT4-enriched vesicles. Identification of novel proteins regulated by insulin and diabetes.

In adipocytes and muscle, insulin stimulates the translocation of glucose transporter proteins from an intracellular vesicle pool to the plasma membrane. To study the molecular basis of this process, we used the anti-GLUT4 antibody 1F8 to isolate intracellular vesicles from rat adipocytes that are enriched in the muscle/fat glucose transporter isoform. These vesicles were then used as immunogens to generate monoclonal antibodies against their protein components. We isolated an antibody, 3F8, that recognizes three polypeptides, designated GTV3, migrating in the 36-40-kDa range as analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting. These proteins are enriched in GLUT4-containing vesicles, and the two smallest of the polypeptides recognized by 3F8 translocate to the cell surface in response to insulin. GTV3 proteins are also present in plasma membranes of fat cells and liver as well as in a wide number of tissues, red blood cells being the only exception. In adipocytes from streptozotocin-induced diabetic rats, GTV3 protein levels decrease dramatically and return to normal levels when animals are treated with insulin. The localization of GTV3 in glucose transporter-containing vesicles as well as their wide tissue distribution suggests that these proteins may be involved in vesicle mediated transport and regulated trafficking between membrane compartments.

Insulin-regulated glucose uptake in rat adipocytes is mediated by two transporter isoforms present in at least two vesicle populations.

We have recently described a monoclonal antibody (1F8) that recognizes a form of glucose transporter unique to fat and muscle (James, D. E., Brown, R., Navarro, J., and Pilch, P. F. (1988) Nature 333, 183-185), tissues that respond acutely to insulin by markedly increasing their glucose uptake. Here, we report that rat adipocytes possess two immunologically distinct glucose-transporters: one recognized by 1F8, and one reactive with antibodies raised against the human erythrocyte glucose transporter. Immunoadsorption experiments indicate that these glucose transporters reside in different vesicle populations and that both transporter isoforms translocate from intracellular sites to the plasma membrane in response to insulin. The insulin-regulatable transporter resides in a unique vesicle that comprises 3% or less of the low density microsomes of fat cells and has a limited protein composition that does not include the bulk of another translocatable protein, the insulin-like growth factor II receptor. Immunoprecipitation with 1F8 of microsomal glucose transporters photoaffinity labeled with [3H]cytochalasin B brings down 90% of the label. Similarly, immunoprecipitation with 1F8 of glucose transporters from insulin-stimulated plasma membranes photolabeled with 3-[125I]iodo-4-azidophenethylamido-7-O-succinyldeacetyl-f ors kolin, another transporter-selective reagent, results in 75% of the labeled transporter localized in the immunoprecipitate. Thus, insulin action involves the combined effect of translocation from at least two vesicle pools each containing different glucose transporters. The 1F8-reactive transporter comprises the majority of the total transporter pool that is responsible for the insulin-induced increase in glucose transporter number.

Developmental regulation of nodule-specific genes in alfalfa root nodules.

We have cloned alfalfa nodule-specific cDNAs that code for leghemoglobin (Lb), glutamine synthetase (GS), and three unidentified nodulins. Hybrid-select translation of nodule RNA followed by 2-D gel electrophoresis showed that the Lb-specific cDNA corresponded to at least four Lb species of 12 kDa. One of the unidentified cDNA clones (N-32/34) corresponded to at least five polypeptides of 32-34 kDa; a second unidentified cDNA clone (N-14) corresponded to an individual polypeptide of 14 kDa. The in vitro translation product(s) of the RNA hybrid selected by the third unidentified cDNA clone (N-22) formed a single band at 22 kDa on a one-dimensional gel. Northern and dot blot analyses of RNA isolated from wild-type nodules and from defective nodules elicited by a variety of Rhizobium meliloti mutants showed that 1) RNAs corresponding to the Lb, nodule-specific GS, and three unidentified nodulins were coordinately expressed during the course of nodule development, and 2) all five nodulins were expressed in Fix- nodules that contained infection threads and bacteroids but were not expressed in nodules that lacked infection threads and intracellular rhizobia.