BackgroundExosomes are emerging targets for biomedical research. However, suitable methods for the isolation of blood plasma-derived exosomes without impurities have not yet been described.AimTherefore, we investigated the efficiency and purity of exosomes isolated with potentially suitable methods; differential ultracentrifugation (UC) and size exclusion chromatography (SEC).Methods and ResultsExosomes were isolated from rat and human blood plasma by various UC and SEC conditions. Efficiency was investigated at serial UC of the supernatant, while in case of SEC by comparing the content of exosomal markers of various fractions. Purity was assessed based on the presence of albumin. We found that the diameter of the majority of isolated particles fell into the size range of exosomes, however, albumin was also present in the preparations, when 1h UC at 4°C was applied. Furthermore, with this method only a minor fraction of total exosomes could be isolated from blood as deduced from the constant amount of exosomal markers CD63 and TSG101 detected after serial UC of rat blood plasma samples. By using UC for longer time or with shorter sedimentation distance at 4°C, or UC performed at 37°C, exosomal yield increased, but albumin impurity was still observed in the isolates, as assessed by transmission electron microscopy, dynamic light scattering and immunoblotting against CD63, TSG101 and albumin. Efficiency and purity were not different in case of using further diluted samples. By using SEC with different columns, we have found that although a minor fraction of exosomes can be isolated without significant albumin content on Sepharose CL-4B or Sephacryl S-400 columns, but not on Sepharose 2B columns, the majority of exosomes co-eluted with albumin.ConclusionHere we show that it is feasible to isolate exosomes from blood plasma by SEC without significant albumin contamination albeit with low vesicle yield.
Numerous diseases, recently reported to associate with elevated microvesicle/ microparticle (MP) counts, have also long been known to be characterized by accelerated immune complex (IC) formation. The goal of this study was to investigate the potential overlap between parameters of protein complexes (eg, ICs or avidinbiotin complexes) and MPs, which might perturb detection and/or isolation of MPs. In this work, after comprehensive characterization of MPs by electron microscopy, atomic force microscopy, dynamic lightscattering analysis, and flow cytometry, for the first time, we drive attention to the fact that protein complexes, especially insoluble ICs, overlap in biophysical properties (size, light scattering, and sedimentation) with MPs. This, in turn, affects MP quantification by flow cytometry and purification by differential centrifugation, especially in diseases in which IC formation is common, including not only autoimmune diseases, but also hematologic disorders, infections, and cancer. These data may necessitate reevaluation of certain published data on patient-derived MPs and contribute to correct the clinical laboratory assessment of the presence and biologic functions of MPs in health and disease. (Blood. 2011;117(4):e39-e48) IntroductionMembrane vesicles are small subcellular structures surrounded by a phospholipid bilayer. Their release by various cell types is enhanced during activation and apoptosis. 1 They represent heterogeneous structures and can be classified into several groups depending on their size, antigenic features, and mechanism of cellular release. 1 The two best characterized categories include exosomes and microvesicles/microparticles (MPs). Both populations are characterized by the exposure of phosphatidylserine, which allows annexin-V (AX) to bind to these-lipid surfaces. 1 Exosomes are composed of small, 50-to 100-nm-sized structures released on exocytosis of multivesicular bodies. 1 The diameter of MPs, formed by membrane blebbing, is described to be 100 to 1000 nm 2 . However, precise definitions of MPs are still lacking. 1,2 MPs are found in various biologic fluids, including blood plasma, 3 urine, 4 and synovial fluid (SF). 5,6 Numerous flow cytometry (FC) studies using blood plasma have shown correlation of MP counts with human cardiovascular 7 and autoimmune diseases, 8 hematologic disorders, 9 and cancer. 10 Of particular interest, autoimmune diseases were reported to be characterized by elevated levels of MPs. 11 The assessment of exosomes and MPs is complicated by the presence of further known categories of membrane bound subcellular structures, such as apoptotic vesicles, exosome-like vesicles, membrane particles, and ectosomes. 1 There is a substantial size overlap among the aforementioned vesicle categories, and the size distribution of a given vesicle preparation may also be affected by the method used for their isolation. 3,12,13 Recently, attempts have been made to standardize isolation and detection protocols for membrane vesicles. 3,14 Up until now, no systemat...
Prolyl oligopeptidase (POP) has emerged as a drug target for neurological diseases. A flexible loop structure comprising loop A (res. 189-209) and loop B (res. 577-608) at the domain interface is implicated in substrate entry to the active site. Here we determined the kinetic and structural properties of POP with mutations in loop A, loop B and in two additional flexible loops. POP lacking loop A proved to be an inefficient enzyme as did POP with a mutation in loop B (T590C). Both constructs displayed an altered substrate preference profile. Ligand binding become markedly degraded. Conversely, the T202C mutation increased the flexibility of loop A, enhancing the catalytic efficiency beyond that of the native enzyme. The T590C mutation in loop B increased the preference for shorter peptides, indicating a role in substrate gating. Loop A and the His loop housing the catalytic histidine are disordered in the H680A mutant crystal structure, implying coordinated structural dynamics of these loops. A 17-mer peptide could not inhibit variants possessing malfunctioning loop A. This substrate may bind non-productively to an exosite involving loop A or to an open enzyme form. Biophysical studies suggest that mammalian POP resides in a predominantly closed conformational state, especially at physiological conditions. The flexible loop A, loop B and His loop system at the active site is the main regulator of substrate gating and specificity and represents a new inhibitor target.
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