Active Soluble Form Research Articles

Expression, purification, and biological activity evaluation of cathepsin L in mammalian cells.

Cathepsin L (CTSL) could cleave and activate SARS-CoV-2 Spike protein to promote viral entry, making it a hopeful therapeutic target for COVID-19 prevention and treatment. So CTSL inhibitors are considered to be a promising strategy to SARS-CoV-2 infection. CTSL has previously been expressed in inclusion body in Escherichia coli. In order to prepare CTSL with high purity and activity in soluble active form, we transformed HEK-293T cells with a recombinant mammalian expression plasmid. CTSL was purified to a purity about 95%, found to migrate at approximately 43kDa and exhibited substrate specificity against Z-Phe-Arg-AMC with specific activity of no less than 85081 U/mg, characteristic of active CTSL. Although eukaryotic purified CTSL is commercially available, our study for the first time reported the details of the expression, purification, and characterization of active, recombinant CTSL in eukaryocyte system, which laid an experimental foundation for the establishment of high-throughput screening model for anti-coronavirus drugs targeting CTSL.

Discovery of novel TACE inhibitors using graph convolutional network, molecular docking, molecular dynamics simulation, and Biological evaluation.

The increasing utilization of deep learning models in drug repositioning has proven to be highly efficient and effective. In this study, we employed an integrated deep-learning model followed by traditional drug screening approach to screen a library of FDA-approved drugs, aiming to identify novel inhibitors targeting the TNF-α converting enzyme (TACE). TACE, also known as ADAM17, plays a crucial role in the inflammatory response by converting pro-TNF-α to its active soluble form and cleaving other inflammatory mediators, making it a promising target for therapeutic intervention in diseases such as rheumatoid arthritis. Reference datasets containing active and decoy compounds specific to TACE were obtained from the DUD-E database. Using RDKit, a cheminformatics toolkit, we extracted molecular features from these compounds. We applied the GraphConvMol model within the DeepChem framework, which utilizes graph convolutional networks, to build a predictive model based on the DUD-E datasets. Our trained model was subsequently used to predict the TACE inhibitory potential of FDA-approved drugs. From these predictions, Vorinostat was identified as a potential TACE inhibitor. Moreover, molecular docking and molecular dynamics simulation were conducted to validate these findings, using BMS-561392 as a reference TACE inhibitor. Vorinostat, originally an FDA-approved drug for cancer treatment, exhibited strong binding interactions with key TACE residues, suggesting its repurposing potential. Biological evaluation with RAW 264.7 cell confirmed the computational results, demonstrating that Vorinostat exhibited comparable inhibitory activity against TACE. In conclusion, our study highlights the capability of deep learning models to enhance virtual screening efforts in drug discovery, efficiently identifying potential candidates for specific targets such as TACE. Vorinostat, as a newly identified TACE inhibitor, holds promise for further exploration and investigation in the treatment of inflammatory diseases like rheumatoid arthritis.

Structural analysis of PLD3 reveals insights into the mechanism of lysosomal 5' exonuclease-mediated nucleic acid degradation.

The phospholipase D (PLD) family is comprised of enzymes bearing phospholipase activity towards lipids or endo- and exonuclease activity towards nucleic acids. PLD3 is synthesized as a type II transmembrane protein and proteolytically cleaved in lysosomes, yielding a soluble active form. The deficiency of PLD3 leads to the slowed degradation of nucleic acids in lysosomes and chronic activation of nucleic acid-specific intracellular toll-like receptors. While the mechanism of PLD phospholipase activity has been extensively characterized, not much is known about how PLDs bind and hydrolyze nucleic acids. Here, we determined the high-resolution crystal structure of the luminal N-glycosylated domain of human PLD3 in its apo- and single-stranded DNA-bound forms. PLD3 has a typical phospholipase fold and forms homodimers with two independent catalytic centers via a newly identified dimerization interface. The structure of PLD3 in complex with an ssDNA-derived thymidine product in the catalytic center provides insights into the substrate binding mode of nucleic acids in the PLD family. Our structural data suggest a mechanism for substrate binding and nuclease activity in the PLD family and provide the structural basis to design immunomodulatory drugs targeting PLD3.

Expression Strategy of Soluble Recombinant Human TGF-β3 in Escherichia coli: sfGFP -Fusion Tag

Transforming growth factor-beta 3 (TGF-β3) is an important cytokine involved in various biological processes. TGF-β3 is used as a scar-reducing antifibrotic agent for acute and chronic wounds and fibrosing disorders. TGF-β3, a valuable therapeutic protein, is produced recombinantly in different expression systems. TGF-β3, produced in the Escherichia coli (E. coli) expression system, widely used due to its various advantages in recombinant production, is commercially available. However, the main problem encountered in protein expression in E. coli cells is the formation of an inclusion body. Various approaches have been developed to solve this problem. The use of a fusion tag is one of the most powerful strategies used to obtain protein in the soluble active form in the E. coli expression system. Superfolder GFP (sfGFP) is one of the fusion tags used to increase the solubility of the fusion partner in E. coli. In this study, TGF-β3 with sfGFP fusion tag (sfGFP-TGFβ3) was successfully produced in soluble form in E. coli BL21 (DE3) in high yield and purity for the first time. Purified protein was identified by western blot and SDS-PAGE. 20 mg of protein with 98% purity was obtained from 1 L of bacterial culture. It was determined that the obtained high purity protein did not have a cytotoxic effect on BJ normal human skin fibroblast cells. The impact of sfGFP-TGFβ3 fusion protein on wound healing was evaluated with in vitro scratch wound healing assay. The results showed that the sfGFP-TGFβ3 fusion protein produced in soluble form in the E. coli expression system has the potential to support the wound healing process.

Refolding of Proteins Expressed as Inclusion Bodies in E. coli.

Microbial-based biotherapeutics that are produced in Escherichia coli (E. coli) can be generated intracellularly in the form of inclusion bodies (IBs) or in soluble active form in periplasmic space or extracellularly. Overexpression of these biotherapeutics in E. coli leads to formation of insoluble aggregates called inclusion bodies. These IBs contain misfolded and inactive form of proteins which need to be refolded to obtain a functionally active form of proteins. Here, we discuss refolding of E. coli-based recombinant human granulocyte colony-stimulating factor (GCSF), expressed as IBs, and highlight some of the key features associated with the refolding kinetic reaction.

Using reagents in the form of inverse microemulsion for lead and zinc sulfide flotation

The paper presents the results of studies on the use of collecting agents in the form of an inverse microemulsion (IМE) of the «water in oil» type (i.e. suspended water droplets are in the oil phase) for the flotation extraction of lead and zinc minerals. Lead and zinc concentrates, lead-zinc ore were used as initial samples for flotation. The content of galena in the lead concentrate was 74.7 %, and the content of sphalerite in zinc was 78.7 %. Basic collecting agents in the IМE composition were potassium butyl xanthate (PBX) and kerosene. A nonionic surfactant (NSA) was used for IМE stabilization. Casein was used as additives to main reagents to remove the negative effect of osmotic pressure during the IМE preparation. Casein was transformed into the active soluble form using sodium sulfide. The particle size in the inverse microemulsion was 12.38 nm. In flotation tests, the following options for feeding reagents to the flotation pulp were studied: IМE, IМE + frother, potassium butyl xanthate + frother. The T-92 reagent was used as a frother. PBX consumption as part of IME and in the traditional feeding was 26 g/ton. The results of laboratory tests showed that the method of feeding flotation reagents in the form of IМE leads to both an increase in the flotation rate of lead and zinc sulfides and an increase in their recovery into a foam product. In addition to the increased flotation speed, tests with the use of IМE in the bulk lead-zinc ore flotation cycle showed an increase in extraction into the ultimate concentrate by 10.8 % for lead, by 38.5 % for zinc, in comparison with the traditional feeding of reagents (collector + frother). An increased selectivity of the IМE effect in relation to zinc sulfides, in comparison with lead sulfides, was noted. The flotation rate coefficient of sphalerite is 7.8 times greater than that of galena. An increase in extraction into the ultimate zinc concentrate is also higher and amounted to 16.78 %, while for the lead concentrate it is 1.9 % under the same conditions.

Role of molecular and chemical chaperon in assisting refolding of BMP2 in E. coli

Bone morphogenetic protein 2 (BMP2) when expressed in bacteria forms inclusion bodies (IBs) due to its complex disulfide-rich structure. Chaperons are already well known for their role in assisting protein folding. In our studies, we have used two E. coli strains, BL21(DE3) and SHuffle® T7 cells for overexpressing BMP2 in soluble fraction. We observed that SHuffle® T7 cells successfully expressed soluble functionally active BMP2 in presence of molecular and chemical chaperones at low temperature. The combination of chemical and molecular chaperons further increases the yield of protein. The best-suited chaperon system for overexpression of BMP2 is GroES-GroEL at low temperature. The soluble functionally active BMP2 is confirmed by its binding to its receptor ALK3 through Native PAGE and ELISA assay using BMP2 specific antibody. It is possible to obtain BMP2 expression in soluble active form by using molecular and chemical chaperons which work synergistically in bacterial cells to fold disulphide-rich proteins at low temperature in easy and time saving steps (18 ̊C).

Neutralization of black widow spider (Latrodectus mactans) venom with rabbit polyclonal serum hyperimmunized with recombinant alpha-latrotoxin fragments

Alpha-latrotoxin (ɑLTx) is the component responsible for causing the pathophysiology in patients bitten by spiders from the genus Latrodectus, commonly known as black widow spiders. The current antivenom used to treat these envenomations in Mexico is produced using the venom of thousands of spiders, obtained through electrical stimulation. This work aimed to produce this protein as well as two of its fragments in a bacterial model, to evaluate their use as immunogens to produce neutralizing hyperimmune sera, in rabbits.ɑLTx is a 130 kDa protein which has not yet been obtained in a soluble active form using bacterial models. In the present work, ɑLTx and two of its fragments, ankyrin domain and amino terminal domain (LTxAnk and LTxNT) were produced in bacteria and solubilized from inclusion bodies using N-lauroyl sarcosine. These three proteins were used for hyperimmunization in order to evaluate their potential as immunogens for the production of neutralizing hyperimmune sera against the complete venom of Latrodectus mactans.The hyperimmune sera obtained using the complete ɑLTx as well as the LTxNT, was capable of preventing death of mice envenomated with 3 LD50s of venom, both in preincubation and rescue experiments. Conversely, the serum obtained using the LTxAnk fragment, generated only partial protection and a delay in the time of death, even with a maximum dose of 450 μL. We therefore conclude that the produced proteins show great potential for their use as immunogens and should be further tested in large animals, such as horses.

The Serine Protease CD26/DPP4 in Non-Transformed and Malignant T Cells.

Simple SummaryThe transmembrane serine protease CD26/Dipeptidylpeptidase 4 modulates T-cell activation, proliferation, and effector function. Due to their remarkable tumoricidal properties CD26-positive T cells are considered promising candidates for T cell-based immunotherapies while in cutaneous T cell lymphoma CD26/DPP4 expression patterns are established markers for diagnosis and possibly prognosis. With a focus on T cells, we review current knowledge on the regulation of CD26/DPP4 expression and release, its implication in T-cell effector function and the suitability CD26/DPP4 as a diagnostic and/or prognostic factor in T-cell malignancies.CD26/Dipeptidylpeptidase 4 is a transmembrane serine protease that cleaves off N-terminal dipeptides. CD26/DPP4 is expressed on several immune cell types including T and NK cells, dendritic cells, and activated B cells. A catalytically active soluble form of CD26/DPP4 can be released from the plasma membrane. Given its wide array of substrates and interaction partners CD26/DPP4 has been implicated in numerous biological processes and effects can be dependent or independent of its enzymatic activity and are exerted by the transmembrane protein and/or the soluble form. CD26/DPP4 has been implicated in the modulation of T-cell activation and proliferation and CD26/DPP4-positive T cells are characterized by remarkable anti-tumor properties rendering them interesting candidates for T cell-based immunotherapies. Moreover, especially in cutaneous T-cell lymphoma CD26/DPP4 expression patterns emerged as an established marker for diagnosis and treatment monitoring. Surprisingly, besides a profound knowledge on substrates, interaction partners, and associated signal transduction pathways, the precise role of CD26/DPP4 for T cell-based immune responses is only partially understood.

The Use of New Parameters to Optimize the Composting Process of Different Organic Wastes

The correct development of the composting process is essential to obtain a product of high value from organic wastes. Nowadays, some composting mixture parameters (i.e., air-filled porosity, moisture and the C/N ratio) are used to optimize the composting process, but their suitability is still debated because the literature reports contrasting results. This paper aimed to find other parameters that control the correct development of composting. The relationship between these and the compost quality was then verified. Twelve different composting mixtures were prepared using different organic wastes and bulking agents and were aerobically treated in a 300 L composter. The physico-chemical and chemical parameters of initial mixtures were analyzed, with particular regard to the total and water-extractable forms of organic C and N and their ratios and correlated with the temperature measured during composting. A positive correlation between temperature parameters during the active phase and soluble forms of N in the initial mixtures was found. A high total organic C to soluble N ratio in the composting mixtures was correlated with the low quality of the compost produced. Based on the results, a minimum content of WEN (water-extractable N) (0.4% w/w) or a TOC/WEN (total organic C/WEN) ratio in the range of 40–80 was recommended to ensure the correct development of the process and to produce compost of high quality.

Molecular cloning and characterization of NAD+ dependent isocitrate dehydrogenase enzyme from Shewanella putrefaciens

Isocitrate dehydrogenase (IDH) is a fundamental enzyme for carbon metabolism in the Krebs cycle. This enzyme is required for oxidation-reduction reactions in both eukaryotic and prokaryotic cells and plays a critical role in their growth and pathogenesis. In this study, we cloned the gene encoding NAD+ dependent isocitrate dehydrogenase from Shewanella putrefaciens. The expression of recombinant protein was induced with 0.5 mM of IPTG. His-tagged IDH overexpressed in E. coli was purified and characterized. The expressed IDH enzyme was purified in an active soluble form. The molecular weight of the enzyme was confirmed with Western blotting. High sequence homology was observed with IDH sequences of other Shewanella strains and remarkable sequence homology was found with other bacteria reported in the database. The open reading frame of the gene encoding IDH of S. putrefaciens was 1069 bp in length, which codes a polypeptide composed of 356 amino acids and with a 38 KDa molecular weight. The optimum enzyme activity was obtained at pH 8. We cloned, purified and characterized Shewanella putrefaciens isocitrate dehydrogenase enzyme (SpIDH). The recombinant enzyme demonstrated a specific activity of Vmax 4.6±0.3 μM/min and Km 334 μM using NAD+ as a cofactor.

A model for COVID-19-induced dysregulation of ACE2 shedding by ADAM17

The angiotensin Converting Enzyme 2 (ACE2) receptor is a key component of the renin-angiotensin-aldesterone system (RAAS) that mediates numerous effects in the cardiovascular system. It is also the cellular point of contact for the coronavirus spike protein. Cleavage of the receptor is both important to its physiological function as well as being necessary for cell entry by the virus. Shedding of ACE2 by the metalloprotease ADAM17 releases a catalytically active soluble form of ACE2, but cleavage by the serine protease TMPRSS2 is necessary for virion internalization. Complicating the issue is the observation that circulating ACE2 can also bind to the virus effectively blocking attachment to the membrane-bound receptor. This work investigates the possibility that the inflammatory response to coronavirus infection can abrogate shedding by ADAM17, thereby favoring cleavage by TMPRSS2 and thus cell entry by the virion.

Soluble Programmed Death Ligand-1 (sPD-L1): A Pool of Circulating Proteins Implicated in Health and Diseases.

Simple SummaryThe interaction of programmed cell death ligand-1 (PDL1) with its receptor PD1 inhibits T-cell responses. Blockade of this interaction with monoclonal antibodies leads to major antitumor effects. However, not all cancer patients respond well to anti-(PD-1/PD-L1) immunotherapy. The PD-L1 protein is expressed at the cell plasma membrane (mPD-L1), at the surface of exosomes (exoPD-L1), in cell nuclei (nPD-L1) and as a soluble circulating protein (sPD-L1). The aim of our analysis was to highlight the multiple variants of sPD-L1 generated either by the proteolytic cleavage of m/exoPD-L1 or by the alternative splicing of PD-L1 pre-mRNA. The objective was also to underline the presence and role of circulating sPD-L1 isoforms in multiple cancer indications and many other diseases (including chronic inflammatory and viral diseases), and under non-pathological conditions (pregnancy). sPD-L1 often represents a general marker of an inflammatory status. The pool of sPD-L1 proteins is an integral part of the highly dynamic PD-1/PD-L1 signaling pathway.Upon T-cell receptor stimulation, the Programmed cell Death-1 receptor (PD-1) expressed on T-cells can interact with its ligand PD-L1 expressed at the surface of cancer cells or antigen-presenting cells. Monoclonal antibodies targeting PD-1 or PD-L1 are routinely used for the treatment of cancers, but their clinical efficacy varies largely across the variety of tumor types. A part of the variability is linked to the existence of several forms of PD-L1, either expressed on the plasma membrane (mPD-L1), at the surface of secreted cellular exosomes (exoPD-L1), in cell nuclei (nPD-L1), or as a circulating, soluble protein (sPD-L1). Here, we have reviewed the different origins and roles of sPD-L1 in humans to highlight the biochemical and functional heterogeneity of the soluble protein. sPD-L1 isoforms can be generated essentially by two non-exclusive processes: (i) proteolysis of m/exoPD-L1 by metalloproteases, such as metalloproteinases (MMP) and A disintegrin and metalloproteases (ADAM), which are capable of shedding membrane PD-L1 to release an active soluble form, and (ii) the alternative splicing of PD-L1 pre-mRNA, leading in some cases to the release of sPD-L1 protein isoforms lacking the transmembrane domain. The expression and secretion of sPD-L1 have been observed in a large variety of pathologies, well beyond cancer, notably in different pulmonary diseases, chronic inflammatory and autoimmune disorders, and viral diseases. The expression and role of sPD-L1 during pregnancy are also evoked. The structural heterogeneity of sPD-L1 proteins, and associated functional/cellular plurality, should be kept in mind when considering sPD-L1 as a biomarker or as a drug target. The membrane, exosomal and soluble forms of PD-L1 are all integral parts of the highly dynamic PD-1/PD-L1 signaling pathway, essential for immune-tolerance or immune-escape.

Characterization of the β-KDO Transferase KpsS, the Initiating Enzyme in the Biosynthesis of the Lipid Acceptor for Escherichia coli Polysialic Acid.

Antiphagocytic capsular polysaccharides are key components of effective vaccines against pathogenic bacteria. Neisseria meningitidis groups B and C, as well as Escherichia coli serogroups K1 and K92, are coated with polysialic acid capsules. Although the chemical structure of these polysaccharides and the organization of the associated gene clusters have been described for many years, only recently have the details of the biosynthetic pathways been discovered. The polysialic acid chains are synthesized by polysialyltransferases on a proposed phosphatidylglycerol lipid acceptor with a poly keto-deoxyoctulosonate (KDO) linker. Synthesis of this acceptor requires at least three enzymes in E. coli K1: KpsS, KpsC, and NeuE. In this report, we have characterized the β-KDO glycosyltransferase KpsS, the first enzyme in the pathway for lipid acceptor synthesis. After purification of KpsS in a soluble active form, we investigated its function and substrate specificity and showed that KpsS can transfer a KDO residue to a fluorescently labeled phosphatidylglycerol lipid. The enzyme tolerated various lengths of fatty acid acyl chains on the phosphatidylglycerol, including fluorescent tags, but exhibited a preference for phosphatidylglycerol diacylated with longer fatty acid chains as indicated by the smaller Kd and Km values for substrates with chains with more than 14 members. Additional structural analysis of the KpsS product confirmed that KpsS transfers KDO from CMP-KDO to the 1-hydroxyl of phosphatidylglycerol to form a β-KDO linkage.

An improved production and purification protocol for recombinant soluble human fibroblast activation protein alpha

Fibroblast activation protein alpha (FAP) is a cell-surface expressed type II glycoprotein that has a unique proteolytic activity. FAP has active soluble forms that retain the extracellular portion but lack the transmembrane domain and cytoplasmic tail. FAP expression is normally very low in adult tissue but is highly expressed by activated fibroblasts in sites of tissue remodelling. Thus, FAP is a potential biomarker and pharmacological target in liver fibrosis, atherosclerosis, cardiac fibrosis, arthritis and cancer. Understanding the biological significance of FAP by investigating protein structure, interactions and activities requires reliable methods for the production and purification of abundant pure and stable protein. We describe an improved production and purification protocol for His6-tagged recombinant soluble human FAP. A modified baculovirus expression construct was generated using the pFastBac1 vector and the gp67 secretion signal to produce abundant active soluble recombinant human FAP (residues 27–760) in insect cells. The FAP purification protocol employed ammonium sulphate precipitation, ion exchange chromatography, immobilised metal affinity chromatography and ultrafiltration. High purity was achieved, as judged by gel electrophoresis and specific activity. The purified 82 kDa FAP protein was specifically inhibited by a FAP selective inhibitor, ARI-3099, and was inhibited by zinc with an IC50 of 25 μM. Our approach could be adopted for producing the soluble portions of other type II transmembrane glycoproteins to study their structure and function.

Gastruloid Development Competence Discriminates Different States of Pluripotency.

SummaryFloating spheroidal aggregates of mouse embryonic stem cells can develop into polarized/elongated organoids, namely gastruloids. We set up a high-performing assay to measure gastruloid formation efficiency (GFE), and found that GFE decreases as pluripotency progresses from naive (GFE ≥ 95%) to primed (GFE = 0) state. Specifically, we show that primed EpiSCs fail to generate proper cell aggregates, while early-primed EpiLCs aggregate but eventually fail to develop into elongated gastruloids. Moreover, we characterized proline-induced cells (PiCs), a LIF-dependent reversible early-primed state of pluripotency, and show that PiCs are able to generate gastruloids (GFE ∼ 50%) and are also competent to differentiate into primordial germ cell-like cells. Thus, we propose the GFE assay as a valuable functional tool to discriminate different states of the pluripotency continuum.

Optimization of expression and purification of mitochondrial HSP 40 (Tid1-L) chaperone: Role of mortalin and tid1 in the reactivation and amyloid inhibition of proteins.

Stimulation of complex chaperone activity may be a viable means of therapy for neurodegenerative diseases. These chaperons execute reactivation of thermally and chemically aggregated protein substrates by cooperating with their partner co-chaperons. We optimized the expression and purification conditions of Tid1-L chaperone. Expression of Tid1-L in E. coli resulted in the formation of inclusion bodies which was further purified to soluble active form using 8 M urea and Ni-NTA column. Also, we investigated the events of the reactivation and disaggregation using aggregated G6PDH, luciferase and insulin as substrates. Incubation of aggregated/denatured enzymes with mortalin but not with Tid1 and/or Mge1 resulted in the initiation of the disaggregation reaction albeit very insignificantly. Under the same conditions coincubating the samples with chaperon and its assisted partners Tid1-L and nucleotide exchange factor Mge1 led to (40%) increase in enzyme activity of G6PDH. Similarly, luciferase activity was synergistically enhanced in the presence of mortlain/Tid1-L/Mge1 chaperones machinery. Chaperone-dependent disaggregation of thermally aggregated insulin showed that addition of Hsp70 and Hsp40 chaperones resulted in fast-track of renaissance reaction and inhibition of amyloid. The present study results conclude the quality of cell-control involves interaction of chaperon Hsp70 and its co-chaperones leading to complex formation with chemically/thermally aggregated substrate eventually causing its reactivation and disaggregation.

Generation and Function of Non-cell-bound CD73 in Inflammation

Extracellular adenine nucleotides participate in cell-to-cell communication and modulate the immune response. The concerted action of ectonucleotidases CD39 and CD73 plays a major role in the local production of anti-inflammatory adenosine, but both ectonucleotidases are rarely co-expressed by human T cells. The expression of CD39 on T cells increases upon T cell activation and is high at sites of inflammation. CD73, in contrast, disappears from the cellular membrane after activation. The possibility that CD73 could act in trans would resolve the conundrum of both enzymes being co-expressed for the degradation of ATP and the generation of adenosine. An enzymatically active soluble form of CD73 has been reported, and AMPase activity has been detected in body fluids of patients with inflammation and cancer. It is not yet clear how CD73, a glycosylphosphatidylinositol (GPI)-anchored protein, is released from the cell membrane, but plausible mechanisms include cleavage by metalloproteinases and shedding mediated by cell-associated phospholipases. Importantly, like many other GPI-anchored proteins, CD73 at the cell membrane is preferentially localized in detergent-resistant domains or lipid rafts, which often contribute to extracellular vesicles (EVs). Indeed, CD73-containing vesicles of different size and origin and with immunomodulatory function have been found in the tumor microenvironment. The occurrence of CD73 as non-cell-bound molecule widens the range of action of this enzyme at sites of inflammation. In this review, we will discuss the generation of non-cell-bound CD73 and its physiological role in inflammation.

Degranulation of human cytotoxic lymphocytes is a major source of proteolytically active soluble CD26/DPP4.

Dipeptidyl peptidase 4 (DPP4, CD26) is a serine protease detected on several immune cells and on epithelial cells of various organs. Besides the membrane-bound enzyme, a catalytically active soluble form (sCD26/DPP4) is detected in several body fluids. Both variants cleave off dipeptides from the N-termini of various chemokines, neuropeptides, and hormones. CD26/DPP4 plays a fundamental role in the regulation of blood glucose levels by inactivating insulinotropic incretins and CD26/DPP4 inhibitors are thus routinely used in diabetes mellitus type 2 therapy to improve glucose tolerance. Such inhibitors might also prevent the CD26/DPP4-mediated inactivation of the T-cell chemoattractant CXCL10 released by certain tumors and thus improve anti-tumor immunity and immunotherapy. Despite its implication in the regulation of many (patho-)physiological processes and its consideration as a biomarker and therapeutic target, the cellular source of sCD26/DPP4 remains highly debated and mechanisms of its release are so far unknown. In line with recent reports that activated T lymphocytes could be a major source of sCD26/DPP4, we now demonstrate that CD26/DPP4 is stored in secretory granules of several major human cytotoxic lymphocyte populations and co-localizes with effector proteins such as granzymes, perforin, and granulysin. Upon stimulation, vesicular CD26/DPP4 is rapidly translocated to the cell surface in a Ca2+-dependent manner. Importantly, activation-induced degranulation leads to a massive release of proteolytically active sCD26/DPP4. Since activated effector lymphocytes serve as a major source of sCD26/DPP4, these results might explain the observed disease-associated alterations of sCD26/DPP4 serum levels and also indicate a so far unknown role of CD26/DPP4 in lymphocyte-mediated cytotoxicity.

Expression and purification of a new lectin from mussel Mytilus trossulus

The gene of mtl from the mussel Mytilus trossulus was cloned into pET-40b(+) expression vector. After expression in E. coli using designed MX-medium an instable soluble form of MTL was obtained. The developed isolation method of the recombinant protein in “semi-denatured” conditions allowed obtaining an active soluble form of the homogenous lectin from the mussel M. trossulus (r-MTL). Both of the lectins had similar antigenic and spatial structures.