Leena Valmu

Analysis of proinflammatory activity of highly purified eukaryotic recombinant HMGB1 (amphoterin)

HMGB1 (amphoterin) is a 30‐kDa heparin‐binding protein that mediates transendothelial migration of monocytes and has proinflammatory cytokine‐like activities. In this study, we have investigated proinflammatory activities of both highly purified eukaryotic HMGB1 and bacterially produced recombinant HMGB1 protens. Mass analyses revealed that recombinant eukaryotic HMGB1 has an intrachain disulphide bond. In mass analysis of tissue‐derived HMGB1, two forms were detected: the carboxyl terminal glutamic acid residue lacking form and a full‐length form. Cell culture studies indicated that both eukaryotic and bacterial HMGB1 proteins induce TNF‐α secretion and nitric oxide release from mononuclear cells. Affinity chromatography analysis revealed that HMGB1 binds tightly to proinflammatory bacterial substances. A soluble proinflammatory substance was separated from the bacterial recombinant HMGB1 by chloroform‐methanol treatment. HMGB1 interacted with phosphatidylserine in both solid‐phase binding and cell culture assays, suggesting that HMGB1 may regulate phosphatidylserine‐dependent immune reactions. In conclusion, HMGB1 polypeptide has a weak proinflammatory activity by itself, and it binds to bacterial substances, including lipids, that may strengthen its effects.

Comparative Metaproteomics and Diversity Analysis of Human Intestinal Microbiota Testifies for Its Temporal Stability and Expression of Core Functions

The human intestinal tract is colonized by microbial communities that show a subject-specific composition and a high-level temporal stability in healthy adults. To determine whether this is reflected at the functional level, we compared the faecal metaproteomes of healthy subjects over time using a novel high-throughput approach based on denaturing polyacrylamide gel electrophoresis and liquid chromatography–tandem mass spectrometry. The developed robust metaproteomics workflow and identification pipeline was used to study the composition and temporal stability of the intestinal metaproteome using faecal samples collected from 3 healthy subjects over a period of six to twelve months. The same samples were also subjected to DNA extraction and analysed for their microbial composition and diversity using the Human Intestinal Tract Chip, a validated phylogenetic microarray. Using metagenome and single genome sequence data out of the thousands of mass spectra generated per sample, approximately 1,000 peptides per sample were identified. Our results indicate that the faecal metaproteome is subject-specific and stable during a one-year period. A stable common core of approximately 1,000 proteins could be recognized in each of the subjects, indicating a common functional core that is mainly involved in carbohydrate transport and degradation. Additionally, a variety of surface proteins could be identified, including potential microbes-host interacting components such as flagellins and pili. Altogether, we observed a highly comparable subject-specific clustering of the metaproteomic and phylogenetic profiles, indicating that the distinct microbial activity is reflected by the individual composition.

Phosphorylation of the Potyvirus Capsid Protein by Protein Kinase CK2 and Its Relevance for Virus Infection

We reported previously that the capsid protein (CP) of Potato virus A (PVA) is phosphorylated both in virus-infected plants and in vitro. In this study, an enzyme that phosphorylates PVA CP was identified as the protein kinase CK2. The α-catalytic subunit of CK2 (CK2α) was purified from tobacco and characterized using in-gel kinase assays and liquid chromatography–tandem mass spectrometry. The tobacco CK2α gene was cloned and expressed in bacterial cells. Specific antibodies were raised against the recombinant enzyme and used to demonstrate the colocalization of PVA CP and CK2α in infected tobacco protoplasts. A major site of CK2 phosphorylation in PVA CP was identified by a combination of mass spectrometric analysis, radioactive phosphopeptide sequencing, and mutagenesis as Thr-242 within a CK2 consensus sequence. Amino acid substitutions that affect the CK2 consensus sequence in CP were introduced into a full-length infectious cDNA clone of PVA tagged with green fluorescent protein. Analysis of the mutant viruses showed that they were defective in cell-to-cell and long-distance movement. Using in vitro assays, we demonstrated that CK2 phosphorylation inhibited the binding of PVA CP to RNA, suggesting a molecular mechanism of CK2 action. These results suggest that the phosphorylation of PVA CP by CK2 plays an important regulatory role in virus infection.

Leukocyte integrins and inflammation

Abstract. Leukocyte adhesion is of pivotal functional importance. Without adequate adhesion, T lymphocytes and natural killer cells are not cytotoxic, B cells cannot develop into antibody secreting plasma cells, leukocytes do not home into inflamed tissues and myeloid cells are not able to phagocytize or exhibit chemotactic responses. During evolution several leukocyte adhesion molecules have developed belonging to a few molecular families. Among these, the leukocyte-specific integrins (β2 integrins, CD11/CD18 molecules) are among the most important. Much progress has taken place during the past few years, and at present we have a considerable knowledge of their structure and function. Inflammation is critically dependent on integrin activity, and its regulation forms the topic of this short review.

Cell Surface Structures Influence Lung Clearance Rate of Systemically Infused Mesenchymal Stromal Cells

The promising clinical effects of mesenchymal stromal/stem cells (MSCs) rely especially on paracrine and nonimmunogenic mechanisms. Delivery routes are essential for the efficacy of cell therapy and systemic delivery by infusion is the obvious goal for many forms of MSC therapy. Lung adhesion of MSCs might, however, be a major obstacle yet to overcome. Current knowledge does not allow us to make sound conclusions whether MSC lung entrapment is harmful or beneficial, and thus we wanted to explore MSC lung adhesion in greater detail. We found a striking difference in the lung clearance rate of systemically infused MSCs derived from two different clinical sources, namely bone marrow (BM‐MSCs) and umbilical cord blood (UCB‐MSCs). The BM‐MSCs and UCB‐MSCs used in this study differed in cell size, but our results also indicated other mechanisms behind the lung adherence. A detailed analysis of the cell surface profiles revealed differences in the expression of relevant adhesion molecules. The UCB‐MSCs had higher expression levels of α4 integrin (CD49d, VLA‐4), α6 integrin (CD49f, VLA‐6), and the hepatocyte growth factor receptor (c‐Met) and a higher general fucosylation level. Strikingly, the level of CD49d and CD49f expression could be functionally linked with the lung clearance rate. Additionally, we saw a possible link between MSC lung adherence and higher fibronectin expression and we show that the expression of fibronectin increases with MSC culture confluence. Future studies should aim at developing methods of transiently modifying the cell surface structures in order to improve the delivery of therapeutic cells. STEM CELLS2013;31:317–326

Extracellular membrane vesicles from umbilical cord blood-derived MSC protect against ischemic acute kidney injury, a feature that is lost after inflammatory conditioning

Background Mesenchymal stromal cells (MSC) are shown to have a great therapeutic potential in many immunological disorders. Currently the therapeutic effect of MSCs is considered to be mediated via paracrine interactions with immune cells. Umbilical cord blood is an attractive but still less studied source of MSCs. We investigated the production of extracellular membrane vesicles (MVs) from human umbilical cord blood derived MSCs (hUCBMSC) in the presence (MVstim) or absence (MVctrl) of inflammatory stimulus. Methods hUCBMSCs were cultured in serum free media with or without IFN-γ and MVs were collected from conditioned media by ultracentrifugation. The protein content of MVs were analyzed by mass spectrometry. Hypoxia induced acute kidney injury rat model was used to analyze the in vivo therapeutic potential of MVs and T-cell proliferation and induction of regulatory T cells were analyzed by co-culture assays. Results Both MVstim and MVctrl showed similar T-cell modulation activity in vitro, but only MVctrls were able to protect rat kidneys from reperfusion injury in vivo. To clarify this difference in functionality we made a comparative mass spectrometric analysis of the MV protein contents. The IFN-γ stimulation induced dramatic changes in the protein content of the MVs. Complement factors (C3, C4A, C5) and lipid binding proteins (i.e apolipoproteins) were only found in the MVctrls, whereas the MVstim contained tetraspanins (CD9, CD63, CD81) and more complete proteasome complex accompanied with MHCI. We further discovered that differently produced MV pools contained specific Rab proteins suggesting that same cells, depending on external signals, produce vesicles originating from different intracellular locations. Conclusions We demonstrate by both in vitro and in vivo models accompanied with a detailed analysis of molecular characteristics that inflammatory conditioning of MSCs influence on the protein content and functional properties of MVs revealing the complexity of the MSC paracrine regulation.

The binding specificity of the marker antibodies Tra-1-60 and Tra-1-81 reveals a novel pluripotency-associated type 1 lactosamine epitope

The expression of the epitopes recognized by the monoclonal antibodies Tra-1-60 and Tra-1-81 is routinely used to assess the pluripotency status of human embryonic stem cells (hESCs) and induced pluripotent stem (iPS) cells. Although it is known that the epitopes recognized by Tra-1-60 and Tra-1-81 are carbohydrates, the exact molecular identity of these epitopes has been unclear. Glycan array analysis with more than 500 oligosaccharide structures revealed specific binding of Tra-1-60 and Tra-1-81 to two molecules containing terminal type 1 lactosamine: Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAc and Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ1-6(Galβ1-3GlcNAcβ1-3)Galβ1-4Glc. The type 1 disaccharide in itself was not sufficient for binding, indicating that the complete epitope requires an extended tetrasaccharide structure where the type 1 disaccharide is β1,3-linked to type 2 lactosamine. Our mass spectrometric analysis complemented with glycosidase digestions of hESC O-glycans indicated the presence of the extended tetrasaccharide epitope on an O-glycan with the likely structure Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ1-6(Galβ1-3)GalNAc. Thus, the present data indicate that the pluripotency marker antibodies Tra-1-60 and Tra-1-81 recognize the minimal epitope Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAc, which is present in hESCs as a part of a mucin-type O-glycan structure. The exact molecular identity of Tra-1-60 and Tra-1-81 is important for the development of improved tools to characterize the pluripotent phenotype.

Glycomics of bone marrow-derived mesenchymal stem cells can be used to evaluate their cellular differentiation stage

Human mesenchymal stem cells (MSCs) are adult multipotent progenitor cells. They hold an enormous therapeutic potential, but at the moment there is little information on the properties of MSCs, including their surface structures. In the present study, we analyzed the mesenchymal stem cell glycome by using mass spectrometric profiling as well as a panel of glycan binding proteins. Structural verifications were obtained by nuclear magnetic resonance spectroscopy, mass spectrometric fragmentation, and glycosidase digestions. The MSC glycome was compared to the glycome of corresponding osteogenically differentiated cells. More than one hundred glycan signals were detected in mesenchymal stem cells and osteoblasts differentiated from them. The glycan profiles of MSCs and osteoblasts were consistently different in biological replicates, indicating that stem cells and osteoblasts have characteristic glycosylation features. Glycosylation features associated with MSCs rather than differentiated cells included high-mannose type N-glycans, linear poly-N-acetyllactosamine chains and α2-3-sialylation. Mesenchymal stem cells expressed SSEA-4 and sialyl Lewis x epitopes. Characteristic glycosylation features that appeared in differentiated osteoblasts included abundant sulfate ester modifications. The results show that glycosylation analysis can be used to evaluate MSC differentiation state.

Glycosylated F4 (K88) fimbrial adhesin FaeG expressed in barley endosperm induces ETEC-neutralizing antibodies in mice

The F4-positive enterotoxigenic Escherichia coli (ETEC) strains are a frequent cause of porcine post-weaning diarrhea. Orally administered F4 fimbriae or FaeG, the major subunit and adhesin of F4, induce a protective mucosal immune response in F4 receptor-positive piglets. Feed plants carrying immunogenic subunit proteins can offer great advantages for oral vaccination of domestic animals. Here, we describe high-level endosperm-specific production (1% of total soluble proteins) of FaeG in the crop plant barley. The endoplasmic reticulum-targeted recombinant endospermic FaeG (erFaeG) was shown to be heterogeneously glycosylated. The erFaeG showed resistance at digestive conditions simulating piglet gastric fluid. Glycosylation did not abolish the immunogenic character of the FaeG protein, since erFaeG was able to induce F4 fimbria-specific antibodies in mice. Biological activity of these anti-F4 antibodies was demonstrated in vitro by blocking the attachment of the F4+ ETEC to the F4 receptors present on porcine intestinal enterocytes.

The cytoskeletal association of CD11/CD18 leukocyte integrins in phorbol ester-activated cells correlates with CD18 phosphorylation

Leukocyte adhesion is a regulated process, which involves CD11/CD18 leukocyte integrins. CD11/CD18 acidity may be regulated intracellularly, and the CD18 polypeptide has previously been shown to become phosphorylated on serine and threonine after phorbol ester activation of T cells. Increased adhesiveness is believed to be mediated by regulating the overall avidity of cellular contact. CD11/CD18 integrins have earlier been reported to interact with several cytoskeletal proteins. We have now studied the involvement of the CD18 phosphorylation in cytoskeletal associations. We have investigated the distribution of phosphorylated CD18 between soluble, cytoskeletal and nuclear fractions of T cell detergent lysates. A significant amount of phosphorylated CD18 polypeptides was observed to fraction along with the cytoskeleton, while the majority of the cell surface CD18 molecules remained in the soluble fraction. Putative candidates for this altered cytoskeletal binding of CD11/CD18 were shown to be talin and filamin, which were observed to bind to CD18 cytoplasmic peptides and co‐precipitate with CD18. The importance of the CD18 cytoplasmic domain in the regulation of the leukocyte adhesion was further strengthened by inhibition of phorbol ester‐induced T cell adhesion with a phosphorylated lipopeptide corresponding to the cytoplasmic portion of the CD18. These results indicate that the induced CD18 phosphorylation and the altered cytoskeletal binding of the phosphorylated integrin complex may contribute to the increased avidity of CD11/CD18‐mediated leukocyte adhesion.