Raija Sormunen

Cloning of a novel bacteria-binding receptor structurally related to scavenger receptors and expressed in a subset of macrophages.

A novel murine plasma membrane protein has been identified in subpopulations of macrophages. It has an intracellular N-terminal domain, a transmembrane domain, and an extracellular region with a short spacer, an 89 Gly-Xaa-Yaa repeat-containing collagenous domain, and a C-terminal cysteine-rich domain. In situ hybridization and immunohistochemical staining have localized the protein to a subset of macrophages in the marginal zone of the spleen and the medullary cord of lymph nodes. No expression was observed in macrophages of liver or lung. Transfected COS cells synthesized a native trimeric plasma membrane protein that bound labeled bacteria and acetylated LDL, but not yeast or Ficoll. The results suggest that the novel protein is a macrophage-specific membrane receptor with a role in host defense, as it shows postnatal expression in macrophages, which are considered responsible for the binding of bacterial antigens and phagocytosis.

Inactivation of the Lysyl Oxidase Gene Lox Leads to Aortic Aneurysms, Cardiovascular Dysfunction, and Perinatal Death in Mice

Background—The lysyl oxidases are extracellular copper enzymes that initiate the crosslinking of collagens and elastin, 5 human isoenzymes having been characterized so far. The crosslinks formed provide the tensile strength and elastic properties for various extracellular matrices, including vascular walls. We studied the role of the first described isoenzyme Lox by inactivating its gene in mice. Methods and Results—Murine Lox gene was disrupted by routine methods. Lox−/− mice died at the end of gestation or as neonates, necropsy of the live-born pups revealing large aortic aneurysms. In light microscopy, hazy and unruffled elastic lamellae in the Lox−/− aortas were observed, and electron microscopy of the aortic walls of the Lox−/− fetuses showed highly fragmented elastic fibers and discontinuity in the smooth muscle cell layers in Lox−/− fetuses. The wall of the aorta in the Lox−/− fetuses was significantly thicker, and the diameter of the aortic lumen was significantly smaller than that in the Lox+/+ aortas. In Lox−/− fetuses, Doppler ultrasonography revealed increased impedance in the umbilical artery, descending aorta, and intracranial artery blood velocity waveforms, decreased mean velocities across cardiac inflow and outflow regions, and increased pulsatility in ductus venosus blood velocity waveforms. Conclusions—Lox has an essential role in the development and function of the cardiovascular system. Inactivation of the Lox gene causes structural alterations in the arterial walls, leading to abnormalities in the cardiovascular functions. Alterations in LOX activity may also play a critical role in certain human cardiovascular diseases.

The impact of disparate isolation methods for extracellular vesicles on downstream RNA profiling

Despite an enormous interest in the role of extracellular vesicles, including exosomes, in cancer and their use as biomarkers for diagnosis, prognosis, drug response and recurrence, there is no consensus on dependable isolation protocols. We provide a comparative evaluation of 4 exosome isolation protocols for their usability, yield and purity, and their impact on downstream omics approaches for biomarker discovery. OptiPrep density gradient centrifugation outperforms ultracentrifugation and ExoQuick and Total Exosome Isolation precipitation in terms of purity, as illustrated by the highest number of CD63-positive nanovesicles, the highest enrichment in exosomal marker proteins and a lack of contaminating proteins such as extracellular Argonaute-2 complexes. The purest exosome fractions reveal a unique mRNA profile enriched for translation, ribosome, mitochondrion and nuclear lumen function. Our results demonstrate that implementation of high purification techniques is a prerequisite to obtain reliable omics data and identify exosome-specific functions and biomarkers.

Heparan sulfate chains of perlecan are indispensable in the lens capsule but not in the kidney

Mice lacking exon 3 of perlecan (Hspg2) gene were generated by gene targeting. Exon deletion does not alter the expression or the reading frame but causes loss of attachment sites for three heparan sulfate (HS) side chains. Hspg2Δ3/Δ3 mice are viable and fertile but have small eyes. Apoptosis and leakage of cellular material through the lens capsule are observed in neonatal lenses, and lenses degenerate within 3 weeks of birth. Electron microscopy revealed altered structure of the lens capsule through which cells had formed extensions. No kidney malfunction, such as protein uria, was detected in Hspg2Δ3/Δ3 mutant mice, nor were ultrastructural changes observed in the glomerular basement membranes (BMs). To achieve further depletion in the HS content of the BMs, Hspg2Δ3/Δ3 mice were bred with collagen XVIII null mice. Lens defects were more severe in the newborn Col18a1−/− × Hspg2Δ3/Δ3 mice and degeneration proceeded faster than in Hspg2Δ3/Δ3 mice. The results suggest that in the lens capsule, HS chains have a structural function and are essential in the insulation of the lens from its environment and in regulation of incoming signals.

Overexpression of peroxiredoxins I, II, III, V, and VI in malignant mesothelioma

Peroxiredoxins (Prxs) are a recently characterized group of thiol‐containing proteins with efficient antioxidant capacity, capable of consuming hydrogen peroxide in living cells. Altogether six distinct Prxs have been characterized in mammalian tissues. Their expression was investigated in histological samples of mesothelioma and in cell lines established from the tumours of mesothelioma patients. Four cases with histopathologically healthy pleura from non‐smokers were used as controls. Healthy pleural mesothelium was negative or very weakly positive for all Prxs. In mesothelioma, the most prominent reactivity was observed with Prxs I, II, V, and VI. Prx I was highly or moderately expressed in 25/36 cases, the corresponding figures for Prxs II–VI being 27/36 (Prx II), 13/36 (Prx III), 2/36 (Prx IV), 24/36 (Prx V), and 30/36 (Prx VI). Positive staining was observed both in the cytosolic and the nuclear compartment, with the exception of Prx III, which showed no nuclear reactivity. The staining pattern of Prxs III and V was granular. Immunoelectron microscopic localization of Prxs was in accordance with the immunohistochemical findings, showing diffuse cytoplasmic localization for Prxs I, II, IV, and VI and distinct mitochondrial labelling for Prxs III and V. There was no significant association between the extent of staining and different Prxs. It appeared that Prxs may not have prognostic significance, but being prominently expressed in most mesotheliomas these proteins, at least in theory, may play a role in the primary drug resistance of this disease. Copyright

Double-stranded RNA Is Internalized by Scavenger Receptor-mediated Endocytosis in Drosophila S2 Cells

Double-stranded RNA (dsRNA) fragments are readily internalized and processed by Drosophila S2 cells, making these cells a widely used tool for the analysis of gene function by gene silencing through RNA interference (RNAi). The underlying mechanisms are insufficiently understood. To identify components of the RNAi pathway in S2 cells, we developed a screen based on rescue from RNAi-induced lethality. We identified Argonaute 2, a core component of the RNAi machinery, and three gene products previously unknown to be involved in RNAi in Drosophila: DEAD-box RNA helicase Belle, 26 S proteasome regulatory subunit 8 (Pros45), and clathrin heavy chain, a component of the endocytic machinery. Blocking endocytosis in S2 cells impaired RNAi, suggesting that dsRNA fragments are internalized by receptor-mediated endocytosis. Indeed, using a candidate gene approach, we identified two Drosophila scavenger receptors, SR-CI and Eater, which together accounted for more than 90% of the dsRNA uptake into S2 cells. When expressed in mammalian cells, SR-CI was sufficient to mediate internalization of dsRNA fragments. Our data provide insight into the mechanism of dsRNA internalization by Drosophila cells. These results have implications for dsRNA delivery into mammalian cells.

Lysyl Oxidase Is Essential for Normal Development and Function of the Respiratory System and for the Integrity of Elastic and Collagen Fibers in Various Tissues

Lysyl oxidases, a family comprising LOX and four LOX-like enzymes, catalyze crosslinking of elastin and collagens. Mouse Lox was recently shown to be crucial for development of the cardiovascular system because null mice died perinatally of aortic aneurysms and cardiovascular dysfunction. We show here that Lox is also essential for development of the respiratory system and the integrity of elastic and collagen fibers in the lungs and skin. The lungs of E18.5 Lox(-/-) embryos showed impaired development of the distal and proximal airways. Elastic fibers in E18.5 Lox(-/-) lungs were markedly less intensely stained and more disperse than in the wild type, especially in the mesenchyme surrounding the distal airways, bronchioles, bronchi, and trachea, and were fragmented in pulmonary arterial walls. The organization of individual collagen fibers into tight bundles was likewise abnormal. Similar elastic and collagen fiber abnormalities were seen in the skin. Lysyl oxidase activity in cultured Lox(-/-) skin fibroblasts and aortic smooth muscle cells was reduced by approximately 80%, indicating that Lox is the main isoenzyme in these cells. LOX abnormalities may thus be critical for the pathogenesis of several common diseases, including pulmonary, skin, and cardiovascular disorders.

An Endoplasmic Reticulum Transmembrane Prolyl 4-Hydroxylase Is Induced by Hypoxia and Acts on Hypoxia-inducible Factor α

Prolyl 4-hydroxylases (P4Hs) act on collagens (C-P4Hs) and the oxygen-dependent degradation domains (ODDDs) of hypoxia-inducible factor α subunits (HIF-P4Hs) leading to degradation of the latter. We report data on a human P4H possessing a transmembrane domain (P4H-TM). Its gene is also found in zebrafish but not in flies and nematodes. Its sequence more closely resembles those of the C-P4Hs than the HIF-P4Hs, but it lacks the peptide substrate-binding domain of the C-P4Hs. P4H-TM levels in cultured cells are increased by hypoxia, and P4H-TM is N-glycosylated and is located in endoplasmic reticulum membranes with its catalytic site inside the lumen, a location differing from those of the HIF-P4Hs. Despite this, P4H-TM overexpression in cultured neuroblastoma cells reduced HIF-α ODDD reporter construct levels, and its small interfering RNA increased HIF-1α protein level, in the same way as those of HIF-P4Hs. Furthermore, recombinant P4H-TM hydroxylated the two critical prolines in HIF-1α ODDD in vitro, with a preference for the C-terminal proline, whereas it did not hydroxylate any prolines in recombinant type I procollagen chains.

Abnormal glycosylation and altered Golgi structure in colorectal cancer: dependence on intra‐Golgi pH

Abnormal glycosylation of cellular glycoconjugates is a common phenotypic change in many human tumors. Here, we explore the possibility that an altered Golgi pH may also be responsible for these cancer‐associated glycosylation abnormalities. We show that a mere dissipation of the acidic Golgi pH results both in increased expression of some cancer‐associated carbohydrate antigens and in structural disorganization of the Golgi apparatus in otherwise normally glycosylating cells. pH dependence of these alterations was confirmed by showing that an acidification‐defective breast cancer cell line (MCF‐7) also displayed a fragmented Golgi apparatus, whereas the Golgi apparatus was structurally normal in its acidification‐competent subline (MCF‐7/AdrR). Acidification competence was also found to rescue normal glycosylation potential in MCF‐7/AdrR cells. Finally, we show that abnormal glycosylation is also accompanied by similar structural disorganization and fragmentation of the Golgi apparatus in colorectal cancer cells in vitro and in vivo. These results suggest that an inappropriate Golgi pH may indeed be responsible for the abnormal Golgi structure and lowered glycosylation potential of the Golgi apparatus in malignant cells.

Pxmp2 Is a Channel-Forming Protein in Mammalian Peroxisomal Membrane

Background Peroxisomal metabolic machinery requires a continuous flow of organic and inorganic solutes across peroxisomal membrane. Concerning small solutes, the molecular nature of their traffic has remained an enigma. Methods/Principal Findings In this study, we show that disruption in mice of the Pxmp2 gene encoding Pxmp2, which belongs to a family of integral membrane proteins with unknown function, leads to partial restriction of peroxisomal membrane permeability to solutes in vitro and in vivo. Multiple-channel recording of liver peroxisomal preparations reveals that the channel-forming components with a conductance of 1.3 nS in 1.0 M KCl were lost in Pxmp2 −/− mice. The channel-forming properties of Pxmp2 were confirmed with recombinant protein expressed in insect cells and with native Pxmp2 purified from mouse liver. The Pxmp2 channel, with an estimated diameter of 1.4 nm, shows weak cation selectivity and no voltage dependence. The long-lasting open states of the channel indicate its functional role as a protein forming a general diffusion pore in the membrane. Conclusions/Significance Pxmp2 is the first peroxisomal channel identified, and its existence leads to prediction that the mammalian peroxisomal membrane is permeable to small solutes while transfer of “bulky” metabolites, e.g., cofactors (NAD/H, NADP/H, and CoA) and ATP, requires specific transporters.