Low Density Lipoprotein Receptor Class A Repeats Are O-Glycosylated in Linker Regions

The low density lipoprotein receptor (LDLR) is crucial for cholesterol homeostasis and deficiency in LDLR functions cause hypercholesterolemia. LDLR is a type I transmembrane protein that requires O-glycosylation for stable expression at the cell surface. It has previously been suggested that LDLR O-glycosylation is found N-terminal to the juxtamembrane region. Recently we identified O-glycosylation sites in the linker regions between the characteristic LDLR class A repeats in several LDLR-related receptors using the “SimpleCell” O-glycoproteome shotgun strategy. Herein, we have systematically characterized O-glycosylation sites on recombinant LDLR shed from HEK293 SimpleCells and CHO wild-type cells. We find that the short linker regions between LDLR class A repeats contain an evolutionarily conserved O-glycosylation site at position −1 of the first cysteine residue of most repeats, which in wild-type CHO cells is glycosylated with the typical sialylated core 1 structure. The glycosites in linker regions of LDLR class A repeats are conserved in LDLR from man to Xenopus and found in other homologous receptors. O-Glycosylation is controlled by a large family of polypeptide GalNAc transferases. Probing into which isoform(s) contributed to glycosylation of the linker regions of the LDLR class A repeats by in vitro enzyme assays suggested a major role of GalNAc-T11. This was supported by expression of LDLR in HEK293 cells, where knock-out of the GalNAc-T11 isoform resulted in the loss of glycosylation of three of four linker regions.     

Glycosylation Modulates Melanoma Cell α2β1 and α3β1 Integrin Interactions with Type IV Collagen

Although type IV collagen is heavily glycosylated, the influence of this post-translational modification on integrin binding has not been investigated. In the present study, galactosylated and nongalactosylated triple-helical peptides have been constructed containing the α1(IV)382–393 and α1(IV)531–543 sequences, which are binding sites for the α2β1 and α3β1 integrins, respectively. All peptides had triple-helical stabilities of 37 °C or greater. The galactosylation of Hyl³⁹³ in α1(IV)382–393 and Hyl⁵⁴⁰ and Hyl⁵⁴³ in α1(IV)531–543 had a dose-dependent influence on melanoma cell adhesion that was much more pronounced in the case of α3β1 integrin binding. Molecular modeling indicated that galactosylation occurred on the periphery of α2β1 integrin interaction with α1(IV)382–393 but right in the middle of α3β1 integrin interaction with α1(IV)531–543. The possibility of extracellular deglycosylation of type IV collagen was investigated, but no β-galactosidase-like activity capable of collagen modification was found. Thus, glycosylation of collagen can modulate integrin binding, and levels of glycosylation could be altered by reduction in expression of glycosylation enzymes but most likely not by extracellular deglycosylation activity.     

Diversity of Channels Generated by Different Combinations of Epithelial Sodium Channel Subunits

The epithelial sodium channel is a multimeric protein formed by three homologous subunits: α, β, and γ; each subunit contains only two transmembrane domains. The level of expression of each of the subunits is markedly different in various Na⁺ absorbing epithelia raising the possibility that channels with different subunit composition can function in vivo. We have examined the functional properties of channels formed by the association of α with β and of α with γ in the Xenopus oocyte expression system using two-microelectrode voltage clamp and patch-clamp techniques. We found that αβ channels differ from αγ channels in the following functional properties: (a) αβ channels expressed larger Na⁺ than Li⁺ currents (I_Na+/I_Li+ 1.2) whereas αγ channels expressed smaller Na⁺ than Li⁺ currents (I_Na+/I_Li+ 0.55); (b) the Michaelis Menten constants (K _m) of activation of current by increasing concentrations of external Na⁺ and Li⁺ of αβ channels were larger (K _m > 180 mM) than those of αγ channels (K _m of 35 and 50 mM, respectively); (c) single channel conductances of αβ channels (5.1 pS for Na⁺ and 4.2 pS for Li⁺) were smaller than those of αγ channels (6.5 pS for Na⁺ and 10.8 pS for Li⁺); (d) the half-inhibition constant (K _i) of amiloride was 20-fold larger for αβ channels than for αγ channels whereas the K _i of guanidinium was equal for both αβ and αγ. To identify the domains in the channel subunits involved in amiloride binding, we constructed several chimeras that contained the amino terminus of the γ subunit and the carboxy terminus of the β subunit. A stretch of 15 amino acids, immediately before the second transmembrane domain of the β subunit, was identified as the domain conferring lower amiloride affinity to the αβ channels. We provide evidence for the existence of two distinct binding sites for the amiloride molecule: one for the guanidium moiety and another for the pyrazine ring. At least two subunits α with β or γ contribute to these binding sites. Finally, we show that the most likely stoichiometry of αβ and αγ channels is 1α:1β and 1α:1γ, respectively.     

The epithelial sodium channel in the Australian lungfish,Neoceratodus forsteri (Osteichthyes: Dipnoi)

Epithelial sodium channel (ENaC) is a Na⁺-selective, aldosterone-stimulated ion channel involved in sodium transport homeostasis. ENaC is rate-limiting for Na⁺ absorption in the epithelia of osmoregulatory organs of tetrapods. Although the ENaC/degenerin gene family is proposed to be present in metazoans, no orthologues or paralogues for ENaC have been found in the genome databases of teleosts. We studied full-length cDNA cloning and tissue distributions of ENaCα, β and γ subunits in the Australian lungfish, Neoceratodus forsteri, which is the closest living relative of tetrapods. Neoceratodus ENaC (nENaC) comprised three subunits: nENaCα, β and γ proteins. The nENaCα, β and γ subunits are closely related to amphibian ENaCα, β and γ subunits, respectively. Three ENaC subunit mRNAs were highly expressed in the gills, kidney and rectum. Amiloride-sensitive sodium current was recorded from Xenopus oocytes injected with the nENaCαβγ subunit complementary RNAs under a two-electrode voltage clamp. nENaCα immunoreactivity was observed in the apical cell membrane of the gills, kidney and rectum. Thus, nENaC may play a role in regulating sodium transport of the lungfish, which has a renin–angiotensin–aldosterone system. This is interesting because there may have been an ENaC sodium absorption system controlled by aldosterone before the conquest of land by vertebrates.     

Reduced IRE1α mediates apoptotic cell death by disrupting calcium homeostasis via the InsP3 receptor

The endoplasmic reticulum (ER) is not only a home for folding and posttranslational modifications of secretory proteins but also a reservoir for intracellular Ca²⁺. Perturbation of ER homeostasis contributes to the pathogenesis of various neurodegenerative diseases, such as Alzheimer''s and Parkinson diseases. One key regulator that underlies cell survival and Ca²⁺ homeostasis during ER stress responses is inositol-requiring enzyme 1α (IRE1α). Despite extensive studies on this ER membrane-associated protein, little is known about the molecular mechanisms by which excessive ER stress triggers cell death and Ca²⁺ dysregulation via the IRE1α-dependent signaling pathway. In this study, we show that inactivation of IRE1α by RNA interference increases cytosolic Ca²⁺ concentration in SH-SY5Y cells, leading to cell death. This dysregulation is caused by an accelerated ER-to-cytosolic efflux of Ca²⁺ through the InsP3 receptor (InsP3R). The Ca²⁺ efflux in IRE1α-deficient cells correlates with dissociation of the Ca²⁺-binding InsP3R inhibitor CIB1 and increased complex formation of CIB1 with the pro-apoptotic kinase ASK1, which otherwise remains inactivated in the IRE1α–TRAF2–ASK1 complex. The increased cytosolic concentration of Ca²⁺ induces mitochondrial production of reactive oxygen species (ROS), in particular superoxide, resulting in severe mitochondrial abnormalities, such as fragmentation and depolarization of membrane potential. These Ca²⁺ dysregulation-induced mitochondrial abnormalities and cell death in IRE1α-deficient cells can be blocked by depleting ROS or inhibiting Ca²⁺ influx into the mitochondria. These results demonstrate the importance of IRE1α in Ca²⁺ homeostasis and cell survival during ER stress and reveal a previously unknown Ca²⁺-mediated cell death signaling between the IRE1α–InsP3R pathway in the ER and the redox-dependent apoptotic pathway in the mitochondrion.     

IKKalpha, a critical regulator of epidermal differentiation and a suppressor of skin cancer

IκB kinase α (IKKα), one of the two catalytic subunits of the IKK complex involved in nuclear factor κB (NF-κB) activation, also functions as a molecular switch that controls epidermal differentiation. This unexpected function requires IKKα nuclear translocation but does not depend on its kinase activity, and is independent of NF-κB signalling. Ikkα^–/– mice present with a hyperproliferative and undifferentiated epidermis characterized by complete absence of a granular layer and stratum corneum. Ikkα-deficient keratinocytes do not express terminal differentiation markers and continue to proliferate even when subjected to differentiation-inducing stimuli. This antiproliferative function of IKKα is also important for the suppression of squamous cell carcinogenesis. The exact mechanisms by which nuclear IKKα controls keratinocyte proliferation and differentiation remained mysterious for some time. Recent studies, however, have revealed that IKKα is a major cofactor in a TGFβ–Smad2/3 signalling pathway that is Smad4 independent. This pathway controls cell cycle withdrawal during keratinocyte terminal differentiation. Although these are not the only functions of nuclear IKKα, this multifunctional protein is a key regulator of keratinocyte and epidermal differentiation and a critical suppressor of skin cancer.     

Selective inhibition of protein kinase C β2 attenuates the adaptor P66Shc-mediated intestinal ischemia–reperfusion injury

Apoptosis is a major mode of cell death occurring during ischemia–reperfusion (I/R) induced injury. The p66^Shc adaptor protein, which is mediated by PKCβ, has an essential role in apoptosis under oxidative stress. This study aimed to investigate the role of PKCβ₂/p66^Shc pathway in intestinal I/R injury. In vivo, ischemia was induced by superior mesenteric artery occlusion in mice. Ruboxistaurin (PKCβ inhibitor) or normal saline was administered before ischemia. Then blood and gut tissues were collected after reperfusion for various measurements. In vitro, Caco-2 cells were challenged with hypoxia–reoxygenation (H/R) to simulate intestinal I/R. Translocation and activation of PKCβ₂ were markedly induced in the I/R intestine. Ruboxistaurin significantly attenuated gut damage and decreased the serum levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Pharmacological blockade of PKCβ₂ suppressed p66^Shc overexpression and phosphorylation in the I/R intestine. Gene knockdown of PKCβ₂ via small interfering RNA (siRNA) inhibited H/R-induced p66^Shc overexpression and phosphorylation in Caco-2 cells. Phorbol 12-myristate 13-acetate (PMA), which stimulates PKCs, induced p66^Shc phosphorylation and this was inhibited by ruboxistaurin and PKCβ₂ siRNA. Ruboxistaurin attenuated gut oxidative stress after I/R by suppressing the decreased expression of manganese superoxide dismutase (MnSOD), the exhaustion of the glutathione (GSH) system, and the overproduction of malondialdehyde (MDA). As a consequence, ruboxistaurin inhibited intestinal mucosa apoptosis after I/R. Therefore, PKCβ₂ inhibition protects mice from gut I/R injury by suppressing the adaptor p66^Shc-mediated oxidative stress and subsequent apoptosis. This may represent a novel therapeutic approach for the prevention of intestinal I/R injury.     

Role of UDP-N-Acetylglucosamine (GlcNAc) and O-GlcNAcylation of Hyaluronan Synthase 2 in the Control of Chondroitin Sulfate and Hyaluronan Synthesis

Hyaluronan (HA) is a glycosaminoglycan present in most tissue microenvironments that can modulate many cell behaviors, including proliferation, migration, and adhesive proprieties. In contrast with other glycosaminoglycans, which are synthesized in the Golgi, HA is synthesized at the plasma membrane by one or more of the three HA synthases (HAS1–3), which use cytoplasmic UDP-glucuronic acid and UDP-N-acetylglucosamine as substrates. Previous studies revealed the importance of UDP-sugars for regulating HA synthesis. Therefore, we analyzed the effect of UDP-GlcNAc availability and protein glycosylation with O-linked N-acetylglucosamine (O-GlcNAcylation) on HA and chondroitin sulfate synthesis in primary human aortic smooth muscle cells. Glucosamine treatment, which increases UDP-GlcNAc availability and protein O-GlcNAcylation, increased synthesis of both HA and chondroitin sulfate. However, increasing O-GlcNAcylation by stimulation with O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate without a concomitant increase of UDP-GlcNAc increased only HA synthesis. We found that HAS2, the main synthase in aortic smooth muscle cells, can be O-GlcNAcylated on serine 221, which strongly increased its activity and its stability (t_½ >5 h versus ∼17 min without O-GlcNAcylation). S221A mutation prevented HAS2 O-GlcNAcylation, which maintained the rapid turnover rate even in the presence of GlcN and increased UDP-GlcNAc. These findings could explain the elevated matrix HA observed in diabetic vessels that, in turn, could mediate cell dedifferentiation processes critical in vascular pathologies.     

Evidence of photosymbiosis in Palaeozoic tabulate corals

Coral reefs form the most diverse of all marine ecosystems on the Earth. Corals are among their main components and owe their bioconstructing abilities to a symbiosis with algae (Symbiodinium). The coral–algae symbiosis had been traced back to the Triassic (ca 240 Ma). Modern reef-building corals (Scleractinia) appeared after the Permian–Triassic crisis; in the Palaeozoic, some of the main reef constructors were extinct tabulate corals. The calcium carbonate secreted by extant photosymbiotic corals bears characteristic isotope (C and O) signatures. The analysis of tabulate corals belonging to four orders (Favositida, Heliolitida, Syringoporida and Auloporida) from Silurian to Permian strata of Europe and Africa shows these characteristic carbon and oxygen stable isotope signatures. The δ¹⁸O to δ¹³C ratios in recent photosymbiotic scleractinians are very similar to those of Palaeozoic tabulates, thus providing strong evidence of such symbioses as early as the Middle Silurian (ca 430 Ma). Corals in Palaeozoic reefs used the same cellular mechanisms for carbonate secretion as recent reefs, and thus contributed to reef formation.     

Subunit Composition Determines the Single Channel Kinetics of the Epithelial Sodium Channel

We have further characterized at the single channel level the properties of epithelial sodium channels formed by coexpression of α with either wild-type β or γ subunits and α with carboxy-terminal truncated β (β_T) or γ (γ_T) subunits in Xenopus laevis oocytes. αβ and αβ_T channels (9.6 and 8.7 pS, respectively, with 150 mM Li⁺) were found to be constitutively open. Only upon inclusion of 1 μM amiloride in the pipette solution could channel activity be resolved; both channel types had short open and closed times. Mean channel open probability (P _o) for αβ was 0.54 and for αβ_T was 0.50. In comparison, αγ and αγ_T channels exhibited different kinetics: αγ channels (6.7 pS in Li⁺) had either long open times with short closings, resulting in a high P _o (0.78), or short openings with long closed times, resulting in a low P _o (0.16). The mean P _o for all αγ channels was 0.48. αγ_T (6.6 pS in Li⁺) behaved as a single population of channels with distinct kinetics: mean open time of 1.2 s and closed time of 0.4 s, with a mean P _o of 0.6, similar to that of αγ. Inclusion of 0.1 μM amiloride in the pipette solution reduced the mean open time of αγ_T to 151 ms without significantly altering the closed time. We also examined the kinetics of amiloride block of αβ, αβ_T (1 μM amiloride), and αγ_T (0.1 μM amiloride) channels. αβ and αβ_T had similar blocking and unblocking rate constants, whereas the unblocking rate constant for αγ_T was 10-fold slower than αβ_T. Our results indicate that subunit composition of ENaC is a main determinant of P _o. In addition, channel kinetics and P _o are not altered by carboxy-terminal deletion in the β subunit, whereas a similar deletion in the γ subunit affects channel kinetics but not P _o.     

Cloning,Expression, and Characterization of the Squid Na+–Ca2+ Exchanger (NCX-SQ1)

We have cloned the squid neuronal Na⁺–Ca²⁺ exchanger, NCX-SQ1, expressed it in Xenopus oocytes, and characterized its regulatory and ion transport properties in giant excised membrane patches. The squid exchanger shows 58% identity with the canine Na⁺–Ca²⁺ exchanger (NCX1.1). Regions determined to be of functional importance in NCX1 are well conserved. Unique among exchanger sequences to date, NCX-SQ1 has a potential protein kinase C phosphorylation site (threonine 184) between transmembrane segments 3 and 4 and a tyrosine kinase site in the Ca²⁺ binding region (tyrosine 462). There is a deletion of 47 amino acids in the large intracellular loop of NCX-SQ1 in comparison with NCX1. Similar to NCX1, expression of NCX-SQ1 in Xenopus oocytes induced cytoplasmic Na⁺-dependent ⁴⁵Ca²⁺ uptake; the uptake was inhibited by injection of Ca²⁺ chelators. In giant excised membrane patches, the NCX-SQ1 outward exchange current showed Na⁺-dependent inactivation, secondary activation by cytoplasmic Ca²⁺, and activation by chymotrypsin. The NCX-SQ1 exchange current was strongly stimulated by both ATP and the ATP-thioester, ATPγS, in the presence of F⁻ (0.2 mM) and vanadate (50 μM), and both effects reversed on application of a phosphatidylinositol-4′,5′-bisphosphate antibody. NCX1 current was stimulated by ATP, but not by ATPγS. Like NCX1 current, NCX-SQ1 current was strongly stimulated by phosphatidylinositol-4′,5′-bisphosphate liposomes. In contrast to results in squid axon, NCX-SQ1 was not stimulated by phosphoarginine (5–10 mM). After chymotrypsin treatment, both the outward and inward NCX-SQ1 exchange currents were more strongly voltage dependent than NCX1 currents. Ion concentration jump experiments were performed to estimate the relative electrogenicity of Na⁺ and Ca²⁺ transport reactions. Outward current transients associated with Na⁺ extrusion were much smaller for NCX-SQ1 than NCX1, and inward current transients associated with Ca²⁺ extrusion were much larger. For NCX-SQ1, charge movements of Ca²⁺ transport could be defined in voltage jump experiments with a low cytoplasmic Ca²⁺ (2 μM) in the presence of high extracellular Ca²⁺ (4 mM). The rates of charge movements showed “U”-shaped dependence on voltage, and the slopes of both charge–voltage and rate–voltage relations (1,600 s⁻¹ at 0 mV) indicated an apparent valency of −0.6 charges for the underlying reaction. Evidently, more negative charge moves into the membrane field in NCX-SQ1 than in NCX1 when ions are occluded into binding sites.     

Evaluation of Host Suitability in Prunus for Criconemella xenoplax

Methods were developed for screening Prunus selections for host suitability to Criconemella xenoplax. The relative host suitability of selections was based upon a doubling accumulation value (β) that was defined as the number of degree-days (base 9 C) required for doubling of an increment of the initial nematode population. The β value characteristic for C. xenoplax (139 ± 8 degree-days) on suitable hosts was similar to the average β value determined for several peach rootstocks known to be suitable hosts. The β values were 144 ± 21 for Halford, 141 ± 16 for Lovell, and 138 ± 10 for Nemaguard. A higher value for β could indicate poorer host suitability or resistance of a selection to C. xenoplax. All of 369 Prunus accessions tested, including eight accessions that had survived well on a field site infested with C. xenoplax, were suitable hosts. Apparently, resistance to C. xenoplax was not a factor in survival of the accessions planted in the field. Seedlings from P. besseyi, P. pumila ''Mando'', and two interspecific hybrids, Redcoat and Sapalta IR 549-1, failed to support nematode population increase in 44-81% of tests conducted, but all selections supported population increase in some tests. These accessions may have resistance mechanisms that are active only under specific conditions.     

A new species of plasmodiidae (Coccidia: Hemosporidia) from the blood of the skink Scincus hemprichii (Scincidae: Reptilia) in Saudi Arabia

Fallisia arabica n. sp. was described from peripheral blood smears of the Skink lizard, Scincus hemprichii from Jazan Province in the southwest of Saudi Arabia. Schizogony and gametogony take place within neutrophils in the peripheral blood of the host. Mature schizont is rosette shaped 17.5 ± 4.1 × 17.0 ± 3.9 μm, with a L/W ratio of 1.03(1.02–1.05) μm and produces 24(18–26) merozoites. Young gametocytes are ellipsoidal, 5.5 ± 0.8 × 3.6 ± 0.5 μm, with a L/W of 1.53(1.44–1.61) μm. Mature macrogametocytes are ellipsoidal, 9.7 ± 1.2 × 7.8 ± 1.0 μm, with a L/W of 1.24(1.21–1.34) μm and microgametocytes are ellipsoidal, 7.0 ± 1.1 × 6.8 ± 0.9 μm. with a L/W of 1.03(1.01–1.10) μm. In comparison to the described Fallisia species, this new taxon has rosette schizonts and is larger than F. dominicensis, in Hispaniola, F. bipocrati, F. poecilopi, in Panama, F. thecadactyli in Venezuela, and F. effusa, F. simplex, F. modesta, in Brazil. F. arabica has fewer merozoites than F. effusa, F. poecilopi, F. thecadactyli and F. siamense in Thailand. This new species has more merozoites than F. dominicensis and F. modesta. All of these species belong to diverse saurian families (Agamidae, Gekkonidae, Polychrotidae, Scincidae and Teiidae) parasitize only thrombocytes or lymphocytes and some species parasitize immature erythroid cells and leucocytes.     

Computational analyses of protein coded by rice (Oryza sativa japonica) cDNA (GI: 32984786) indicate lectin like Ca2+ binding properties for Eicosapenta Peptide Repeats (EPRs)

Eicosapenta peptide repeats (EPRs) occur exclusively in flowering plant genomes and exhibit very high amino acid residue conservation across occurrence. DNA and amino acid sequence searches yielded no indications about the function due to absence of similarity to known sequences. Tertiary structure of an EPR protein coded by rice (Oryza sativa japonica) cDNA (GI: 32984786) was determined based on ab initio methodology in order to draw clues on functional significance of EPRs. The resultant structure comprised of seven α-helices and thirteen anti-parallel β-sheets. Surface-mapping of conserved residues onto the structure deduced that (i) regions equivalent to β α4- the primary function of EPR protein could be Ca²⁺ binding, and (iii) the putative EPR Ca²⁺ binding domain is structurally similar to calcium-binding domains of plant lectins. Additionally, the phylogenetic analysis showed an evolving taxa-specific distribution of EPR proteins observed in some GNA-like lectins.     

Mouse Macrophage Galactose-type Lectin (mMGL) is Critical for Host Resistance against Trypanosoma cruzi Infection

The C-type lectin receptor mMGL is expressed exclusively by myeloid antigen presenting cells (APC) such as dendritic cells (DC) and macrophages (Mφ), and it mediates binding to glycoproteins carrying terminal galactose and α- or β-N-acetylgalactosamine (Gal/GalNAc) residues. Trypanosoma cruzi (T. cruzi) expresses large amounts of mucin (TcMUC)-like glycoproteins. Here, we show by lectin-blot that galactose moieties are also expressed on the surface of T. cruzi. Male mMGL knockout (-/-) and wild-type (WT) C57BL/6 mice were infected intraperitoneally with 10⁴ T. cruzi trypomastigotes (Queretaro strain). Following T. cruzi infection, mMGL-/- mice developed higher parasitemia and higher mortality rates compared with WT mice. Although hearts from T. cruzi-infected WT mice presented few amastigote nests, mMGL-/- mice displayed higher numbers of amastigote nests. Compared with WT, Mφ from mMGL-/- mice had low production of nitric oxide (NO), interleukin (IL)-12 and tumor necrosis factor (TNF)-α in response to soluble T. cruzi antigens (TcAg). Interestingly, upon in vitro T. cruzi infection, mMGL-/- Mφ expressed lower levels of MHC-II and TLR-4 and harbored higher numbers of parasites, even when mMGL-/- Mφ were previously primed with IFN-γ or LPS/IFN-γ. These data suggest that mMGL plays an important role during T. cruzi infection, is required for optimal Mφ activation, and may synergize with TLR-4-induced pathways to produce TNF-α, IL-1β and NO during the early phase of infection.