BBA - General Subjects (v.1810, #2)

Recognition roles of the carbohydrate glycotopes of human and bovine lactoferrins in lectin–N-glycan interactions by Meng-Hsiu Yen; Albert M. Wu; Zhangung Yang; Yu-Ping Gong; En-Tzu Chang (139-149).
Lactoferrin is an iron-binding protein belonging to the transferrin family. In addition to iron homeostasis, lactoferrin is also thought to have anti-microbial, anti-inflammatory, and anticancer activities. Previous studies showed that all lactoferrins are glycosylated in the human body, but the recognition roles of their carbohydrate glycotopes have not been well addressed.The roles of human and bovine lactoferrins involved in lectin–N-glycan recognition processes were analyzed by enzyme-linked lectinosorbent assay with a panel of applied and microbial lectins.Both native and asialo human/bovine lactoferrins reacted strongly with four Man-specific lectins — Concanavalia ensiformis agglutinin, Morniga M, Pisum sativum agglutinin, and Lens culinaris lectin. They also reacted well with PA-IIL, a LFuc>Man-specific lectin isolated from Pseudomonas aeruginosa. Both human and bovine lactoferrins also recognized a sialic acid specific lectin-Sambucus nigra agglutinin, but not their asialo products. Both native and asialo bovine lactoferrins, but not the human ones, exhibited strong binding with a GalNAc>Gal-specific lectin-Wisteria floribunda agglutinin. Human native lactoferrins and its asialo products bound well with four Gal>GalNAc-specific type-2 ribosome inactivating protein family lectins-ricin, abrin-a, Ricinus communis agglutinin 1, and Abrus precatorius agglutinin (APA), while the bovine ones reacted only with APA.This study provides essential knowledge regarding the different roles of bioactive sites of lactoferrins in lectin–N-glycan recognition processes.► Recognition roles of N-glycans of lactoferrin (Lf) are examined with applied lectins. Both human and bovine Lf (hLf /bLf) react strongly with four Man- specific lectins. Native Lfs react well with Sambucus nigra agglutinin, a sialic acid specific lectin. However, hLf and bLf differ in binding intensity with Gal- specific lectins. These data indicate the recognition roles and glycotopes of N-glycans of hLf and bLf.
Keywords: Lactoferrin; Iron-binding glycoprotein; N-glycans; Lectin binding; Carbohydrate glycotope;

Human galectin-3 (Mac-2 antigen) is a cell-type-specific multifunctional effector owing to selective binding of distinct cell-surface glycoconjugates harboring β-galactosides. The structural basis underlying the apparent preferences for distinct glycoproteins and for expression is so far unknown.We strategically combined solid-phase assays on 43 natural glycoproteins with a new statistical approach to fully flexible computational docking and also processed the proximal promoter region in silico.The degree of branching in N-glycans and clustering of core 1 O-glycans are positive modulators for avidity. Sialylation of N-glycans in α2–6 linkage and of core 1 O-glycans in α2–3 linkage along with core 2 branching was an unfavorable factor, despite the presence of suited glycans in the vicinity. The lectin–ligand contact profile was scrutinized for six natural di- and tetrasaccharides enabling a statistical grading by analyzing flexible docking trajectories. The computational analysis of the proximal promoter region delineated putative sites for Lmo2/c-Ets-1 binding and new sites with potential for RUNX binding.These results identify new features of glycan selectivity and ligand contact by combining solid-phase assays with in silico work as well as of reactivity potential of the promoter.Protein–carbohydrate recognition is involved in diverse medical processes. Using an adhesion/growth-regulatory human lectin as model, ligand contact is computationally processed by flexible ligand docking. Binding assays with glycoproteins take analysis of selectivity to the level of natural ligands delineating relevance of branching.Display Omitted► Degree of N-glycan branching/core 1 O-glycan clustering enhances galectin-3 binding. ► Core 2 O-glycan branching, core 1 O-glycan α2,3-sialylation and N-glycan α2,6-sialylation reduce affinity. ► Flexible ligand docking with a statistical approach quantitates transient contacts. ► Computational promoter analysis identifies new sites with potential to explain the cell-type-specific profile.
Keywords: Glycosylation; Lectin; Macrophage; Modeling; Mucin; Sialylation;

Pore-forming activity of BAD is regulated by specific phosphorylation and structural transitions of the C-terminal part by Lisa Polzien; Angela Baljuls; Heide-Marie Roth; Jochen Kuper; Roland Benz; Kristian Schweimer; Mirko Hekman; Ulf R. Rapp (162-169).
BAD protein (Bcl-2 antagonist of cell death) belongs to the BH3-only subfamily of proapoptotic proteins and is proposed to function as the sentinel of the cellular health status. Physiological activity of BAD is regulated by phosphorylation, association with 14-3-3 proteins, binding to membrane lipids and pore formation. Since the functional role of the BAD C-terminal part has not been considered so far, we have investigated here the interplay of the structure and function of this region.The structure of the regulatory C-terminal part of human BAD was analyzed by CD spectroscopy. The channel-forming activity of full-length BAD and BAD peptides was carried out by lipid bilayer measurements. Interactions between proteins and peptides were monitored by the surface plasmon resonance technique.In aqueous solution, C-terminal part of BAD exhibits a well-ordered structure and stable conformation. In a lipid environment, the helical propensity considerably increases. The interaction of the C-terminal segment of BAD with the isolated BH3 domain results in the formation of permanently open pores whereby the phosphorylation of serine 118 within the BH3 domain is necessary for effective pore formation. In contrast, phosphorylation of serine 99 in combination with 14-3-3 association suppresses formation of channels. C-terminal part of BAD controls BAD function by structural transitions, lipid binding and phosphorylation. Conformational changes of this region upon membrane interaction in conjunction with phosphorylation of the BH3 domain suggest a novel mechanism for regulation of BAD.Multiple signaling pathways mediate inhibition and activation of cell death via BAD.► C-terminal region of BAD controls its function by structural transitions. ► C-terminal part of BAD is per se sufficient to form ion channels. ► Phosphorylation of serine 118 is essential for the pore formation. ► Serine 99 controls pore formation via interaction with 14-3-3 proteins.
Keywords: Protein interaction; Phosphorylation; Structure; Channel; Bcl-2 proteins;

Evaluation of antioxidant activity of vanillin by using multiple antioxidant assays by Akihiro Tai; Takeshi Sawano; Futoshi Yazama; Hideyuki Ito (170-177).
Vanillin, a compound widely used in foods, beverages, cosmetics and drugs, has been reported to exhibit multifunctional effects such as antimutagenic, antiangiogenetic, anti-colitis, anti-sickling, and antianalgesic effects. However, results of studies on the antioxidant activity of vanillin are not consistent.We systematically evaluated the antioxidant activity of vanillin using multiple assay systems. DPPH radical-, galvinoxyl radical-, and ABTS• +-scavenging assays, ORAC assay and an oxidative hemolysis inhibition assay (OxHLIA) were used for determining the antioxidant activity.Vanillin showed stronger activity than did ascorbic acid and Trolox in the ABTS• +-scavenging assay but showed no activity in the DPPH radical- and galvinoxyl radical-scavenging assays. Vanillin showed much stronger antioxidant activity than did ascorbic acid and Trolox in the ORAC assay and OxHLIA. In the ABTS• +-scavenging assay, ORAC assay and OxHLIA, vanillin reacted with radicals via a self-dimerization mechanism. The dimerization contributed to the high reaction stoichiometry against ABTS• + and AAPH-derived radicals to result in the strong effect of vanillin. Oral administration of vanillin to mice increased the vanillin concentration and the antioxidant activity in plasma. These data suggested that antioxidant activity of vanillin might be more beneficial than has been thought for daily health care.Based on the results of the present study, we propose the addition of antioxidant capacity to the multifunctionality of vanillin.►Vanillin shows no activity in the DPPH and galvinoxyl radical-scavenging assays. ►Vanillin shows strong activity in the ABTS• +-scavenging assay. ►Vanillin shows strong antioxidant activity in the ORAC assay and OxHLIA. ►Vanillin reacts with radicals via a self-dimerization mechanism. ►Oral administration of vanillin to mice increases the antioxidant activity in plasma.
Keywords: Vanillin; Antioxidant; Self-dimerization; ABTS radical cation; ORAC; Oxidative hemolysis inhibition assay;

Low inorganic phosphate (Pi) availability triggers metabolic responses to maintain the intracellular phosphate homeostasis in plants. One crucial adaptive mechanism is the immediate cleavage of Pi from phosphorylated substrates; however, phosphohydrolases that function in the cytosol and putative substrates have not been characterized yet. One candidate gene is Arabidopsis thaliana At1g73010 encoding an uncharacterized enzyme with homology to the haloacid dehalogenase (HAD) superfamily.This work reports the molecular cloning of At1g73010, its expression in Escherichia coli, and the enzymatic characterisation of the recombinant protein (33.5 kD). The Mg2+-dependent enzyme named AtPPsPase1 catalyzes the specific cleavage of pyrophosphate (K m 38.8 μM) with an alkaline catalytic pH optimum. Gel filtration revealed a tetrameric structure of the soluble cytoplasmic protein. Modelling of the active site and assay of the recombinant protein variant D19A demonstrated that the enzyme shares the catalytic mechanism of the HAD superfamily including a phosphorylated enzyme intermediate.The tight control of AtPPsPase1 gene expression underlines its important role in the Pi starvation response and suggests that cleavage of pyrophosphate is an immediate metabolic adaptation reaction.The novel enzyme, the first pyrophosphatase in the HAD superfamily, differs from classical pyrophosphatases with respect to structure and catalytic mechanism. The enzyme function could be used to discover unknown aspects of pyrophosphate metabolism in general.►The AtPPsPase1 gene is highly activated by Pi starvation in Arabidopsis. ►AtPPsPase1 is an intracellular, alkaline phosphatase specific for PPi cleavage. ►Structure and catalysis of AtPPsPase1 differ from classical pyrophosphatases. ►AtPPsPase1 is the initial member of a new subclass in the HAD superfamily of phosphatases.
Keywords: HAD superfamily; Inorganic pyrophosphatase; Nutrient stress; Plant pyrophosphate metabolism; Phosphatase; Phosphate deprivation;

Defects of the CLN3 gene on chromosome 16p12.1 lead to the juvenile form of neuronal ceroid-lipofuscinosis (JNCL, Batten Disease), the most common recessive inherited neurodegenerative disorder in children. Dysregulation of intracellular calcium homeostasis in the absence of a functional CLN3 protein (CLN3P, Battenin) has been linked to synaptic dysfunction and accelerated apoptosis in vulnerable neuronal cells. Prolonged increase of intracellular calcium concentration is considered to be a significant trigger for neuronal apoptosis and cellular loss in JNCL.We examined the potential effect of 41 different calcium channel modulators on intracellular calcium concentration in CLN3 siRNA knock down SH-SY5Y neuroblastoma cells.Six drugs belonging to the group of voltage dependent L-type channel blockers show significant lowering of the increased intracellular calcium levels in CLN3 siRNA knock down cells.Our studies provide important new data suggesting possible beneficial effects of the tested drugs on calcium flux regulated pathways in neuronal cell death. Therapeutic intervention in this untreatable disease will likely require drugs that cross the blood–brain barrier as did all of the positively screened drugs in this study.Better comprehension of the mechanism of neurodegeneration in rare recessive disorders, such as neuronal ceroid-lipofuscinoses, is likely to help to better understand mechanisms involved in more complex genetic neurodegenerative conditions, such as those associated with aging.
Keywords: Juvenile neuronal ceroid-lipofuscinosis; CLN3 protein; Intracellular trafficking; L-type calcium channels; Palmitoyl-protein Δ-9 desaturase;

Mechanism of bone collagen degradation due to KOH treatment by C. Wynnyckyj; S. Omelon; T.L. Willett; K. Kyle; H. Goldberg; M.D. Grynpas (192-201).
The mechanisms underlying the effect of alterations in type I collagen on bone mechanical properties are not well defined. In a previous study, male and female emu tibiae were endocortically treated with 1 M potassium hydroxide (KOH) solution for 1–14 days. This treatment resulted in negligible mass loss (0.5%), collagen loss (0.05%), no differences in geometrical parameters but significant changes in mechanical properties. The objective of this study was to determine the mechanism of collagen degradation due to KOH treatment in order to explain the previously observed mechanical property changes.Bone mineral was assessed using x-ray diffraction (XRD), microhardness and backscattered electron imaging (BSE). Bone collagen was assessed using α-chymotrypsin digestion, differential scanning calorimetry (DSC), gel electrophoresis (SDS-PAGE) and polarized light microscopy (PLM).BSE, microhardness and XRD revealed no changes in bone mineral due to KOH treatment. DSC showed an altered curve shape (lower and broader), indicating a change in collagen organization due to KOH treatment. Decreased α-chain band intensity in 14-day KOH treated groups detected using SDS-PAGE indicated α-chain fragmentation due to KOH treatment. PLM images revealed differences in collagen structure in terms of pattern distribution of preferentially oriented collagen between the periosteal and endocortical regions.These results suggest that endocortical KOH treatment causes in situ collagen degradation, which explains the previously reported altered mechanical properties.Compromising the organic component of bone contributes to an increase in bone fragility.
Keywords: Bone collagen; Collagen degradation; Sex differences; Bone mineral; Potassium hydroxide (KOH);

We focus on temperature- and hydration-dependence of internal molecular motions in stripped human red blood cell (RBC) vesicles, widely used as a model system for more complex biomembranes.We singled out picosecond local motions of the non-exchangeable hydrogen atoms of RBC vesicles by performing elastic and quasielastic incoherent neutron scattering measurements in dry and heavy water (D2O)-hydrated RBC powders.In dry stripped RBCs, hydrogen motions remained harmonic all along the measured temperature range (100–310 K) and mean–square displacements (MSDs) exhibited no temperature transition up to 310 K. In contrast, MSDs of hydrated stripped RBCs (h  ≈ 0.38 g D2O/g dry powder) exhibited a pronounced transition near 260 K, with the sharp rise of anharmonic diffusive motions of hydrogen atoms. This transition at ~ 260 K was correlated with both the onset of nonvibrational (harmonic and nonharmonic) motions and the melting of crystallized hydration water.In conclusion, we have shown that MSDs in human RBC vesicles are temperature-and hydration-dependent. These results provide insight into biomembrane internal dynamics at picosecond timescale and nanometer length scale. Such motions have been shown to act as the “lubricant” of larger conformational changes on a slower, millisecond timescale that are necessary for important biological processes.► Harmonic dynamics of dry erythrocyte vesicles in the temperature range from 100 to 310 K. ► Mean–square displacement temperature transition of hydrated erythrocyte vesicles is near 260 K. ► Nonvibrational motions of hydrated erythrocyte vesicles are at T  > ~ 260 K.
Keywords: Erythrocyte; Vesicle; Membrane protein; Internal dynamics; Dynamical transition; Neutron scattering;

Adsorption of low-density lipoprotein, its oxidation, and subsequent binding of specific recombinant antibodies: An in situ ellipsometric study by Maria M. Stollenwerk; Olof Svensson; Alexandru Schiopu; Bo Jansson; Thomas Arnebrant; Gunilla Nordin Fredrikson (211-217).
Low-density lipoprotein (LDL) particles accumulate in the arterial wall and become oxidized during atherogenesis, leading to the formation of atherosclerotic plaques. The major protein of the LDL particle, apolipoprotein B-100 (apoB-100), becomes fragmented during oxidation and a target for the immune system.In this study we used in situ ellipsometry to monitor the adsorption of LDL to solid silica surfaces and the effects of oxidation on the structure of the adsorbed LDL layer. We additionally investigated the binding kinetics of two recombinant human antibodies with different specificities recognizing epitopes of apoB-100 in surface-bound native and CuCl2-oxidized LDL (oxLDL). The latter process was studied by adsorbing LDL and then adding the antibody and CuCl2 while continuously monitoring adsorbed amount and the thickness of the film. The molar ratios between the antibodies and surface-bound LDL and oxLDL were calculated from these data.Our results indicate that oxidation of surface-bound LDL induces swelling of the layer, accompanied by a slight desorption. We further found that both antibodies were able to recognize LDL and oxLDL in its adsorbed orientation. Quantitative information was obtained on the number of available binding sites on surface-bound LDL and oxLDL for these two antibodies.Using ellipsometry for real-time monitoring of adsorption, in situ oxidation of LDL and binding of specific recombinant antibodies to surface-bound LDL, will open up possibilities to map different conformations and orientations of LDL in the adsorbed state. In situ ellipsometry allows quantification of molar ratios between antibody and oxLDL. ► LDL binding takes place at the solid/liquid interface in a concentration-dependent way. ► OxLDL specific antibodies recognize oxLDL in its adsorbed orientation. ► Two oxLDL specific antibodies recognize diverse number of epitopes on adsorbed oxLDL.
Keywords: Adsorption; Antibody; apoB-100; Ellipsometry; Oxidation; Low-density lipoprotein (LDL);

Deuteration as a tool in investigating the role of protons in cell signaling by Yutaka Hirakura; Takashi Sugiyama; Masahiro Takeda; Masayuki Ikeda; Tohru Yoshioka (218-225).
The mechanisms underlying the inhibitory effects of deuterium oxide (D2O; heavy water) are likely to provide insight into the fundamental significance of hydrogen bonds in biological functions. Previously, to begin elucidating the effect of D2O on physiological functions in living cells, we studied the effects of D2O on voltage-sensitive Ca2+ channels in AtT 20 cells and showed that actin distribution, Ca2+ currents, and β-endorphin release were affected. However, the molecular mechanisms underlying the inhibitory effects of D2O in whole animals and living cells remain obscure, especially in the effects of D2O on the cell signaling.We investigated the molecular mechanisms underlying the inhibitory effects of D2O on the IP3-mediated Ca2+ signaling pathway using Ca2+ imaging and micro-calorimetric measurements in mGluR1-expressing CHO cells.DHPG-induced Ca2+ elevations were markedly reduced in D2O. Moreover, the Ca2+ elevations were completely suppressed in H2O after receptor activation with DHPG in D2O, recovering gradually in H2O medium. Without prior stimulation in D2O, however, DHPG-induced Ca2+ elevations in H2O were not affected. Micro-calorimetric measurements showed reduced total DHPG-evoked heat generation in D2O, while initial heat production and absorption associated with receptor activation were found to be larger. The reduction of DHPG-induced Ca2+ elevation and heat generation in D2O medium may be due to decreased amount of IP3 by the reduced hydrolysis of PIP2.Protein structure changes due to the replacement of hydrogen with deuterium will induce the inhibitory effects of D2O by reduction of the frequency of –OH bonds.► DHPG-evoked Ca2+ influx was reduced quickly when proton was changed to deuteron. ► Hysteresis effect on acute D/H exchange associated with receptor activation. ► Heat production after receptor stimulation was markedly attenuated in D2O. ► Amount of IP3 may be decreased in D2O by the reduced hydrolysis of PIP2.
Keywords: Heavy water; Metabotropic glutamate receptor; Calcium imaging; Hysteresis effect; Heat production;

Induced pluripotent stem (iPS) cells can be generated from somatic cells by introducing the four transcription factors Oct4, Sox2, Klf4, and c-Myc. Given that iPS cell technology may be useful for medical applications, the quality of iPS cells needs to be maintained during prolonged cultivation. However, it is unclear whether there are any differences in stability among different iPS clones.We infected mouse embryonic and adult fibroblasts with retroviruses encoding Oct4, Sox2, Klf4, c-Myc, and green fluorescent protein (GFP). We obtained embryonic stem (ES) cell-like colonies with silenced retroviral transgenes and divided these colonies into two groups: ES cell-like colonies that underwent retroviral silencing (i) on around day 14 (called early iPS) or (ii) on around day 30 (called late iPS), after infection. We compared morphology, proliferation efficiency, pluripotency marker expression, and karyotype between early iPS and late iPS cells.Early iPS cells were more stable than late iPS cells. At passage 20, most of the early iPS clones maintained ES cell-like morphology, expressed pluripotency markers, and showed proliferation efficiency similar to ES cells. Furthermore, early iPS clones derived from both embryonic and adult fibroblasts gave rise to chimeras and could show germ line competency. In contrast, late iPS clones tended to lose their ES cell-like morphology and normal karyotype in prolonged culture.Our results provide useful information on the efficient derivation of stable iPS cells that may be useful for germline transmission in mouse. This study suggests that early completion of full reprogramming allows for superior iPS cell generation.► Different iPS cell clones are generated at different time points. ► We compare the quality among different iPS clones with silenced retroviral factors. ► Superior iPS clones can be efficiently obtained at earlier time points.
Keywords: Induced pluripotent stem cell; Reprogramming; Stability;