Vertebrate Plasma Research Articles

Glycosphingolipids in Osteoarthritis and Cartilage-Regeneration Therapy: Mechanisms and Therapeutic Prospects Based on a Narrative Review of the Literature.

Glycosphingolipids (GSLs), a subtype of glycolipids containing sphingosine, are critical components of vertebrate plasma membranes, playing a pivotal role in cellular signaling and interactions. In human articular cartilage in osteoarthritis (OA), GSL expression is known notably to decrease. This review focuses on the roles of gangliosides, a specific type of GSL, in cartilage degeneration and regeneration, emphasizing their regulatory function in signal transduction. The expression of gangliosides, whether endogenous or augmented exogenously, is regulated at the enzymatic level, targeting specific glycosyltransferases. This regulation has significant implications for the composition of cell-surface gangliosides and their impact on signal transduction in chondrocytes and progenitor cells. Different levels of ganglioside expression can influence signaling pathways in various ways, potentially affecting cell properties, including malignancy. Moreover, gene manipulations against gangliosides have been shown to regulate cartilage metabolisms and chondrocyte differentiation in vivo and in vitro. This review highlights the potential of targeting gangliosides in the development of therapeutic strategies for osteoarthritis and cartilage injury and addresses promising directions for future research and treatment.

Regulation of VWF Cleavage Is Mediated By Coupling ADAMTS13 D- and C- Domain Binding Sites

Introduction: The metalloprotease ADAMTS13 consists of metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), Cys-rich (C), and spacer (S) domains (proximal domains), followed by 7 T and 2 CUB domains (distal domains). ADAMTS13 cleaves the cryptic Tyr1605-Met1606 bond in the A2 domain of von Willebrand factor (VWF) to prevent ultra-large multimeric VWF-induced platelet aggregation. We and others previously described the allosteric regulation of ADAMTS13 by which the distal domains fold back and inhibit the proximal domains. VWF D4-CK domains bind the ADAMTS13 T8-CUB region to disrupt this allostery and accelerate VWF proteolysis. We recently discovered that ADAMTS13 and VWF have not evolved to be optimal enzyme-substrate pairs (Muia, Blood, 2019). For example, c-VWF71 (C-71), a wild-type bovine VWF domain A2-derived peptide substrate, is cleaved extremely well by many vertebrate ADAMTS13. Conversely, similar substrates prepared from wild-type human h-VWF71 (H-71) and other VWF domain A2 sequences mostly tolerated cleavage by their ADAMTS13 or additional few species-specific ADAMTS13. Although unfolded VWF A2 domain engages ADAMTS13 M, D, C, and S exosites for substrate specificity and efficient cleavage, sequence differences among the various vertebrates VWF71 substrates may hold clues about specificity determinants of ADAMTS13 substrate recognition and cleavage. Methods: Multiplex FRET ADAMTS13 assays were performed using 10 novel fluorogenic substrates. Substrates P3-P10 are chimeric substrates between C-71 and H-71; species-specific amino acid residues were swapped sequentially (Fig 1A). Plasmas from various vertebrates have been characterized and reported before. Recombinant ADAMTS13 variants were transiently expressed in HEK293 cells. Assays were performed as previously described and mean results are reported. Results: The chimeric substrates (P3-P10) were designed to substitute wild-type, species-specific amino acid residues at sites known to interact with ADAMTS13 D and C domain exosites. However, amino acid substitutions also occurred at the sites that do not interact directly with any ADAMTS13 exosite. Compared to C-71 (P2), only P3 and P6 exhibited reduced rates of cleavage by all vertebrate plasma containing ADAMTS13 (Fig 1B). The P3 substrate substituted the D binding residues from human VWF71 into C-71, whereas P6 substituted the C binding residues from human VWF71 into C-71. For P3, all ADAMTS13 assayed showed ~30-100% reduction in the rate of cleavage except for armadillo and dog plasma ADAMTS13. Likewise, P6 was cleaved ~0% to 60% slowly relative to P2 by all ADAMTS13. Substituting the D, nb-1(non-binding region 1), and nb-2 sequences from human into C-71, named P8, did not further reduce rates of cleavage compared to P3. These results suggest the nb-1 and nb-2 regions of VWF71 do not participate in protease engagement, consistent with previous studies. We wondered if swapping human VWF D- and C- binding sites to C-71 as well as the bovine's to H-71 would result in ADAMTS13 cleavage rates similar to wild-type sequences. Interestingly, there was no major change in cleavage rates when D- and C- VWF binding sites were of the same species, i.e., P9 ~ P1 and P10 ~ P2, irrespective of the peptide backbone. We also sought to know if allosteric regulation of ADAMTS13 attenuates the binding of these chimeric substrates to ADAMTS13. Pigeon (potently activated by low pH) and human plasma ADAMTS13 were activated ~15.0-28.0 and ~4.8-8.0-fold, respectively when assayed with all 10 peptides. Whether ADAMTS13 was activated or not, the ratio of cleavage for individual substrates to the parental P2 remained relatively constant (~ 0.4-1.3), implying the substrate-dependent activity is independent of allosteric activation of ADAMTS13. Conclusions: The fact that D- and C- VWF binding sites from human and bovine VWF71 cannot be combined without negatively affecting the Tyr-Met bond cleavage may suggest coupling of ADAMTS13 D and C domains to regulate its activity. It is plausible to claim that ADAMTS13 VWF D- and C- binding sites and/or ADAMTS13 D and C domains may have evolved to provide a fail-safe point for allosterically activated ADAMTS13. This may prevent substrate-activated ADAMTS13, which precedes the unraveling of the VWF domain A2, from engaging and cleaving off-targets. We conclude that VWF as a substrate serves both as an ADAMTS13 activator and activity regulator. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Ameliorative Effect of Quercetin against Modification Induced by 5-Fluorouracil on Alpha-2-Macroglobulin

Introduction: Alpha-2-macroglobulin (α2M) is large glycoprotein, found in the plasma of vertebrates and invertebrates, which plays a major role in regulation and transport. In this study, we report the interaction of 5-fluorouracil (5-FU) with α2M and the protective effect of quercetin against 5-FU induced modification of α2M. Methods: Various biochemical and biophysical methods and techniques were employed to determine the binding interaction between α2M-5-FU and the preventive effect of quercetin on 5-FU induced modification of α2M. Fluorescence microscopy was performed to analyze the formation of free radicals by 5-FU and to examine the scavenging effect of quercetin. Results: Our results of antiproteinase activity assay show that the 5-FU-α2M interaction causes functional inactivation of sheep α2M, while the 5-FU-α2M complex after exposure to quercetin regains its native conformation. Intrinsic fluorescence results suggest an increase of 5-FU quenching in the fluorescence intensity, and when the mixture was incubated with quercetin the α2M-FU-quercetin mixture showed the same intensity as that of native protein. The absorption spectra of 5-FU-α2M complex suggest the formation of a complex leading to increase in absorbance. CD and FTIR spectroscopy show 5-FU causes secondary structural alteration of α2M and quercetin provides a protective role against structural modification. Conclusion: Our study suggests that 5-FU produces reactive oxygen species (ROS) in the presence of light and compromises the integrity of α2M. Quercetin quenches the ROS formed by 5-FU and helps the protein to maintain its native conformation. We have demonstrated the protective effect of quercetin for the first time against the toxicity induced by 5-FU on α2M.

The role of uptake and degradation in the regulation of peripheral serotonin dynamics in Gulf toadfish, Opsanus beta

The neurotransmitter serotonin (5-hyroxytryptamine, 5-HT) is involved in a variety of peripheral processes. Arguably most notable is its role as a circulating vasoconstrictor in the plasma of vertebrates. Plasma 5-HT is maintained at constant levels under normal conditions through the processes of cellular uptake, degradation, and excretion, known collectively as clearance. However, the degree to which each individual component of clearance contributes to this whole animal response remains poorly understood. The goal of this experiment was to determine the extent to which transporter-mediated uptake and intracellular degradation contribute to 5-HT clearance in the model teleost Gulf toadfish (Opsanus beta). Fish that were treated with the 5-HT transport inhibitors fluoxetine, buproprion, and decynium-22 had 1.47-fold higher plasma 5-HT concentrations and a 40% decrease in clearance rate compared to control fish. In contrast, fish treated with the MAO inhibitor clorgyline had a 1.54-fold increase in plasma 5-HT with no change in clearance rate. The results show that transporter-mediated 5-HT uptake plays an important role in controlling circulating 5-HT and whole body 5-HT homeostasis.

Bilirubin binding affects the structure and function of alpha-2-macroglobulin

ABSTRACT Bilirubin is an endogenous antioxidant that is a metabolite of the heme in red blood cells (RBC). In blood, bilirubin is associated with albumin to form a water-soluble complex, known as unconjugated bilirubin. Alpha-2-macroglobulin (α2M) is a proteinase inhibitor found in the plasma of vertebrates. In the present study, we have investigated the interaction of photo-illuminated bilirubin with serum α2M using various biophysical and thermodynamic techniques. The binding of bilirubin to α2M leads to various functional and structural changes in α2M protein. The result of ultraviolet (UV) and fluorescence spectroscopy suggests that binding of bilirubin to α2M induces a conformational change in the secondary structure of protein which was corroborated by circular dichroism (CD) and Fourier-transform infrared spectroscopy (FT-IR). This binding leads to the conversion of β-sheet into α-helical conformation and subsequently loss in protein activity. The thermodynamic parameters of bilirubin-α2M binding indicate that the binding is exothermic, and the reaction spontaneous. Our studies show that binding of bilirubin with α2M in the presence of light induces structural and functional modifications in the protein. Bilirubin possesses multiple biological activities, including immunomodulatory property which has not been extensively explored and which may be of interest for further study.

Interaction between aspirin and vitamin C with human serum albumin as binary and ternary systems.

Foods generally contain special ingredients which easily to interact with drugs human intaking, thus affecting drug efficacy and excretion, and even cause adverse reactions. Vitamin C (Vit. C) is abundant in fresh fruits and vegetables. It plays a regulatory role in redox metabolism, and its absence can cause scurvy. Aspirin (ASP) can be used to treat many diseases, is the earliest, common and widely used as antipyretic, analgesic and antirheumatic medicine. Human serum albumin (HSA) is the most abundant protein in vertebrate plasma and has the property of combining and transporting endogenous and exogenous substances. In this paper, the effects of Vit. C on the combination of ASP and HSA were studied by multi-spectra and voltammetric approaches. Fluorescence spectra showed that the quenching mode between Vit. C and HSA is dynamic, and the main binding force is hydrophobic force. The quenching mode between ASP and HSA is static one, and the main binding force is hydrogen bond and van der Waals force. For ternary biological system of (HSA-ASP)-Vit. C, the binding constant decreases compared with HSA-Vit. C system. However, for (HSA-Vit. C)-ASP system, the binding constant does not change when compared with binary system of HSA-ASP. Based on the technology combination of voltammetry, infrared, three-dimensional fluorescence and circular dichroism (CD), it is proved that the existence of ASP will influence the binding process of Vit. C to HSA. It could be concluded that taking Vit. C first doesn't affect the absorption of ASP and may be good for health; in contrast, it is not good to take Vit. C immediately as one have just taken ASP, because the existence of ASP reduce the absorption of Vit. C for human body.

Quercetin-induced inactivation and conformational alterations of alpha-2-macroglobulin: multi-spectroscopic and calorimetric study

Quercetin is a widely used bioflavonoid found in onions, grapes, berries and citrus fruits. Under certain conditions, quercetin acts as a pro-oxidant thereby generating reactive oxygen species and promoting the oxidation of molecules. Our study investigates the effect of quercetin on the structure and function of alpha-2-macroglobulin (α2M) by employing various biophysical techniques and trypsin inhibitory assay. α2M is the major antiproteinase present in the plasma of vertebrates. Results of activity assay indicated that α2M loses its 56% of inhibitory activity on treatment with quercetin in the presence of light. UV spectroscopy reveals hyper chromaticity in absorption spectra of protein on interaction with quercetin suggesting structural change. The intrinsic fluorescence studies showed quenching of α2M spectra in the presence of quercetin, and the mode of quenching was found to be static in nature. Synchronous fluorescence indicated the alteration in the microenvironment of tryptophan residues. CD and FTIR spectroscopy confirms concentration-dependent alterations in secondary structure of α2M instigated by quercetin. The magnitude of binding constant, enthalpy change, entropy change and free energy change during the interaction process was determined by isothermal titration calorimetry. Hydrogen bonding and hydrophobic interaction were the main intermolecular forces involved during the process. This study identifies and signifies the damage induced by quercetin to α2M due to its pro-oxidant action. Communicated by Ramaswamy H. Sarma

Deciphering the binding of dutasteride with human alpha-2-macroglobulin: Molecular docking and calorimetric approach

Dutasteride is a pharmacologically important drug employed to treat prostate cancer. Alpha-2-macroglobulin (α2M) is the primary proteinase inhibitor and is abundant in vertebrate plasma. Previous studies have shown that α2M levels were down regulated in prostate cancer. Our results of functional assay shows 50% decrease in the antiproteolytic potential ofα2Mupon its interaction with dutasteride. Fluorescence quenching revealed that dutasteride binds with α2M via static mechanism, resulting in the formation of dutasteride-α2M complex. Synchronous fluorescence studies suggest alteration in the microenvironment around tryptophan residues. Changes in the UV–visible spectra hints at formation of complex between the drug and protein. Secondary structural perturbations in α2M are confirmed by circular dichroism studies. Molecular docking discloses the involvement of hydrogen bonding during the interaction process and suggests the site of interaction of dutasteride on α2M monomer as Asn173, Lys171, Asp1178, Lys1236, His1182, Lys1177, Ser1180 and Lys1240.Isothermal titration calorimetry affirms the binding process to be spontaneous and exothermic. The results of this study may potentially be important should it be shown that dutasteride interacts with α2M under physiological conditions.

The conserved metalloprotease invadolysin is present in invertebrate haemolymph and vertebrate blood.

ABSTRACTWe identified invadolysin, a novel essential metalloprotease, for functions in chromosome structure, cell proliferation and migration. Invadolysin also plays an important metabolic role in insulin signalling and is the only protease known to localise to lipid droplets, the main lipid storage organelle in the cell. In silico examination of the protein sequence of invadolysin predicts not only protease and lipase catalytic motifs, but also post-translational modifications and the secretion of invadolysin. Here we show that the protease motif of invadolysin is important for its role in lipid accumulation, but not in glycogen accumulation. The lipase motif does not appear to be functionally important for the accumulation of lipids or glycogen. Post-translational modifications likely contribute to modulating the level, localisation or activity of invadolysin. We identified a secreted form of invadolysin in the soluble fraction of invertebrate hemolymph (where we observe sexually dimorphic forms) and also vertebrate plasma, including in the extracellular vesicle fraction. Biochemical analysis for various post-translational modifications demonstrated that secreted invadolysin is both N- and O-glycosylated, but not apparently GPI-linked. The discovery of invadolysin in the extracellular milieu suggests a role for invadolysin in normal organismal physiology.

The evolution of apolipoprotein B and its mRNA editing complex. Does the lack of editing contribute to hypertriglyceridemia?

The evolution of apolipoprotein B (Apob) has been intensely researched due to its importance during lipid transport. Mammalian full-length apob100 can be post-transcriptionally edited by the enzyme apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like complex-one (Apobec1) resulting in a truncated Apob, known as Apob48. Whilst both full-length and truncated forms of Apob are important for normal lipid homeostasis in mammals, there is no evidence for the presence of apob mRNA editing prior to the divergence of the mammals, yet, non-mammalian vertebrates appear to function normally with only Apob100. To date, the majority of the research carried out in non-mammalian vertebrates has focused on chickens with only a very limited number examining apob mRNA editing in fish. This study focused on the molecular evolution of Apobec1 and Apob in order to ascertain if apob mRNA editing occurs in eels, a basal teleost which represents an evolutionarily important animal group. No evidence for the presence of Apobec1 or the ability for eel apob to be edited was found. However, an important link between mutant mice and the evident hypertriglyceridemia in the plasma of non-mammalian vertebrates was made. This study has provided imperative evidence to help bridge the evolutionary gap between fish and mammals and provides further support for the lack of apob mRNA editing in non-mammalian vertebrates.

Thermal performance and acclimatization of a component of snake (Agkistrodon piscivorus) innate immunity.

Complement-an immune protein cascade involved in pathogen lysis-was discovered as a temperature-labile component of vertebrate plasma, yet since that time the thermal performance of complement has not received much attention from a comparative or ecological perspective. We investigated two thermal hypotheses involving the complement system of the cottonmouth snake (Agkistrodon piscivorus). We tested whether complement performance would conform to optimal thermal reaction norms commonly observed in ectotherm ecophysiological studies, predicting that complement efficiency would be maximal at or near the cottonmouth's field body temperatures. We also tested thermal acclimatization of complement performance, by comparing temperature/performance curves from samples collected in three different seasons. Complement efficiency exhibited the same significant positive correlation with temperature in all three seasons. This seasonally invariable temperature-performance relationship may allow easy acquisition of behavioral fever, as well as trade-offs between immune performance and energy balance, ultimately endowing snakes with immunological flexibility not available to endotherms.

Dysregulated Expression of Glycolipids in Tumor Cells: From Negative Modulator of Anti-tumor Immunity to Promising Targets for Developing Therapeutic Agents

Glycolipids are complex molecules consisting of a ceramide lipid moiety linked to a glycan chain of variable length and structure. Among these are found the gangliosides, which are sialylated glycolipids ubiquitously distributed on the outer layer of vertebrate plasma membranes. Changes in the expression of certain species of gangliosides have been described to occur during cell proliferation, differentiation, and ontogenesis. However, the aberrant and elevated expression of gangliosides has been also observed in different types of cancer cells, thereby promoting tumor survival. Moreover, gangliosides are actively released from the membrane of tumor cells, having a strong impact on impairing anti-tumor immunity. Beyond the undesirable effects of gangliosides in cancer cells, a substantial number of cancer immunotherapies have been developed in recent years that have used gangliosides as the main target. This has resulted in successful immune cell- or antibody-responses against glycolipids, with promising results having been obtained in clinical trials. In this review, we provide a general overview on the metabolism of glycolipids, both in normal and tumor cells, as well as examining glycolipid-mediated immune modulation and the main successes achieved in immunotherapies using gangliosides as molecular targets.

Computational Analysis and Polymorphism study of Tumor Suppressor Candidate Gene-3 for Non Syndromic Autosomal Recessive Mental Retardation

Computational Analysis and Polymorphism study of Tumor Suppressor Candidate Gene-3 for Non Syndromic Autosomal Recessive Mental Retardation Mental Retardation (MR) is regarded as a neuronal malfunction characterized by a low Intellectual Quotient (IQ). To date, few genes (GRIK2, TUSC3, TRAPPC9, TECR, ST3GAL3, MED23, MAN1B1, NSUN1 PRSS12, CRBN, CC2D1A) for autosomal-recessive non syndromic MR (NS-ARMR) have been identified and established in various families with MR. The recently reported candidate gene “Tumor Suppressor Candidate Gene-3 (TUSC3)” was selected for computational analysis to explore its potential role in pathology as it is the only gene for MR reported in more than five different familial cases worldwide. TUSC3 gene located at chromosome 8p22 contains 11 exons and encodes a protein involved in the vertebrate plasma membrane magnesium ion transport system. Three dimensional structures of the candidate gene TUSC3 and its mutated structure with Q55X were generated. Template parameters were based on Z-score, E value, resolution and quality. Present study demonstrates the reliability of 3D model which satisfies the overall quality of the model constructed, employing both physiochemical and statistical model evaluation. Previously a homozygous 163C-T transition in exon 2 of the TUSC3 gene, resulting in a gln55-to-ter (Q55X) substitution has been reported. And here we predicted by PHD-SNP that X could be either proline (P) or Aspartic acid (D) causing deleterious effect. As mutation at 55 position was responsible for non-syndromic autosomal recessive mental retardation, so it was assumed that mutation was either by proline or aspartic acid. Further, MUPRO, PROVEAN, I-Mutant, Predict SNP, Meta SNP, Panther, SIFT, SNPs 3D confirmed that deleterious mutation was Q55P transition... .

An Adaptable Spectrin/Ankyrin-Based Mechanism for Long-Range Organization of Plasma Membranes in Vertebrate Tissues.

Ankyrins are membrane-associated proteins that together with their spectrin partners are responsible for micron-scale organization of vertebrate plasma membranes, including those of erythrocytes, excitable membranes of neurons and heart, lateral membrane domains of columnar epithelial cells, and striated muscle. Ankyrins coordinate functionally related membrane transporters and cell adhesion proteins (15 protein families identified so far) within plasma membrane compartments through independently evolved interactions of intrinsically disordered sequences with a highly conserved peptide-binding groove formed by the ANK repeat solenoid. Ankyrins are coupled to spectrins, which are elongated organelle-sized proteins that form mechanically resilient arrays through cross-linking by specialized actin filaments. In addition to protein interactions, cellular targeting and assembly of spectrin/ankyrin domains also critically depend on palmitoylation of ankyrin-G by aspartate-histidine-histidine-cysteine 5/8 palmitoyltransferases, as well as interaction of beta-2 spectrin with phosphoinositide lipids. These lipid-dependent spectrin/ankyrin domains are not static but are locally dynamic and determine membrane identity through opposing endocytosis of bulk lipids as well as specific proteins. A partnership between spectrin, ankyrin, and cell adhesion molecules first emerged in bilaterians over 500 million years ago. Ankyrin and spectrin may have been recruited to plasma membranes from more ancient roles in organelle transport. The basic bilaterian spectrin-ankyrin toolkit markedly expanded in vertebrates through gene duplications combined with variation in unstructured intramolecular regulatory sequences as well as independent evolution of ankyrin-binding activity by ion transporters involved in action potentials and calcium homeostasis. In addition, giant vertebrate ankyrins with specialized roles in axons acquired new coding sequences by exon shuffling. We speculate that early axon initial segments and epithelial lateral membranes initially were based on spectrin-ankyrin-cell adhesion molecule assemblies and subsequently served as "incubators," where ion transporters independently acquired ankyrin-binding activity through positive selection.

Purification, characterization and molecular cloning of alpha-2-macroglobulin in cobia, Rachycentron canadum

Alpha-2-macroglobulin (α-2-M) is a broad spectrum protease inhibitor which is abundant in the plasma of vertebrates and several invertebrates. The α-2-M was purified from cobia (Rachycentron canadum) plasma by a four-step procedure: poly ethylene glycol fractionation, affinity chromatography, hydrophobic interaction chromatography and ion exchange chromatography on Fast Protein liquid chromatography system in the present study. It migrated as one protein band with a molecular mass of about 360 kDa in the native state, whereas in SDS-PAGE it was about 180 kDa under non-reducing condition. This result revealed that the native protein was a dimer. In addition, it was cleaved into two different fragments of molecular mass about 93 and 87 kDa when reduced by dithiothreitol (DTT). The anti-protease activity of the purified α-2-M was apparently decreased as temperature elevated above 50 °C. The α-2-M exhibited highest protease inhibitory activity at pH 9. The results indicate that the α-2-M is a heat-labile and alkaline protease inhibitor. The purified α-2-M exhibited more than 50％ protease inhibitory activity against extracellular products (ECP) of Vibrio alginolytius isolated from diseased cobia. It seems that the protease activities in ECP may be affected by the plasma α-2-M. The protease inhibitory activities of cobia plasma or purified α-2-M were decreased when incubated with 10 mM methylamine for 30 min.The α-2-M cDNA consisted of 4611 bp with an open reading frame of 4374 bp had been cloned from cobia liver. This sequence contained thioester domain (GCGEQ) and thirteen predicted N-linked glycosylation sites. In addition, the amino acid sequence of thioester domain and genes of adjacent regions of cobia α-2-M were further compared with sequences of known fish species in GenBank. The unweighted pair group method using arithmetic average (UPGMA) was employed to construct the phylogenetic trees of α-2-M among different fish species (freshwater fish, sea water fish and primitive fish), and all these fish species were then clustered into three groups. The cobia α-2-M was closer to that of sea water fish than that of freshwater fish compared basing on its similarity of amino acid sequence and phylogenetic analysis of the partial gene.

Capability of ganglioside GM1 in modulating interactions, structure, location and dynamics of peptides/proteins: biophysical approaches: interaction of ganglioside GM1 with peptides/proteins.

Gangliosides, are glycosphingolipids, present in all vertebrate plasma membranes with particular abundance in nerve cell membrane. Gangliosides can act as portals for antimicrobial peptides, hormones, viruses, lectins, toxins and pathogens. They are strategically positioned on the outer membrane and hence can participate in a large number of recognition processes. Their abundance in nerve cell membrane makes them "likely" receptor candidates for neuropeptides. In this review we outline our work in the area of GM1-peptide/protein interaction. We have explored the effect of GM1 containing micelles/bicelles on structures of peptides, proteins as well as on denatured proteins. It has been observed that the peptides that are disordered or having random coil structure in aqueous solution, attained an ordered three-dimensional structure when interact with GM1. It is also observed that denatured proteins undergo refolding in presence of ganglioside. Peptides/proteins show stronger interaction with membrane lipid bilayer in presence of ganglioside than that without ganglioside. This review mainly focuses on capability of ganglioside GM1 in modulating interaction, structural, location and dynamics of peptides/proteins using a number of biophysical techniques-solution NMR, DOSY, CD, fluorescence etc.

Characterization of alpha-2-macroglobulin from groupers

Alpha-2-macroglobulin (α-2-M) is a protease inhibitor broadly present in the plasma of vertebrates and invertebrates, and is an important non-specific humoral factor in defence system of the animals. This study conducted the immuno-analysis and mass spectrometric analysis methods to investigate the characteristics of the protease inhibitor, α-2-M, among groupers and related species. Rabbit antiserum to the purified α-2-M of Epinephelus coioides was used in different immunological methods to determine the immune cross-reactions of the α-2-M in samples. Plasma of Epinephelus bruneus, Epinephelus fuscoguttatus, Epinephelus lanceolatus, and Epinephelus quoyanus exhibited high protease inhibitory activities by BAPNA-trypsin assay. To purify the α-2-M protein, plasma protein of grouper E. coioides was first precipitated by using PEG 6000, then Blue Sepharose 6 Fast Flow, DEAE Sephacel, Con A Separose 4B and Phenyl Sepharose High Performance columns were used on FPLC system for purification. The molecular mass of grouper plasma α-2-M was determined as a 180 kDa protein on non-reduced SDS-PAGE. In addition, it was determined as 97 and 80 kDa protein on reduced SDS-PAGE. Enzymatic and chemical deglycosylation of glycogen revealed that the contents of glycogen in 97 and 80 kDa subunits were 12.4% and 15%, respectively, and were all belonging to N-linked type.Only one precipitation arc was visualized in all plasma of Epinephelus spp. using the rabbit antiserum to the purified α-2-M of E. coioides, on crossed immunoelectrophoresis (CIE) gels. The plasma of Epinephelus spp. and seawater fish species showed stronger responses than freshwater fish species while that of other animal species showed no response by dot-blot assay. One single band was detected on Native PAGE-Western blotting assay, one single 180 kDa band was detected on non-reduced SDS-PAGE-Western blotting, and four bands (80, 97, 160, 250 kDa) were detected on reduced SDS-PAGE when various grouper plasma was performed respectivity. However, no band was detected using plasma from the freshwater fish species and other animal species. Thus, further indicates that the protein structure of α-2-M of Epinephelus spp. was closely related among seawater fish species. In addition the identity of the two subunits was identified using LC/MS/MS which was similar to α-2-M of grass carp (Ctenopharyngodon idella) and bluegill sunfish (Lepomis macrochirus) on the protein hit.

The exon 29 c.3535A>T in the alpha-2-macroglobulin gene causing aberrant splice variants is associated with mastitis in dairy cattle

Alpha-2-macroglobulin (A2M) binds proteases, thereby acting as defense barriers against pathogens in the plasma and tissues of vertebrates and invertebrates. Quantitative real-time polymerase chain reaction (PCR) and the isobaric tags for relative and absolute quantitation method were used to determine the expression levels of A2M mRNA and proteins in mastitis-infected mammary tissues. A2M mRNA and protein expression were significantly higher in mastitis-infected mammary tissues than those in healthy tissues. We also identified 23 novel A2M splice variants in the bovine mammary tissues using reverse transcription PCR combined with clone sequencing. These splice variants predominantly affected the bait region, the inhibitory region, and the thioester region of the protein, which have the functional key roles in inhibiting the proteases of pathogens. Genomic sequencing analysis revealed a nonsynonymous c.3535A>T single-nucleotide polymorphism (SNP) in exon 29, which is located within a putative exonic splice enhancer and may be the reason why the A2M gene produces the aberrant splice variant A2M-AS4. Our findings suggest that the A2M gene can play its role by alternative splicing mechanism and it may be of significance against mastitis. This study provides clues to better understand the function of the bovine A2M gene and the effects of the exonic SNP on the production of aberrant splice variants.

Evidence that muscle cells do not express the histidine-rich glycoprotein associated with AMP deaminase but can internalise the plasma protein

Histidine-rich glycoprotein (HRG) is synthesized by liver and is present at relatively high concentration in the plasma of vertebrates. We have previously described the association of a HRG-like molecule to purified rabbit skeletal muscle AMP deaminase (AMPD). We also provided the first evidence for the presence of a HRG-like protein in human skeletal muscle where a positive correlation between HRG content and total determined AMPD activity has been shown. In the present paper we investigate the origin of skeletal muscle HRG. The screening of a human skeletal muscle cDNA expression library using an anti-HRG antibody failed to reveal any positive clone. The RT-PCR analysis, performed on human skeletal muscle RNA as well as on RNA from the rhabdomyosarcoma (RD) cell line, failed to show any mRNA specific for the plasma HRG or for the putative muscle variant. When the RD cells were incubated with human plasma HRG, a time-dependent increase of the HRG immunoreactivity was detected both at the plasma membrane level and intracellularly. The internalisation of HRG was inhibited by the addition of heparin. The above data strongly suggest that skeletal muscle cells do not synthesize the muscle variant of HRG but instead can actively internalise it from plasma.

Hepatic α-Tocopherol Transfer Protein: Ligand-Induced Protection from Proteasomal Degradation

There are eight naturally occurring forms of the dietary antioxidant vitamin E. Of these, only α-tocopherol is retained at high levels in vertebrate plasma and tissues. This selectivity is achieved in part by the action of the hepatic α-tocopherol transfer protein (TTP), which facilitates the selective incorporation of dietary α-tocopherol into circulating lipoproteins. We examined the effects of vitamin E on TTP expression in cultured hepatocytes. Treatment with vitamin E precipitated a time- and dose-dependent increase in the steady-state levels of TTP. This stabilization was caused by α-tocopherol-induced attenuation of the ubiquitination of TTP and its subsequent degradation by the proteasome. In vitro, vitamin E protected TTP from proteolytic degradation by trypsin, suggesting ligand-induced changes in protein conformation. Cell fractionation studies showed that TTP is distributed between the cytosolic and membranous organelle fraction, and that tocopherol induced the translocation of some TTP from the cytosol to the organelle fraction. Furthermore, vitamin E markedly attenuated the degradation of organelle-bound TTP. These findings suggest that vitamin E imparts a distinct conformation on TTP that is associated with localization to a specific cellular compartment, where the protein is less susceptible to proteasomal degradation.