Anionic Proteins Research Articles

A newly identified secretory phospholipase A2 group XIIA homolog (LcPLA2XIIA) in Larimichthys crocea exhibits antimicrobial and antitumor activities

The phospholipase A2 (PLA2) superfamily has attracted increasing attention in recent years due to the multiple physiological and pathological functions exerted by its members. Up to date, the knowledge about the biological role of PLA2XIIA subfamily members remains limited. In this study, a new member of PLA2XIIA subfamily, LcPLA2XIIA, was characterized in large yellow croaker. Different from most members of the PLA2 superfamily with positive charge, LcPLA2XIIA encodes an anionic protein, which is similar to other members of PLA2XIIA subfamily. LcPLA2XIIA is highly expressed in the intestine, and afterwards, it is up-regulated after with Pseudomonas plecoglossicida or Staphylococcus aureus. LcPLA2XIIA exhibits strong inhibitory activity against these two bacteria. The results indicate that LcPLA2XIIA plays an important role in the antimicrobial immune responses of large yellow croaker. LcPLA2XIIA displays strong binding activity to all the tested bacteria. It specifically interacts with LTA, a unique component on the surface of Gram-positive bacteria. It also significantly promotes bacterial agglutination in the presence of Ca2+. These findings reveal that the binding and agglutinating abilities of LcPLA2XIIA to bacteria contribute greatly to its antibacterial activity. In addition, LcPLA2XIIA significantly inhibits the proliferation of infectious hematopoietic necrosis virus instead of recombinant human adenovirus type 5. It also suppresses the growth of human colorectal adenocarcinoma cells by inducing apoptosis, but it has no obvious inhibitory effect on the growth of epithelioma papulosum cyprinid cells. This study provides new insights into the antibacterial activity, and the mechanism of LcPLA2XIIA in large yellow croaker, and antiviral and antitumor functions of PLA2XIIA subfamily members.

Photoresponsive protamine ionic complex towards a smart hemostatic biomaterial

Protamine (PA) is the only licensed antidote for reversing heparin anticoagulation by electrostatically binding with heparin. Efforts have been made on designing various heparin-scavengers, while, it remains a great challenge for gaining the external-stimuli responsive PA-release material. In this study, a generic strategy is developed for fabricating photoresponsive protein materials with the designed azobenzene-containing surfactant. For the first time, based on the isomerization of azobenzene, both cationic and anionic proteins could be phase change biomaterials which are capable of transiting to isotropic state under UV irradiation at room temperature. The formation of isotropic state could set the proteins free from the binding state, activating their intrinsic biological functions. Employing this mechanism, one smart PA material for inhibiting heparin is developed, which could effectively photo-modulate the heparin concentration by turning on-and-off the free state of PA from the binding state. With good biocompatibility, the PA material addresses photoresponsive hemostatic activity in biological studies, confirming its great potential clinical values. This work provides a new designing strategy for gaining photocontrollable hemostasis materials, also opening new opportunities for developing photoresponsive protein drugs and biomedical materials.

Multistep Array-Based Sensing of Bioanalytes Using Modified MoS2, Fluorescence Proteins, and Cucurbituril.

One pot sensor by multiplexing in the array is an attractive system for rapid discrimination of multiple analytes. Multiplexing can be achieved in two ways, i.e., using multiple signal transducers or adding sequential agents to the sensor media. Herein, we have used a combination of both multichannel and sequential ON-OFF strategies for the discrimination of different bioanalytes. The sensor array was constructed by implementing positively charged MoS2 as a receptor and different fluorescent proteins possessing distinguishable emission profiles as signal transducers. The sensing setup was constructed with the interaction between oppositely charged MoS2 and the host-guest combination between a cationic headgroup of MoS2 and Cucurbit [7] uril (CB7) to alter the fluorescence of signal transducers in situ noncovalently. Electrodynamic analysis and optical assays suggest that the electrostatic interaction played a major role in the modulation of the fluorescence outcomes in the array. Both cationic and anionic proteins were discriminated at a 50 nM concentration. The detection limit of the sensor array by using β-gal protein was found to be 1 nM. The sensor array was further implemented for the discrimination of normal and diseased cell lines and lysates, which indicates the versatile detection ability of this reported sensor array.

Chitosan in biomedicine: A comprehensive review of recent developments

A naturally occurring biopolymer, chitosan has found widespread application in the fields of biomedical research and healthcare enhancement. Through a thorough analysis of recent research findings and technological advancements, this review seeks to provide a comprehensive overview of the latest developments and potential uses of chitosan in the field of biomedicine. Microbe cells' anionic lipopolysaccharides and surface proteins interact with the polycationic composition, releasing intracellular components and resulting in cell death. The degree of antimicrobial activity is highly dependent on the particular type of microbe being targeted. Chitosan is highly susceptible to enzymatic hydrolytic breakdown pathways in the human body, especially when lysozymes are involved. Moreover, adding chitosan derivatives to mice's diet improved their health by increasing their enzymatic activity, which is related to its scavenging properties and shows anti-microbial activity. Furthermore, the assessment of sutures manufactured from this substance emphasizes their noteworthy input to the field of modern wound care.

Biomimetic nanoplatforms constructed from dialkylaminostyryl hetarene dyes and phospholipids exhibiting selective fluorescent response to specific proteins

The present work explores the specificity of supramolecular assemblies comprising dialkylaminostyrylhetarene dye molecules incorporated into phosphatidylcholine (PC) or phosphatidylserine (PS) aggregates. In PS-based assemblies, the dyes demonstrate a concentration-dependent fluorescent response, distinguishing anionic proteins such as bovine serum albumin (BSA) and pepsin from lysozyme (LYZ) in aqueous solutions. Conversely, no significant response is observed when the dyes are incorporated into the well-organized bilayers of neutral PC. The fluorescent response arises from the binding of dyes to proteins, leading to the detachment of dye molecules from the assemblies, rather than from the binding of proteins to the assemblies, although the latter process is facilitated by electrostatic attraction. Thus, both the poor ordering of PS molecules and the interfacial arrangement of the dyes are prerequisites for the fluorescent response of dye-PS aggregates. The structure of the dyes significantly impacts the spectral features of dye-PS and dye-protein assemblies. An optimal dye structure has been identified for the recognition of BSA, with a limit of detection (LOD) of 10.8 nM.

Engineering Biomimetic Protein Camouflage for Delivering Peptide/siRNA Nanocomplexes.

For cationic nanoparticles, the spontaneous nanoparticle-protein corona formation and aggregation in biofluids can trigger unexpected biological reactions. Herein, we present a biomimetic strategy for camouflaging the cationic peptide/siRNA nanocomplex (P/Si) with single or dual proteins, which exploits the unique properties of endogenous proteins and stabilizes the cationic P/Si complex for safe and targeted delivery. An in-depth study of the P/Si protein corona (P/Si-PC) formation and protein binding was conducted. The results provided insights into the biochemical and toxicological properties of cationic nanocomplexes and the rationales for engineering biomimetic protein camouflages. Based on this, the human serum albumin (HSA) and apolipoprotein AI (Apo-AI) ranked within the top 20 abundant protein species of P/Si-PC were selected to construct biomimetic HSA-dressed P/Si (P/Si@HSA) and dual protein (HSA and Apo-AI)-dressed P/Si (P/Si@HSA_Apo), given that the dual-protein camouflage plays complementary roles in efficient delivery. A branched cationic peptide (b-HKR) was tailored for siRNA delivery, and their nanocomplexes, including the cationic P/Si and biomimetic protein-dressed P/Si, were produced by a precise microfluidic technology. The biomimetic anionic protein camouflage greatly enhanced P/Si biostability and biocompatibility, which offers a reliable strategy for overcoming the limitation of applying cationic nanoparticles in biofluids and systemic delivery.

Protein analysis using capillary electrophoresis coupled to mass spectrometry through vibrating sharp-edge spray ionization.

Capillary electrophoresis (CE) interfaced to mass spectrometry (MS) with electrospray ionization typically incorporates acidic additives or organic solvents to assist in ionization. Vibrating sharp-edge spray ionization (VSSI) is a voltage-free method to interface CE and MS that does not require these additives, making it appealing for protein analyses. CE-VSSI nanoflow sheath separations are performed with low ionic strength aqueous solutions in the sheath to reduce suppression. Serine is also included in the sheath to reduce analyte adduction. Proteins are detected in the 2.5-10µM range, corresponding to an injected mass range of 0.1-1.2ng. The anionic proteins β-lactoglobulin and transferrin are resolved using an unmodified fused silica capillary because they do not exhibit nonspecific surface adsorption. Conversely, separations of cationic proteins cytochrome c, ribonuclease A, and α-chymotrypsinogen A in an unmodified capillary require acidic background electrolytes to overcome adsorption. Alternatively, a semipermanent coating comprised self-assembled lipids overcomes surface adsorption at a neutral pH. Separations with zwitterionic and hybrid cationic coatings are complete within 15 or 6min, respectively. The dimeric form of triosephosphate isomerase was observed at a 60µM, corresponding to a mass of 19ng, by dropping the temperature of the MS inlet.

Chemical control of excited-state reactivity of the anionic green fluorescent protein chromophore.

Controlling excited-state reactivity is a long-standing challenge in photochemistry, as a desired pathway may be inaccessible or compete with other unwanted channels. An important example is internal conversion of the anionic green fluorescent protein (GFP) chromophore where non-selective progress along two competing torsional modes (P: phenolate and I: imidazolinone) impairs and enables Z-to-E photoisomerization, respectively. Developing strategies to promote photoisomerization could drive new areas of applications of GFP-like proteins. Motivated by the charge-transfer dichotomy of the torsional modes, we explore chemical substitution on the P-ring of the chromophore as a way to control excited-state pathways and improve photoisomerization. As demonstrated by methoxylation, selective P-twisting appears difficult to achieve because the electron-donating potential effects of the substituents are counteracted by inertial effects that directly retard the motion. Conversely, these effects act in concert to promote I-twisting when introducing electron-withdrawing groups. Specifically, 2,3,5-trifluorination leads to both pathway selectivity and a more direct approach to the I-twisted intersection which, in turn, doubles the photoisomerization quantum yield. Our results suggest P-ring engineering as an effective approach to boost photoisomerization of the anionic GFP chromophore.

The role of solvation on the conformational landscape of α-synuclein.

Native ion mobility mass spectrometry has been used extensively to characterize ensembles of intrinsically disordered protein (IDP) conformers, but the extent to which the gaseous measurements provide realistic pictures of the solution conformations for such flexible proteins remains unclear. Therefore, we systematically studied the relationship between the solution and gaseous structural ensembles by measuring electrospray charge state and collision cross section (CCS) distributions for cationic and anionic forms of α-synuclein (αSN), an anionic protein in solution, as well as directly probed gas phase residue to residue distances via ion/ion reactions between gaseous α-synuclein cations and disulfonic acid linkers that form strong electrostatic bonds. We also combined results from in-solution protein crosslinking identified from native tandem mass spectrometry (MS/MS) with an initial αSN ensemble generated computationally by IDPConformerGenerator to generate an experimentally restrained solution ensemble of αSN. CCS distributions were directly calculated for the solution ensembles determined by NMR and compared to predicted gaseous conformers. While charge state and collision cross section distributions are useful for qualitatively describing the relative structural dynamics of proteins and major conformational changes induced by changes to solution states, the predicted and measured gas phase conformers include subpopulations that are significantly different than those expected from completely "freezing" solution conformations and preserving them in the gas phase. However, insights were gained on the various roles of solvent in stabilizing various conformers for extremely dynamic proteins like α-synuclein.

Biochemical and Biophysical Characterization of Tau and α-Linolenic Acid Vesicles In Vitro.

Alzheimer's disease (AD) is characterized by the abnormal accumulation of disordered protein, that is, extracellular senile plaques of amyloid-β (Aβ) and intracellular neurofibrillary tangles of Tau. Tau protein has gained the attention in recent years owing to the ability to propagate in a "prion-like" nature. The disordered protein Tau possesses a high positive charge, which allows its binding to anionic proteins and factors. The native disorder of proteins attends the β-sheet structure from its random-coiled conformation upon charge compensation by various polyanionic agents such as heparin, RNA, etc. Anionic lipids such as arachidonic acid (AA) and oleic acid (OA) are also one of the factors which can induce aggregation of Tau in physiological conditions. The free units of Tau protein can bind to lipid membranes through its repeat domain (RD), the anionic side chains of the membrane lipids induce aggregation of Tau by reducing the activation barrier. In this study, we investigated the role of α-linolenic acid (ALA) as an inducing agent for Tau aggregation in vitro conditions. Omega-3 fatty acids bear a capacity to reduce the pathology of Tau by downregulating the Tau phosphorylation pathway. We have studied by using various biochemical or biophysical methods the potency of ALA as an aggregating agent for Tau. We have implemented different techniques such as SDS-PAGE, transmission electron microscopy, CD spectroscopy to evaluated higher-order aggregates of Tau upon induction by ALA.

Isoelectric point (pI)-based phase separation (pI-BPS) purification of elastin-like polypeptides (ELPs) containing charged, biologically active fusion proteins (ELP-FPs).

Elastin-like polypeptides (ELPs) are peptide-based biomaterials with residue sequence (VPGXG)n where X is any residue except proline. ELPs are a useful modality for delivering biologically active proteins (growth factors, protease inhibitors, anti-inflammatory peptides, etc.) as fusion proteins (ELP-FP). ELP-FPs are particularly cost-effective because they can be rapidly purified using Inverse Temperature Cycling (ITC) via the reversible formation and precipitation of entropically driven aggregates above a transition temperature (Tt ). When ELP fusion proteins (ELP-FPs) contain significant charge density at physiological pH, electrostatic repulsion between them severely inhibits aggregate formation. The literature does not currently describe methods for purifying ELP-FPs containing charged proteins on either side of the ELP sequence as fusion partners without organic solvents. Here, the isoelectric point (pI) of ELP-FPs is discussed as a means of neutralizing surface charges on ELP-FPs and increasing ITC yield to dramatically high levels. We use pI-based phase separation (pI-BPS) to purify ELP-FPs containing cationic and anionic fusion proteins. We report a dramatic increase in protein yield when using pI-BPS for purification of ELP-FPs. Proteins purified by this method also retain the functional activity of the protein present in the ELP-FP. Techniques developed here enable significant diversification of possible fusion proteins delivered by ELPs as ELP-FPs by allowing them to be produced and purified at higher quantities and yields.

Genome-wide identification and expression analysis of SLAC1 gene family in tomato (Solanum lycopersicum) and the function of SlSLAC1–6 under cold stress

The effects of cold stress on plants are mainly reflected in enzyme activity, membrane system, cell water loss, etc., leading to cell metabolism disorder, and even death. The SLAC1 gene family encodes S-type anionic channel proteins, which play an essential role in plant response to environmental stimuli by regulating stomatal opening and closure of guard cells. Therefore, the tomato SLAC1 gene family was used as the object of this study to explore its function and mechanism in tomato cold resistance. Firstly, bioinformatics analysis showed that most of the promoter regions of the SLAC1 gene family members contained stress-related cis-acting elements. Transcriptomic data and qRT-PCR experiments result show that SlSLAC1–6 might be a key gene in the resistance of the family. After cold treatment, the increase of antioxidant enzymes in SLAC1–6 silenced plants resulted in the inability of plants to maintain normal osmotic pressure of cells and significantly reduced the cold tolerance of tomato plants compared with the control (pTRV2:00) tomato plants. In addition, the stomatal aperture of the SLAC1–6 silenced plant was significantly more extensive than that of the pTRV2:00 tomato plants. The results showed that SlSLAC1–6 was a positive regulator of tomato cold resistance. In conclusion, our study provides a more extensive analysis of the tomato SLAC1 gene family and will provide a reference for future research on the mechanism of the SlSLAC1–6 gene in tomato cold resistance.

Protein Release by Controlled Desorption from Transiently Cationic Nanoparticles.

Therapeutic release from hydrogels is traditionally controlled by encapsulation within nanoparticles; however, this strategy is limited for the release of proteins due to poor efficiency and denaturation. To overcome this problem, we designed an encapsulation-free release platform where negatively charged proteins are adsorbed to the exterior of transiently cationic nanoparticles, thus allowing the nanoparticles to be formulated separately from the proteins. Release is then governed by the change in nanoparticle surface charge from positive to neutral. To achieve this, we synthesized eight zwitterionic poly(lactide-block-carboxybetaine) copolymer derivatives and formulated them into nanoparticles with differing surface chemistry. The nanoparticles were colloidally stable and lost positive charge at rates dependent on the hydrolytic stability of their surface ester groups. The nanoparticles (NPs) were dispersed in a physically cross-linked hyaluronan-based hydrogel with one of three negatively charged proteins (transferrin, panitumumab, or granulocyte-macrophage colony-stimulating factor) to assess their ability to control release. For all three proteins, dispersing NPs within the gels resulted in significant attenuation of release, with the extent modulated by the hydrolytic stability of the surface groups. Release was rapid from fast-hydrolyzing ester groups, reduced with slow-hydrolyzing bulky ester groups, and very slow with nonhydrolyzing amide groups. When positively charged lysozyme was loaded into the nanocomposite gel, there was no significant attenuation of release compared to gel alone. These data demonstrate that electrostatic interactions between the protein and NP are the primary driver of protein release from the hydrogel. All released proteins retained bioactivity as determined with in vitro cell assays. This release strategy shows tremendous versatility and provides a promising new platform for controlled release of anionic protein therapeutics.

Molecular insights into alginate β-lactoglobulin A multivalencies-The foundation for their amorphous aggregates and coacervation.

For improved control of biomaterial property design, a better understanding of complex coacervation involving anionic polysaccharides and proteins is needed. Here, we address the initial steps in condensate formation of β-lactoglobulin A (β-LgA) with nine defined alginate oligosaccharides (AOSs) and describe their multivalent interactions in structural detail. Binding of AOSs containing four, five, or six uronic acid residues (UARs), either all mannuronate (M), all guluronate (G), or alternating M and G embodying the block structural components of alginates, was characterized by isothermal titration calorimetry, nuclear magnetic resonance spectroscopy (NMR), and molecular docking. β-LgA was highly multivalent exhibiting binding stoichiometries decreasing from five to two AOSs with increasing degree of polymerization (DP) and similar affinities in the mid micromolar range. The different AOS binding sites on β-LgA were identified by NMR chemical shift perturbation analyses and showed diverse compositions of charged, polar and hydrophobic residues. Distinct sites for the shorter AOSs merged to accommodate longer AOSs. The AOSs bound dynamically to β-LgA, as concluded from saturation transfer difference and 1 H-ligand-targeted NMR analyses. Molecular docking using Glide within the Schrödinger suite 2016-1 revealed the orientation of AOSs to only vary slightly at the preferred β-LgA binding site resulting in similar XP glide scores. The multivalency coupled with highly dynamic AOS binding with lack of confined conformations in the β-LgA complexes may help explain the first steps toward disordered β-LgA alginate coacervate structures.

Development of emulsion-based edible inks for 3D printing applications: Pickering emulsion gels

Emulsions gels can be used as edible inks for 3D food printing applications because of their tunable composition and viscoelastic properties. Oil-in-water emulsion gels can be created by crosslinking the oil droplets using chemical or physical approaches. For food applications, chemical crosslinking agents are undesirable because they have safety concerns. In this study, Pickering emulsions gels (PEGs) were therefore fabricated by physically crosslinking anionic protein nanoparticle-coated oil droplets with cationic chitosan. Chitosan formed electrostatic bridges between neighboring oil droplets, as well as a network structure in the surrounding aqueous phase. The viscoelastic properties of PEGs could be tuned by adjusting the droplets and chitosan concentrations. These soft materials exhibited physicochemical attributes that made them highly suitable as edible inks for 3D printing applications, including the ability to maintain specific shapes, to form smooth surfaces, and to give a high print fidelity. Moreover, the tribological behavior of these PEGs was similar to that of oil in the mixed and hydrodynamic regimes, indicating that they exhibited good lubrication properties. Overall, this study has demonstrated the suitability of PEGs to be used as edible inks for creating novel food materials by additive manufacturing.

Protein-Templated Core/Shell Au Nanostructures for Intracellular Reactive Oxygen Species Detection by SERS

Core/shell gold “raspberry” nanostructures capable of multiple therapeutic functionalities were synthesized using a template composed of monodispersed anionic protein (bovine serum albumin) nanoparticles coated with a cationic biopolymer (poly-l-lysine). The nanostructures exhibited high photothermal conversion efficiency when exposed to a near-infrared (NIR) laser, which led to significant cellular inhibition of A549 human lung cancer cells due to intracellular hyperthermia. The raspberry structures also provided hot spots for surface-enhanced Raman scattering (SERS) ratiometric sensing of intracellular reactive oxygen species (ROS) when modified with the Raman reporter molecule 4-aminothiophenol (4-ATP). ROS was detected in A549 lung cancer cells upon photothermal heating of internalized nanostructures, enabling a possible mechanism for feedback on therapeutic efficacy. This was confirmed by adding the antioxidant N-acetylcysteine (NAC) and using a complementary fluorescence technique, which showed that the amount of detectable intracellular ROS decreased. These safe-by-design gold raspberry nanostructures could be promising for simultaneous therapeutic applications and monitoring therapeutic efficacy.

Purification of RgpA from external outer membrane vesicles of Porphyromonas gingivalis

IntroductionPurification of native gingipains is challenging because these proteases are frequently associated with the cell surface, which affects yield. This study aimed to purify native Arg-gingipain (RgpA) from Porphyromonas gingivalis Outer Membrane Vesicles (OMV). MethodsNative RgpA was purified from P. gingivalis strain ATCC33277 OMV using a strategy including ultracentrifugation, sonication, and successive anionic and cationic fast protein liquid chromatography (FPLC). The presence and purity of the protease were confirmed by SDS-PAGE and detection of protease activity using fluorogenic substrates. Rat antibodies produced against the unique adhesin hemagglutinin (H1) domain of RgpA (amino acids 719–865) were titrated by ELISA at a 1:100 dilution using whole P. gingivalis lysate as an antigen and western blotting to detect a 75 kDa band corresponding to RgpA. ResultsDouble anionic-cationic FLPC yielded prominent peaks with evident amidolytic gingipain activity of the appropriate molecular weight, as confirmed by western blotting. The final RgpA yield from 1 L of bacterial culture with colony forming unit (CFU) (Log10) 7.4 ± 0.08/mL was of 12.6% (2 mg/mL), with 3.2 FU/μg of amidolytic activity. ConclusionsThis protocol allows purification of native RgpA from OMV that retains protease activity.

Different Influences of Biotinylation and PEGylation on Cationic and Anionic Proteins for Spheroid Penetration and Intracellular Uptake to Cancer Cells.

To better understand the effects of PEGylation and biotinylation on the delivery efficiency of proteins, the cationic protein lysozyme (LZ) and anionic protein bovine serum albumin (BSA) were chemically conjugated with poly(ethylene glycol) (PEG) and biotin-PEG to primary amine groups of proteins using N-hydroxysuccinimide reactions. Four types of protein conjugates were successfully prepared: PEGylated LZ (PEG-LZ), PEGylated BSA (PEG-BSA), biotin-PEG-conjugated LZ (Bio-PEG-LZ), and biotin-PEG-conjugated BSA (Bio-PEG-BSA). PEG-LZ and Bio-PEG-LZ exhibited a lower intracellular uptake than that of LZ in A549 human lung cancer cells (in a two-dimensional culture). However, Bio-PEG-BSA showed significantly improved intracellular delivery as compared to that of PEG-BSA and BSA, probably because of favorable interactions with cells via biotin receptors. For A549/fibroblast coculture spheroids, PEG-LZ and PEG-BSA exhibited significantly decreased tissue penetration as compared with that of unmodified proteins. However, Bio-PEG-BSA showed tissue penetration comparable to that of unmodified BSA. In addition, citraconlyated LZ (Cit-LZ) showed reduced spheroid penetration as compared to that of LZ, probably owing to a decrease in protein charge. Taken together, chemical conjugation of targeting ligands-PEG to anionic proteins could be a promising strategy to improve intracellular delivery and in vivo activity, whereas modifications of cationic proteins should be more delicately designed.

Pathogenesis of the obstetric antiphospholipid syndrome: the key role of beta 2 glycoprotein I

Antiphospholipid syndrome (APS) is defined by recurrent pregnancy morbidity and/or vascular thrombosis associated with the persistent presence of antibodies against anionic phospholipid-binding proteins. Beta 2 glycoprotein I (β2GPI) and prothrombin (PT) are the major antigens for antiphospholipid antibodies (aPL) detectable by functional coagulation [lupus anticoagulant (LA)] or solid-phase assays [anti-β2GPI-dependent cardiolipin (aCL) and anti-β2GPI]. β2GPI-dependent aPL are responsible for the positivity of the three classification laboratory criteria. While medium/high titers of antibodies against β2GPI are risk factors for both the vascular and the obstetric manifestations of APS, persistent low titers are also associated with pregnancy complications. There is evidence from animal models of aPL-dependent fetal loss and from in vitro systems that β2GPI-dependent aPL can be pathogenic. β2GPI is physiologically found in large quantities at the placental level being available for the specific antibodies circulating in the maternal blood. Once bound to the protein, the antibodies trigger a local inflammation via the activation of the complement cascade and affect trophoblast and decidual function. The final result is represented by defective placentation, while thrombotic events are apparently less important. β2GPI is a pleiotropic molecule with scavenging properties towards several molecules including apoptotic material and displays anti-oxidant activity. These functions may explain the β2GPI placental localization in an area of intensive tissue remodeling and low oxygen tension. Since β2GPI interacts also with the complement and the coagulation cascade, its binding with specific antibodies may affect the physiology of placentation in several ways.

Lactophorin in Camel Milk Undergoing Specific Proteolysis and Exhibiting Potent Anticancer Action against Human Colon and Breast Cancer Cells Through ROS Generation

Background: Camel milk has been recognized for its health benefits since ancient times and has recently been attracting increasing attention as a form of medical treatment for diverse human diseases. Studies on the health benefits of camel milk attributed its medicinal effects to nutritional status, but the molecular mechanisms of proteins involved in such effects remain unknown. Objectives: The aim of this study was to explore the anticancer properties of camel milk proteins (CMPs). Methods: CMPs were fractionated into camel casein proteins (CCPs) and camel whey proteins (CWPs). The CWP exhibited the most potent anticancer activity against colon (HCT-116) and breast (MCF-7) cancer cells. The CWP was further fractionated into cationic and anionic proteins using HiTrap cationic (SP-XL) and anionic (QFF) exchange columns. Results: QFF-bound proteins (QFF-B) exhibited the strongest anticancer activities against both cancer cells. QFF-B proteins produced three peaks (P1~P3) on RP-HPLC, whereas P3 showed superior anticancer activity. The cytotoxic effects of CWP and QFF-B proteins are associated with increased production of intracellular ROS and subsequent apoptosis in both cancer cells. MALDI-TOF-MS identified lactophorin, glycation-dependent cell adhesion molecule1 (GlyCAM-1), and its three driven fragments as dominant peptides in QFF-B, while RP-HPLC-P3 contained two of them with molecular masses of 8080.3 and 9395.6 Da. The two peptides, both derived from the C-terminal of lactophorin, were the most representative peptides in the most active protein fractions (QFF-B and RP-HPLC-P3). Conclusion: The results highlight for the first time that lactophorin is the major anti-cancer ingredient in camel milk and its unique C-terminal peptides present potential candidacy as anticancer agents in nutraceutical and pharmacological applications.