Porcine Erythrocytes Research Articles

TurboID screening of ApxI toxin interactants identifies host proteins involved in Actinobacillus pleuropneumoniae-induced apoptosis of immortalized porcine alveolar macrophages

Actinobacillus pleuropneumoniae (APP) is a gram-negative pathogenic bacterium responsible for porcine contagious pleuropneumonia (PCP), which can cause porcine necrotizing and hemorrhagic pleuropneumonia. Actinobacillus pleuropneumoniae-RTX-toxin (Apx) is an APP virulence factor. APP secretes a total of four Apx toxins, among which, ApxI demonstrates strong hemolytic activity and cytotoxicity, causing lysis of porcine erythrocytes and apoptosis of porcine alveolar macrophages. However, the protein interaction network between this toxin and host cells is still poorly understood. TurboID mediates the biotinylation of endogenous proteins, thereby targeting specific proteins and local proteomes through gene fusion. We applied the TurboID enzyme-catalyzed proximity tagging method to identify and study host proteins in immortalized porcine alveolar macrophage (iPAM) cells that interact with the exotoxin ApxI of APP. His-tagged TurboID-ApxIA and TurboID recombinant proteins were expressed and purified. By mass spectrometry, 318 unique interacting proteins were identified in the TurboID ApxIA-treated group. Among them, only one membrane protein, caveolin-1 (CAV1), was identified. A co-immunoprecipitation assay confirmed that CAV1 can interact with ApxIA. In addition, overexpression and RNA interference experiments revealed that CAV1 was involved in ApxI toxin-induced apoptosis of iPAM cells. This study provided first-hand information about the proteome of iPAM cells interacting with the ApxI toxin of APP through the TurboID proximity labeling system, and identified a new host membrane protein involved in this interaction. These results lay a theoretical foundation for the clinical treatment of PCP.

Improved lipid saponification for chromatographic quantification of fatty acids in porcine erythrocytes – an important lipidomic biomarker of the effectiveness of dietary fat supplementation in pigs as a large animal model for human studies

Improved lipid saponification for chromatographic quantification of fatty acids in porcine erythrocytes – an important lipidomic biomarker of the effectiveness of dietary fat supplementation in pigs as a large animal model for human studies

Structural basis of membrane skeleton organization in red blood cells

The spectrin-based membrane skeleton is a ubiquitous membrane-associated two-dimensional cytoskeleton underneath the lipid membrane of metazoan cells. Mutations of skeleton proteins impair the mechanical strength and functions of the membrane, leading to several different types of human diseases. Here, we report the cryo-EM structures of the native spectrin-actin junctional complex (from porcine erythrocytes), which is a specialized short F-actin acting as the central organizational unit of the membrane skeleton. While an α-/β-adducin hetero-tetramer binds to the barbed end of F-actin as a flexible cap, tropomodulin and SH3BGRL2 together create an absolute cap at the pointed end. The junctional complex is strengthened by ring-like structures of dematin in the middle actin layers and by patterned periodic interactions with tropomyosin over its entire length. This work serves as a structural framework for understanding the assembly and dynamics of membrane skeleton and offers insights into mechanisms of various ubiquitous F-actin-binding factors in other F-actin systems.

Molecular Simulation Study on the Interaction between Porcine CR1-like and C3b.

The molecular basis of porcine red blood cell immune adhesion function stems from the complement receptor type 1-like (CR1-like) on its cell membrane. The ligand for CR1-like is C3b, which is produced by the cleavage of complement C3; however, the molecular mechanism of the immune adhesion of porcine erythrocytes is still unclear. Here, homology modeling was used to construct three-dimensional models of C3b and two fragments of CR1-like. An interaction model of C3b-CR1-like was constructed by molecular docking, and molecular structure optimization was achieved using molecular dynamics simulation. A simulated alanine mutation scan revealed that the amino acids Tyr761, Arg763, Phe765, Thr789, and Val873 of CR1-like SCR 12-14 and the amino acid residues Tyr1210, Asn1244, Val1249, Thr1253, Tyr1267, Val1322, and Val1339 of CR1-like SCR 19-21 are key residues involved in the interaction of porcine C3b with CR1-like. This study investigated the interaction between porcine CR1-like and C3b using molecular simulation to clarify the molecular mechanism of the immune adhesion of porcine erythrocytes.

PSVII-16 Galactosylated chitosan-oligosaccharides have anti-adhesive effect against enterotoxigenic Escherichia coli in piglets

Abstract Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea in piglets. In vitro, high molecular weight β-galactosylated chitosan-oligosaccharides (Gal-COS) had strong anti-adhesive activity against ETEC-expressing K88 fimbriae (ETEC K88) binding to porcine erythrocytes. This study assessed the effects of Gal-COS differing in structure on anti-adhesive properties against ETEC in a small intestinal segment perfusion (SISP) model in 8 piglets (BW 10 kg; 5-wk old). With 10 jejunal segments in each pig, 5 segments were infected with ETEC K88, and the other 5 segments were infused with saline (non-ETEC). Every 2 paired segments (ETEC or non-ETEC) from the same pig were treated for 8 h with 64 ml of 10 g L-1 of one of the following test products: 1) α-Gal-COS; 2) β-Gal-COS; 3) exopolysaccharides produced by Lactobacillus reuteri; and 4) raffinose in a double 4 × 4 Latin square with a saline control. Infection by ETEC K88 was verified by quantitative PCR. Net fluid loss was calculated as difference of fluid loss between ETEC segment and its paired non-ETEC segment. Data were analyzed using the mixed model with segment and test product as fixed effects, and pig as random effect. Number of eubacterial rRNA genes was 10-fold greater (P < 0.001) in ETEC segments than non-ETEC segments, indicating that ETEC K88 accounted for > 90% of bacterial gene counts. Test product did not affect (P > 0.10) the number of ETEC bacteria in the outflow fluid. Furthermore, net fluid loss caused by ETEC tended (P = 0.08) to be decreased by β-Gal-COS compared to all other treatments. In conclusion, the in vivo SISP model confirmed that Gal-COS had anti-diarrheal effects, indicating that β-Gal-COS is a potential feed additive to reduce the ETEC-induced diarrhea in piglets.

Potentiation of β-lactam antibiotics and β-lactam/β-lactamase inhibitor combinations against MDR and XDR Pseudomonas aeruginosa using non-ribosomal tobramycin-cyclam conjugates.

To develop a multifunctional adjuvant molecule that can rescue β-lactam antibiotics and β-lactam/β-lactamase inhibitor combinations from resistance in carbapenem-resistant Pseudomonas aeruginosa clinical isolates. Preparation of adjuvant was guided by structure-activity relationships, following standard protocols. Susceptibility and chequerboard studies were assessed using serial 2-fold dilution assays. Toxicity was evaluated against porcine erythrocytes, human embryonic kidney (HEK293) cells and liver carcinoma (HepG2) cells via MTS assay. Preliminary in vivo efficacy was evaluated using a Galleria mellonella infection model. Conjugation of tobramycin and cyclam abrogates the ribosomal effects of tobramycin but confers a potent adjuvant property that restores full antibiotic activity of meropenem and aztreonam against carbapenem-resistant P. aeruginosa. Therapeutic levels of susceptibility, as determined by CLSI susceptibility breakpoints, were attained in several MDR clinical isolates, and time-kill assays revealed a synergistic dose-dependent pharmacodynamic relationship. A triple combination of the adjuvant with ceftazidime/avibactam (approved), aztreonam/avibactam (Phase III) and meropenem/avibactam enhances the efficacies of β-lactam/β-lactamase inhibitors against recalcitrant strains, suggesting rapid access of the combination to their periplasmic targets. The newly developed adjuvants, and their combinations, were non-haemolytic and non-cytotoxic, and preliminary in vivo evaluation in G. mellonella suggests therapeutic potential for the double and triple combinations. Non-ribosomal tobramycin-cyclam conjugate mitigates the effect of OprD/OprF porin loss in P. aeruginosa and potentiates β-lactam/β-lactamase inhibitors against carbapenem-resistant clinical isolates, highlighting the complexity of resistance to β-lactam antibiotics. Our strategy presents an avenue to further preserve the therapeutic utility of β-lactam antibiotics.

Molecular detection of Mycoplasma suis in extensive pig production systems in the State of Maranhão, northeast Brazil.

Mycoplasma suis is a bacterium that causes hemoplasmosis in pigs. This agent is capable of adhering to the surface of porcine erythrocytes, inducing structural changes on these cells. In Brazil, there are few reports about the disease, its causal agent, and the economic impact of this pathogen on pig production systems and farm sanitation. The present study aimed to investigate the occurrence of M. suis in extensive swine farms located in the counties of Itapecuru Mirim, Santa Rita and Rosario, State of Maranhão, northeast Brazil. For such purpose, 64 blood samples of pigs from these facilities were tested for M. suis using a 16S rRNA gene-based quantitative real-time PCR (qPCR); 82.3%, 65.2% and 25% of blood samples of swine from farms in the cities of Itapecuru Mirim, Santa Rita and Rosario were positive for M. suis by qPCR, respectively. This study shows, for the first time, that M. suis circulates in pig populations from the state of Maranhão, Northeast Brazil.

In vitro observation: the GFP-E. coli adhering to porcine erythrocytes can be removed by porcine alveolar macrophages.

Although the activation of pathogen phagocytosis via complement system has been studied, erythrocyte-phagocyte interactions in pigs are not clearly understood. Therefore, we sought to investigate the ability of porcine erythrocytes to clear immune complexes (ICs) by using laser confocal microscopy and flow cytometry to observe the immune adhesion of porcine erythrocytes to fluorescent bacilli and the immune presentation process of transferring fluorescent bacilli to macrophages. Isolated porcine alveolar macrophages (PAMs) had uniform morphology and size, and a survival rate of 97.2%. The phagocytosis rate was 98.8%. After WT E. coli was labeled with Fluorescein Isothiocyanate (FITC), the bacteria showed a bright green fluorescence, and the labeling rate was 92.3%. When laser confocal microscopy was utilized to observe the co-incubation system of porcine erythrocytes, PAM, and fluorescent E. coli, the fluorescence intensity of bacilli decreased with increasing observation time and even disappeared. Flow Cytometry examination showed that the average fluorescence intensity of PAMs co-incubated with porcine erythrocytes adhered to WT-E. coli-FITC, was significantly higher than that of normal PAMs. Furthermore, when porcine erythrocytes adhered to WT E. coli were incubated with PAMs, the surface mean fluorescence intensity of porcine erythrocytes was significantly higher than that of the blank control group. This shows that PAMs can competitively bind to the oposinized E. coli adhered to the surface of porcine erythrocytes, and these oposinized pathogens can enter macrophages by the process of phagocytosis, which promoting the internalization of ICs or pathogens. During this process, the physical morphology of porcine erythrocytes was not damaged, but the levels of its main functional protein CR1-like were reduced.

New Rabbit Erythrocyte Specific Mycelial Lectins from Fusarium sp. with Complex Saccharide Specificity

Eight Fusarium sp. namely, F. acutatum, F. globosum, F. graminearum, F. lactis, F. nivale, F. proliferatum, F. pseudoanthophilum and F. robustum were screened for the presence of lectins by hemagglutination activity using human ABO, porcine, ovine, goat and rabbit erythrocytes. Mycelial extracts of all the fungal cultures except F. graminearum displayed unique lectin activity with only rabbit erythrocytes. Enzymatic treatment of rabbit erythrocytes with neuraminidase has significantly enhanced the titre of all the lectin positive extracts of fungal cultures. In contrast, most of the lectins showed a decline in lectin activity with protease treated rabbit erythrocytes. Saccharide specificity studies have shown that majority of the lectins were inhibitory towards O-acetyl sialic acids. None of the lectins from Fusarium sp. were inhibited by dextran, meso-inositol and N-acetyl-D-glucosamine. Most of the fungal cultures displayed highest hemagglutination activity during 10th day of growth in broth cultures. The unique saccharide specificity of Fusarium sp. lectins can be used for elucidating their clinical role in glycobiology research.

Invitro free radical scavenging capacity of dimethylglycine sodium salt and its protective ability against oleic acid hydroperoxide-induced oxidative damage in IPEC-J2 cells.

The aim of the present study was to evaluate the invitro free radical scavenging capacity of dimethylglycine sodium (DMG‑Na) and its protective ability against oleic acid hydroperoxide (OAHPx)‑induced oxidative damage in IPEC‑J2 cells. Initially, the free radical scavenging activities of water‑soluble pigments (DMG‑Na, betalain, capsanthin and cyanidin‑3‑rutinoside) were measured and compared with those of Trolox. Subsequently, freshly collected swine blood was mixed with heparin and centrifuged to obtain erythrocytes. In order to induce the free radical chain oxidation in erythrocytes, the aqueous peroxyl radicals were generated by thermal decomposition of 2,2'‑azobis(2‑amidinopropane) dihydrochloride (AAPH) in oxygen. A 2% suspension of porcine erythrocytes in PBS buffer were pre‑incubated for 30min at 37˚C with DMG‑Na (32µM), followed by incubation with or without AAPH (75mM) for 5h with gentle shaking. Additionally, IPEC‑J2 cells were randomly assigned to four groups (n=6per group): Cells treated with phosphate buffered saline (PBS); cells treated with DMG‑Na (32µM); cells treated with oleic acid hydroperoxides (OAHPx, 20µM; TO group); cells treated with DMG‑Na (32µM) followed by OAHPx (20µM; DTO group). The cells were cultured in Dulbecco's modified Eagle's medium, Ham's F‑12mixture, 1.5mM HEPES, 5% (v/v) fetal bovine serum, 1% (v/v) insulin‑transferrin‑selenium mixture, 1% (v/v) penicillin‑streptomycin mixture and 2.5µg/ml fungizone (37˚C, 5% CO2). The results showed that DMG‑Na exerted the strongest free radical scavenging capacity at 0.32M from 0.08‑0.64M, and that it could prevent AAPH‑induced porcine erythrocyte hemolysis by increasing its antioxidant capacity (P<0.05). The results also demonstrated that antioxidant capacity and antioxidant‑associated gene expression increased in the DTO group relative to the TO group (P<0.05), indicating that DMG‑Na prevented the OAHPx‑induced oxidative damage in IPEC‑J2 cells by improving the antioxidant capacity and antioxidant‑associated gene expression.

Identification of erythrocyte membrane proteins interacting with Mycoplasma suis GAPDH and OSGEP

Mycoplasma suis (M. suis) is an uncultivable haemotrophic mycoplasma that parasitizes the red blood cells of a wide range of domestic and wild animals. Adhesion of M. suis to host erythrocytes is crucial for its unique RBC-dependent lifecycle. MSG1 protein (now named as GAPDH) with homology to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was the first identified adhesion protein of M. suis. In this study, we found that O-sialoglycoprotein endopeptidase (OSGEP) is another M. suis protein capable of binding porcine erythrocytes. Recombinant OSGEP expressed in E. coli demonstrated surface localization similar to GAPDH. Purified rOSGEP bound to erythrocyte membrane preparations in a dose-dependent manner and this adhesion could be specifically inhibited by anti-rOSGEP antibodies. E. coli transformants expressing OSGEP on their surface were able to adhere to porcine erythrocytes. Furthermore, using far-western and pull-down assays, we determined the host membrane proteins that interacted with OSGEP and GAPDH were Band3 and glycophorin A (GPA). In conclusion, our studies indicated that OSGEP and GAPDH could interact with both Band3 and GPA to mediate adhesion of M. suis to porcine erythrocytes.

Structure and function relationships of the binding of β- and ɑ-galactosylated oligosaccharides to K88 fimbriae of enterotoxigenic Escherichia coli

Chitosan-oligosaccharides (COS) are suitable acceptors for β-galactosidase, and galactosylated-COS prevent adhesion of enterotoxigenic Escherichia coli (ETEC) K88 to porcine erythrocytes. The structure and functional relationship of oligosaccharide binding to ETEC K88 fimbriae was investigated. β- or α-galacto-oligosaccharides (βGOS and αGOS), β-galactosylated melibiose, α-galactosylated lactose and β-/ɑ-galactosylated COS were produced with β-/α-galactosidase using lactose or melibiose as galactosyl donor. Oligosaccharides were fractionated with cation exchange and size exclusion chromatography; their ability to prevent pathogen adhesion was measured with a hemagglutination assay, ELISA with ETEC cells, or with purified K88 fimbriae. High molecular weight β-GalCOS, β-galactosylated melibiose, and βGOS had strong anti-adhesion activity. Other oligosaccharides had weak or no anti-adhesive effects against ETEC K88. The ability of oligosaccharides to prevent binding of ETEC K88 or purified K88 fimbriae decreased with decreasing molecular weight. An avenue for valorization of lactose from whey to produce oligosaccharides with specific biological activity is suggested.

Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry Characterization of Gangliosides in Biological Samples.

The hydrophilic interaction liquid chromatography (HILIC) coupled to a negative-ion electrospray ionization tandem mass spectrometry (ESI-MS/MS) method has been developed for the identification of a wide range of gangliosides in biological samples. Gangliosides consist of a backbone of sphingoid base and a polar oligosaccharide chain containing at least one sialic acid. Gangliosides are extracted by chloroform-methanol-water mixture, where an upper aqueous layer containing gangliosides and other polar lipid subclasses is further purified by C18 solid-phase extraction. The optimization of chromatographic conditions includes the column selection, mobile-phase composition, pH value, buffer type, and concentration with the goal to achieve the best chromatographic resolution and MS sensitivity. The identification of gangliosides and other polar lipids is based on accurate m/z values of [M-H]- ions and fragment ions as well measured by high-resolution MS. The detailed interpretation of MS/MS spectra enables the generalization of fragmentation pathways, which is then used for the differentiation of a, b, and c series of gangliosides. The structural assignment is further confirmed by agreement with the predicted retention behavior in HILIC mode on the basis of the correlation among the ganglioside retention, the number of saccharide units, and their sequence. The final HILIC/ESI-MS/MS method is applied for the analysis of porcine brain, human kidney, lungs, plasma, and erythrocytes resulting in unambiguous identification of 145 ganglioside species from 19 subclasses, which represents the highest number of reported gangliosides. Moreover, 71 sulfatides and 59 polar phospholipids (phosphatidylserines, phosphatidylinositols, lysophosphatidylinositols, and phosphatidylglycerols) are detected within a 15 min run.

Enzymatic Synthesis and Purification of Galactosylated Chitosan Oligosaccharides Reducing Adhesion of Enterotoxigenic Escherichia coli K88.

Enterotoxigenic Escherichia coli (ETEC) K88 causes diarrhea in weaned piglets and represent a suitable model system for ETEC causing childhood diarrhea. This study aimed to evaluate the effects of oligosaccharides against ETEC K88 adhesion to porcine erythrocytes with two bioassays. Galactosylated chitosan-oligosaccharides (Gal-COS) were synthesized through transgalactosylation by β-galactosidase. Fractions 2-5 of Gal-COS were obtained through cation exchange and size exclusion chromatography. Fractions 2-5 of acetylated Gal-COS were obtained through chemical acetylation followed by size exclusion chromatography. Gal-COS F2 containing the largest oligosaccharides had the highest antiadhesion activity with the minimum inhibitory concentration of 0.22 g/L, followed by F3 and F4. Acetylation of Gal-COS decreased their ability to reduce ETEC K88 adhesion. The composition of active oligosaccharides was determined with LC-MS. Galactosylation of COS produces oligosaccharides which reduce ETEC K88 adhesion; moreover, resulting oligosaccharides match the composition of human milk oligosaccharides, which prevent adhesion of multiple pathogens.

Mapping of hemoglobin in erythrocytes and erythrocyte ghosts using two photon excitation fluorescence microscopy.

The present study describes utilization of two photon excitation fluorescence (2PE) microscopy for visualization of the hemoglobin in human and porcine erythrocytes and their empty membranes (i.e., ghosts). High-quality, label- and fixation-free visualization of hemoglobin was achieved at excitation wavelength 730 nm by detecting visible autofluorescence. Localization in the suspension and spatial distribution (i.e., mapping) of residual hemoglobin in erythrocyte ghosts has been resolved by 2PE. Prior to the 2PE mapping, the presence of residual hemoglobin in the bulk suspension of erythrocyte ghosts was confirmed by cyanmethemoglobin assay. 2PE analysis revealed that the distribution of hemoglobin in intact erythrocytes follows the cells’ shape. Two types of erythrocytes, human and porcine, characterized with discocyte and echinocyte morphology, respectively, showed significant differences in hemoglobin distribution. The 2PE images have revealed that despite an extensive washing out procedure after gradual hypotonic hemolysis, a certain amount of hemoglobin localized on the intracellular side always remains bound to the membrane and cannot be eliminated. The obtained results open the possibility to use 2PE microscopy to examine hemoglobin distribution in erythrocytes and estimate the purity level of erythrocyte ghosts in biotechnological processes.

Identification and Characterization of Glycopeptides from Egg Protein Ovomucin with Anti-Agglutinating Activity against Porcine K88 Enterotoxigenic Escherichia coli Strains.

Ovomucin is a glycoprotein from egg white with potential to act as an anti-adhesive agent against infectious diseases. This study aimed to determine whether ovomucin or ovomucin hydrolysates could prevent adhesion of two porcine K88 enterotoxigenic Escherichia coli (ETEC) strains. Adhesion was assessed in vitro using a hemagglutination assay. Ovomucin hydrolysates, but not intact ovomucin, prevented adhesion of ETEC to porcine erythrocytes. The ovomucin hydrolysate prepared by acid protease II exhibited the best anti-agglutinating activity against both strains; this hydrolysate was fractionated by cation exchange chromatography and reverse-phase high-performance liquid chromatography (HPLC). The most active fractions, F3(9) and F7(1), with minimal inhibitory concentrations of 0.03 and 0.25 g/L against strains ECL13795 and ECL13998, respectively, were subjected to structural characterization. Six identified glycopeptides were all derived from α-ovomucin, composed of a pentasaccharide core of two N-acetylglucosamine and three mannose residues (GlcNAc2Man3) and a bisecting N-acetylglucosamine (GlcNAc). The terminal β-linked galactose from these glycopeptides could be one of the binding sites for K88ac fimbriae.

Comparative studies on osmosis based encapsulation of sodium diclofenac in porcine and outdated human erythrocyte ghosts

The objective of our study was to develop controlled drug delivery system based on erythrocyte ghosts for amphiphilic compound sodium diclofenac considering the differences between erythrocytes derived from two readily available materials – porcine slaughterhouse and outdated transfusion human blood. Starting erythrocytes, empty erythrocyte ghosts and diclofenac loaded ghosts were compared in terms of the encapsulation efficiency, drug releasing profiles, size distribution, surface charge, conductivity, surface roughness and morphology. The encapsulation of sodium diclofenac was performed by an osmosis based process – gradual hemolysis. During this process sodium diclofenac exerted mild and delayed antihemolytic effect and increased potassium efflux in porcine but not in outdated human erythrocytes. FTIR spectra revealed lack of any membrane lipid disorder and chemical reaction with sodium diclofenac in encapsulated ghosts. Outdated human erythrocyte ghosts with detected nanoscale damages and reduced ability to shrink had encapsulation efficiency of only 8%. On the other hand, porcine erythrocyte ghosts had encapsulation efficiency of 37% and relatively slow drug release rate. More preserved structure and functional properties of porcine erythrocytes related to their superior encapsulation and release performances, define them as more appropriate for the usage in sodium diclofenac encapsulation process.

Biomembranes from slaughterhouse blood erythrocytes as prolonged release systems for dexamethasone sodium phosphate.

The present study investigated preparation of bovine and porcine erythrocyte membranes from slaughterhouse blood as bio-derived materials for delivery of dexamethasone-sodium phosphate (DexP). The obtained biomembranes, i.e., ghosts were characterized in vitro in terms of morphological properties, loading parameters, and release behavior. For the last two, an UHPLC/-HESI-MS/MS based analytical procedure for absolute drug identification and quantification was developed. The results revealed that loading of DexP into both type of ghosts was directly proportional to the increase of drug concentration in the incubation medium, while incubation at 37°C had statistically significant effect on loaded amount of DexP (P < 0.05). The encapsulation efficiency was about fivefold higher in porcine compared to bovine ghosts. Insight into ghosts' surface morphology by field emission-scanning electron microscopy and atomic force microscopy confirmed that besides inevitable effects of osmosis, DexP inclusion itself had no observable additional effect on the morphology of the ghosts carriers. DexP release profiles were dependent on erythrocyte ghost type and amount of residual hemoglobin. However, sustained DexP release was achieved and shown over 3 days from porcine ghosts and 5 days from bovine erythrocyte ghosts. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1046-1055, 2016.

Complement receptor activity of recombinant porcine CR1-like protein expressed in a eukaryotic system.

Primate complement receptor type 1 (CR1) protein, a single-chain transmembrane glycoprotein, plays an important role in immune adherence and clearing complement-opsonized immune complexes. Here, the mRNA of the porcine primate-like complement receptor (CR1-like) gene was analyzed, and two domain sequences with potential functions were cloned into the pwPICZalpha vector for expression in Pichia pastoris. The recombinant proteins were purified with both Protein Pure Ni-NTA resin and strong anion exchange resin. The activities of the purified recombinant proteins were evaluated by SDS-PAGE, western blotting, and complement receptor assays. The results indicated that two domains of the CR1-like protein, CCP36 and CCP811 with molecular weights of 29.8kDa and 30kDa, respectively, were successfully expressed in P. pastoris. These two recombinant proteins possess some of the functions of the primate CR1 protein. Using these two proteins coupled with an antibody blocking technique, we also showed that CR1-like is expressed on natural porcine erythrocytes.

The immune adherence receptor CR1-like existed on porcine erythrocytes membrane.

In the present study, we obtain a mouse anti-porcine complement receptor type 1 (CR1)-like monoclonal antibody (McAb) and use this McAb to verify the existence of CR1-like protein on porcine erythrocytes. Our results confirm that CR1-like protein is localized on the surface of porcine erythrocytes. Mouse immunoglobulin G inhibited the binding of serum-opsonized green fluorescent protein-expressing Escherichia coli to porcine erythrocytes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicates that CR1-like McAb reacts with biochemically-purified porcine erythrocyte membrane fractions, with a clear band at 135 kDa to 140 kDa. We postulate that the 135 kDa to 140 kDa membrane protein is the equivalent of the porcine erythrocyte CR1-like protein.