Butyrophilin 1a1 (Btn1a1), which is a member of the Ig superfamily, is highly expressed in the lactating mammary gland and is secreted into milk in association with lipid droplets. To determine the potential function of Btn1a1 in milk secretion, we ablated Btn1a1 in mice and analyzed the lactation phenotype of homozygous (Btn1a1 ؊/؊ ) animals. Two mutant mouse lines were generated in which expression of Btn1a1 was either disrupted or eliminated, respectively. The regulated secretion of milk-lipid droplets was severely compromised in both mutant mouse lines in comparison to wild-type animals. Large pools of triacylglycerol accumulated in the cytoplasm of secretory cells, and lipid droplets escaped from the apical surface with disrupted outer membranes. Luminal spaces became engorged with unstable lipid droplets, which coalesced to form large aggregates. The amount of lipid (wt͞vol) was elevated, on average by 50%, during the first 10 days of lactation, and the diameter of the droplets was up to seven times larger than the normal diameter. In contrast, there was no significant difference between wild-type and null animals in the relative amounts of skim-milk proteins secreted from Golgi-derived secretory vesicles. Approximately half the pups suckling Btn1a1 ؊/؊ animals died within the first 20 days, and weaning weights for the surviving pups were 60 -80% of those suckling wild-type mice. Thus, expression of Btn1a1 is essential for the regulated secretion of milk-lipid droplets. We speculate that Btn1a1 functions either as a structural protein or as a signaling receptor by binding to xanthine dehydrogenase͞oxidase.

The etiology of multiple sclerosis (MS) is believed to involve environmental factors, but their identity and mode of action are unknown. In this study, we demonstrate that Ab specific for the extracellular Ig-like domain of myelin oligodendrocyte glycoprotein (MOG) cross-reacts with a homologous N-terminal domain of the bovine milk protein butyrophilin (BTN). Analysis of paired samples of MS sera and cerebrospinal fluid (CSF) identified a BTN-specific Ab response in the CNS that differed in its epitope specificity from that in the periphery. This effect was statistically significant for the Ab response to BTN76–100 (p = 0.0026), which cosequestered in the CSF compartment with Ab to the homologous MOG peptide MOG76–100 in 34% of MS patients (n = 35). These observations suggested that intratheccal synthesis of Ab recognizing BTN peptide epitopes in the CNS was sustained by molecular mimicry with MOG. Formal evidence of molecular mimicry between the two proteins was obtained by analyzing MOG-specific autoantibodies immunopurified from MS sera. The MOG-specific Ab repertoire cross-reacts with multiple BTN peptide epitopes including a MOG/BTN76–100-specific component that occurred at a higher frequency in MS patients than in seropositive healthy controls, as well as responses to epitopes within MOG/BTN1–39 that occur at similar frequencies in both groups. The demonstration of molecular mimicry between MOG and BTN, along with sequestration of BTN-reactive Ab in CSF suggests that exposure to this common dietary Ag may influence the composition and function of the MOG-specific autoimmune repertoire during the course of MS.

We investigated the expression of butyrophilin in eukaryotic cells with a view to determining the number of mRNA species, the incorporation of the peptide chain into microsomes, and the topology of the processed protein in biological membranes. Butyrophilin is synthesized from a single sized mRNA in both bovine and murine lactating mammary tissue and associates with microsomal membranes with a type I topology (N exo ⅐C cyto ) via a single hydrophobic anchor in the middle of the sequence. Several isoelectric variants of the protein were detected in cellular membranes from lactating bovine mammary tissue and in the milk-fat-globule membrane. We found no evidence for soluble forms of butyrophilin in postmicrosomal supernatants. The 66-kDa protein appears to be subjected to limited proteolysis, giving rise to a 62-kDa fragment lacking the C terminus and to other more minor fragments of lower M r in the milk-fat-globule membrane. Antipeptide antibodies to epitopes within the N-and C-terminal domains were used to show that butyrophilin retains a type I topology in plasma membranes when expressed in insect cells from a baculovirus vector, and in secreted milk-fat globules. These data do not agree with previous suggestions that butyrophilin may exist in cytoplasmic soluble forms, or be reorganized in the plane of the lipid bilayer during secretion in lipid droplets from mammary cells. The results are discussed with reference to the role butyrophilin may play as the principal scaffold for the assembly of a complex with xanthine oxidase and other proteins that functions in the budding and release of milk-fat globules from the apical surface during lactation.Several years ago we cloned a cDNA encoding the major milk-fat-globule membrane (MFGM) 1 protein, butyrophilin (BTN) (1) with a view to understanding the function of this mammary-specific protein in milk-fat secretion (for reviews of this process, see Refs. 2-4). From the derived amino acid sequence we predicted that BTN is an integral protein with a single membrane anchor and a glycosylated exoplasmic N terminus (type I orientation) (1). Subsequent comparisons with more recently cloned cDNAs have established that BTN is a member of the immunoglobulin superfamily (5) with two Nterminal immunoglobulin domains, one of the intermediate type (IgI) (6) toward the N terminus and one of the constant C1 type (7) toward the membrane bilayer (8). A large proportion of the C-terminal region comprises the RFP or B30.2 domain, a sequence which is present in a subfamily of zinc-finger proteins (9, 10), and a group of recently identified BTN-like genes (11, 12). Several workers have proposed that BTN may function as an integral receptor for cytoplasmic fat droplets and that budding of the droplets at the cell surface is initiated by interactions between the cytoplasmic tail of BTN and other proteins, notably the redox enzyme xanthine oxidase (1, 13-16). However, the assumption that BTN behaves as an integral protein in mammary epithelial cells has been confounded by several observati...

Autoantibodies directed against conformation-dependent epitopes of the extracellular domain of the myelin oligodendrocyte glycoprotein (MOG(Igd)) play a major role in the immunopathogenesis of demyelination in experimental autoimmune encephalomyelitis. We now demonstrate that one or more genes encoded within the MHC selectively censor the ability of H-2(b) mice to mount this conformation-dependent autoantibody response, while leaving T and B cell responses to linear MOG(Igd) epitopes intact. This novel form of selective B cell unresponsiveness discriminates between pathogenic and nonpathogenic Ab responses to MOG and determines whether or not Ab-dependent effector mechanisms play an important role in the pathogenesis of MOG-induced experimental autoimmune encephalomyelitis in the mouse.

Butyrophilin 1A1 (BTN1A1) and xanthine oxidoreductase (XOR) are highly expressed in the lactating mammary gland and are secreted into milk associated with the milk fat globule membrane (MFGM). Ablation of the genes encoding either protein causes severe defects in the secretion of milk lipid droplets, suggesting that the two proteins may function in the same pathway. Therefore, we determined whether BTN1A1 and XOR directly interact using protein binding assays, surface plasmon resonance analysis, and gel filtration. Bovine XOR bound with high affinity in a pH-and salt-sensitive manner (K D ‫؍‬ 101 ؎ 31 nM in 10 mM HEPES, 150 mM NaCl, pH 7.4) to the PRY/SPRY/B30.2 domain in the cytoplasmic region of bovine BTN1A1. Binding was stoichiometric, with one XOR dimer binding to either two BTN1A1 monomers or one dimer. XOR bound to BTN1A1 orthologs from mice, humans, or cows but not to the cytoplasmic domains of the closely related human paralogs, BTN2A1 or BTN3A1, or to the B30.2 domain of human RoRet (TRIM 38), a protein in the TRIM family. Analysis of the protein composition of the MFGM of wild type and BTN1A1 null mice showed that most of the XOR in mice lacking BTN1A1 was released from the MFGM in a soluble form when the milk lipid droplets were disrupted to prepare membrane, compared with wild-type mice, in which most of the XOR remained membrane-bound. Thus BTN1A1 functions in vivo to stabilize the association of XOR with the MFGM by direct interactions through the PRY/ SPRY/B30.2 domain. The potential significance of BTN1A1/ XOR interactions in the mammary gland and other tissues is discussed. Members of the butyrophilin (BTN)3 gene family are attracting increasing attention because they may play multifunctional roles in diverse physiologies, including lactation (1, 2), selection and regulation of T-cells in the immune system (3-6), and modulation of autoimmune disease (7-9). BTN proteins have the canonical structures of cell surface receptors, which, after an N-terminal signal sequence, generally comprise two exoplasmic Ig folds (10, 11), a membrane anchor and a cytoplasmic domain consisting of a stem region, a PRY/SPRY/B30.2 domain (12, 13), and a cytoplasmic tail at the C terminus (14).The eponymous BTN1A1 protein has been linked to the secretion of milk lipid droplets because it is highly expressed in the mammary epithelium during lactation and is incorporated into the surface membrane coat surrounding cytoplasmic lipid droplets (the milk fat globule membrane (MFGM)) as they bud into milk from the apical surface (15). Furthermore, ablation of the Btn1a1 gene disrupts lipid secretion, causing the accumulation of large pools of triacylglycerol in the cytoplasm of Btn1a1 null mice (1). In a different context, dietary exposure to BTN1A1 in dairy products has been associated with modulation of the autoimmune disease multiple sclerosis because of structural similarities between the IgI fold of BTN1A1 (16) and the IgV fold of myelin oligodendrocyte glycoprotein (MOG) (17) an antigen on the myelin nerve sheath that is...

BackgroundRicin A chain (RTA) and Pokeweed antiviral proteins (PAPs) are plant-derived N-glycosidase ribosomal-inactivating proteins (RIPs) isolated from Ricinus communis and Phytolacca Americana respectively. This study was to investigate the potential production amenability and sub-toxic antiviral value of novel fusion proteins between RTA and PAPs (RTA-PAPs). In brief, RTA-Pokeweed antiviral protein isoform 1 from seeds (RTA-PAPS1) was produced in an E. coli in vivo expression system, purified from inclusion bodies using gel filtration chromatography and protein synthesis inhibitory activity assayed by comparison to the production of a control protein Luciferase. The antiviral activity of the RTA-PAPS1 against Hepatitis B virus (HBV) in HepAD38 cells was then determined using a dose response assay by quantifying supernatant HBV DNA compared to control virus infected HepAD38 cells. The cytotoxicity in HepAD38 cells was determined by measuring cell viability using a tetrazolium dye uptake assay. The fusion protein was further optimized using in silico tools, produced in an E. coli in vivo expression system, purified by a three-step process from soluble lysate and confirmed in a protein synthesis inhibition activity assay.ResultsResults showed that RTA-PAPS1 could effectively be recovered and purified from inclusion bodies. The refolded protein was bioactive with a 50% protein synthesis inhibitory concentration (IC50) of 0.06 nM (3.63 ng/ml). The results also showed that RTA-PAPS1 had a synergetic activity against HBV with a half-maximal response concentration value (EC50) of 0.03 nM (1.82 ng/ml) and a therapeutic index of > 21,818 with noticeable steric hindrance. Results also showed that the optimized protein ricin A chain mutant-Pokeweed antiviral protein isoform 1 from leaves (RTAM-PAP1) could be recovered and purified from soluble lysates with gain of function on protein synthesis inhibition activity, with an IC50 of 0.03 nM (1.82 ng/ml), and with minimal, if any, steric hindrance.ConclusionsCollectively, our results demonstrate that RTA-PAPs are amenable to effective production and purification in native form, possess significant gain of function on protein synthesis inhibition and anti-HBV activities in vitro with a high therapeutic index and, thus, merit further development as potential potent antiviral agents against chronic HBV infection to be used as a standalone or in combination with existent therapies.

SUMMARYContinuous cultures of pluripotent parenchymal hepatocytes were derived from the epiblasts of 8-day-old pig blastocysts. The cells were polygonal and had phase-contrast dark, granular cytoplasm with prominent nuclei and nucleoli.

The deadly pandemic named COVID-19, caused by a new coronavirus (SARS-CoV-2), emerged in 2019 and is still spreading globally at a dangerous pace. As of today, there are no proven vaccines, therapies, or even strategies to fight off this virus. Here, we describe the in silico docking results of a novel broad range anti-infective fusion protein RTAM-PAP1 against the various key proteins of SARS-CoV-2 using the latest protein-ligand docking software. RTAM-PAP1 was compared against the SARS-CoV-2 B38 antibody, ricin A chain, a pokeweed antiviral protein from leaves, and the lectin griffithsin using the special CoDockPP COVID-19 version. These experiments revealed novel binding mechanisms of RTAM-PAP1 with a high affinity to numerous SARS-CoV-2 key proteins. RTAM-PAP1 was further characterized in a preliminary toxicity study in mice and was found to be a potential therapeutic candidate. These findings might lead to the discovery of novel SARS-CoV-2 targets and therapeutic protein structures with outstanding functions.