Characteristics of lactoferrin receptor in bovine intestine: higher binding activity to the epithelium overlying Peyer's patches

Several lines of evidence have recently demonstrated the occurrence of specific lactoferrin (Lf) receptors in different cells. We report here, for the first time, the characteristics of binding, and distribution of Lf receptors in the bovine intestinal tract with special emphasis on the epithelium overlying Peyer's patches (EOPP). Brush-border membrane vesicles (BBMV) were prepared from the mucosa of duodenum, jejunum, ileum, colon, EOPP in jejunum and EOPP in ileum. Receptor binding assays were carried out using 125I-labelled bovine Lf. Specific and saturable Lf receptors were found in BBMV of all the intestinal segments examined. Non-linear regression and Scatchard plot analyses clearly revealed that EOPP had the highest binding maximal (Bmax), and lowest in colon. The maximum dissociation constant (Kd) 3.74 microm was in the ileum. We found that bovine transferrin competed with Lf for the same binding site of receptors. In contrast, no binding of bovine serum albumin occurred. It was concluded that Lf receptors in the mucosal lining are attributable to mediate multifunctional activities of Lf in the gut, especially in the EOPP.     

The detection of bovine lactoferrin binding protein on Trypanosoma brucei

Trypanosoma brucei, the causative agent of sleeping sickness in humans, requires transferrin (TF) for growth. Therefore, T. brucei has a TF receptor that allows it to obtain iron from TF. Lactoferrin (LF), a member of the TF family protein, is an iron-binding protein that is found in most biological fluids of mammals. LF has been shown to interact with some bacteria species by specific receptor-ligand binding. We examined the ability of T. brucei to bind bovine LF (bLF) by using a fluorescence test, streptavidin-biotin (SAB) microplate analysis, and far Western blotting using a biotin-streptavidin system. We found that bLF bound to components of T. brucei, and that bLF hydrolysate disrupted the sites responsible for binding to parasite proteins. Furthermore, bLF, human LF, bovine TF, and ovotransferrin bound same proteins of T. brucei, which exhibited molecular masses of 40 and 43 kDa. The N-terminal amino acid sequence of the 40 kDa bLF binding protein was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH).     

Bovine lactoferrin binds to insulin-like growth factor-binding protein-3

Insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) has been shown to have IGF independent actions that appear to be mediated by specific IGFBP-3 binding proteins located on cell membranes. We show here using Western ligand blotting, a number of mammary membrane proteins that bind ¹²⁵I-labeled rhIGFBP-3. Immunoprecipitation studies demonstrated that the >70 kDa protein was identified from bovine mammary microsomes as bovine lactoferrin (bLf). In addition to being a secretory protein, Lf is tightly associated with cellular membranes. Labeled rhIGFBP-3 was shown to bind to commercially purchased and processed apo- or holo-human or bLf, but not bovine transferrin (bTf). Binding of [¹²⁵I]rhIGFBP-3 to other positively charged proteins was not detected nor was binding to rhIGFBP-5 or other mammary-secreted IGFBPs observed. Reciprocal specific binding of [¹²⁵I]bLf to rhIGFBP-3 was shown, but [¹²⁵I]bTf did not show binding to rhIGFBP-3. While [¹²⁵I]rhIGF-II does not bind to bLf, unlabeled rhIGF-II was shown to compete with [¹²⁵I]bLf for rhIGFBP-3 binding. More detailed analysis by dot blot showed that Lf competes (ED₅₀=3 μg/ml) or displaces (ED₅₀=1 mg/ml) bound [¹²⁵I]rhIGF-II from dot blotted rhIGFBP-3. In vitro studies with a bovine primary mammary epithelial cell culture showed that all-trans-retinoic acid stimulates the appearance of bovine IGFBP-3 and bLf in the conditioned media and that [¹²⁵I]rhIGFBP-3 could be utilized to detect conditioned media bLf. These findings reveal a novel role for bLf, binding to IGFBP-3 and perhaps disassociating IGFBP-3:IGF when in high concentration.     

The detection of bovine lactoferrin binding protein on Toxoplasma gondii

Lactoferrin (LF), a member of the transferrin (TF) protein family, is an iron-binding protein that is known to interact with bacteria through a specific receptor. We examined the binding of bovine LF (bLF), bovine TF (bTF), and ovotransferrin (OTF) by Toxoplasma gondii using a fluorescence test and the streptavidin-biotin (SAB) method using biotin-streptavidin, and found that bLF, bTF, and OTF bound to the protein components of T. gondii. Furthermore, we confirmed that bLF, bTF, and OTF bound a 42 kDa soluble protein of T. gondii by far Western blot method. These results demonstrated that bLF binding proteins are present on T. gondii.     

Receptor-mediated transport of lactoferrin into the cerebrospinal fluid via plasma in young calves

Milk, especially colostrum, contains different kinds of macromolecules abundantly, such as immunoglobulin G (IgG), lactoferrin (Lf), transferrin (Tf), and growth factors. These are essential for the development and maintenance of health, which greatly depends on the absorption and transportation of macromolecules to the target organs. To evaluate the macromolecular transport, and concentrations in plasma and cerebrospinal fluid (CSF), colostrum was fed to newborn calves followed by milk and milk replacer, and maintained up to the 4th week under farm conditions. Plasma and CSF were collected at different times, and were analyzed for Lf, Tf, IgG and iron concentrations. Lf, Tf and IgG concentrations were steeply increased in plasma and CSF after colostrum feeding, and fluctuating patterns were observed during the experiments. Furthermore, intraduodenal administration of bovine Lf alone in young calf experiments revealed that the Lf concentration reached a peak at 4 hr, and was 7 and 4 times higher than preadministration in plasma and CSF, respectively. To explore the transport mechanism of Lf into CSF in young calves, epithelial membranes of the choroid plexus were prepared and a binding assay for Lf receptors (Lf-R) was carried out with 125I-Lf. The saturation kinetics revealed that the Bmax of epithelial membranes was 26.15 nmol/mg protein with a Kd of 0.11 microM, which also showed that Lf-R is saturable and specific. Scatchard plot transformation showed the presence of a single type of Lf-R in the choroid plexus. These results suggest that Lf is transported into the CSF through receptor mediated transcytosis in young calves, and that Lf may play an important role(s) in brain function.     

Characterization of two proteins of Staphylococcus aureus isolated from bovine clinical mastitis with homology to glyceraldehyde-3-phosphate dehydrogenase

Staphylococcus aureus is the most common causative agent of bovine mastitis and vaccines developed to control this disease showed limited protection due in part to the lack of common antigens among the mastitis isolates. We isolated and identified two genes encoding proteins with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity from a S. aureus strain isolated from bovine clinical mastitis. The GapB and GapC proteins share considerable homology to the GapB and GapC products of human strains of S. aureus. These two proteins could be distinguished by their different GAPDH activities and binding to bovine transferrin properties. Both gapB and gapC genes were conserved in 11 strains tested, and the GapC protein was present on the surface of all S. aureus strains.     

Non-genomic steroid receptors in the bovine ovary

Over the last few years, rapid and physiologically important non-genomic actions of all classes of steroid hormones have been described in many cell types. A putative non-genomic membrane progesterone receptor (NGPR) was the first, and so far the only, non-genomic steroid receptor cloned. Two homologous NGPR proteins have been identified in the human, and a similar protein in the bovine and rat. Various detection methods have been used to identify putative NGPRs in a range of tissues: however, different methods often yield quite different molecular weights, and probably detect distinct moieties. We describe some properties of the specific cell-surface membrane binding sites for [3H]-progesterone in enriched cell membrane preparations of bovine luteal and follicular cells. Similar binding sites were also detected in cell-membranes of some (but not all) bovine tissues. Western blots of detergent extracts of bovine luteal membranes identified a protein (85kDa) that reacted with an antiserum to the N-terminal peptide of porcine NGPR. Activity was low in native non-denatured extracts, but increased dramatically in a dose-dependent manner following pretreatment with the cholesterol-complexing agent, digitonin. This protein was co-precipitated by antisera to caveolin. In contrast, a specific monoclonal antibody to the ligand binding domain of the genomic progesterone receptor (Mab C262) detected two proteins (M(r), 55 and 60kDa) in luteal membrane detergent extracts. Immunostaining of these proteins by Mab C262 was abolished by digitonin concentration-dependent manner in non-denatured extracts. However, both proteins were unaffected by digitonin in fully denatured detergent extracts, suggesting that digitonin induced a conformational change in the native protein that prevented binding of Mab C262 to its epitope. Our data suggest the presence of a complex of two or more distinct membrane-associated progesterone-binding proteins in bovine luteal membranes. Moreover, their conformations are specifically affected by removal of bound cholesterol.     

Role of complement S protein (vitronectin) in adherence of Streptococcus dysgalactiae to bovine epithelial cells.

The binding of bovine complement S protein (vitronectin) to Streptococcus dysgalactiae isolates from cattle with mastitis and the S protein's role in streptococcal adherence to bovine epithelial cells were investigated. All 25 clinical isolates of S dysgalactiae interacted with bovine S protein. None of the other streptococcal species tested bound to bovine S protein. The S protein-binding sites were saturable and highly sensitive to trypsin. The binding of bovine S protein to S dysgalactiae isolates was specific and could not be inhibited by other plasma proteins, such as fibronectin, albumin, fibrinogen, alpha 2-macroglobulin, or IgG. Similarly, streptococcal binding of bovine S protein was not influenced by the synthetic peptide Gly-Arg-Gly-Asp-Ser, which constituted the host cell attachment sequence of S protein. In adherence experiments, prior binding of bovine S protein to S dysgalactiae enhanced streptococcal adherence to bovine epithelial cells. The enhancing effects by bovine S protein were abolished when the respective binding sites on the streptococci were digested by trypsin. Thus, bovine S protein could be an important mediator of adherence of S dysgalactiae to bovine epithelial cells.     

Small molecule lactoferrin with an inflammatory effect but no apparent antibacterial activity in mastitic mammary gland secretion

We have identified various lactoferrin (Lf) molecules in mastitic mammary gland secretions (MGSs), and these Lf molecules were examined for their physiological function in MG. These Lf molecules were isolated by Con A affinity chromatography, and then analyzed by various electrophoresis methods and N-terminal amino acid sequencing. The low Con A affinity Lf was found to have low molecular peptides as compared with the 86 kDa of the high Con A affinity Lf, which is usually detected in healthy MGSs. The N-terminal amino acid sequence of each of the small molecular Lfs were confirmed as fragments of 86 kDa Lf. This low Con A affinity Lf stimulated spleen adherent cells to produce more O(2)(-) than 86 kDa Lf. Furthermore, the low Con A affinity Lf showed low antibacterial activity against E. coli and S. aureus, and had decreased iron-binding capacity in comparison with 86 kDa Lf. Moreover, the 86 kDa Lf could stimulate bovine T cells or macrophages to produce IFN-gamma, IL-4, IL-6, and IL-1alpha. However low Con A affinity Lf induced the production of TNFalpha, but not physiological T cell or macrophage cytokines. It was also found that when the healthy MGs of dry cows were injected with the low Con A affinity Lf, there was an increase in polymorphonuclear cells together with TNFalpha, MCP-1, and IL-8 production. These results suggested that low Con A affinity Lf in mastitic MGSs differed from 86 kDa Lf in physiological characteristics, and, that it induced an inflammatory reaction in MGs.     

Bovine intelectins: cDNA sequencing and expression in the bovine intestine

Intelectins (Itlns) are lectins with potential roles in innate immunity, capable of binding bacteria via galactofuranose residues. Itlns also function as intestinal receptors for the antimicrobial glycoprotein lactoferrin (Lf). Since Lf binds strongly to enterohemorrhagic Escherichia coli O157:H7 (EHEC), we aimed to determine the expression of Lf receptor in terminal rectum, the site of predilection of EHEC in cattle. We sequenced two bovine intelectins (Itln1 and Itln2) and showed that both were expressed in abomasum and rectum, but expression appeared minimal in the jejunum. There was significantly higher expression of Itln2 in terminal rather than proximal rectum. Lactoferrin was expressed in all samples examined. Thus, we have demonstrated two novel bovine Itlns and shown that they are expressed along with Lf in the gastrointestinal tract, where they may interact with microbial pathogens.     

Lactoferrin interaction with retinoid signaling: cell growth and apoptosis in mammary cells

Lactoferrin (Lf) is a multifunctional iron-binding protein that was first identified in mammary secretions, but is synthesized by most mammalian tissues. The protein has a signal sequence that dictates secretion; it also has a nuclear localization sequence that facilitates entry into the cell nucleus. The mechanism of the latter action is currently unknown, but is thought to occur via a Lf receptor. Lactoferrin content of mammary tissue and secretions varies with developmental state; it is synthesized in mammary tissue at high levels during both pregnancy and involution, and during mammary infections. Using fluorescent (FITC)-labeled holo-bLf, we show that bovine primary epithelial cells and MCF-7 breast cancer cells do not translocate the exogenously added Lf to the nucleus after culture in serum free media (SFM). However, the supplementation of SFM with 1 μM all-trans retinoic acid (atRA) caused breast cancer cells to gain the capacity to take up labeled bLf into the cell nucleus. Primary bovine mammary cells (MeBo) exhibited similar capacity in culture. This suggests that in addition to Lf, one or more components modulated by atRA, are necessary for nuclear translocation to occur. Transfection experiments with atRA treated MCF-7 cells containing retinoic acid response element reporter constructs showed that the extracellular application of lactoferrin alters reporter gene expression. Lactoferrin increased a DR5 luciferase response element in a dose-dependent manner only when atRA was applied. Immunocytochemical markers for the cell cycle (Ki67) and apoptotic events (Caspase-3 and PARP-85) showed that lactoferrin alters the atRA-induced phenotype, blocking apoptosis and maintaining cell cycle activity in both MCF-7 and MeBo cells in the presence of 1 μM atRA. We propose that nuclear lactoferrin interacts with retinoic acid signaling pathways in cells and alters/blocks the signals so that cells remain in the cell cycle and/or do not enter the apoptotic pathway.     

Characteristic transfer of colostrum-derived biologically active substances into cerebrospinal fluid via blood in natural suckling neonatal pigs

The characteristic transfer of colostral components into cerebrospinal fluid (CSF) via serum after natural suckling has been studied by sodium dodecyl sulphate (SDS) electrophoresis, two-dimensional electrophoresis, immunoblot and enzyme linked immunosorbent assay methods in non-suckling pigs. Total protein concentrations in the serum increased immediately after first suckling, reached a peak value at 12 h, corresponding to a 2.3-fold compared with pre-suckling level. The protein concentration in CSF also increased and reached peak value at 6 h corresponding to 1.6-fold compared with presuckling level. IgG in serum not detected before suckling, increased steeply after suckling, IgG, IgM and IgA transported into the serum were observed in completely intact form by immunoblot method. The IgG transported into serum was quickly transferred into CSF after natural suckling in contrast to the case of bovine IgG. Serum concentration of transferrin was maintained at high level before suckling and was not changed by suckling. Transferrin also detected in CSF was not changed by suckling. Bovine lactoferrin (Lf) administered into the intestinal lumen was transported into serum (0.01%) and also detected in CSF after 6 h as undegraded form (3.1%). Thus, homologous IgG and bovine Lf are transported into CSF, suggesting that the transport of macromolecules into CSF is selective in neonatal pigs.     

Characterization of leptin binding in bovine kidney membranes

The interactions of leptin with its receptor and other leptin binding sites is not well described or understood. We have used Scatchard analysis of saturation binding data to characterize the affinity of leptin for binding sites in bovine kidney membranes. 125I-Leptin was used in saturation studies, over a range of concentrations from 50 pM to 9 nM. 125I-Leptin differentiated a high affinity binding site from an abundant low affinity site. The high affinity/low density binding site (putative leptin receptor) had K(d)=0.098 nM and B(max)=46.2f mol/mg protein. An additional class of low affinity, highly abundant sites with an apparent K(d)=175 nM, and B(max)=574 fmol/mg protein was characterized. The association and dissociation kinetics for 125I-leptin binding were also studied. Dissociation of the leptin-receptor complex was very rapid, and this necessitated the use of a specially developed separation method for radioligand binding studies (precipitation with PEG and filtration). Competitive displacement of 125I-leptin by mouse and human leptin and polyclonal anti-bovine leptin antibodies was dose-dependent. Specificity of binding was shown as bound 125I-leptin was not displaced by insulin or control antibodies. These data indicate that leptin binds the bovine leptin receptor with high affinity and that a pool of leptin is bound to abundant cell membrane-associated proteins. These observations are consistent with the plasma concentration range for leptin and imply that free leptin concentration in the tissues may be partially buffered by cell-associated and bound forms in plasma. Thus, acute changes in leptin secretion may have little effect at the leptin receptor. The development of leptin agonists/antagonists should facilitate further characterization of leptin binding and clarify the role of abundant low affinity binding sites at the leptin axis.     

Binding of bovine IgG2a and IgG2b allotypes to protein A, protein G, and Haemophilus somnus IgBPs.

Immunoglobulin binding proteins (IgBPs) are thought to be virulence factors which enable pathogens to evade the host's immune response. Since bovine IgG2 is important in protection against pyogenic infections, the binding characteristics of Staphylococcus aureus protein A (PrA), streptococcal protein G (PrG), or Haemophilus somnus high molecular weight IgBPs to the two bovine IgG2 allotypes were examined. For PrA or PrG binding of IgG2, guinea pig red blood cells coated with specific IgG2a or IgG2b antibodies were used in a competitive binding inhibition assay with unlabeled and horseradish peroxidase-labeled PrA or PrG. To determine which sizes of H. somnus. IgBPs bind to the two IgG2 allotypes, immunoblots with H. somnus culture supernatant were probed with anti-DNP IgG2a and IgG2b. This detects only Fc binding because anti-DNP does not cross-react with H. somnus antigens. Both IgG2 allotypes bound equally well to PrA and PrG. However, IgG2b but not IgG2a bound to H. somnus high molecular weight IgBPs. The lack of differential binding of bovine IgG2 allotypes to PrA and PrG means that these IgBPs can be considered to be unbiased reagents in assays for detection of bovine IgG2 or for immunoaffinity purification. The differential binding of H. somnus IgBPs to the IgG2 allotypes indicates that animals having one allotype may be more resistant to H. somnus infection than animals having the other allotype.     

Acquisition of haemoglobin-bound iron by Histophilus somni

Histophilus somni is an important pathogen of cattle and sheep. H. somni requires iron and can use ruminant transferrins as iron sources for growth. Here, we investigated the abilities of bovine (strains 649 and 2,336) and ovine (strains 9L and 3384Y) isolates of H. somni to acquire iron from haemoglobins. Using growth assays, the bovine isolates were shown to acquire iron from bovine haemoglobin, but not from ovine, porcine or human haemoglobins; the ovine isolates, however, failed to use any of these haemoglobins as iron sources for growth. In solid phase binding assays, the bovine isolates, grown under iron-restricted conditions in the presence of bovine haemoglobin, bound not only bovine but also ovine and human haemoglobins. Competition binding assays indicated that all three haemoglobins were bound by the same receptor(s) and SDS-PAGE of membrane fractions revealed that expression of haemoglobin-binding activity was associated with the production of an approximately 120-kDa outer membrane protein. PCR approaches allowed the amplification and sequencing of hgbA, and also hugX and hugZ homologues from strains 649, 9L and 3384Y. While hgbA of strain 649 was predicted to encode an HgbA precursor that is processed to yield a mature, 123.9-kDa haemoglobin-binding protein, the hgbA genes of strains 9L and 3384Y were predicted to give rise to truncated products. RT-PCR experiments revealed that in strain 649, hugX, hugZ and hgbA are co-transcribed and iron-regulated and additional sequencing suggested that in strain 2336, expression of HgbA is subject to phase variation involving a poly C tract within hgbA.     

Evaluation of the protective effect of bovine lactoferrin against lipopolysaccharides in a bovine mammary epithelial cell line

Lactoferrin (Lf) is a non-haem iron-binding glycoprotein with a molecular weight of about 80 kDa, synthesized by glandular epithelial cells and stored in the secondary granules of neutrophils. The physiological significance of Lf is related to non-specific immune defence against pathogens, immunomodulatory activity, iron homeostasis, antioxidant properties and regulation of cell growth. Lf is a bioactive component of the mammary secretions and its modulatory and defensive functions do affect the newborn and the mammary gland as well. In this work a bovine mammary epithelial cell line (BME-UV1) was used as an in vitro model of the bovine mammary epithelium to examine the protective role of exogenous bovine Lf (bLf) against the cytotoxic damage induced by bacterial lipopolysaccharides (LPS) and the endogenous bLf mRNA expression after LPS exposure. In the in vitro model used, exogenous bLf exerts a protective effect against endotoxin cytotoxicity, which could be mediated by the LPS-neutralizing capability of bLf. In addition, in BME-UV1 cells the response to LPS exposure does not involve bLf mRNA expression, suggesting that this cell line lack of functional LPS-responsive elements.     

人初乳与牛初乳中乳脂肪球膜蛋白质组成的对比研究

通过十二烷基硫酸钠-聚丙烯酰氨凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDS-PAGE)将人初乳与牛初乳中乳脂肪球膜蛋白进行分离并结合液-质联用技术鉴定,在人初乳乳脂肪球膜蛋白中鉴定出l 076种蛋白质,牛初乳乳脂肪球膜蛋白中鉴定出682种蛋白质,其中人初乳中有757种特异性表达蛋白质,牛初乳中有363种特异性表达蛋白质,两者有319种相同表达蛋白质.通过基因本体(gene ontolpgy,GO)功能注释分析发现,人初乳乳脂肪球膜蛋白在生物过程中发挥的作用高于牛初乳,尤其表现在细胞组成组织功能上;在分子功能上,人初乳乳脂肪球膜蛋白主要体现在结合作用方面:在细胞组成上,与牛初乳乳脂肪球膜蛋白相比人初乳乳脂肪球膜参与的细胞组成均较多,其中在细胞胞内区的组成上参与最多.通过京都基因与基因组百科全书系统(Kyoto Encyclopedia of Genes and Genomes,KEGG)代谢通路分析可知,人初乳乳脂肪球膜蛋白中有15种蛋白参与了与消化吸收相关的KEGG通路-酶酵解.     

Bovine CD18 identified as a species specific receptor for Pasteurella haemolytica leukotoxin.

Ligand blotting of Pasteurella haemolytica leukotoxin (LKT) susceptible BL3 bovine lymphoma cell membranes with LKT detected two putative receptors with Mr of 95 and 100 kDa, whereas no LKT binding to membrane proteins was detected for LKT non-susceptible human leukemic cells. Anti-bovine CD18 and CD11a/CD18 mAb recognized 95 and 100kDa bands from BL3 cell membranes. CD18 isolated from BL3 cell membranes bound LKT. Pre-incubation of BL3 cells with anti-bovine CD18 or CD11a/CD18 mAb caused partial inhibition of LKT-induced leukolysis. Therefore, we propose that bovine CD18 acts as a species-specific leukocyte receptor for P. haemolytica LKT.     

Correlation between the ability of Haemophilus paragallinarum to acquire ovotransferrin-bound iron and the expression of ovotransferrin-specific receptors.

To investigate the mechanisms of iron acquisition in avian haemophili, strains of Haemophilus paragallinarum and H. avium were tested for siderophore production and utilization of transferrin iron for growth. No evidence of siderophore production was detected in either of these species using a functional screening assay. H. paragallinarum, but not strains of H. avium, was able to acquire iron from 30% saturated chicken and turkey transferrins but not from human, porcine, or bovine transferrins. In response to iron limitation, H. paragallinarum expressed four iron-regulated outer-membrane proteins of 53, 62, 66, and 94 kilodaltons (kDa). Only the 53- and 94-kDa proteins were detected in the H. avium strains. Using affinity methods, the 94- and 53-kDa proteins were isolated specifically by chicken or turkey transferrin, indicating that they may be equivalent to transferrin binding proteins (TBP1 and TBP2, respectively) isolated from other bacterial species. The isolation of the 62- and 66-kDa proteins in association with TBP1 and TBP2 under less stringent washing conditions only in H. paragallinarum implicates these proteins in the iron acquisition process.