John D. Lippolis

Bovine Milk Fat Globule Membrane Proteome

Milk fat globule membranes (MFGM) were isolated from the milk of mid-lactation Holstein cows. The purified MFGM were fractionated using 1-dimensional SDS gels. Tryptic peptides from gel slices were further fractionated on a micro-capillary high performance liquid chromatograph connected to a nanospray-tandem mass spectrometer. Analysis of the data resulted in 120 proteins being identified by two or more unique peptide sequences. Of these 120 proteins, 71% are membrane associated proteins with the remainder being cytoplasmic or secreted proteins. Only 15 of the proteins identified in the cow MFGM were the same as proteins identified in previous mouse or human MFGM proteomic studies. Thus, the bulk of the proteins identified are new for bovine MFGM proteomics. The proteins identified were associated with membrane/protein trafficking (23%), cell signalling (23%), unknown functions (21%), fat transport/metabolism (11%), transport (9%), protein synthesis/folding (7%), immune proteins (4%) and milk proteins (2%). The proteins associated with cell signalling or membrane/protein trafficking may provide insights into MFGM secretion mechanisms. The finding of CD14, toll like receptor (TLR2), and TLR4 on MFGM suggests a direct role for the mammary gland in detecting an infection.

Prevalence of subclinical hypocalcemia in dairy herds.

The prevalence of subclinical hypocalcemia in the transition cow is unknown. Cows with subclinical hypocalcemia have no clinical signs of hypocalcemia but may be more susceptible to other diseases. The objective of this study was to determine the prevalence of subclinical hypocalcemia in the US dairy herds. As a part of the United States Department of Agricultures National Animal Health Monitoring System 2002 Dairy study, serum samples were collected from 1462 cows within 48 h of parturition. The samples were sorted by lactation number: 1st (n=454), 2nd (n=447), 3rd (n=291), 4th (n=166), 5th (n=72), and 6th (n=32). Subclinical hypocalcemia (<2.0 mM) increased with age and was present in 25%, 41%, 49%, 51%, 54%, and 42% of 1st-6th lactation cows, respectively. Cows with serum calcium concentrations >2.0 mM had significantly lower serum non-esterified fatty acids indicating better energy balance than those with subclinical hypocalcemia. Subclinical hypocalcemia may make cows more susceptible to secondary diseases but more research will be required to determine if this is true.

Developmental changes in the milk fat globule membrane proteome during the transition from colostrum to milk.

Shotgun proteomics, using amine-reactive isobaric tags (iTRAQ), was used to quantify protein changes in milk fat globule membranes (MFGM) that were isolated from d 1 colostrum and compared with MFGM from d 7 milk. Eight Holstein cows were randomly assigned to 2 groups of 4 cow sample pools for a simple replication of this proteomic analysis using iTRAQ. The iTRAQ labeled peptides from the experiment sample pools were fractionated by strong cation exchange chromatography followed by further fractionation on a microcapillary high performance liquid chromatograph connected to a nanospray-tandem mass spectrometer. Data analysis identified 138 bovine proteins in the MFGM with 26 proteins upregulated and 19 proteins downregulated in d 7 MFGM compared with colostrum MFGM. Mucin 1 and 15 were upregulated greater than 7-fold in MFGM from d 7 milk compared with colostrum MFGM. The tripartite complex of proteins of adipophilin, butyrophilin, and xanthine dehydrogenase were individually upregulated in d 7 MFGM 3.4-, 3.2-, and 2.6-fold, respectively, compared with colostrum MFGM. Additional proteins associated with various aspects of lipid transport synthesis and secretion such as acyl-CoA synthetase, lanosterol synthase, lysophosphatidic acid acyltransferase, and fatty acid binding protein were upregulated 2.6- to 5.1-fold in d 7 MFGM compared with colostrum MFGM. In contrast, apolipoproteins A1, C-III, E, and A-IV were downregulated 2.6- to 4.3-fold in d 7 MFGM compared with colostrum MFGM. These data demonstrate that quantitative shotgun proteomics has great potential to provide new insights into mammary development.

Null mutation in the gene encoding plasma membrane Ca2+-ATPase isoform 2 impairs calcium transport into milk.

The means by which calcium is transported into the milk produced by mammary glands is a poorly understood process. One hypothesis is that it occurs during exocytosis of secretory products via the Golgi pathway, consistent with the observation that the SPCA1 Ca2+-ATPase, which is expressed in the Golgi, is induced in lactating mammary tissue. However, massive up-regulation of the PMCA2bw plasma membrane Ca2+-ATPase also occurs during lactation and is more strongly correlated with increases in milk calcium, suggesting that calcium may be secreted directly via this pump. To examine the physiological role of PMCA2bw in lactation we compared lactating PMCA2-null mice to heterozygous and wild-type mice. Relative expression levels of individual milk proteins were unaffected by genotype. However, milk from PMCA2-null mice had 60% less calcium than milk from heterozygous and wild-type mice, the total milk protein concentration was lower, and an indirect measure of milk production (litter weights) suggested that the PMCA2-null mice produce significantly less milk. In contrast, lactose was higher in milk from PMCA2-null mice during early lactation, but by day 12 of lactation there were no differences in milk lactose between the three genotypes. These data demonstrate that the activity of PMCA2bw is required for secretion of much of the calcium in milk. This major secretory function represents a novel biological role for the plasma membrane Ca2+-ATPases, which are generally regarded as premier regulators of intracellular Ca2+.

Bovine milk exosome proteome.

Exosomes are 40-100 nm membrane vesicles of endocytic origin, secreted by cells and are found in biological fluids including milk. These exosomes are extracellular organelles important in intracellular communication, and immune function. Therefore, the proteome of bovine milk exosomes may provide insight into the complex processes of milk production. Exosomes were isolated from the milk of mid-lactation cows. Purified exosomes were trypsin digested, subjected offline high pH reverse phase chromatography and further fractionated on a nanoLC connected to tandem mass spectrometer. This resulted in identification of 2107 proteins that included all of the major exosome protein markers. The major milk fat globule membrane (MFGM) proteins (Butyrophilin, Xanthine oxidase, Adipophilin and Lactadherin) were the most abundant proteins found in milk exosomes. However, they represented only 0.4-1.2% of the total spectra collected from milk exosomes compared to 15-28% of the total spectra collected in the MFGM proteome. These data show that the milk exosome secretion pathway differs significantly from that of the MFGM in part due to the greatly reduced presence of MFGM proteins. The protein composition of milk exosomes provides new information on milk protein composition and the potential physiological significance of exosomes to mammary physiology.

Bovine milk proteome: Quantitative changes in normal milk exosomes, milk fat globule membranes and whey proteomes resulting from Staphylococcus aureus mastitis

UNLABELLED Milk protein expression in healthy cows and cows with mastitis will provide information important for the dairy food industry and immune function in the mammary gland. To facilitate protein discovery, milk was fractioned into whey, milk fat globule membranes (MFGM) and exosomes from healthy and Staphylococcus aureus infected cows. Amine-reactive isobaric tags (iTRAQ) were used to quantify protein changes between milk fractions isolated from healthy and S. aureus infected cows. 2971 milk proteins were identified with a false discovery rate of 0.1%. Greater than 300 milk proteins associated with host defense were identified and 94 were significantly differentially regulated in S. aureus infected milk compared to their uninfected controls. These differentially regulated host defense proteins were selectively segregated in the 3 milk compartments examined. An example of this segregation of host defense proteins was the partitioning and high concentration of proteins indicative of neutrophil extracellular traps (NETs) formation in the MFGM preparations from S. aureus infected milk as compared to exosomes or whey. Protein composition changes found in milk exosomes, MFGM and whey during an infection provides new and comprehensive information on milk protein composition in general as well as changes occurring during an infection. BIOLOGICAL SIGNIFICANCE The significance of this study is the identification and quantification of the individual components of the neutrophil extracellular traps (NET) functional proteome in an apparent stable complex with MFGM and/or milk fat globules during an intra-mammary infection. NETs could be functionally relevant in intra-mammary infection, as it is known that during an infection neutrophils ingest large amounts of milk fat that down regulates many of their traditional immune functions. Thus the presence of NETs in milk fat provides new insights to mammary immune function and suggests a role for NETs in clinical mastitis. These in vivo NETs can now be tested to determine if they retain functional antimicrobial activity when primarily associated with milk fat. Then we can estimate their real world functional relevance during an intra-mammary infection, which is one key to understanding clinical mastitis in dairy cows.

Modulation of the bovine innate immune response by production of 1α,25-dihydroxyvitamin D3 in bovine monocytes

In cattle, the kidney has been the only known site for production of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] from 25-hydroxyvitamin D(3) [25(OH)D(3)] by 1alpha-hydroxylase (1alpha-OHase). Based on human studies, it was hypothesized that bovine monocytes could produce 1,25(OH)(2)D(3) upon activation and 1,25(OH)(2)D(3) would regulate expression of vitamin D-responsive genes in monocytes. First, the effects of 1,25(OH)(2)D(3) on bovine monocytes isolated from peripheral blood were tested. Treatment of nonstimulated monocytes with 1,25(OH)(2)D(3) increased expression of the gene for the vitamin D 24-hydroxylase (24-OHase) enzyme by 51+/-13 fold, but 1,25(OH)(2)D(3) induction of 24-OHase expression was blocked by lipopolysaccharide (LPS) stimulation. In addition, 1,25(OH)(2)D(3) increased the gene expression of inducible nitric oxide synthase and the chemokine RANTES (regulated upon activation, normal T-cell expressed and secreted) in LPS-stimulated monocytes 69+/-13 and 40+/-12 fold, respectively. Next, the ability of bovine monocytes to express 1alpha-OHase and produce 1,25(OH)(2)D(3) was tested. Activation of monocytes with LPS, tripalmitoylated lipopeptide (Pam3CSK4), or peptidoglycan caused 43+/-9, 17+/-3, and 19+/-3 fold increases in 1alpha-OHase gene expression, respectively. Addition of 25(OH)D(3) to LPS-stimulated monocytes enhanced expression of inducible nitric oxide synthase and RANTES and nitric oxide production in a dose-dependent manner, giving evidence that activated monocytes convert 25(OH)D(3) to 1,25(OH)(2)D(3). In conclusion, bovine monocytes produce 1,25(OH)(2)D(3) in response to toll-like receptor signaling, and 1,25(OH)(2)D(3) production in monocytes increased the expression of genes involved in the innate immune system. Vitamin D status of cattle might be important for optimal innate immune function because 1,25(OH)(2)D(3) production in activated monocytes and subsequent upregulation of inducible nitric oxide synthase and RANTES expression was dependent on 25(OH)D(3) availability.

Mammary gland involution is associated with rapid down regulation of major mammary Ca2+-ATPases

Sixty percent of calcium in milk is transported across the mammary cells apical membrane by the plasma membrane Ca(2+)-ATPase 2 (PMCA2). The effect of abrupt cessation of milk production on the Ca(2+)-ATPases and mammary calcium transport is unknown. We found that 24 h after stopping milk production, PMCA2 and secretory pathway Ca(2+)-ATPases 1 and 2 (SPCA1 and 2) expression decreased 80-95%. PMCA4 and Sarco/Endoplasmic Reticulum Ca(2+)-ATPase 2 (SERCA2) expression increased with the loss of PMCA2, SPCA1, and SPCA2 but did not increase until 72-96 h of involution. The rapid loss of these Ca(2+)-ATPases occurs at a time of high mammary tissue calcium. These results suggest that the abrupt loss of Ca(2+)-ATPases, required by the mammary gland to regulate the large amount of calcium associated with milk production, could lead to accumulation of cell calcium, mitochondria Ca(2+) overload, calcium mediated cell death and thus play a part in early signaling of mammary involution.

CENTENNIAL PAPER: Proteomics in animal science

Proteomics holds significant promise as a method for advancing animal science research. The use of this technology in animal science is still in its infancy. The ability of proteomics to simultaneously identify and quantify potentially thousands of proteins is unparalleled. In this review, we will discuss basic principles of doing a proteomic experiment. In addition, challenges and limitations of proteomics will be considered, stressing those that are unique to animal sciences. The current proteomic research in animal sciences will be discussed, and the potential uses for this technology will be highlighted.

In Vivo Activation of the Intracrine Vitamin D Pathway in Innate Immune Cells and Mammary Tissue during a Bacterial Infection

Numerous in vitro studies have shown that toll-like receptor signaling induces 25-hydroxyvitamin D3 1α-hydroxylase (1α-OHase; CYP27B1) expression in macrophages from various species. 1α-OHase is the primary enzyme that converts 25-hydroxyvitamin D3 to 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Subsequently, synthesis of 1,25(OH)2D3 by 1α-OHase in macrophages has been shown to modulate innate immune responses of macrophages. Despite the numerous in vitro studies that have shown 1α-OHase expression is induced in macrophages, however, evidence that 1α-OHase expression is induced by pathogens in vivo is limited. The objective of this study was to evaluate 1α-OHase gene expression in macrophages and mammary tissue during an in vivo bacterial infection with Streptococcus uberis. In tissue and secreted cells from the infected mammary glands, 1α-OHase gene expression was significantly increased compared to expression in tissue and cells from the healthy mammary tissue. Separation of the cells by FACS9 revealed that 1α-OHase was predominantly expressed in the CD14+ cells isolated from the infected mammary tissue. The 24-hydroxylase gene, a gene that is highly upregulated by 1,25(OH)2D3, was significantly more expressed in tissue and cells from the infected mammary tissue than from the healthy uninfected mammary tissue thus indicating significant local 1,25(OH)2D3 production at the infection site. In conclusion, this study provides the first in vivo evidence that 1α-OHase expression is upregulated in macrophages in response to bacterial infection and that 1α-OHase at the site of infection provides 1,25(OH)2D3 for local regulation of vitamin D responsive genes.