The bovine milk proteome: what s in it and how can it be manipulated?

Transcription

1 Bovine milk protein The bovine milk proteome: what s in it and how can it be manipulated? Sabrina Greenwood Bovine Milk Proteins (30 35g/L) 80% Casein Casein composition αs1 Casein 32% αs2 Casein 8% Κ Casein 8% β Casein 32% Department of Animal and Veterinary Sciences 20% Whey proteins Whey protein composition β Lactoglobulin 10 12% α Lactalbumin 3 4% > 1000 Other proteins 4 7% Low abundance proteins ( Other ) Thousands identified Examples include: Lactadherin Lactoperoxidase Lactoferrin Some proteins are mammary epithelial cell (MEC) origin, others originate from different tissues and are taken up via the bloodstream Exosome Image from Shennan & Peaker Physiol. Rev. 80: 925. Adapted from de la Torre Gomez et al Slide courtesy of R. Scuderi. Front. Genet. Doi.org/ /fgene Adapted from Shennan & Peaker Physiol. Rev. 80: 925 Adapted from Hernell et al J. Peds. S60.

2 Protein in a milk sample: Visualization after centrifugation GO cellular component analysis of skim milk from early lactation dairy cows synapse, 0.70% cell junction, 2.70% Cream layer Milk fat globule membrane (MFGM) associated whey proteins Skim milk High abundance proteins Caseins (α s1, α s2, β, κ) α Lactalbumin β Lactoglobulin Low abundance proteins extracellular region, 20.10% organelle, 20.80% membrane, 14.50% cell part, 30.20% macromolecular complex, 8.00% extracellular matrix, 3.00% Cell pellet Cellular debris and exosomes Tacoma et al J. Proteomics. 103: 200. GO cellular component comparison across milk fractions 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 10.10% 15.60% 12.50% 14.40% 6.00% 24.30% 15.30% 25.80% 33.50% 34.90% Skim milk Comparison adapted from Greenwood & Honan symposium review In review. Skim milk: Nissen et al Proteomics. 12: 2866; Reinhardt et al J. Proteomics. 82: 141; Boggs et al DiB. 8: 52; and Tacoma et al J. Proteomics. 103: 200. MFGM: Reinhardt et al J. Proteomics. 82: 141; Yang et al., DiB. 3: 12; and Zhang et al PLoS ONE 10(2); e MFGM Synapse Cell junction Extracellular matrix Macromolecular complex Membrane Extracellular region Organelle Cell part GO biological process enrichments in skim milk from early lactation dairy cows Tacoma et al J. Proteomics. 103: 200. biological adhesion, 3.00% locomotion, 1.60% multicellular organismal process, 6.40% immune system process, 2.90% growth, 0.10% metabolic process, 20.40% developmental process, 5.90% cell killing, 0.10% response to stimulus, 10.20% cellular process, 24.90% cellular component organization or biogenesis, 8.10% localization, 7.40% reproduction, 0.40% biological regulation, 8.70%

3 metabolic process, 20.40% biological adhesion, 3.00% locomotion, 1.60% multicellular organismal process, 6.40% immune system cell killing, 0.10% process, 2.90% growth, 0.10% developmental process, 5.90% Early lactation response to biological stimulus, regulation, 10.20% 8.70% cellular component organization or biogenesis, 8.10% cellular process, 24.90% localization, 7.40% reproduction, 0.40% Tacoma et al J. Proteomics. 103: 200. metabolic process, 12.70% biological adhesion, 3.90% multicellular organismal process, 9.80% developmental process, 4.90% Colostrum immune system process, 11.80% response to stimulus, 7.80% biological regulation, 10.80% Tacoma et al J. Dairy Sci. 100: cellular process, 16.70% localization, 13.70% cellular component organization or biogenesis, 7.80% Milk bioactive proteins and peptides Not all milk proteins and peptides are bioactive, but the list is growing quickly Bioactive indicates that the compound (protein) can act on the body of the consumer to exert a biological effect Some milk proteins are considered bioactive as the complete protein (example osteopontin) Digestion of milk proteins (example β casein) by digestive proteolytic enzymes yields mono, di, tri, poly peptides and these peptides can be bioactive Examples of Bioactive proteins and peptides Opioid activity Examples of Bioactive proteins and peptides Opioid activity Examples: segments from alpha casein, kappa casein, alphalactalbumin, lactoferrin, bovine serum albumin Gut development and digestive function Examples: lactoferrin, amylase, bile salt stimulated lipase Immunomodulating and antimicrobial activity Examples: alpha casein fragments, lactoferrin, osteopontin, lactoperoxidase, haptocorrin, zinc alpha 2 glycoprotein (ZAG) From Clare & Swaisgood J. Dairy Sci. 83: 1187 Clare & Swaisgood J. Dairy Sci. 83: 1187; Haschke et al Annals Nutr. Metab. 69: 17.

4 Variation of casein and whey fractions Species comparisons Breed comparisons Stage of lactation differences Milk processing Mastitis Diet Energy restriction Gross nutrient profile Nutrient fractions An example of high abundance proteins affected by diet Nutrient composition, % DM DM, % CP,% of DM RDP RUP ADF NDF e NFC NE L (Mcal/lbs) Tacoma et al JDS. 100: An example of high abundance proteins affected by diet Table 2. Daily dry matter intake (DMI), milk yield and milk components of Holstein dairy cattle fed diets with either a high RDP: RUP ratio (RDP) or low RDP: RUP (RUP) ratio Treatment 1 SE P value RDP RUP Treatment Period DMI (kg/d) Milk yield (kg/d) Milk components yield (kg/d) Fat Protein Milk components (%) Fat Protein Somatic cell count ( 1000) Milk urea nitrogen (mg/dl) <0.01 An example of high abundance proteins affected by diet Table 6. High abundance milk proteins from Holstein dairy cattle fed diets with either a high RDP: RUP ratio (RDP) or low RDP: RUP (RUP) ratio Diet 1 SE P value RDP RUP Treatment Period CN (mg/ml skim milk) β-cn κ-cn Total α-cn α-s α-s Total CN < Whey (mg/ ml skim milk) α-la β-lga β-lgb Total α-la, β-lga, β-lgb Tacoma et al JDS. 100: Tacoma et al JDS. 100:

5 An example of low abundance proteins in skim milk altered by diet An example of low abundance proteins in skim milk altered by diet Ingredient (kg DM/ day): Control GM Corn silage Concentrate Grass silage Mash Grape Marc (GM) Beet Pulp/ soybean hulls (BP) Dry matter (%) andfom (% DM) Crude protein (% DM) Non fiber carbohydrate (% DM) Ether extract (% DM) GM CT content (g CT intake total) Contains wheat midds, pellet steam flaked corn, soybean meal, distillers grains, fine corn meal 2 Contains Ground fine corn grain (28%), Canola meal solvent (12.5%), soybean meal solvent (8.9%), and Rumensin (270 mg/day) Scuderi et al., in review. Accession number Protein Control GM SE P Value Q03247 Apolipoprotein E Q3MHN2 Complement component C P17697 Clusterin P18892 Butyrophilin subfamily 1 member A F1MM32 Sulfhydryl oxidase Q4GZT4 ATP binding cassette sub family G member F1MIT3 von Willebrand factor A domain containing protein M0QW03 TPA: prolactin like protein P01035 Cystatin C P02769 Serum albumin F1MMW8 Serum amyloid A protein F1MNV5 Kininogen P26201 Platelet glycoprotein F1N6D4 Sodium dependent phosphate transport protein 2B F1MUP9 synaptic vesicle membrane protein VAT 1 homolog Q0V8M0 Protein KRI1 homolog Least square means reported for Control and GM groups expressed as relative abundance. SE; standard error Scuderi et al., in review. Affected (>1.5 fold) by dry period length (0 vs 60 d), in week 2. Acyl CoA synthetase NADH cytochrome b5 reductase Affected (>1.5 fold) by DIM (week 14 vs week 2 of 60DP) Changed due to an improvement in EB (red increased as EB improved, proteins in green decreased as EB improved) EB impacts milk profile Milk serum metabolites affected by EB: Lower abundance if improved EB From Lu et al J. Proteome Res. 12: Summary The functional profiles of the various protein fractions are different Many influencing factors, including diet and physiologic states that are commonplace on dairy farms Moving forward, there is opportunity to use these To enhance the quality of milk for human consumers To enhance the healthfulness of milk for calves As animal health biomarkers

6 Thank you Funding USDA NIFA HATCH Grant number VT H02009 National Institutes of Health Grant P20GM (The Vermont Genetics Network Proteomics Facility) Acknowledgments Rink Tacoma, MS graduate Ricky Scuderi, MS graduate Dave Ebenstein, past Greenwood lab technician Dr. Ying Wai Lam, Director of The Vermont Genetics Network Proteomics Facility Julia Fields and Bethany Ahlers, past technicians at The Vermont Genetics Network Proteomics Facility Paul R. Miller Research and Educational Center staff and CREAM students, assistance during animal trials UVM s Paul R. Miller Research and Educational Center