Characterisation of Host Defence Proteins in Milk Using a Proteomic Approach Grant Smolenski,† Stephen Haines,‡ Fiona Y.-S. Kwan,§ Jude Bond,§ Vicki Farr,†,| Stephen R. Davis,†,⊥ Kerst Stelwagen,† and Thomas T. Wheeler*,† Dairy Science and Technology Section, AgResearch, Ruakura Research Centre, East Street, Hamilton, New Zealand, Growth and Development Section, AgResearch, Invermay Campus, Puddle Alley, Mosgiel, New Zealand, and AgResearch-Victoria University of Wellington Proteomics Facility, School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand Received July 11, 2006
Besides providing nutrition to the newborn, milk also protects the neonate and the mammary gland against infection. As well as the six major proteins, bovine milk contains minor proteins, not all of which have been characterized. In this study, we have subjected bovine skim milk, whey, and milk fat globule membrane (MFGM) fractions to both direct liquid chromatography-tandem mass spectrometry (LC-MS/MS), and two-dimensional electrophoresis (2-DE) followed by matrix assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry (MS) of individual protein spots to better characterize the repertoire of minor milk proteins, particularly those involved with host defense. Milk from peak lactation as well as during the period of colostrum formation and during mastitis were analyzed to gain a more complete sampling of the milk proteome. In total, 2903 peptides were detected by LC-MS and 2770 protein spots by 2-DE. From these, 95 distinct gene products were identified, comprising 53 identified through direct LC-MS/MS and 57 through 2-DE-MS. The latter were derived from a total of 363 spots analyzed with 181 being successfully identified. At least 15 proteins were identified that are involved in host defense. These results demonstrate that the proteome of milk is more complex than has previously been reported and a significant fraction of minor milk proteins are involved in protection against infection. Keywords: bovine milk protein • proteomics • 2D electrophoresis • dairy cow
IgA and IgM immunoglobulins in addition to the major milk proteins.2 These confer passive immunity to the newborn.
Introduction Production of milk is a defining feature of mammals. Milk is the sole source of nutrition for the newborn and very young offspring, as well as being an important means of transferral of immunity to pathogens from the mother to the newborn. The major constituents of milk are a small number of very abundant proteins, triacylglycerides, and simple sugars, predominantly lactose. The type of major milk proteins, their amino acid sequence, and their relative abundance varies considerably between species.1 Bovine milk contains six major milk proteins (RS1-, RS2-, β- and κ-caseins, β-lactoglobulin, and R-lactalbumin). The milk secreted immediately after birth (colostrum) has a distinct protein composition, containing IgG, * To whom correspondence should be addressed: Dairy Science and Technology Section, AgResearch, Ruakura Research Centre, Private Bag 3123, Hamilton, New Zealand. E-mail:
[email protected]; Phone: 647-838-5196; Fax: 64-7-838-5628. † Ruakura Research Centre. ‡ Invermay Campus. § Victoria University of Wellington. | Present address: Sensortec Ltd, Waikato Innovation Park, Ruakura Road, PO Box 11004, Hamilton, New Zealand. ⊥ Present address: ViaLactia Biosciences (NZ) Ltd, PO Box 109-185, Newmarket, Auckland, New Zealand. 10.1021/pr0603405 CCC: $37.00
2007 American Chemical Society
The bovine mammary gland has an architecture that is susceptible to infection. The major milk components are synthesized by mammary epithelial cells, which secrete them into the alveolar lumen. A system of branching ducts connects the alveoli to the gland cistern and ultimately to the teat, where milk is expressed upon suckling or milking. This open ductal tree structure makes the mammary gland inherently susceptible to microbial invasion, particularly at the onset of lactation and at weaning/drying off.3 Moreover, the rich nutrient content of milk and body temperature provides optimal growing conditions for microbes, once inside the mammary gland. To counteract this, the gland must have a robust host defense system, one aspect of which is the secretion of antimicrobial proteins and peptides into milk.4 A second host defense function of milk is to protect the neonate against pathogens. Thus, milk contains antimicrobial and immunomodulatory proteins that are active in the digestive tract of the newborn.5-7 Cow’s milk is of great human nutritional and economic significance, yet its repertoire of minor proteins has been only incompletely characterized. Besides the major milk proteins, cow’s milk contains low levels of serum-derived proteins such Journal of Proteome Research 2007, 6, 207-215
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research articles as albumin,8,9 enzymes such as plasmin,10 immunoglobulins, and complement proteins,11 growth factors such as the IGF family,12 and lactoferrin, an iron binding protein that also has antimicrobial properties.13 Some minor milk proteins are known to be present at elevated levels during the period of colostrum secretion and during drying off.14,15 Proteins associated with inflammation have also been detected in milk during infection.16 A range of additional biologically active proteins and peptides have been identified in milk, including some with antimicrobial properties.17 However, these studies are likely to provide only an incomplete picture of the full repertoire of bovine milk host defense proteins. There have been few reports surveying the proteome of milk, and these have not focused on the host defense function of cow’s milk,18,19 or have been limited in their resolution and sensitivity.20,21 The aim of this study was therefore to characterize the proteome of cow’s milk, including from the period of colostrum secretion and during mastitis, using a combination of direct liquid chromatographymass spectrometry (LC-MS), two-dimensional electrophoresis (2-DE) followed by mass spectrometry (MS), and fractionation of milk in order to gain a better understanding of the range of minor proteins present, particularly those involved with host defense.
Experimental Section Milk Samples. Raw bovine milk was obtained from a healthy cow at 1 day after calving (colostrum), from another cow at 10 weeks after calving (peak lactation), and from a third cow during a natural clinical infection of the mammary gland with Streptococcus uberis (mastitic milk). The milk samples were obtained from New Zealand Friesian dairy cows grazed on pasture and milked twice daily. The milk samples were centrifuged at 1500 × g for 20 min immediately after collection, and the fat layer was removed before storage at -20 °C (skim milk). Whey and milk fat globule membrane (MFGM) fractions of each of the above samples were prepared as follows. An aliquot of the skim milk was centrifuged at 100 000 × g for 60 min to pellet the casein micelles, and the clear supernatant (whey) removed and stored at -20 °C. MFGM fraction was produced by sonication and subsequent high-speed centrifugation (100 000 × g for 90 min at 30 °C) of the washed and resuspended fat layer produced by the initial centrifugation, as previously described.22 The pellet from the high-speed centrifugation was resuspended in water, and stored at -20 °C. The protein concentration of all the samples was estimated with a Coomassie blue binding assay based on that of Bradford23 using a commercially produced reagent (Bio-Rad). Direct LC-MS of Milk Samples. For direct LC-MS analysis of the samples, aliquots of skim milk, whey, and MFGM fraction from each of colostrum, peak lactation milk, and mastitic milk were digested with trypsin (Promega cat # V5111) following the method of Kintner and Sherman.24 Each sample was analyzed in triplicate and the peptides reported were those detected above the threshold parameters in all replicates. In brief, aliquots that contained 1 mg protein were freeze-dried and were then reconstituted to a concentration of 10 mg/mL in a 6 M solution of urea in 100 mM Tris. HCl, pH 7.8. Reduction was performed with dithiothreitol (5 µL, 200 mM in 100 mM Tris.HCl, pH 7.8), followed by alkylation using iodoacetamide (20 µL, 200 mM in 100 mM Tris.HCl, pH 7.8), each for 1 h at ambient temperature. Excess iodoacetamide was then consumed by incubation with a second aliquot (20 µL) of dithiothreitol for 1 h at ambient temperature. After dilution with 208
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water (775 µL), samples were incubated overnight at 37 °C in the presence of a 1:50 ratio of trypsin (Promega, cat # V5111). The samples were then acidified by the addition of 10 µL acetic acid and injected onto an Aqua 2.0 × 150 mm C18 reverse phase HPLC column (Phenomenex, U.S.A.) at a flow rate of 200 µL/min. Separation of tryptic digests was performed using a WellChrom HPLC system (Knauer) that comprised a K-1001 pump, K-1500 solvent organizer, high pressure mixer, and a Degasys inline vacuum degasser (Uniflow). A linear gradient was employed from 100% A to 60% B over 90 min, where A was 0.5% acetic acid and B was 80% acetonitrile containing 0.5% acetic acid. Analysis by LC-MS employed an LCQ Duo ion trap mass spectrometer (Thermo Finnigan) fitted for electrospray ionization. The instrument was set to positive ion mode, with data acquired using the “triple play” method (full scan MS over the range 50 to 2000 m/z, followed by data-dependent Zoom and MS/MS scans of the most abundant ions). Individual peptides present in the sample were analyzed using TurboSEQUEST (BioWorks v3.1, ThermoFinnigan) using an indexed subset of the NCBI non-redundant protein sequence database, based on the presence of the keywords “Homo sapiens”, “Bos taurus”, “Ovis aries”, “Capra”, “Mus muscularis”, and “Rattus” in the database entry. Proteins were accepted as being present in the sample if the presence of four or more distinct peptides were detected, each having Xcorr scores of greater than 1.65 for a +1 ion, 2.2 for a +2 ion, or 3.7 for a +3 ion. When three or less peptides for a given protein were identified, it was accepted as being present if these peptides had a delta correlation score of greater than 0.1, and an Sp score of greater than 400, if they were unique to the identified protein, and if at least 75% of the total number of predicted b and y ions were detected in the MS/MS spectrum. The data presented in the supplementary tables contains amino acid sequence together with the amino acids N- and C-terminal to that peptide’s sequence, the precursor mass and charge, and probability scores, thus meeting a previously published set of guidelines for the reporting of one-peptide hits.25 The falsepositive rate for the protein identifications was estimated by subjecting 542 dta files to a reversed mammalian protein database using the BioWorks v3.2 software package. This resulted in hits for 16 of the dta files, a false positive rate of 2.9%. 2-D Electrophoresis. Samples were subjected to 2-DE using flat bed iso-electric focusing in precast IPG strips (18 cm, pH 3-10, Amersham Biosciences) in the first dimension and discontinuous SDS electrophoresis gels in the second dimension using the procedure outlined in the Amersham 2-DE user manual. Each sample was analyzed in triplicate. All the spots chosen for MS analysis were present in all the replicate gels. A volume of sample solution equivalent to the target protein load was made up to 120 µL with 2-D lysis solution (8 M urea, 3 M thiourea, 65 mM DTT, 65 mM CHAPS), to which was added 225 µL rehydration solution (8 M urea, 2% (w/v) CHAPS, 6 mM DTT) and 5 µL of DeStreak reagent (Amersham). Gels were loaded with 40 µg of skim milk, 40 µg of whey, or 250 µg of MFGM proteins. This mixture (350 µL) was used to passively rehydrate the IPG strip. Focusing was carried out using an IEF Cell System (Bio-Rad) for 17 h at 20 °C to a total of 62 830 Vh. Upon completion of the first dimension, strips were incubated with gentle shaking in two changes of equilibration buffer, containing 0.25% (w/v) dithiothreitol for 20 min for the first wash and 2.5% (w/v) iodoacetamide for 20 min for the second
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wash. The equilibration buffer contained 50 mM Tris. HCl, pH 6.8, 6 M urea, 30% (v/v) glycerol, and 2% (w/v) SDS. For the second dimension, strips were transferred to the tops of discontinuous 12.5% (w/v) polyacrylamide gels, incorporating a stacking buffer.26 Gels were run for 5 h at 30 mA per gel, then fixed and stained with a colloidal Coomassie G-250 stain as previously described.27 After staining, the 2-D gels were washed in a solution containing 5% (v/v) methanol, the image captured using a GS-800 calibrated densitometer (Bio-Rad), and the spots of interest excised as described below. Spot detection was performed using the PDQuest v6.0 software package (Bio-Rad). Identification of Proteins from 2-D Gel Spots. Selected proteins detected as spots on the 2-D gels were subjected to in-gel trypsin digestion. The gel pieces were excised as approximately 1-3 mm2 pieces using a scalpel, rinsed with water, and destained with three washes of a solution containing 50 mM ammonium bicarbonate, pH 7.8 and 50% (v/v) acetonitrile, for a minimum of 3 h. The gel pieces were then dried under vacuum and rehydrated with modified trypsin (Promega, cat # V5111) in 50 mM ammonium bicarbonate, pH 7.8. After 15 min, 10-25 µL of 50 mM ammonium bicarbonate, pH 7.8, was added and the mixture was incubated overnight at 37 °C with shaking. The peptides were then extracted, first with 20 µL of 25 mM ammonium bicarbonate, pH 7.8, twice more with 20 µL of a solution containing 50% (v/v) acetonitrile and 0.25% (v/v) TFA, and then finally with 20-100 µL acetonitrile. For LCion trap MS, the pooled extracts were lyophilised then resuspended in 25 µL 5% (v/v) formic acid/50% (v/v) acetonitrile. A portion (5 µL) of this peptide mixture was injected onto a 75 µm id × 15 cm PepMap reverse phase C18 HPLC column (LC Packings, San Francisco, CA) and resolved at a flow rate of 400 nL/min with a linear gradient from 2 to 80% B (acetonitrile + 0.1% (v/v) formic acid) over 50 min. Solvent A was 0.1% (v/v) aqueous formic acid. Tandem MS was performed in-line with the eluate using an ion trap mass spectrometer fitted with a nanospray interface (LCQ Deca, ThermoFinnigan, San Jose, CA). The mass spectrometer was operated in the positive ion mode and the mass range acquired was m/z 300-1800. Data was acquired using a top 2 experiment in data-dependent mode with dynamic exclusion enabled. Data analysis and identification of proteins was performed as described above. For matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) MS, the resuspended peptide mixtures were concentrated and desalted using a microscale reverse phase matrix packed into a pipet tip (ZipTip, Millipore) using the manufacturer’s instructions. The peptides were eluted from the tip with 3 µL of 100% (v/v) acetonitrile, dried under vacuum and stored at -20 °C. The peptides were then resuspended in a 2 µL aliquot of matrix (12 mg/mL), freshly prepared by vortexing 13 mg R-cyano-4-hydroxycinnamic acid per mL of a solution containing 50% (v/v) acetonitrile and 0.2% (v/v) TFA. This peptide-matrix solution mixture was spotted onto a target plate and air-dried. The samples were then analyzed by MALDITOF MS using a Voyager DE-PRO mass spectrometer (Applied Biosystems) fitted with a 337 nm laser and operated in reflectron mode. Spectral data was collected in the range of 700-3500 daltons using positive ionization. Masses were internally calibrated using at least two known trypsin autolysis peaks (m/z ) 805.4167, 906.5048, 1020.5034, 2163.0566, 2273.1599 for bovine trypsin). Calibrated peak mass lists were applied to peptide mass fingerprint searches using either one or both of two search engines; ProFound (genomic solutions http://
65.219.84.5/service/prowl/profound.html) and MASCOT (Matrix Science http://www.matrixscience.com/). Searches were performed against the mammalian subset of the non-redundant NCBI protein sequence database allowing for up to 200 ppm error tolerance and up to one missed trypsin cleavage site. For both search engines, cysteine iodoacetylation (carbamidomethyl, +57) was selected as a fixed modification, whereas oxidation on methionine (+16) and lysine acetylation (+42) were selected as variable modifications. Proteins were identified by MASCOT using the probability based MOWSE score (scores with a p < 0.05 are considered significant), or with ProFound (if the p value was less than 0.001). Scores, sequence coverage, and the number and sequence of detected peptides are presented in the supplementary data tables.
Results LC-MS Analysis of Milks. Nine bovine milk, colostrum, and MFGM samples were prepared for direct LC-ion trap MS analysis as described in the Methods in order to assess the range of proteins present. The samples comprised skim milk, whey, and MFGM preparations from each of three samples: peak lactation milk, colostrum, and milk from a cow with mastitis. Each sample was digested with trypsin and subjected to LC-ion trap mass spectrometry in triplicate to identify the range of proteins and peptides present in the samples. In total 2903 peptides were detected. As expected, most corresponded to the six major bovine milk proteins (RS1-casein, RS2-casein, β-casein, κ-casein, β-lactoglobulin, and R-lactalbumin). However, 146 peptides corresponded to the products of 53 distinct additional genes (Table 1). Among them are proteins that have been previously identified in milk, such as lactoferrin, lactoperoxidase, and immunoglobulins, as well as proteins previously not identified in milk. Some of these proteins were identified only in specific fractions or in specific types of milk. The 53 identified minor milk proteins were each assigned to one of eight functional categories, according to their Gene Ontology classifications28 (see Table 1), to gain an understanding of the physiological roles that these proteins may play in milk. Host defense/immune related, enzyme, structural, transport, DNA binding and signal transduction proteins were approximately equally represented (19, 13, 17, 15, 11, and 11% respectively), whereas no chaperonins were detected. An additional 13% of the proteins were of unknown or unclassified function. 2-D Electrophoresis of Milks. An alternative technological approach, 2-D electrophoresis, was used to identify minor milk proteins in the samples. Individual proteins were resolved by 2-D electrophoresis, visualized by Coomassie blue staining and subsequently identified by either MALDI-TOF or LC-ion trap tandem MS as described in the Methods. The pattern of spots obtained for eight of the nine samples is shown in Figure 1. Some samples produced relatively simple patterns of spots (panels A-D), whereas others resulted in more complex patterns with many more minor proteins visualized (panels E-H). In total, 2770 distinct protein spots were detected in 2-D gels loaded with the nine samples after Coomassie blue staining followed by analysis with the PDQuest 2-D gel analysis package. Of these, 363 were selected for analysis by MS, based on their abundance, position relative to other spots on the gel, and nonobvious identity. In total, 181 spots were successfully identified, using either MALDI-TOF (152 out of 331 spots analyzed) or LCion trap (29 out of 32 spots analyzed). Of these, 37 were identified as isoforms of major milk proteins, while 144 spots Journal of Proteome Research • Vol. 6, No. 1, 2007 209
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Table 1. Unique Gene Products Identified as Minor Proteins in Bovine Milk protein no.
no. spotsa (peptides)
1 2 3 4 5 6 7 8 9 10 11 12 13
1 1 13 (3) 10 (6) 5 (15) 1 (1) 4 2 4 (2) 1 1
AAB22943 NP_694762 P60712 NP_776405 NP_851335 P20000 BAC86721 NP_786978 NP_777141 P15497 XP_137955 NP_776651 NP_777109
14 15 16
4 (2) 6 (13) 1
AAP78964 CAB07533 NP_033927
17 18 19 20
1 1 6 (1)
BAB17767 NP_776946 NP_777250 BAA08224
21 22 23 24 25 26 27
1 (1) (1) (1) 1 2 (1)
AAB64304 BAC27438 CAB63941 NP_001013764 Q29092 NP_776474 AAC53095
28 29 30 31 32 33 34 35 36 37
6 (12) (1) 1 6 2 (1) 1 (1) 1 1
NP_776738 P29701 P02672 P02676 P12799 AAH58808 NP_776758 AAH39263 NP_058710 P10096
38 39
1 12 (5)
AAC50556 S65138
40
2 (3)
NP_777253
41 42 43 44
2 (3) 2 1
AAC72250 BAC56329 NP_776770 NP_071705
45 46 47 48 49 50 51 52 53 54 55 56 57 58 59
1 1 (2) 1 (1) (8) (1) 2 (9) 1 (9) (1) 1 1 (1) 1 (1)
AAA18337 BAA32525 NP_778235 NP_078982 NP_057000 AAC98391 AAN07166 AAB37381 AAN60017 AAQ88452 NP_851355 CAA82315 BAA34178 P13645 BAB13412
GenBank acc. no.
protein name
14-3-3 protein zeta chain 5-oxoprolinase (ATP-hydrolyzing) actin, cytoplasmic 1 (beta-actin). adipose differentiation-related protein Albumin (precursor) aldehyde dehydrogenase (NAD) 2 precursor ankyrin 3, node of Ranvier (ankyrin G) annexin 1 annexin A2 apolipoprotein A-I apolipoprotein B ARP3 (actin-related protein 3, yeast) homolog ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit beta-2-microglobulin precursor (lactollin) butyrophilin, subfamily 1, member A1 capping protein (actin filament) muscle Z-line, alpha 1 casein kinase 1, alpha 1 coronin, actin binding protein, 1A cathelicidin 1 (Bactenecin 1) CD36 antigen [collagen type I receptor, thrombospondin receptor] Chitinase-like protein 1 (CLP-1) DEAD (Asp-Glu-Ala-Asp) box polypeptide 54 deleted in malignant brain tumors 1 diacylglycerol kinase kappa endoplasmin precursor (GRP94/GP96) enolase 1 eukaryotic translation initiation factor 4, gamma 2 fatty acid binding protein, heart-type (MDGI) fetuin fibrinogen alpha chain fibrinogen beta chain precursor fibrinogen gamma- B chain precursor gene model 440, (NCBI) glucose regulated protein 58kD glutamate receptor, ionotropic, delta 1 glutathione S-transferase, mu 1 glyceraldehyde-3-phosphate dehydrogenase (GAPDH) glycerol-3-phosphate dehydrogenase 2 glycoprotein antigen MGP57/53 (Lactadherin/bP47 protein) glycosylation-dependent cell adhesion molecule 1 (lactophorin/PP3) guanine nucleotide binding protein, beta 2 H3 histone, family 3A heat shock 70 kDa protein 8 heat shock 70kD protein 5 (glucose-regulated protein) heat shock protein 27 heat shock protein 70 kDa protein 1A histone 2, H2ab zinc finger protein 668 hypothetical/unnamed protein LOC51063 immunoglobulin IgA immunoglobulin IgD immunoglobulin IgG immunoglobulin IgM IRTA2 isocitrate dehydrogenase 1 (NADP+), soluble keratin 9 keratin complex 2, basic, gene 6a keratin, type I cytoskeletal 10 KIAA1586 protein
condition presentc
milk fraction presentd
2D-M 2D-M dL, 2D-M, 2D-L dL, 2D-M, 2D-L dL, 2D-M 2D-M dL 2D-M 2D-M 2D-M dL 2D-M 2D-M
M C C, L, M M, C, L C, M, L M M M L, C M, L L, M M M
M M S, W, M M S, W M S M M S, M W, M M M
S E ST U T E ST ST ST T T ST E
dL, 2D-L dL, 2D-M 2D-M
C C, L, M M
M, W M M
D/I U ST
2D-M 2D-M dL, 2D-M, 2D-L dL
L M M C
W M M M
S ST D/I T
2D-M dL dL dL 2D-M 2D-M dL
L C, L M L C M L
W W M W M S, M W
U B U S C E S
dL, 2D-L dL 2D-M 2D-M 2D-M dL 2D-M dL 2D-M 2D-M
C, L, M C M M M M M C L M
M W M M M S, M M M M M
T E ST ST ST ST C S E E
2D-M dL, 2D-M
M M, C, L
M M
E S
dL, 2D-M, 2D-L
M, C, L
W, M
ST
2D-M dL 2D-M 2D-M
L, C M L, M M
M M M M
S B C C
2D-M 2D-M dL 2D-M dL dL dL dL, 2D-M dL, 2D-M dL 2D-M 2D-M dL 2D-M dL
C M M C M C L, M M, L, C M, L, C M L M C C M
M M M W M W W, M S, W, M S, W, M S M W W W M
C C B B T D/I D/I D/I D/I D/I E ST ST ST B
detection-ID methodb
Gene Ontology classe
a Number of 2D gel spots identified as the gene product, or number of peptides identified as the gene product through direct LC-MS. One peptide hits should be regarded as tentative (see Methods for threshold parameters). b 2D electrophoresis followed by MALDI-TOF (2D-M), direct LC-MS (dL), or 2D electrophoresis followed by LC-MS (2D-L). c Milk from peak lactation (L), colostrum (C), and milk from a mastitic cow (M). d Milk fraction in which the protein was identified is denoted; skim milk (S), whey (W), and milk fat globule membrane (M). e The functional classification to which the protein was assigned. Categories are (B) DNA binding protein, (C) chaperone, (D/I) defense and immunity, (E) enzymes, (S) signalling, (ST) structural, (T) transport, and (U) unknown function.
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Host Defence Proteins in Milk Table 1. (Continued) protein no.
no. spotsa (peptides)
GenBank acc. no.
60 61 62
2 (5) (1) 2
AAA30617 NP_776358 BAA07085
63 64 65
1 (1) (1)
P21758 CAC81810 AAH30946
66 67 68 69 70
(3) 2 (1) (3) (1)
NP_004534 AAA36383 NP_080407 NP_776998 NP_573512
71 72
1 (7) 1
P81265 NP_776560
73 74 75 76 77 78
1 (1) (1) 1 (3) 1 (5) 3 (8)
NP_001029744 XP_216318 NP_766387 XP_611685 XP_593653 NP_777076
79 80
(1) (1)
BAB14869 NP_776307
81 82 83 84 85
1 (1) (1) (1) (1) (1)
P42819 XP_218444 XP_224295 XP_219779 NP_777086
86 87 88 89 90 91 92 93 94 95
(1) 5 (2) 4 1 4 1 (1) (18) (1) (1)
BAC55524 NP_803450 AAH24038 NP_776642 NP_776394 NP_003365 AAB33562 CAA67117 XP_291202 BAA33727
protein name
lactoferrin lactoperoxidase lymphocyte cytosolic protein 1 (65K macrophage protein/L-plastin) macrophage scavenger receptor types I and II mucin 1 NADH dehydrogenase (ubiquinone) flavoprotein 2 nebulin nucleobindin 1 PDZ and LIM domain 7 peptidoglycan recognition protein peroxisome proliferator-activated receptor gamma coactivator 1 beta polymeric-immunoglobulin receptor precursor procollagen-proline, 2-oxoglutarate 4-dioxygenase prohibitin ribosomal protein L7 RIKEN cDNA 4932418K24 gene S100 calcium binding protein (calgranulin B) S100 calcium binding protein A11 (calgizzarin) S100 calcium binding protein A12 (calgranulin C) SCY1-like 2 (H. sapien) serine (or cysteine) proteinase inhibitor, clade A serum amyloid A protein similar to FKSG27 (predicted) similar to KIAA0853 protein (predicted) similar to KIAA2026 protein (predicted) solute carrier family 34 (sodium phosphate), member 2 titin/conectin transferrin tubulin, beta, 2 villin 2 vimentin voltage-dependent anion channel 1 WC1 xanthine dehydrogenase zinc finger protein 479 zinc metallopeptidase (STE24 homolog, yeast)
were classed as minor milk proteins. These 144 spots corresponded to 57 distinct gene products, and they are listed in Table 1. Supprisingly, only 15 distinct gene products were detected by both direct LC-MS and 2DE-MS (Table 1). Thus, combining the results from the two technologies resulted in a set of minor milk proteins derived from 95 distinct genes. Each protein within the set of 57 identified from 2-D gels was assigned to one of eight functional categories using the same approach as described above for those identified by LCMS. The category having the greatest number of proteins was the structural proteins (15 out of 57, 26%). Three other categories were significantly represented: these are enzymes (9 out of 57, 16%), transport proteins (8 out of 57, 14%), and host defense/immune-related proteins (8 out of 57, 14%). Interestingly, six distinct chaperone proteins were identified, comprising 10% of the total number of identified proteins. All of these were found in the MFGM from mastitic milk.
Discussion The investigation reported here is, to our knowledge, the most comprehensive characterization to date of minor proteins
detection-ID methodb
condition presentc
milk fraction presentd
Gene Ontology classe
dL, 2D-M dL 2D-M
L, M L, C M
S, W, M W M
T E D/I
2D-M dL dL
L M C
M M W
D/I ST E
dL 2D-M dL dL dL
C C, M M M M
W W M S, M M
ST D/I U D/I B
dL, 2D-M 2D-M
C, L, M M
W, M M
D/I E
2D-M dL dL 2D-L 2D-L 2D-L
M M M M M M
M M S M M M
S ST E T T T
dL dL
M C
M W
S E
dL, 2D-M dL dL dL dL
C, M M L M M
M S W W M
U S U U T
dL dL, 2D-M 2D-M 2D-M 2D-M 2D-M dL dL dL dL
M C, M, L M, C M M M L C, L, M C C, L
M W, S M M M M S M S W
ST T ST ST ST S D/I E B E
in bovine milk. It is the first report that applies both direct LCtandem MS and 2-DE-MS to a range of bovine milk samples and subfractions derived from them. The majority of the minor milk proteins reported in previous proteomic studies of milk29-32 were also found to be present in bovine milk in the current study. However, in part as a result of the parallel analysis strategy employed, this study also identified many additional proteins that have not been previously reported to be in bovine milk. The complexity of the milk proteome that has become apparent from this study highlights the multiple functions of milk, which besides providing nutrition to the neonate, also promotes the health and development of the newborn and helps to maintain optimal mammary gland function. A significant proportion of the identified minor milk proteins have a function associated with host defense. These are listed in Table 2. In total, 13 proteins were detected that were classified as host-defense using the Gene Ontology criteria (see Table 1). The Gene Ontology classifications, however, do not fully account for the multiple functions of some milk proteins. For example, lactoferrin, lactoperoxidase and xanthine dehyJournal of Proteome Research • Vol. 6, No. 1, 2007 211
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Figure 1. Two-dimensional gels loaded with 40 µg protein from (A) skim milk from peak lactation, (B) whey from peak lactation, (C) whey from colostrum, (D) whey from a cow with mastitis, (E) skim milk from a cow with mastitis, (F) MFGM fraction from a cow with mastitis, (G) MFGM from colostrum, and (H) MFGM from peak lactation. The gels were stained with colloidal Coomassie blue. Spots denoted by letters are (A) RS1-casein, (B) RS2-casein, (C) β-casein, (D) κ-casein, (E) R-lactalbumin, and (F) β-lactoglobulin as identified by comparison with previously published 2d gels, or in this study by western blotting and/or mass spectrometry. The encircled spots were subjected to mass spectrometry. The number associated with some encircled spots denotes its identity (protein number in Table 1). 212
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research articles
Host Defence Proteins in Milk Table 2. Defence-Related Proteins protein no.
acc. no.
protein
19 21 25
NP_777250 AAB64304 Q29092
cathelicidin 1 (Bactenecin 1) Chitinase-like protein 1 (CLP-1) endoplasmin precursor (GRP94/GP96)
34 43
NP_776758 NP_776770
glucose regulated protein 58kD heat shock 70 kDa protein 8
44
NP_071705
45 46
AAA18337 BAA32525
50 51 52 53 54 60 61 62
AAC98391 AAN07166 AAB37381 AAN60017 AAQ88452 AAA30617 NP_776358 BAA07085
63
P21758
67 69 76
AAA36383 NP_776998 XP_611685
77
XP_593653
78
NP_777076
81 93
P42819 CAA67117
heat shock 70kD protein 5 (glucose-regulated protein) heat shock protein 27 heat shock protein 70 kDa protein 1A immunoglobulin IgA immunoglobulin IgD immunoglobulin IgG immunoglobulin IgM IRTA2 lactoferrin lactoperoxidase lymphocyte cytosolic protein 1 (65K macrophage protein/L-plastin) macrophage scavenger receptor types I and II nucleobindin 1 peptidoglycan recognition protein S100 calcium binding protein A9 (calgranulin B) S100 calcium binding protein A11 (calgizzarin) S100 calcium binding protein A12 (calgranulin C) serum amyloid A protein xanthine dehydrogenase
drogenase are not classed as host defense-related, despite clearly having such a role.4,33,34 The S100 calcium binding protein (CaBP) family, classed by Gene Ontology as transport proteins, may also have a role in host defense.35 Our study identified three members of the S100 CaBP family in mastitic MFGM, two of which have also been identified recently in colostrum.19 The chaparonins have recently been described to also have a role in pathogen recognition36 and in this study 6 were identified in mastitic samples. Thus, the number of defense related proteins is likely to be an underestimate. The 24 defense-related proteins listed in Table 2 include some that have been previously identified in milk, such as the immunoglobulins. This is the first report showing that the neutrophilassociated proteins cathelicidin, peptidoglycan recognition protein, lymphocyte cytosolic protein 1, and macrophage scavenger receptor types I and II are present in cows’ milk. These proteins are most likely secreted from neutrophils that have infiltrated the mammary gland. The number of neutrophils in milk increases significantly during mammary gland infection. Two lymphocyte proteins, IRTA2 and nucleobindin, were also identified in milk as part of this study. These proteins have also been implicated in host defense processes.37,38 Their origin is most likely lymphocytes that have migrated into the mammary gland. The functional significance of these proteins in host defense could be tested by further studies involving their direct effect on microbes or in modulating the responsiveness of immune cells to their presence.
function
reference
antimicrobial peptide eosinophil chemotactic properties, participates in the assembly of antibody molecules and signalling molecule for polymorphonuclear neutrophils regulates signalling by interacting with stat3 activated through proinflammatory response mechanisims enhancing MMP-9 expression in monocytic cells Upregulation in macrophages upon IL-4 stimulation
40 41 42
inhibitor of neutrophil apoptosis stress response (refolding and degredation of denatured proteins) antigen recognition antigen recognition antigen recognition antigen recognition B-cell immunoglobulin super-family receptor iron binding and antimicrobial peptide “lactoferricin” oxidative peroxidase activity regulation of neutrophil integrin function mediate the binding, internalization, and processing of negatively charged macromolecules promotes production of DNA-specific antibodies innate immunity pattern recognition molecule associated with S100A8 and implicated in inflammatory response Upregulation associated with proinflammatory response Antimicrobial peptide “calcitermin” involved in acute phase cytokine signalling superoxide anion, hydrogen oxide and peroxynitrite production
43 44
45 46 47
48 49 50 51 52 53 54 55 56 35 57 34
Somewhat surprisingly, this study also identified a number of DNA-binding proteins, enzymes, transport proteins and structural proteins that would not be expected to be present in milk. One possible explanation for this could be that they are the consequence of the shedding and subsequent lysis of somatic cells in milk. These somatic cells could be the result of epithelial cells sloughed from the mammary gland, or they could be due to the well-documented recruitment of neutrophils and other circulating immune cells to the gland in response to infection. We have observed that a proportion of these cells are embedded in the cream layer after initial centrifugation of milk. This may be the reason why an increased abundance of major cellular proteins is observed in the MFGM fraction, particularly in the mastitic milk. It is not clear whether these proteins play a significant functional role in the mammary gland or neonatal digestive tract. The MFGM protein fraction is significantly more complex compared with the other milk fractions. This most likely reflects the diversity of proteins associated with this specialized membrane, which is derived from the apical plasma membrane of the mammary epithelial cell39 as well as the presence of somatic cells in the fat layer as described above. It is possible that the apical membrane, among its many functions, represents a first line of defense against pathogen colonisation at the mucosal surface of the mammary alveolus. Therefore, it is not surprising that many of the host defense related proteins identified in this study were associated with the MFGM. Furthermore, there appear to be significant differences in the Journal of Proteome Research • Vol. 6, No. 1, 2007 213
research articles repertoire of MFGM proteins between the milks taken from the healthy and the mastitic cow. Verification of these apparent responses to infection will require further investigation with a larger number of samples. There are likely to be more proteins present in milk than were detected in this study. Both technical approaches employed (2-DE and LC-MS/MS) are limitedsthe former by the sensitivity of Coomassie blue staining and the latter by the resolution of capillary LC and the sensitivity of the mass spectrometer used. These two methods identified quite distinct sets of proteins, with only 15 distinct gene products out of 95 being detected by both methods. This low degree of overlap would not be expected if each method alone had detected a high proportion of the total number of proteins present in milk. Moreover, only 35% of the spots cut out of the gels were successfully identified. The success rate for protein identification was likely to be limited by the incompleteness of bovine protein databases, the omission of microbial databases from the searches, and the relatively high threshold parameters used to define a positive identification. Whereas some of the unidentified spots are likely to represent additional isoforms of the 95 gene products already identified, others may represent additional “novel” milk proteins. Therefore, it seems likely that the use of additional samples, more sensitive protein stains for 2-DE, a more thorough and comprehensive analysis of spots using multiple configurations of MS, and more powerful multidimensional LC procedures in line with a higher performance mass spectrometer and advanced data analysis techniques such as de novo peptide sequencing could well reveal the presence of many additional proteins. Peptide and protein matches were attempted against a database that was species restricted and excluded microbial sequences, because we wished to identify only proteins of bovine origin since this study is focused on the diversity of bovine derived gene products in the mammary gland rather than understanding host-pathogen interactions. Thus, additional microbial-derived milk proteins could be identified in a future expanded study by employing an expanded protein database.
Conclusion This study has shown that milk is a more complex biological fluid than has previously been assumed and that a significant proportion of this complexity is devoted to defense of the mammary gland and/or the neonate against pathogens. This study therefore provides a springboard from which to understand the role of those milk proteins in preventing and combating infections in the mammary gland and neonate and to explore their utility as the basis for novel pharmaceuticals and functional food ingredients.
Acknowledgment. We acknowledge the assistance of Marita Broadhurst, Brendan Haigh, and Paul Harris in the preparation of the manuscript. Tandem MS identification of 2D gel spots was performed by Janine Cooney and Dwayne Jensen, HortResearch, Ruakura. MALDI-TOF was carried out by the VUW-AgResearch Proteomics Facility at Victoria University of Wellington. We acknowledge the role of Dr. T. W. Jordan in establishing and maintaining this facility. References (1) Forsyth, I.; Hayden, T. J. Comparative endocrinology of mammary growth and lactation. In Comparative Aspects of Lactation; Peaker, M., Ed.; Academic Press: London, 1977; Vol. 41, pp 135-163.
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Smolenski et al. (2) Farrell, H. M., Jr.; Jimenez-Flores, R.; Bleck, G. T.; Brown, E. M.; Butler, J. E.; Creamer, L. K.; Hicks, C. L.; Hollar, C. M.; Ng-KwaiHang, K. F.; Swaisgood, H. E. Nomenclature of the proteins of cows’ milk-sixth revision. J. Dairy Sci. 2004, 87 (6), 1641-1674. (3) Fetherston, C. M.; Lee, C. S.; Hartmann, P. E. Mammary gland defense: the role of colostrum, milk and involution secretion. Adv. Nutr. Res. 2001, 10, 167-198. (4) Sordillo, L. M.; Shafer-Weaver, K.; DeRosa, D. Immunobiology of the mammary gland. J. Dairy Sci. 1997, 80 (8), 1851-1865. (5) Goldman, A. S. The immune system of human milk: antimicrobial, antiinflammatory and immunomodulating properties. J. Pediatr. Infect. Dis. 1993, 12 (8), 664-671. (6) Garofalo, R. P.; Goldman, A. S. Expression of functional immunomodulatory and anti-inflammatory factors in human milk. Clin. Perinatol. 1999, 26 (2), 361-377. (7) Reiter, B. Review of the progress of dairy science: antimicrobial systems in milk. J. Dairy Res. 1978, 45 (1), 131-147. (8) Stelwagen, K.; Davis, S. R.; Farr, V. C.; Eichler, S. J.; Politis, I. Effect of once daily milking and concurrent somatotropin on mammary tight junction permeability and yield of cows. J. Dairy Sci. 1994, 77 (10), 2994-3001. (9) Lacy-Hulbert, S. J.; Woolford, M. W.; Nicholas, G. D.; Prosser, C. G.; Stelwagen, K. Effect of milking frequency and pasture intake on milk yield and composition of late lactation cows. J. Dairy Sci. 1999, 82 (6), 1232-1239. (10) Nielsen, S. S. Plasmin system and microbial proteases in milk: characteristics, roles, and relationship. J. Agric. Food Chem. 2002, 50 (22), 6628-6634. (11) Korhonen, H.; Marnila, P.; Gill, H. S. Milk immunoglobulins and complement factors. Br. J. Nutr. 2000, 84 Suppl 1, S75-80. (12) Prosser, C. G. Insulin-like growth factors in milk and mammary gland. J. Mammary Gland Biol. Neoplasia 1996, 1 (3), 297-306. (13) Ward, P. P.; Uribe-Luna, S.; Conneely, O. M. Lactoferrin and host defense. Biochem. Cell Biol. 2002, 80 (1), 95-102. (14) Hartmann, P. E. Changes in the composition and yield of the mammary secretion of cows during the initiation of lactation. J. Endocrinol. 1973, 59 (2), 231-247. (15) Hurley, W. L. Mammary gland function during involution. J. Dairy Sci. 1989, 72 (6), 1637-1646. (16) Eckersall, P. D.; Young, F. J.; McComb, C.; Hogarth, C. J.; Safi, S.; Weber, A.; McDonald, T.; Nolan, A. M.; Fitzpatrick, J. L. Acute phase proteins in serum and milk from dairy cows with clinical mastitis. Vet. Rec. 2001, 148 (2), 35-41. (17) Clare, D. A.; Swaisgood, H. E. Bioactive milk peptides: a prospectus. J. Dairy Sci. 2000, 83 (6), 1187-1195. (18) Galvani, M.; Hamdan, M.; Righetti, P. G. Two-dimensional gel electrophoresis/matrix-assisted laser desorption/ionisation mass spectrometry of commercial bovine milk. Rapid Commun. Mass Spectrom. 2001, 15 (4), 258-264. (19) Palmer, D. J.; Kelly, V. C.; Smit, A. M.; Kuy, S.; Knight, C. G.; Cooper, G. J. Human colostrum: identification of minor proteins in the aqueous phase by proteomics. Proteomics 2006, 6 (7), 2208-2216. (20) Yamada, M.; Murakami, K.; Wallingford, J. C.; Yuki, Y. Identification of low-abundance proteins of bovine colostral and mature milk using two-dimensional electrophoresis followed by microsequencing and mass spectrometry. Electrophoresis 2002, 23 (78), 1153-1160. (21) Hogarth, C. J.; Fitzpatrick, J. L.; Nolan, A. M.; Young, F. J.; Pitt, A.; Eckersall, P. D. Differential protein composition of bovine whey: a comparison of whey from healthy animals and from those with clinical mastitis. Proteomics 2004, 4 (7), 20942100. (22) Stelwagen, K.; Politis, I.; Guo, M. R.; Kindstedt, P. S.; Davis, S. R.; Farr, V. C. Mammary-derived growth inhibitor in bovine milk: Effect of milking frequency and somatotropin administration. Canadian J. Anim. Sci. 1994, 74, 695-698. (23) Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248-254. (24) Kintner, M. a. S. N. E. Protein Sequencing and Identification Using Tandem Mass Spectrometry; John Wiley & Sons: New York, 2000. (25) Carr, S.; Aebersold, R.; Baldwin, M.; Burlingame, A.; Clauser, K.; Nesvizhskii, A. The need for guidelines in publication of peptide and protein identification data: Working Group on Publication Guidelines for Peptide and Protein Identification Data. Mol. Cell Proteomics 2004, 3 (6), 531-533. (26) Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227 (5259), 680-685.
research articles
Host Defence Proteins in Milk (27) Neuhoff, V.; Arold, N.; Taube, D.; Ehrhardt, W. Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis 1988, 9 (6), 255-262. (28) Xie, H.; Wasserman, A.; Levine, Z.; Novik, A.; Grebinskiy, V.; Shoshan, A.; Mintz, L. Large-scale protein annotation through gene ontology. Genome Res. 2002, 12 (5), 785-794. (29) Cavaletto, M.; Giuffrida, M. G.; Conti, A. The proteomic approach to analysis of human milk fat globule membrane. Clin. Chim. Acta 2004, 347 (1-2), 41-48. (30) Murakami, K.; Lagarde, M.; Yuki, Y. Identification of minor proteins of human colostrum and mature milk by twodimensional electrophoresis. Electrophoresis 1998, 19 (14), 25212527. (31) Fortunato, D.; Giuffrida, M. G.; Cavaletto, M.; Garoffo, L. P.; Dellavalle, G.; Napolitano, L.; Giunta, C.; Fabris, C.; Bertino, E.; Coscia, A.; Conti, A. Structural proteome of human colostral fat globule membrane proteins. Proteomics 2003, 3 (6), 897-905. (32) Charlwood, J.; Hanrahan, S.; Tyldesley, R.; Langridge, J.; Dwek, M.; Camilleri, P. Use of proteomic methodology for the characterization of human milk fat globular membrane proteins. Anal. Biochem. 2002, 301 (2), 314-324. (33) Clare, D. A.; Catignani, G. L.; Swaisgood, H. E. Biodefense properties of milk: the role of antimicrobial proteins and peptides. Curr. Pharm. Des. 2003, 9 (16), 1239-1255. (34) Martin, H. M.; Hancock, J. T.; Salisbury, V.; Harrison, R. Role of xanthine oxidoreductase as an antimicrobial agent. Infect. Immun. 2004, 72 (9), 4933-4939. (35) Cole, A. M.; Kim, Y. H.; Tahk, S.; Hong, T.; Weis, P.; Waring, A. J.; Ganz, T. Calcitermin, a novel antimicrobial peptide isolated from human airway secretions. FEBS Lett 2001, 504 (1-2), 5-10. (36) Triantafilou, K.; Triantafilou, M.; Dedrick, R. L. A CD14independent LPS receptor cluster. Nat. Immunol. 2001, 2 (4), 338-345. (37) Hatzivassiliou, G.; Miller, I.; Takizawa, J.; Palanisamy, N.; Rao, P. H.; Iida, S.; Tagawa, S.; Taniwaki, M.; Russo, J.; Neri, A.; Cattoretti, G.; Clynes, R.; Mendelsohn, C.; Chaganti, R. S.; DallaFavera, R. IRTA1 and IRTA2, novel immunoglobulin superfamily receptors expressed in B cells and involved in chromosome 1q21 abnormalities in B cell malignancy. Immunity 2001, 14 (3), 277289. (38) Kubota, T.; Miyauchi, M.; Miura, K.; Hirokawa, G.; Awaya, A.; Miyasaka, N.; Kurosawa, Y.; Kanai, Y.; Maruyama, K. Upregulation of nucleobindin expression in human-activated lymphocytes and non-Hodgkin’s lymphoma. Pathol. Int. 1998, 48 (1), 22-28. (39) Heid, H. W.; Keenan, T. W. Intracellular origin and secretion of milk fat globules. Eur. J. Cell Biol. 2005, 84 (2-3.), 245-258. (40) Ramanathan, B.; Davis, E. G.; Ross, C. R.; Blecha, F. Cathelicidins: microbicidal activity, mechanisms of action, and roles in innate immunity. Microbes Infect. 2002, 4 (3), 361-372. (41) Owhashi, M.; Arita, H.; Hayai, N. Identification of a novel eosinophil chemotactic cytokine (ECF-L) as a chitinase family protein. J. Biol. Chem. 2000, 275 (2), 1279-1286. (42) Radsak, M. P.; Hilf, N.; Singh-Jasuja, H.; Braedel, S.; Brossart, P.; Rammensee, H. G.; Schild, H. The heat shock protein Gp96 binds to human neutrophils and monocytes and stimulates effector functions. Blood 2003, 101 (7), 2810-2815. (43) Guo, G. G.; Patel, K.; Kumar, V.; Shah, M.; Fried, V. A.; Etlinger, J. D.; Sehgal, P. B. Association of the chaperone glucose-regulated protein 58 (GRP58/ER-60/ERp57) with Stat3 in cytosol and plasma membrane complexes. J. Interferon Cytokine Res. 2002, 22 (5), 555-563.
(44) Zugel, U.; Kaufmann, S. H. Role of heat shock proteins in protection from and pathogenesis of infectious diseases. Clin. Microbiol. Rev. 1999, 12 (1), 19-39. (45) Welch, J. S.; Escoubet-Lozach, L.; Sykes, D. B.; Liddiard, K.; Greaves, D. R.; Glass, C. K. TH2 cytokines and allergic challenge induce Ym1 expression in macrophages by a STAT6-dependent mechanism. J. Biol. Chem. 2002, 277 (45), 42821-428219. (46) Sheth, K.; De, A.; Nolan, B.; Friel, J.; Duffy, A.; Ricciardi, R.; MillerGraziano, C.; Bankey, P. Heat shock protein 27 inhibits apoptosis in human neutrophils. J. Surg. Res. 2001, 99 (1), 129-133. (47) Kol, A.; Lichtman, A. H.; Finberg, R. W.; Libby, P.; Kurt-Jones, E. A. Cutting edge: heat shock protein (HSP) 60 activates the innate immune response: CD14 is an essential receptor for HSP60 activation of mononuclear cells. J. Immunol. 2000, 164 (1), 1317. (48) Miller, I.; Hatzivassiliou, G.; Cattoretti, G.; Mendelsohn, C.; Dalla-Favera, R. IRTAs: a new family of immunoglobulinlike receptors differentially expressed in B cells. Blood 2002, 99 (8), 2662-2669. (49) Yamauchi, K.; Wakabayashi, H.; Shin, K.; Takase, M. Bovine lactoferrin: benefits and mechanism of action against infections. Biochem. Cell Biol. 2006, 84 (3), 291-296. (50) Silanikove, N.; Shapiro, F.; Shamay, A.; Leitner, G. Role of xanthine oxidase, lactoperoxidase, and NO in the innate immune system of mammary secretion during active involution in dairy cows: manipulation with casein hydrolyzates. Free Radic. Biol. Med. 2005, 38 (9), 1139-1151. (51) Chen, H.; Mocsai, A.; Zhang, H.; Ding, R. X.; Morisaki, J. H.; White, M.; Rothfork, J. M.; Heiser, P.; Colucci-Guyon, E.; Lowell, C. A.; Gresham, H. D.; Allen, P. M.; Brown, E. J. Role for plastin in host defense distinguishes integrin signaling from cell adhesion and spreading. Immunity 2003, 19 (1), 95-104. (52) Emi, M.; Asaoka, H.; Matsumoto, A.; Itakura, H.; Kurihara, Y.; Wada, Y.; Kanamori, H.; Yazaki, Y.; Takahashi, E.; Lepert, M. et al. Structure, organization, and chromosomal mapping of the human macrophage scavenger receptor gene. J. Biol. Chem. 1993, 268 (3), 2120-2125. (53) Kanai, Y.; Miura, K.; Uehara, T.; Amagai, M.; Takeda, O.; Tanuma, S.; Kurosawa, Y. Natural occurrence of Nuc in the sera of autoimmune-prone MRL/lpr mice. Biochem. Biophys. Res. Commun. 1993, 196 (2), 729-736. (54) Girardin, S. E.; Philpott, D. J. Mini-review: the role of peptidoglycan recognition in innate immunity. Eur. J. Immunol. 2004, 34 (7), 1777-1782. (55) Gebhardt, C.; Breitenbach, U.; Tuckermann, J. P.; Dittrich, B. T.; Richter, K. H.; Angel, P. Calgranulins S100A8 and S100A9 are negatively regulated by glucocorticoids in a c-Fos-dependent manner and overexpressed throughout skin carcinogenesis. Oncogene 2002, 21 (27), 4266-4276. (56) Spach, K. M.; Pedersen, L. B.; Nashold, F. E.; Kayo, T.; Yandell, B. S.; Prolla, T. A.; Hayes, C. E. Gene expression analysis suggests that 1,25-dihydroxyvitamin D3 reverses experimental autoimmune encephalomyelitis by stimulating inflammatory cell apoptosis. Physiol. Genomics. 2004, 18 (2), 141-151. (57) Lee, H. Y.; Kim, M. K.; Park, K. S.; Shin, E. H.; Jo, S. H.; Kim, S. D.; Jo, E. J.; Lee, Y. N.; Lee, C.; Baek, S. H.; Bae, Y. S. Serum amyloid A induces contrary immune responses via formyl peptide receptor-like 1 in human monocytes. Mol. Pharm. 2006, 70 (1), 241-248
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