Proteomic Evaluation of Chicken Brush-Border Membrane during the

Proteomic Evaluation of Chicken Brush-Border Membrane during the Early Posthatch Period Elizabeth R. Gilbert,† Patricia M. Williams,† William K. Ray,‡ Huifeng Li,§ Derek A. Emmerson,| Eric A. Wong,† and Kenneth E. Webb, Jr.*,† Departments of Animal and Poultry Sciences and Biochemistry, Virginia Tech, Blacksburg, Virginia 24061, Shanxi Agricultural University, Shanxi Province, China, and Aviagen, Huntsville, Alabama 35805 Received April 20, 2010

The chicken small intestine undergoes structural and functional changes during the early posthatch period to accommodate the transition from a lipid-rich diet inside the egg to a carbohydrate- and proteinbased diet. Many of the enterocyte brush-border membrane-associated proteins responsible for mediating changes in nutrient utilization are unknown. The objective of this study was to conduct a proteomic analysis of chicken small intestine during the early posthatch period. We isolated brushborder membrane at day of hatch and days 1, 3, 7, and 14 posthatch from the small intestine of 2 genetic lines of broilers that differ in growth performance, and performed 2D gel-electrophoresis. A total of 1693 spots were analyzed by matrix-assisted laser desorption/ionization-tandem time-of-flight mass spectrometry (MALDI-TOF/TOF). In total, 132 different proteins were identified and grouped according to biological function. Of these, there were 10 nutrient transporters, 9 digestive enzymes, and 17 proteins associated with cytoskeletal structure and microvilli organization. The remaining proteins were classified as basolateral membrane (3), endosomal/membrane trafficking (8), signaling (14), metabolic (33), degradative (5), stress-related (5), protein synthesis machinery/mitochondria/nucleus (19), immunologic (1), or unknown (8). Of the spots in which proteins were identified, there were 10 that showed an effect of broiler genetic line on protein spot density (P < 0.001) and 19 spots showing a correlation of broiler genetic line x age (P < 0.001). Identification of brush-border membrane-associated proteins is an important step in furthering our understanding of digestion and absorption in the chicken. Keywords: chicken • brush-border • intestine • MALDI • mass spectrometry

Introduction The final stages of intestinal digestion and nutrient absorption occur at the brush-border membrane (BBM) of enterocytes that line the villi.1 Villi enhance the surface area while BBMs maximize the surface area for contact with luminal contents. The BBM is equipped with a diverse assortment of enzymes and nutrient transporters, as well as a complex network of cytoskeletal proteins that anchor the membrane and contribute to its characteristic “ruffled” appearance.2 While many of these proteins were catalogued for mammals,3,4 there are no studies to date that involve characterization of the BBM proteome in an avian species. Very little is known about the digestive and absorptive machinery in the chicken gut, particularly during the early posthatch period. At hatch, there is a transition from a lipidrich diet to a carbohydrate- and protein-based diet. The timing * To whom correspondence should be addressed. Dr. Kenneth E. Webb, Jr., Virginia Tech, Department of Animal and Poultry Sciences, 3470 Litton Reaves Hall, Blacksburg, VA 24061. Tel.: (540) 231-9157. Fax: (540) 231-3010. E-mail: [email protected]. † Department of Animal and Poultry Sciences, Virginia Tech. ‡ Department of Biochemistry, Virginia Tech. § Shanxi Agricultural University. | Aviagen.

4628 Journal of Proteome Research 2010, 9, 4628–4639 Published on Web 07/23/2010

of gene expression must be carefully orchestrated to set the framework for consumption of feed. The first 2 weeks posthatch represent a period in which digestion and/or absorption at the BBM is a bottleneck to nutrient assimilation and much of this may be due to the developmental regulation of pancreatic and intestinal enzymes in the small intestine.5-7 A greater understanding of digestion and absorption in the bird is needed. We have conducted several early posthatch studies using 2 genetically selected broiler lines, Line A and Line B.8-10 These lines originate from the same genetic stock but have been selected on different diets for at least 10 generations, leading to a divergence in growth characteristics. We hypothesize that this divergence in growth may be attributable to differences in nutrient assimilation at the intestinal level during the early posthatch period. The objective of this study was to conduct a proteomic evaluation of the BBM during the first 2 weeks posthatch in Line A and Line B broilers.

Methods Animals. All animal procedures were approved by the Institutional Animal Care and Use Committee at Virginia Tech. The birds used for this experiment were from 2 broiler lines selected under different nutritional environments. A description 10.1021/pr1003533

 2010 American Chemical Society

Chicken Brush-Border Membrane Proteome

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Figure 1. Master image of gel stained with SYPRO Ruby following 2-dimensional gel electrophoresis of chicken intestinal brush-border membrane. The first dimension was carried out using pH 3-10 immobilized pH gradient strips and the second dimension was performed using 8-16% acrylamide gels. Annotated spots are those showing a significant effect of genetic line and/or genetic line × age interaction at P < 0.001, as shown in Tables 10 and 11 and Figures 2-5.

of these lines is reported elsewhere.8,9 Line A was selected on corn- and soy-based diets and lower relative amino acid concentrations. Line B was fed wheat-based diets and amino acid concentrations that were 15-20% higher than for Line A. On the corn-based diet, Line A birds are heavier at market age, and when crude protein levels in the diet are reduced, this difference becomes accentuated.8 Eggs were obtained from Aviagen (Huntsville, AL). Day-of-hatch (DOH) chicks from each genetic line were randomly assigned to heated floor pens with wood shavings and given ad libitum access to a corn- and soybased diet, as previously described.8 Chicks were killed by cervical dislocation at DOH (before feeding), and 1, 3, 7, and 14 days posthatch (D1, D3, D7, and D14, respectively). Total intestine was removed and separated into the duodenum, jejunum, and ileum.8 Segments were rinsed with ice-cold PBS, minced with a razor blade, and separated into aliquots; frozen in liquid nitrogen; and stored at -80 °C. The sex of the birds was determined by PCR as previously described,8 using liver DNA and tyrosinase- and W chromosome-specific primers. Reagents. Reagents were purchased from Thermo Fisher Scientific (Waltham, MA) as electrophoresis- or molecular biology-grade unless otherwise specified. Brush-Border Membrane Isolation. Brush-border membrane isolation was performed as previously described, based

on modification of several protocols.11-13 Frozen tissue was added to ice-cold homogenization buffer (100 mM mannitol, 2 mM HEPES/Tris pH 7.4, 0.1 mM phenylmethylsulfonyl fluoride (PMSF)) and homogenized at full speed for four 30 s periods, and buffer was added to achieve a 5% homogenate. The HEPES was biotech grade (Sigma Aldrich; St. Louis, MO) and PMSF was purchased from Pierce. An aliquot of homogenate was removed and stored at -80 °C for subsequent protein and marker enzyme assays. The remaining homogenate was centrifuged at 500g for 12 min and pellets were discarded. A 1 M MgCl2 solution was added to the remaining supernatant to a final concentration of 10 mM, followed by gentle mixing for 20 min. The suspension was centrifuged at 3000g for 15 min and the supernatant was removed and centrifuged again at 3000g for 15 min. The supernatant was centrifuged at 30 000g for 30 min. The resultant pellet was resuspended in 20 mL of buffer (100 mM mannitol, 2 mM HEPES/Tris pH 7.4, 1 mM MgSO4, and 0.1 mM PMSF). The suspension was homogenized for 1 min and centrifuged at 30 000g for 30 min. The final pellet containing BBM was resuspended in buffer (300 mM mannitol, 20 mM HEPES/Tris pH 7.4, and 0.1 mM MgSO4). The suspension was passed through a 25-gauge needle 15 times. The resulting suspension was aliquoted and stored at -80 °C. Journal of Proteome Research • Vol. 9, No. 9, 2010 4629

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Table 1. Grouping of Identified Proteins (Total 132) by Function

group (description)a

1 (Transporters) 2 (Brush-border digestive enzymes) 3 (Cytoskeletal, motility and cell recognition) 4 (Tight junction) 5 (Basolateral membrane) 6 (Endosomal/membrane trafficking) 7 (Signaling) 8 (Metabolic) 9 (Degradative proteins) 10 (Stress-related proteins) 11 (Protein synthesis assembly, mitochondria, nucleus, others) 12 (Immunologic) 13 (Unknown)

number of proteinsb

Table 2. Identified Transporter Proteins and Brush-Border Digestive Enzymes

percent of total proteinsc

10 9

7.6 6.8

17

12.9

0 3 8

0 2.2 6.1

14 33 5 5 19

10.6 25. 3.8 3.8 14.4

1 8

0.8 6.1

a

List of categories.4 Proteins categorized based on GO annotation from Protein Knowledgebase (UniProtKB); http://uniprot.org. b Number of different proteins identified in each category. c Percent (%) that protein category contributes to total number of proteins.

Protein concentrations were determined using the protein 2-D Quant kit (GE Healthcare; Waukesha, WI) following the manufacturer’s protocol with bovine serum albumin serving as a standard. Sucrase activity was used as a marker for enrichment of BBM.14 The BBM enrichment factor was calculated as a ratio of sucrase activity/mg protein in the BBM fraction divided by the sucrase activity/mg protein in the whole intestinal homogenate. Sucrase activity was assayed as previously described.15 Acceptable enrichment factors were >15. Isoelectric Focusing. Brush-border membrane fractions were resuspended in rehydration buffer (8.1 M urea, 2 M thiourea, 4% CHAPS, 0.2% CA (Biolyte) (3-10), 2 mM TBP, and 0.05 M DTT). Sample pooling consisted of 2 composites for each treatment group and each composite was composed of 4 male birds. Composite samples were applied to wells in isoelectric focusing (IEF) trays (17 cm, Bio-Rad; Hercules, CA) in Protean IEF cell units (Bio-Rad). The pH 3-10 nonlinear immobilized pH gradient (IPG) strips were applied to the wells followed by an overlay of mineral oil (Bio-Rad). The strips were rehydrated, focused and then equilibrated in 3 successive steps on an orbital shaker: (1) 30 min (buffer 1; 4% SDS, 0.37 M, Tris-Cl, pH 8.8, 20% glycerol), (2) 30 min (2% weight/volume DTT in buffer 1), (3) 30 min (2.5% iodoacetamide in buffer 1). Gel Electrophoresis and Staining. Twelve 8-16% gradient gels were poured using a casting chamber (Bio-Rad) and gradient-former. The 8% gel contained 8% acryl/bis, 0.38 M Tris, pH 8.8, 2% SDS, 0.045% ammonium persulfate (APS), and 0.023% Temed. The 16% gel contained 8% acryl/bis, 0.38 M Tris, pH 8.8, 2% SDS, 10% glycerol, 0.045% APS, and 0.023% Temed. The IPG strips were submerged between plates after a heated 0.5% agarose gel solution containing bromophenol blue was loaded onto the top of each polymerized gel. Plates were transferred to a Protean plus Dodeca Cell (Bio-Rad) tank chilled to 15 °C containing 1× TGS buffer (0.0125 M Tris, 0.096 M glycine, 0.05% SDS). Electrophoresis was performed for approximately 8 h (time for dye front to migrate to within 1 cm 4630

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protein name

Calbindin Chloride intracellular channel protein 5 Fatty acid binding protein 1, liver Fatty acid binding protein 2, intestinal Predicted: similar to intestinal 15 kDa protein, I-15P Predicted: similar to chloride intracellular channel protein 4 Predicted: similar to MGC84171 protein, chloride channel Predicted: similar to vacuolar H-ATP synthase subunit B osteoclast Voltage-dependent anion selective channel protein 2 V-type ATP synthase catalytic subunit A Angiotensin-converting enzyme (ACE) Dipeptidyl peptidase 4 Membrane alanine aminopeptidase, Aminopeptidase N (APN) Predicted: similar to alpi-prov protein (alkaline phosphatase) Predicted: similar to maltase glucoamylase Predicted: similar to maltase glucoamylase, alpha-glucosidase partial Predicted: similar to N-benzoyl-L-tyrosyl-p-aminobenzoic acid, meprin A Sucrase-isomaltase, intestinal Xaa-Pro dipeptidase a

group

number of spotsa

1 1

5 3

1

3

1

1

1

3

1

3

1

1

1

1

1

1

1

2

2

1

2 2

1 55

2

55

2

99

2

124

2

36

2

1

2

3

Number of spots in which the protein was identified.

of the gel’s edge). Gels were washed (10% methanol, 7% glacial acetic acid) on an orbital shaker for 30 min, followed by staining with SYPRO Ruby (Bio-Rad) stain in a dark box overnight. Gels were washed for 1 h (10% methanol, 7% glacial acetic acid) and scanned using PDQuest 2D analysis software (Molecular Imager Fx, Bio-Rad). Protein Spot Analysis and Excision. In total, 1693 protein spots were identified and a matched set was generated using PDQuest 2D Analysis software. A total of 60 gels was included in the analysis (2 genetic lines × 3 intestinal segments × 5 timepoints × 2 composites) and spots were analyzed by densitometry. All 1693 spots were excised (Proteome Works Spot Cutter; Bio-Rad) and transferred to sterile microtiter plates containing 0.1% acetic acid. Gel cores were destained for 12 h in 25 mM ammonium bicarbonate-50% acetonitrile and further dehydrated in 100% acetonitrile. Cores were rehydrated

research articles

Chicken Brush-Border Membrane Proteome in 10 µg/mL of trypsin (Sigma-Aldrich, St. Louis, MO) in 25 mM ammonium bicarbonate and incubated for 16 h at 37 °C. Matrix-Assisted Laser Desorption/Ionization Tandem Time-of-Flight (MALDI-TOF/TOF). Matrix-assisted laser desorption/ionization was performed after digestion. Samples were brought to near dryness and reconstituted in 2 mg/mL freshly prepared R-cyano-4-hydroxycinnamic acid (SigmaAldrich) in 0.5% formic, 40% acetonitrile supplemented with 10 mM freshly prepared ammonium phosphate. The sample/ matrix mixture was spotted onto a polished stainless steel MALDI plate and air-dried. Data were acquired utilizing an Applied Biosystems 4800 MALDI TOF/TOF. An MS scan for the m/z range of 800-4000, averaging data from 1000 laser shots, was acquired in reflector positive ion mode. The 4000 Series explorer software was then used to generate a list of at most nine peaks above a minimum signal-to-noise ratio for subsequent MS/MS analysis. MS/MS scans were the average of 1500 laser shots acquired in MS/MS 1 kV positive ion mode. Protein Identification. Protein identifications were obtained utilizing Mascot Daemon v. 2.2.2 (Matrix Science, Inc., Boston, MA) to automatically submit peak lists to a local Mascot Server v. 2.2 (Matrix Science, Inc., Boston, MA) search engine. Two separate searches for each peak list were performed using two reverse concatenated databases derived from Gallus gallus specific protein databases available from the NCBI Web site ftp://ftp.ncbi.nih.gov/genomes/Gallus_gallus/protein/ containing only annotated proteins (protein.fsa.gz, 37 058 sequences) or containing ab initio protein predictions (Gnomon_prot. fsa.gz, 83 750 sequences) generated utilizing the NCBI eukaryotic gene prediction tool Gnomon. Both protein databases were based on v. 1 build 2 (release date 11/30/06) of the chicken genomic sequences database. Searches of MS/MS data utilized a semitrypsin protease specificity with the possibility of two missed cleavages, a peptide mass tolerance of (400 ppm, a peptide fragment mass tolerance of (0.2 Da and the possibility of carbamidomethylation at cysteine, carbamylation at lysine, deamidation at asparagine and glutamine, oxidation of methionine, histidine and tryptophan, and pyroglutamic acid formation from N-terminal glutamines. Results for both sets of searches were then imported into Scaffold Q+ v. 3.00.02 (Proteome Software, Inc., Portland, OR) utilizing Java 1.6.0_18, amd64, 64 bit, and simultaneously searched using the same parameters described above utilizing a second search engine, X Tandem. When protein IDs were limited by requiring a minimum peptide and protein probability of 50%, the software reported a peptide false discovery rate of 2.6%. Of the 132 proteins identified, five were from a reversed sequence giving a protein false discovery rate of just under 3%. The tandem MS spectra corresponding to protein identifications based upon a single unique peptide were validated manually by ensuring the spectra contained at least four consecutive -y or -b ions matching the predicted amino acid sequence. Where mass spectrometry data was not adequate to determine which particular isoform of a protein was present in a protein spot, all possible isoforms are grouped together as a single protein ID. We compared sequences of all identified proteins to eliminate redundancies. Statistical Analysis. A total of 1693 protein spots was identified in each gel and a densitometric analysis was performed using PDQuest 2D Analysis software to quantify protein spot density. A total of 60 gels was included in the analysis (2 genetic lines × 3 intestinal segments × 5 time-points × 2 composites). Density values of each protein spot were analyzed

Table 3. Identified Proteins Involved in Cytoskeletal Assembly, Cell Motility, and Cell Recognition protein name

group

number of spotsa

Actin Annexin A2 Epithelial cell adhesion molecule Coactosin-like protein Destrin Galectin-3 Keratin, type II cytoskeletal cochleal Na(+)/H(+) exchange regulatory cofactor NHE-RF1 Plastin (isoforms 1,3) Predicted: similar to galectin-2 related protein isoforms 1-5 Predicted: similar to K12 keratin Predicted: similar to Ras-related C3 botulinum toxin substrate 1, Rac2 Predicted: similar to tubulin alpha-3,-7 chain, isotype M Radixin/Ezrin Tubulin alpha chain (3, 7, 8) Tubulin beta chain (2, 3) Villin-1

3 3 3

145 29 3

3 3 3 3

1 9 4 9

3

4

3 3

4 33

3

7

3

2

3

6

3 3 3 3

65 5 12 115

a


using the Proc Mixed procedure of SAS (Cary, NC). An alpha level of P < 0.001 was considered significant. The statistical model included the main effects of age, broiler genetic line and intestinal segment, 2-way interactions, and the 3-way interaction on protein spot density. The bird composite was designated as the experimental unit. Significant 2-way interactions of age × broiler genetic line on protein spot density were further evaluated with linear pairwise comparisons within age using the “slice” function of SAS. For the purpose of this paper, only effects involving genetic line and age will be discussed.

Results and Discussion Summary of Complete Protein Identification List. In total, 1693 spots were identified and matched across gels (Figure 1). We identified 132 different proteins. Proteins were categorized by function using GO annotation in a manner similar to the method described by Donowitz et al.4 As shown in Table 1, the category representing the greatest fraction of proteins was Group 8 (25%), comprising proteins involved in metabolism. Protein synthesis assembly site, mitochondria, and nuclear (Group 11; 14%), cytoskeletal, motility and cell recognition (Group 3; 13%), and signaling (Group 7; 11%) proteins comprised the next largest fractions. The complete lists of proteins within each category are shown in Tables 2-9. Transporters accounted for 8% of identified proteins, consisting mainly of ion channels and fatty-acid binding proteins. Digestive enzymes accounted for 7% of the total proteins identified, consisting of a variety of proteolytic and carbohydrolase enzymes, as well as alkaline phosphatase (alpi-prov). Proteins with unknown function comprised 6% of the total number of proteins. Summary of Proteins Showing Significant Changes. To pare down the list of protein spots showing significant changes in Journal of Proteome Research • Vol. 9, No. 9, 2010 4631

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Table 4. Identified Basolateral and Endosomal/Membrane Trafficking Proteins group

Leucine-rich repeat-containing protein 1 Sodium/potassium transporting ATPase subunit alpha-1 precursor Sodium/potassium transporting ATPase subunit beta-1 Alpha-centractin Apolipoprotein A-1 preproprotein Clathrin heavy chain 1 Major vault protein Predicted: similar to annexin XIIIb isoform 1 and 2 Predicted: similar to N-ethylmaleimide-sensitive factor Predicted: similar to PDZK1 Predicted: similar to syndet isoform 1 and2

5

1

5

1

5

1

6 6

1 3

6 6 6

2 6 12

6

1

6 6

28 1


Table 5. Identified Signaling Proteins protein name

group

number of spotsa

14-3-3 protein beta/alpha 14-3-3 protein epsilon 14-3-3 protein gamma Albumin precursor Annexin A5 Annexin A6 Calcium-binding protein p22 Calmodulin Cytidine deaminase Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 Predicted: similar to annexin A8, VAC beta Predicted: similar to cytidine deaminase Rho GDP-dissociation inhibitor 1 Thioredoxin

7 7 7 7 7 7 7 7 7 7

7 4 1 4 4 2 2 4 4 6

7

7

7

2

7

1

7

1

a


density across gels containing BBM protein from birds of different genetic lines and ages, we used an alpha level of P < 0.001 to denote significance, and focused our efforts on characterizing proteins that exhibited differences in spot densities which correlated to genetic line and/or age. A total of 26 spots showed a genetic line effect and/or age × line interaction after spots with multiple proteins were excluded (Tables 10 and 11; Figures 2-5). Protein Spots Showing Differences in Density That Correlated to Genetic Line. Of the protein spots that showed an effect of broiler genetic line on density (10), 4 were identified as villin-1, 2 as maltase-glucoamylase, 1 as cognin-prolyl-4hydroxylase, 1 as chloride intracellular channel protein 4, 1 as annexin A5, and 1 as galectin-3 (Table 10). Villin-1 and maltaseglucoamylase were 2 of the most abundant proteins identified in this study and were also developmentally regulated. These 4632


number of spotsa

protein name

group

Adenosine deaminase Alpha-enolase Arylamine N-acetyltransferase, pineal gland isozyme NAT-3 Dihydrolipoyl dehydrogenase mitochondrial Fructose-bisphosphate aldolase B Glutathione S-transferase Glyceraldehyde-3-phosphate dehydrogenase Hematopoietic prostaglandin D synthase Inosine-5′-monophosphate dehydrogenase 2 Lambda-crystallin homologue Lactate dehydrogenase B Malate dehydrogenase, cytoplasmic Nucleoside diphosphate kinase Phosphatidyl inositol-5-phosphate 4-kinase type -2 alpha Phosphoglycerate kinase Phosphoglycerate mutase 1 Predicted: similar to aflatoxin aldehyde reductase Predicted: similar to aldehyde dehydrogenase 9 family, A1 Predicted: similar to aldose reductase Predicted: similar to calcium binding protein Predicted: similar to cytosolic NADP-dependent isocitrate dehydrogenase Predicted: similar to esterase D/ formylglutathione hydrolase-like Predicted: similar to galactose mutarotase (aldose 1-epimerase) Predicted: similar to phosphopantothenecysteine synthetase Predicted: similar to pyridoxal kinase Predicted: similar to retinol-binding protein II (CRBP II) Predicted: similar to transketolase Prostaglandin reductase 1 Pyruvate kinase muscle isozyme Retinal dehydrogenase 1 Ribose-phosphate pyrophosphokinase 1 Serine/threonine-protein phosphatase PP1-beta catalytic subunit Triosephosphate isomerase

8 8 8

1 3 1

8

1

8 8 8

5 6 9

8

3

8

1

8 8 8 8 8

1 2 1 3 1

8 8 8

2 2 1

8

6

8 8

2 1

8

2

8

2

8

1

8

1

8 8

1 4

8 8 8 8 8

2 1 6 10 1

8

1

8

2

number of spotsa

protein name

a

Table 6. Identified Metabolic Proteins

a


proteins are important in cytoskeletal structure and digestive function, respectively. Changes in abundance of villin-1 and maltase-glucoamylase may have important implications for digestive and absorptive function of the enterocyte. One of the maltase-glucoamylase proteins was greater in Line A while the other was more abundant in Line B birds. The molecular weights of the 2 proteins were different from the predicted molecular weight and may represent distinct isoforms. Three of the 4 villin proteins were greater in Line A birds and all of these had observed molecular weights that were similar to the predicted. The villin-1 that was greater in Line B birds had an

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Chicken Brush-Border Membrane Proteome Table 7. Identified Degradative and Stress-Related Proteins protein name

Cathepsin B precursor Cytosolic nonspecific peptidase Lysozyme C precursor Predicted: similar to MGC83094 Proteasome subunit beta type-1 Heat shock cognate 71 kDa protein Heat shock cognate protein HSP 90 beta Heat shock protein beta-1 Heat shock protein HSP 90-alpha Peroxiredoxin (1, 2, 4, natural killer cell enhancing factor isoform 4) a

group

number of spotsa

9 9

1 1

9 9

1 1

9

1

10

11

10

4

10 10

2 3

10

11


Table 8. Identified Proteins Associated with Protein Synthesis Assembly, Mitochondria, Nucleus, or Other protein name

group

number of spotsa

40S ribosomal protein S27a Annexin A11 ATP synthase subunit beta, mitochondrial Cytochrome b5 Elongation factor 1-alpha Elongation factor 2 Elongation factor Tu, mitochondrial Heterogeneous nuclear ribonucleoprotein H3 Peptidylprolyl isomerase A Predicted: similar to chaperonin-containing TCP-1 complex gamma chain Predicted: similar to cognin/ prolyl-4-hydroxylase-protein disulfide isomerase Predicted: similar to protein disulfide-isomerase Protein disulfide-isomerase A3 precursor T-complex protein 1 subunit beta T-complex protein 1 subunit epsilon T-complex protein 1 subunit eta T-complex protein 1 subunit theta T-complex protein 1 subunit zeta WD repeat-containing protein

11 11 11

5 4 1

11 11 11 11

1 2 1 1

11

1

11 11

2 1

11

2

11

2

11

2

11

1

11

1

11

1

11

2

11

2

11

3

a


observed molecular weight that was much smaller than the predicted molecular weight. There was a 2-fold greater abundance of galectin-3 in Line B birds as compared with Line A birds (P < 0.001). Galectin-3

Table 9. Identified Immunologic Proteins and Proteins with Unknown Function protein name

Predicted: similar to natural killer cell enhancement factor isoforms 2, 3 Gene predicted by Gnomon genome contig Gga22_wGA271_22 Hypothetical protein Ovalbumin Predicted: similar to Cg5189-prov protein, partial Predicted: similar to Chain A, Crystal Structure Of Soluble Form Of Clic4 Predicted: similar to mk1AA1258 SH3 domain binding glutamic acid-rich protein like Sorcin a

group

number of spotsa

12

9

13

7

13 13 13

20 3 1

13

1

13

2

13

3

13

4


plays an important role in trafficking glycoproteins, including hydrolases, to the BBM.16 Differences in abundance of membrane trafficking and assembly proteins among genetic lines may suggest that organization of digestive enzyme complexes and transporters is differentially regulated. The chloride intracellular channel protein 4 was 2-fold more abundant in Line A as compared with Line B. The molecular weight from 2DE was 56.7 as compared with the predicted molecular weight of 43.7 kDa, suggesting the presence of posttranslational modifications. This protein is enriched in microvilli and is closely associated with cytoskeletal proteins in the membrane.17 Because of the importance of ion channels in a variety of cellular processes, including maintaining membrane potential, signaling pathways, and secretion and absorption, differences in abundance of a chloride channel may be related to enterocyte membrane function. Cognin (a protein disulfide isomerase) showed an effect of line where there was greater abundance of this protein in Line A birds (1.6-fold). Protein disulfide isomerase heterodimerizes with an unknown protein to form the microsomal triglyceride transfer protein complex, which plays an important role in lipoprotein assembly and lipid trafficking.18 Originally thought to localize to the ER, results from recent studies demonstrate enrichment of this complex in the BBM.18 Annexin A5 was almost 2-fold more abundant in Line B birds. The annexin proteins are critical in regulating membrane-membrane and membrane-cytoskeleton interactions, and may also play an important role in facilitating exocytosis and stabilization of membrane domains.19 Proteins Showing a Correlation of Genetic Line × Age. A total of 19 spots showed a genetic line × developmental stage interaction. Of these, 6 spots were identified as maltase glucoamylase (Table 11; Figure 2). The developmental changes were striking. For maltase-glucoamylase, some of the proteins were greatest at hatch and decreased during the next 2 weeks, while others were low at hatch and rose dramatically (Figure 2). In general, there was greater abundance of maltaseglucoamylase in Line A birds posthatch, suggesting a greater capacity for carbohydrate digestion. Journal of Proteome Research • Vol. 9, No. 9, 2010 4633

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Table 10. Protein Spots Showing an Effect of Genetic Line on Density (P < 0.001) protein

spota

MW (2DE)b

MW Prc

line Ad

line Be

A/Bf

SEMg

Annexin A5 Chloride Intracellular Channel Protein 4 Galectin-3 Maltase Glucoamylase Maltase Glucoamylase Cognin/prolyl-4-hydroxylase Villin-1 Villin-1 Villin-1 Villin-1

4211 2403 9020 1817 4814 2401 615 1601 5103 5623

35.11 56.69 17.59 185.00 238.00 61.25 98.50 99.00 32.43 97.50

36.2 43.7 35.4 205.7 205.7 45.1 92.5 92.5 92.5 92.5

110628 277227 1576975 730032 145747 647264 1069032 553302 368249 7403491

208274 140184 3409605 300066 294802 399773 424367 219468 556057 4660257

0.53 1.98 0.46 2.43 0.49 1.62 2.52 2.52 0.66 1.59

16992 25212 290196 64630 25731 43624 112952 54607 23421 450263

a Unique identifier for each protein spot. b Molecular weight of protein in kDa determined from second dimension of 2D gel electrophoresis by MW standard. c Predicted molecular weight in kDa from Protein Bank G. gallus ID entry. d Mean density (density units) of protein in Line A birds. e Mean density (density units) of protein in Line B birds. f Ratio of Line A spot density to Line B spot density density. g Pooled standard error of the mean (SEM).

Table 11. Spots Showing an Interaction of Genetic Line × Age (P < 0.001) protein

spota

MW (2DE)b

MW Prc

Maltase-glucoamylase Maltase-glucoamylase Maltase-glucoamylase Maltase-glucoamylase Maltase-glucoamylase Maltase-glucoamylase Annexin A2 Annexin VIII Annexin A5 Annexin XIIIb isoform 2 Alpi-prov Ezrin/radixin Villin-1 Villin-1 Villin-1 Actin, Meprin A subunit alpha Galectin-2 related Galectin-2 related

1817 2818 3822 4814 5719 5731 8301 2209 4211 5220 5301 1504 7215 7705 5636 9124 1718 6020 6116

185 192 201 238 115 112 39 34 35.1 35.3 52.2 76 35.7 100 94.5 30 124.7 13 24.5

205.7 205.7 205.7 205.7 205.7 205.7 38.6 36.7 36.2 35.3 84.8 69.4 92.5 92.5 92.5 41.7 80.2 10.9 10.9

a Unique identifier for each protein spot. b Molecular weight of protein in kDa determined from second dimension of 2D gel electrophoresis by MW standard. c Predicted molecular weight in kDa from Protein Bank G. gallus ID entry.

In general, observed molecular weights of proteins in spots showing significant differences were greater than calculated molecular weights of the unmodified protein. These proteins may represent distinct functional isoforms or products of alternative splicing and their differential regulation may point to a difference in nutrient assimilation among Line A and B broiler chicks. Glycosylation plays a key role in regulating protein-protein interactions. Proteins with different glycosylation patterns can behave quite differently and be targeted to different parts of the cell. Brush-border membrane proteins, such as digestive enzymes and nutrient transporters, contain numerous potential glycosylation sites, adding a further layer of complexity to our interpretation of changes in protein abundance and effects on biological activity.20 The annexin proteins were most abundant around hatch, with greater expression in Line B birds (Figure 3). Similarly, the villin-1 and ezrin proteins were most abundant around day of hatch and declined thereafter, with 2 of them greater in Line A birds and 2 greater in Line B birds (Figure 4). Actin increased after hatch and was greater in Line B birds until day 14. The 2 galectin-2 proteins also decreased after hatch, with greater expression in Line B birds. Because the developmental changes 4634


in these proteins were so dramatic (i.e., appearing or disappearing after DOH), they may represent distinct isoforms that are active during a short window of time. It is worth noting that of the 132 proteins identified in this study, the ones that showed changes in abundance that were correlated with genetic line and development were either digestive enzymes or cytoskeletal proteins associated with the microvilli. For the other 2 digestive enzymes showing changes among genetic lines with age (alpi-prov and meprin; Figures 3 and 5, respectively), abundance was greater in Line B birds at hatch, but decreased to negligible levels afterward. Digestion and Absorption-Related Proteins. The purpose of evaluating the intestinal BBM proteome in 2 genetically selected lines of broilers was to identify differences in digestive enzymes and nutrient transporters that may help explain the differences observed in growth performance. In other words, we hypothesized that differences in growth may be attributable to differences in nutrient utilization at the intestinal level. Furthermore, since the chicken BBM has not yet been characterized, identification of proteins associated with digestion and nutrient absorption are important molecular targets for further study. Thus, the remainder of this discussion will focus on nutrient digestion/absorption-related proteins. The Aminopeptidase Enzymes. The relative abundance of membrane-bound digestive enzymes speaks to their functional importance. Aminopeptidase N and maltase-glucoamylase are two enzymes involved in the terminal digestion of peptides and starch, respectively, and were 2 of the most abundant proteins identified in this study. The APN cleaves neutral or basic amino acids from the N-terminal end of peptides, including bioactive peptides such as enkaphalin, and is instrumental in maintaining a balance of absorbable peptides and amino acids, in addition to other known functions.21,22 Interestingly, these data corroborate our findings at the transcript level where APN was measured at quantities much greater than nutrient transporters and similar to housekeeping genes (hundreds of thousands of molecules of mRNA vs thousands or hundreds of mRNA molecules for peptide, amino acid, and glucose transporters).8,9 N-Benzoyl-L-tyrosyl-p-amino-benzoic Acid (Meprin A Subunit Alpha). Meprins are zinc-dependent, multimeric endopeptidases expressed predominantly in the BBM of renal and intestinal cells, with a substrate preference for peptides with more than 7 amino acids.23,24 They cleave a host of proteinaceous factors encountered at the BBM, including proteins that are secreted into the extracellular matrix, hormones, growth factors, cytokines, as well as microbially derived peptides.24 This protein was present at an observed molecular weight and pI


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Figure 2. Interaction of genetic line × age (P < 0.001) on abundance of maltase glucoamylase proteins. Intestinal samples were collected from Line A and Line B broilers at day of hatch and 1,3, 7, and 14 days posthatch. Brush-border membrane was isolated and used for 2D-gel electrophoresis followed by MALDI-TOF/TOF. Proteins were identified from peak lists using Mascot Daemon (Matrix Science; Boston, MA). Values represent mean density units ( SEM. Density was quantified using the PDQuest 2D Analysis Software (Bio-Rad; Hercules, CA).

of 125 kDa as compared with the predicted value of approximately 80 kDa, suggesting post-translational modifications. Other Peptidases. The other identified BBM enzymes involved in peptide digestion were less abundant. The Xaa-Pro dipeptidase plays an important role in collagen catabolism. The dipeptidyl peptidase IV also functions in catabolizing prolinecontaining peptides as well as inactivation of the incretin hormone GLP-1. The products of hydrolysis by dipeptidyl peptidase IV are C-terminal proline-containing dipeptides, a substrate for the aforementioned proline dipeptidases or the intestinal peptide transporter.25 The functional significance of these proteins in chickens is not characterized. Intestinal Saccharidases. Maltase-glucoamylase is a final step in yielding the absorbable monosaccharide glucose from linear starch molecules.26 Interestingly, although sucrase-

isomaltase activity is mutually reinforcing (i.e., it generates monomers from branched starch molecules), it was much less abundant than maltase-glucoamylase. Dietary starches are a mixture of approximately 25% amylase (linear polymer composed of alpha 1-4 linkages) and 75% amylopectin (branched polymer due to additional alpha 1-6 linkages).26 As a result, pancreatic amylase, which hydrolyzes internal alpha 1-4 linkages, yields both linear and branched maltose oligosaccharides that must be hydrolyzed to glucose to generate an absorbable end product. Since the majority of these oligosaccharides would be linear, it is likely that the enrichment for maltase-glucoamylase in the small intestine of the birds sampled in this study reflects a greater abundance of linear oligosaccharides made available from the diet. Indeed, maltaseglucoamylase, at least in mammals, is responsible for almost Journal of Proteome Research • Vol. 9, No. 9, 2010 4635

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Figure 3. Interaction of genetic line × age (P < 0.001) on abundance of annexin A2, AVIII, A5, and XIIIB isoform 2, alpi-prov and ezrin/ radixin proteins. Intestinal samples were collected from Line A and Line B broilers at day of hatch and 1,3, 7, and 14 days posthatch. Brush-border membrane was isolated and used for 2D-gel electrophoresis followed by MALDI-TOF/TOF. Proteins were identified from peak lists using Mascot Daemon (Matrix Science; Boston, MA). Values represent mean density units ( SEM. Density was quantified using the PDQuest 2D Analysis Software (Bio-Rad; Hercules, CA).

all mucosal glucoamylase exohydrolase activity for both amylase and amylopectin substrate, and 20% of all mucosal maltase activity.26 Sucrase-isomaltase is responsible for approximately 80% of all mucosal maltase activity, virtually all sucrase activity, and almost all iso-maltase activity.26 The standard broiler diet is based on corn and soybean meal, and at hatch, the bird transitions from a lipid-rich in-ovo diet to a protein and carbohydrate-based diet. Thus, the timing of gene expression should be orchestrated such that proteins responsible for protein and carbohydrate digestion are up-regulated at hatch. Some of the spots containing maltase glucoamylase increased in abundance after hatch while others decreased (Figure 2). 4636


Alpi-Prov (Alkaline Phosphatase). In the spot that showed changes in density, abundance of alpi-prov was more than 3-fold greater in Line B than Line A at DOH after which abundance dropped to negligible levels in both lines. It was one of the more abundant proteins identified in this study. Given the important role of alkaline phosphatase activity in critical luminal processes including detoxification, organic phosphate digestion, fat absorption, and maintenance of luminal pH, changes in its abundance can provide important clues about animal health and development.27 Transporters and Similarities to Previous Studies. Ten proteins classified as “transporters” were identified, and of


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Figure 4. Interaction of genetic line × age (P < 0.001) on abundance of villin-1 and actin proteins. Intestinal samples were collected from Line A and Line B broilers at day of hatch and 1,3, 7, and 14 days posthatch. Brush-border membrane was isolated and used for 2D-gel electrophoresis followed by MALDI-TOF/TOF. Proteins were identified from peak lists using Mascot Daemon (Matrix Science; Boston, MA). Values represent mean density units ( SEM. Density was quantified using the PDQuest 2D Analysis Software (Bio-Rad; Hercules, CA).

these, none belonged to any of the solute carrier (SLC) families of transporters. Given the shortcomings in proteomics methodology and trade-offs associated with analyzing membrane samples, it is not surprising that these transporters, containing up to 14 transmembrane domains, were not detected in the current study. Other reports of BBM proteomes utilized a similar membrane isolation procedure; however, sample separation and mass spectrometric methods were different from our own.3,4,28 One of the studies that used animal tissue employed blue native PAGE followed by LC-MS/MS,3 while the other employed 2D LC-MS/MS with strong-cation exchange chromatography.4 The third study used proliferating and differentiated Caco-2 cells, which form an apical membrane that structurally and functionally resembles the BBM.28 They used two methods for evaluating membrane composition: (1) SDS-PAGE followed by RP-nLC-MS/MS and (2) NH2-terminusisotopic tagging followed by 2D (ion-exchange and RP)-LC-MS/ MS.28 Our catalogue is in good agreement with Donowitz.4 In particular, the percent of proteins contributed by non-BBM proteins is similar, particularly when comparing by category. While the absolute number of proteins identified was much lower in our study, the relative percentages of transporters, digestive enzymes, and cytoskeletal proteins were similar. In comparison to Babusiak et al.,3 the number of proteins identified in our study was greater: 132 different proteins versus 55 in their study, although their protein list includes several multipass nutrient transporters. Their protein search criteria were relaxed in order to capture as many proteins as possible and they manually verified each hit in order to remove false

positives. Still, identification of only three SLC transporters in that study, the sodium-glucose cotransporter 1 (SGLT1), the intestinal peptide transporter 1 (PepT1), and a sodium-coupled neutral amino acid transporter (SNAT), under-represents multipass proteins. Similarly, Pshezhetsky et al.28 identified only several SLC transporters that were specific to the apical membrane of differentiated Caco-2 cells. We conducted a global gene expression analysis of chicken intestine and identified 162 SLC family members present at the transcript level.10 Thus, it is obvious that attempts made to identify such proteins are drastically missing the target. Future studies should employ novel techniques to improve membrane protein solubility. Chromatographic techniques for improved peptide separation and resolution could enhance detection of multipass membrane proteins. Our results also bear striking similarities to those reported using insect midgut microvilli samples and a variety of PAGEbased techniques.29-31 Aminopeptidase N, actin, and alkaline phosphatase were relatively abundant. The vacuolar ATPase subunits as well as a variety of metabolic proteins including alcohol dehydrogenase, and glutathione transferase were also abundant. Multipass transporters were not identified in these studies either. Others have used 2D SDS-PAGE to examine the proteome of chicken tissue.32 The soluble fraction of pectoralis muscle in layers was examined in 1 day-old chicks and then every 3 days until day 27. Similar to our study, there were dramatic changes occurring during the first 2 weeks posthatch. Actin was the predominant protein in 1 day-old chicks, but disappeared from the soluble fraction in subsequent days. The Journal of Proteome Research • Vol. 9, No. 9, 2010 4637

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Figure 5. Interaction of genetic line × age (P < 0.001) on abundance of meprin A subunit alpha, and galectin-2 proteins. Intestinal samples were collected from Line A and Line B broilers at day of hatch and 1,3, 7, and 14 days posthatch. Brush-border membrane was isolated and used for 2D-gel electrophoresis followed by MALDI-TOF/TOF. Proteins were identified from peak lists using Mascot Daemon (Matrix Science; Boston, MA). Values represent mean density units ( SEM. Density was quantified using the PDQuest 2D Analysis Software (Bio-Rad; Hercules, CA).

list of soluble proteins was dominated by metabolic proteins, similar to the list of metabolic proteins identified in our study. Conclusions and Implications. In summary, we identified 132 different proteins in the BBM of chicken small intestine. In addition to known digestion and absorption-related proteins, we identified numerous proteins with “unknown” function. Enzymes and nutrient transporters that are responsive to changes in developmental stage and genetic background will make excellent molecular targets for future studies aimed at further understanding the relationship between diet composition and digestion and absorption in the chicken small intestine. We identified a number of proteins where spot density was different among genetic lines. These proteins are involved in starch, protein, and fat digestion, maintaining membrane potential, membrane trafficking and cytoskeletal organization of the BBM. In general, these proteins were identified in multiple spots and the observed molecular weights were slightly different from predicted values. Differential abundance of such proteins among broiler lines may indicate differences in post-translational modifications that could influence kinetics of overall digestion and transport at the BBM. Future studies involving regulation of these proteins in chickens during the early posthatch period can address these theories. We must exercise caution in making assumptions about the source of proteins identified in our BBM extracts. Validation studies are necessary to confirm localization and function of 4638


identified proteins. It is our hope that this study will provide a solid foundation for further proteomic studies of the BBM. Abbreviations: APS, ammonium persulfate; BBM, brushborder membrane; DOH, day of hatch; D1, day 1 posthatch; D3, day 3 posthatch; D7, day 7 posthatch; D14, day 14 posthatch; DTT, dithiothreitol; IEF, isoelectric focusing; IPG, immobilized pH gradient; MALDI-TOF/TOF, matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; SDS, sodium dodecyl sulfate; TGS, trisglycine-SDS buffer.

Acknowledgment. This project was supported by National Research Initiative Competitive Grant no. 2005-35206-15271 from the USDA Cooperative State Research, Education, and Extension Service. This research was also funded in part by the John Lee Pratt Animal Nutrition Program at Virginia Tech (awarded to E.R.G.). References (1) Mamajiwalla, S. N.; Fath, K. R.; Burgess, D. R. Curr. Top. Dev. Biol. 1992, 26, 123–143. (2) Shibayama, T.; Carboni, J. M.; Mooseker, M. S. J. Cell. Biol. 1987, 105, 335–344. (3) Babusiak, M.; Man, P.; Petrak, J.; Vyoral, D. Proteomics 2007, 7, 121–129. (4) Donowitz, M.; Singh, S.; Salahuddin, F. F.; Hogema, B. M.; Chen, Y.; Gucek, M.; Cole, R. N.; Ham, A.; Zachos, N. C.; Kovbasnjuk, O.;

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PR1003533

Journal of Proteome Research • Vol. 9, No. 9, 2010 4639

Proteomic Evaluation of Chicken Brush-Border Membrane during the

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