Differential Expression of Vitreous Proteins in Young and Mature New Zealand White Rabbits

<div><p>Different anatomical regions have been defined in the vitreous humor including central vitreous, basal vitreous, vitreous cortex, vitreoretinal interface and zonule. In this study we sought to characterize changes in the proteome of vitreous humor (VH) related to compartments or age in New Zealand white rabbits (NZW). Vitreous humor was cryo-collected from young and mature New Zealand white rabbit eyes, and dissected into anterior and posterior compartments. All samples were divided into 4 groups: Young Anterior (YA), Young Posterior (YP), Mature Anterior (MA) and Mature Posterior (MP) vitreous. Tryptic digests of total proteins were analyzed by liquid chromatography followed by tandem mass spectrometry. Spectral count was used to determine the relative protein abundances and identify proteins with statistical differences between compartment and age groups. Western blotting was performed to validate some of the differentially expressed proteins. Our results showed that 231, 375, 273 and 353 proteins were identified in the YA, YP, MA and MP respectively. Fifteen proteins were significantly differentially expressed between YA and YP, and 11 between MA and MP. Carbonic anhydrase III, lambda crystallin, alpha crystallin A and B, beta crystallin B1 and B2 were more abundant in the anterior region, whereas vimentin was less abundant in the anterior region. For comparisons between age groups, 4 proteins were differentially expressed in both YA relative to MA and YP relative to MP. Western blotting confirmed the differential expression of carbonic anhydrase III, alpha crystallin B and beta crystallin B2. The protein profiles of the vitreous humor showed age- and compartment-related differences. This differential protein profile provides a baseline for understanding the vitreous compartmentalization in the rabbit and suggests that further studies profiling proteins in different compartments of the vitreous in other species may be warranted.</p></div

Venn diagram of proteins identified in anterior or posterior vitreous of young and mature rabbits by LC-MS/MS.

<p>(A) 231 proteins were identified in the anterior vitreous of young rabbit (YA) while 375 proteins were identified in the posterior vitreous of young rabbit (YP), among them were 204 proteins shared by these two groups; (B) The total numbers of proteins detected in the anterior and posterior vitreous of mature rabbit (MA and MP respectively) were 273 and 353, they shared 238 proteins; (C) The number of common proteins shared by YA and MA was 194; (D) 295 proteins were identified in both YP and MP.</p

Differential Expression of Vitreous Proteins in Young and Mature New Zealand White Rabbits - Fig 3

<p>Western blot analysis of anterior and posterior vitreous of young and mature rabbits respectively using antibodies against (from top to bottom): CA3, CRYAB, CRY BB2 and vimentin (A) and Densitometric analysis of CA3, CRYAB, CRYBB2 and vimentin in young anterior vitreous (YA, downward diagonally striped bars) vs. young posterior vitreous (YP, upward diagonally striped bars) (B). (Values are listed as the mean ± SD). Densitometric analysis of CA3, CRYAB, CRYBB2 and vimentin in mature anterior vitreous (MA, downward diagonally striped bars) vs. mature posterior vitreous (MP, upward diagonally striped bars) (C).</p

Differentially expressed proteins between groups determined by G test and Holm-Sidak test.

<p>Differentially expressed proteins between groups determined by G test and Holm-Sidak test.</p

Venn diagram comparing the proteomes of four human vitreous studies (present study versus Yee et al. versus Aretz et al. Versus versus Semba et al. (made with online venn diagram plotter at http://bioinfogp.cnb.csic.es/tools/venny).

<p>Venn diagram comparing the proteomes of four human vitreous studies (present study versus Yee et al. versus Aretz et al. Versus versus Semba et al. (made with online venn diagram plotter at <a href="http://bioinfogp.cnb.csic.es/tools/venny" target="_blank">http://bioinfogp.cnb.csic.es/tools/venny</a>).</p

Generating Recombinant Antibodies against Putative Biomarkers of Retinal Injury

<div><p>Candidate biomarkers, indicative of disease or injury, are beginning to overwhelm the process of validation through immunological means. Recombinant antibodies developed through phage-display offer an alternative means of generating monoclonal antibodies faster than traditional immunization of animals. Peptide segments of putative biomarkers of laser induced injury in the rabbit, discovered through mass spectrometry, were used as targets for a selection against a library of phage-displayed human single-chain variable fragment (scFv) antibodies. Highly specific antibodies were isolated to four of these unique peptide sequences. One antibody against the retinal protein, Guanine Nucleotide-Binding Protein Beta 5 (GBB5), had a dissociation constant ~300 nM and recognized the full-length endogenous protein in retinal homogenates of three different animal species by western blot. Alanine scanning of the peptide target identified three charged and one hydrophobic amino acid as the critical binding residues for two different scFvs. To enhance the utility of the reagent, one scFv was dimerized through a Fragment-crystallizable hinge region (i.e., Fc) and expressed in HEK-293 cells. This dimeric reagent yielded a 25-fold lower detection limit in western blots.</p></div

Five scFvs in a soluble ELISA retain their specificity.

<p>After transferring the scFv coding region from the phagemid into an expression vector, these five scFvs were expressed in soluble form and purified. To demonstrate the specificity of each antibody, the ELISA was performed against all biomarker peptides available.</p

Comparison of scFv and the Fc format of the anti-GBB5-H9.

<p>(A) Target peptides are immobilized on a NeutrAvidin-coated microtiter plate at 25 nM and assayed with 30 nM of monomeric <i>E</i>. <i>coli</i> expressed anti-GBB5 H9 scFv or 10 nM of the HEK-293 expressed bivalent Fc format. The secondary antibody anti-FLAG-HRP is added and subsequent chromogenic reagent. Absorbance is recorded at 405 nm. (B) The GBB5-MBP fusion protein (46 kDa) is detected on PVDF membrane using the anti-GBB5-H9 scFv or Fc format of the H9 clone. Amount loaded is in μg and decreases from left to right. The negative control included is the RGS9-MBP fusion protein at 2μg.</p

Western blot of retinal lysates.

<p>The anti-GBB5-scFv H9 was used to probe retinal lysates from chicken, mouse, and rabbit. The expected size of rabbit and mouse GBB5 is 38.7 kDa, whereas the expected size for the chicken homolog is 38.8 kDa. The left panel is probed with the scFv antibody and the secondary anti-FLAG-HRP antibody. The right panel serves as the negative control (i.e., probed with the secondary antibody only). The total lysates loaded for chicken, mouse, and rabbit are ~35 μg, ~ 50 μg, and ~35 μg, respectively.</p

ELISA of scFv-displayed virions against the GBB5 peptide.

<p>ScFv antibodies, raised against the GBB5 peptide, are displayed on phage particles and used to probe peptides, which are captured on microtiter plate well surface. The phage-displayed scFvs are named according to the well location of the 96-well screen in which they were originally found. Specificity is determined by the intensity of the absorbance signal for the target peptide (GBB5), compared to the background NeutrAvidin (NA), Streptavidin (SA), Phosphate Buffered Saline (PBS) coated wells or unrelated peptides, CACNA1F and CNGA3.</p