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

Liu Y, Bouhenni RA, Dufresne CP, Semba RD, Edward DP - PLoS ONE (2016)

Bottom Line: 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.Western blotting confirmed the differential expression of carbonic anhydrase III, alpha crystallin B and beta crystallin B2.

View Article: PubMed Central - PubMed

Affiliation: Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, United States of America.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

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).
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pone.0153560.g003: 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).

Mentions: WB analysis and densitometry of the blots (Fig 3) were used to confirm results obtained by LC-MS/MS. Similar to LC-MS/MS, CA3 was more abundant in young anterior vitreous than in young posterior vitreous (p = 0.04). The expression of CA3 in mature anterior vitreous was also higher than that in mature posterior vitreous (p = 0.03). CRYBB2 was more abundant in anterior compared to posterior vitreous in both young and mature rabbits (p = 0.02 and 0.04, respectively). The expression of CRYAB was also greater in the anterior vitreous in young and mature rabbits (p = 0.02 and 0.0495, respectively). However, there were no significant differences in the level of vimentin between young anterior and young posterior vitreous (p = 0.08) or mature anterior and mature posterior vitreous (p = 0.08). Although a visible difference was seen in the band thickness of vimentin between mature anterior and mature posterior vitreous in the WB, that difference was not statistically significant.


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

Liu Y, Bouhenni RA, Dufresne CP, Semba RD, Edward DP - PLoS ONE (2016)

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).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835093&req=5

pone.0153560.g003: 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).
Mentions: WB analysis and densitometry of the blots (Fig 3) were used to confirm results obtained by LC-MS/MS. Similar to LC-MS/MS, CA3 was more abundant in young anterior vitreous than in young posterior vitreous (p = 0.04). The expression of CA3 in mature anterior vitreous was also higher than that in mature posterior vitreous (p = 0.03). CRYBB2 was more abundant in anterior compared to posterior vitreous in both young and mature rabbits (p = 0.02 and 0.04, respectively). The expression of CRYAB was also greater in the anterior vitreous in young and mature rabbits (p = 0.02 and 0.0495, respectively). However, there were no significant differences in the level of vimentin between young anterior and young posterior vitreous (p = 0.08) or mature anterior and mature posterior vitreous (p = 0.08). Although a visible difference was seen in the band thickness of vimentin between mature anterior and mature posterior vitreous in the WB, that difference was not statistically significant.

Bottom Line: 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.Western blotting confirmed the differential expression of carbonic anhydrase III, alpha crystallin B and beta crystallin B2.

View Article: PubMed Central - PubMed

Affiliation: Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, United States of America.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus