The zebrafish lens proteome during development and aging

Abstract

Purpose: Changes in lens protein expression during zebrafish development results in a smooth gradient of refractive index necessary for excellent optical function. Age-related changes in crystallin expression have been well documented in mammals but are poorly understood in the zebrafish.

Methods: In the zebrafish lens, a systematic analysis of protein content with age was performed using size exclusion chromatography (SEC) combined with linear trap quadrupole Fourier transform tandem mass spectrometry (LTQ-FT LC-MS/MS; rank-order shotgun) proteomics in lenses of larval, juvenile, and adult zebrafish.

Results: alpha-Crystallins, previously shown to have low abundance in the zebrafish lens, were found to increase dramatically with maturation and aging. SEC determined that beta-crystallin was predominant at 4.5 days. With age, the alpha- and gamma-crystallins increased, and a high molecular weight fraction appeared between six weeks and six months to become the dominant component by 2.5 years. Similarly, shotgun proteomics determined that beta-crystallins were the predominant proteins in the young lens. With age, the proportion of alpha- and gamma-crystallins increased dramatically. After crystallins, calpain 3, membrane, and cytoskeletal proteins were most abundant. Five new beta-crystallins and 13 new gamma-crystallins were identified.

Conclusions: As expected, SEC and proteomics demonstrated changing levels of protein expression with age, especially among the crystallins. The results also confirmed the existence of novel crystallins in the zebrafish genome.

Figure 1

Size exclusion chromatography of zebrafish lens homogenates during development and aging. Absorbance at 280 nm for detection of proteins was plotted versus elution volume (x-axis), which corresponds with molecular size. Individual protein molecular weight standards are shown at the bottom of the graph. High molecular weight aggregates elute early followed by a broad peak of polydisperse α-crystallin oligomers with an average size of 24 subunits. Next, a broad peak of β-crystallin elutes from the column, which forms octamers, tetramers, and dimers, and finally, γ-crystallins, which are monomeric, were observed. The youngest lenses (4.5 days) were dominated by a large broad β-crystallin peak. α-Crystallin and γ-crystallin abundance increased during lens maturation, and a high molecular weight peak, first observed at six months, increased with age to become the largest peak by 2.5 years.

Figure 2

Slit-lamp views of living, anesthetized six-month-old (left panel) and 2.5-year-old (right panel) WIK zebrafish. Minimal light scattering is visible from the cornea, and no light scatter is visible from the lens. Lens transparency was maintained over 2.5 years, demonstrating the clarity of the lens and cornea in the zebrafish at ages up to 2.5 years.

Figure 3

Phylogenetic tree of human and zebrafish β-crystallin genes constructed by Mega 4 with 1000 bootstraps. All zebrafish β-crystallins listed were detected by shotgun proteomics of the zebrafish lens (Table 2). Five novel β-crystallins (unfilled symbols) were detected and named based on their alignment. H, human; Z, zebrafish.

Figure 4

Phylogenetic tree of zebrafish γ-crystallin genes constructed by Mega 4 with 1000 bootstraps. All zebrafish γ-crystallins listed were detected by shotgun proteomics of the zebrafish lens (Table 3). Thirteen novel γ-crystallins (unfilled symbols) were detected and named based on their alignment.

Figure 5

Zebrafish chromosome 9,400 kilobase-pair region containing 35 known and hypothetical γM-crystallin genes. Proteins from 30 genes in this region were found by shotgun proteomic analysis of the zebrafish lens (Table 3). The five genes marked (indicated by asterisk) were not detected but also show sequence similarity to the γM-crystallins. Gene positioning was determined by the NCBI map viewer, Ensembl Genes on Sequence Map. The scale on the left side of the image represents mega base-pairs. The gray line represents the chromosome. Genes on the left side of the gray line are located on the minus strand, and genes on the right side of the gray line are located on the plus strand.