Ординатура / Офтальмология / Английские материалы / Eye, Retina, and Visual System of the Mouse_Chalupa, Williams_2008

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Proteomics, the science surrounding all of a sample’s protein constituents, and the eye lens have a long mutual history (Hoehenwarter et al., 2006b). The lens is a tissue whose function is to concentrate light and help produce a sharp visual image. It is completely avascular and consists mostly of the proteins of one superfamily, the crystallins (Wistow and Piatigorsky, 1988). Their short-range order and precise molecular arrangement in solution establish the refractive index and ultimately its function (Delaye and Tardieu, 1983).

Proteomics has extensively characterized these proteins in many species. A high-definition separation of all the ureasoluble protein constituents of the mouse lens was produced using large-scale carrier ampholyte-based two-dimensional gel electrophoresis (2DE) (Jungblut et al., 1998). This work produced 1,940 distinct dye-stained protein spots, many of which were excised from the gel and analyzed with matrixassisted laser desorption ionization time-of-flight mass spectrometry. It became clear that most of the polypeptides of the eye lens of the mouse are indeed different modified crystallin proteins, or more appropriately crystallin protein species. In-depth studies have been completed in several wildtype and naturally occurring and induced mutant mouse strains and have identified numerous posttranslational modifications in the healthy lens and in the lens affected by cataract, and have elucidated some of the factors implicated in this disease. Future studies should establish the totality of the protein species, resolve proteins specifically involved in the onset and progression of cataract, and give an in-depth understanding of the tissue as a whole.

Major structural proteins of the lens: The crystallins

The proteins of the crystallin superfamily make up 80%– 90% of the entire mass of lens proteins. They were originally classified according to their native size, and the first three letters of the Greek alphabet were assigned to them in descending order of their molecular weight (Mörner, 1893). α-Crystallin is the largest native crystallin, with a molecular weight of 300–>1,000 kd; β-crystallin is the next largest, with a molecular weight of between 40 and 200 kd; and γ- crystallins are the smallest crystallins, with a molecular weight of around 20 kd. Native α- and β-crystallins are multimers composed of several types of monomeric subunits; native γ-crystallins do not polymerize. More recent size

exclusion chromatography has separated the native watersoluble crystallins of the adult lens more precisely into a large high-molecular-weight (HMW) fraction containing mostly α-crystallin and other proteins on the verge of insolubilization, a fraction containing α-crystallin, a higher and a lower molecular weight fraction of β-crystallin, β high (βH) and β low (βL), and a small fraction containing the γS-crystallin monomer, followed by the larger and lowest molecular weight γ-crystallin fraction.

The native crystallin proteins are in various stages of solution and can be thought of as bulk material that fills the otherwise empty lens cells. This material is transparent due to the supramolecular arrangement and short-range order of the proteins. This order is not random but is precisely defined at a molecular level. A disturbance of this homeostasis can lead to unfolding, improper folding, insolubilization, and aggregation of proteins. The insoluble aggregates diffract incident light, resulting in opacification of the lens and visual impairment. This is what is known as cataract (Harding, 1972; Carrell and Lomas, 1997).

The peculiarities of the lens, namely, the little protein biosynthesis and reduced metabolism and the exposure to light and its effects, have imparted a high degree of stability to the crystallins. They are also connected to the stress response (de Jong et al., 1989) next to their function as transparent structure. The protein concentration and the stochiometry of the crystallins in the regions of the lens vary, so a refractive index gradient is created. In most species the refractive index declines from center to periphery, which increases the convexity of the lens and can eliminate spherical and chromatic aberration (Fernald and Wright, 1983). This is especially so in the mouse, which has a lens nucleus with a very high protein content and little water.

The α-crystallins are clearly distinct from the β- and γ- crystallins in molecular evolution. In contrast, the β- and γ- crystallin proteins seem to have a common ancestor. This leads to the assumption of an initial α-crystallin and β-/γ- crystallin gene in an early archetype lens that differentiated into the multiple genes of the current crystallin protein families. The 16 major proteins of the α-, β-, and γ-crystallin protein families are highly conserved and ubiquitous in the lens of mammals (Lubsen et al., 1988), and are also present in many other species. Two in an evolutionary context, more distant members of the γ-crystallin protein family, are also known in the mouse, γNand γS-crystallin (van Rens et al.,

1989; Wistow et al., 2005). In addition, a number of less abundant crystallin proteins with specific functions related to enzymes have been detected in the lens of various species (Wistow, 1993).

α-Crystallin Protein Family α-Crystallin is the most abundant protein in the lens. It constitutes between 30% and 50% of the total mass of lens proteins and is evenly distributed throughout the organ in most species. In the mouse, there are three unmodified α-crystallin polypeptides, αA-crystallin, αB-crystallin, and αA-insert crystallin. αAand αB-crystallin are 57% identical in primary structure. αA-insert crystallin is unique to mammals and common in rodents and is also a product of the αA-crystallin gene. It is produced by alternative splicing of an insert exon in the first intron of the gene (Cohen et al., 1978).

α-Crystallin has never been crystallized, so its threedimensional (3D) structure could not yet be determined by X-ray diffraction. The native proteins’ large size also precludes nuclear magnetic resonance (NMR) measurements. However, the secondary and tertiary structure of the individual α-crystallin gene products are known. Circular dichroism and infrared measurements have determined that they are composed primarily of β strands with little α helix structure (Thomson and Augusteyn, 1989; Farnsworth et al., 1997). They are subdivided into a hydrophobic, globular N-terminal domain, a hydrophilic C-terminal domain in β sheet conformation, and a C-terminal extension. The amino acid composition and tertiary structure of the N-terminal domain are relatively varied in the polypeptides and between species but include three structure-function regions in α- helical conformation (Smith et al., 1996; Pasta et al., 2003). A large part of the C-terminal domain is known as the α- crystallin domain (Caspers et al., 1995) and is conserved in the protein family. The C-terminal extension is variable. We have constructed a tertiary structure model of αAcrystallin of the mouse using ab initio protein structure prediction algorithms (figure 58.1; Hoehenwarter et al., 2006a).

α-Crystallins undergo extensive posttranslational modification beginning at the earliest stages of lens development. These are mainly deamidation, phosphorylation, and N- and C-terminal truncation. The unmodified and modified α-crystallin monomers are the minimal subunits that assemble the functional native protein. Several models have been proposed that agree that they exchange dynamically (van den Oetelaar et al., 1990; Gesierich and Pfeil, 1996) and form small multimers as the building blocks of higher molecular order (Bova et al., 2000). Posttranslational modification of the monomers affects subunit multiand oligomerization (Merck et al., 1992; Bova et al., 2000, Pasta et al., 2003; Thampi and Abraham 2003), quaternary structure dynamics (Bova et al., 2000; Pasta et al., 2003), and protein function

Figure 58.1 Model of αA-crystallin secondary and tertiary structure, colored according to the hydrophobicity of its amino acid residues. The most hydrophobic residues are colored dark blue; the least hydrophobic residues are colored red, as shown in color plate 67. The Rosetta algorithm on the HMMSTR server (www.bioinfo. rpi.edu/~bystrc/hmmstr/server.php) (Bystroff and Shao, 2002) available on the ExPASY (http://au.expasy.org/) home page was used for molecular modeling. Full-length αA-crystallin secondary structure was calculated at 29.5% α helix and 32% β sheet content. The N-terminal globular domain is organized into three helices, displayed as ribbons with hydrophobic side chains buried. Struc- ture-function regions identified earlier (Smith et al., 1996; Pasta et al., 2003) make up the first two of these N-terminal α helices. The highly conserved residues 102–117 of the “α crystallin domain” (Caspers et al., 1995), containing the substantial first part of a DNA-binding motif (Singh et al., 1998), as well as an arginine residue 116 shown to be critical for molecular integrity (Bera et al., 2002), are predicted to have α-helical conformation and are displayed as ribbons. This is consistent with an older 3D model (Farnsworth et al., 1998) and makes the α-helical prediction that is somewhat higher than previous calculations (Farnsworth et al., 1997; Horwitz et al., 1998; Bova et al., 2000) seem plausible. However, it is inconsistent with site-directed spin label studies that demonstrate β sheet conformation for residues 109–120 (Berengian et al., 1997). The model confirms the β sheet secondary structure of residues 67–101, determined to be an alcohol dehydrogenase (ADH) and 1,1′-bi (4-anilino) naphtalene-5,5′-disulfonic acid (bisANS) binding site and to exhibit extensive chaperone activity (Farnsworth and Singh, 2004). See color plate 67.

(Takemoto et al., 1993; Pasta et al., 2003). The small heat shock protein HSP27 can also coassemble with the α- crystallin monomers. Thus, α-crystallin is a polydisperse and highly dynamic protein whose size, structure, and function vary according to the composition of its subunits.

α-Crystallin is a molecular chaperone (Horwitz, 1992; Jakob et al., 1993). The “α-crystallin domain” is implicit in all small heat shock proteins, and it was shown that αBcrystallin is indeed a member of this protein family (Klemenz et al., 1991). The small heat shock proteins interact with denatured proteins, keeping them in solution and in a refoldable conformation independently of ATP. The substrates of α-crystallin in the lens are the β- and γ-crystallins (Wang and Spector, 1994; Bloemendal et al., 2004), some enzymes, and elements of the cytoskeleton, such as the intermediate filament protein vimentin and the beaded filament. It is reasonable to assume that next to its role as transparent structural material, α-crystallin induces proper cytoskeleton architecture and prevents protein insolubilization, and that it is a major factor in upholding the clarity and function of the lens over years.

β- and γ-Crystallin Protein Families The β- and γ- crystallins are the other abundant major proteins in the lens. The families have very similar protein and gene structure and thus presumably have a common ancestor. In the lens of the mouse and most other mammals, there are seven unmodified β-crystallin polypeptides, four relatively acidic, termed βA1 through A4, and three more basic, termed βB1 through βB3. βA1and βA3-crystallin are the products of the same gene and are the result of alternate translation initiation starting points. In the mouse, βA1-crystallin lacks the first 17 N-terminal amino acid residues of βA3-crystallin. There are six highly conserved, unmodified γ-crystallin polypeptides, γA–γF, whose genes are linked in one gene cluster, and two more distantly related polypeptides, γNand γS-crystallin. The primary function of both the β- and γ- crystallins is as transparent material.

Data on the protein structure for both families are abundant. β- and γ-crystallins have similar secondary and tertiary structure. The monomers are composed mostly of β strands organized into four motifs of four antiparallel β strands each. The motifs are of the Greek key type, with the β strands 1, 2, and 4 forming a β sheet and strand 3 in a proximal position. This motif structure leaves many hydrophobic amino acid residue side chains exposed. The isolated β strand 3 interacts with β strand 4 of the β sheet of the neighboring motif, and the two motifs assemble into a complete globular domain with hydrophobic side chains buried. The monomers are subdivided into two globular domains separated by an unorganized stretch of primary structure, the connecting peptide. Also, primary structure can extend beyond the domains, which are then known as either the N- or C- terminal extensions.

The β-crystallin monomers are subunits that assemble the native higher molecular weight β-crystallin protein. The protein can be homoor heteromeric (Bax et al., 1990; Slingsby and Bateman, 1990; Bateman et al., 2003) and is

mostly a dimer (Slingsby and Bateman, 1990) or tetramer; however, further oligomerization is also known (Bateman et al., 2003). The connecting peptide is extended, which mediates the interaction of two globular domains from individual molecules in pseudo-twofold symmetry. The βB-crys- tallins have long unstructured N- and C- terminal extensions, the βA-crystallins have only N-terminal extensions. Their function is not clear, but there is evidence that the extensions are involved in promoting higher-order assembly of β-crys- tallin tetraand oligomers, and that they may act as “spacers” in the supramolecular arrangement of the proteins (Nalini et al., 1994; Bateman et al., 2003).

The native protein is found throughout the fiber cells of the lens; however, the distribution of the individual β- crystallin monomers varies. βB2-crystallin is the major β-crystallin monomer in mammals, and synthesis of the β- crystallins is increased postnatally, so that the native protein is more abundant in the cortex than in the nucleus of the lens.

The γ-crystallin proteins are strictly monomers. The connecting peptide is bent, which brings the two globular domains of one molecule into close proximity and promotes intermolecular domain interaction around a pseudo-twofold axis (Blundell et al., 1981). The two motifs that constitute a domain have adopted a slight asymmetry that allows rows of hydrophobic residues to interdigitate much as in a zipper and creates an extra degree of close packing. The γ-crystal- lins lack N-terminal extensions, and their C-terminal extensions are very short, so the molecules as a whole are very compact, with only limited regions exposed for proteolysis. This guarantees maximum protein stability and allows a very close association, to a high degree excluding water (Mayr et al., 1994). Indeed, the γ-crystallins achieve the highest protein density and are particularly suited for the dehydrated conditions in the nucleus of the lens, where they are most abundant and create the highest refractive index. In this context, the proteins can only conditionally be considered monomeric; it seems more plausible that they have entered a state of macromolecular crowding in an almost completely dehydrated environment (Stevens et al., 1995). The γ-crystallins also have an unusually high content of cysteine residues, which may be involved in molecular bonds with other molecules in this tightly packed arrangement.

Proteomics and eye lens proteomics: An introduction

The high abundance and easy accessibility of the proteins of the lens have made the tissue a frequent subject of proteomics studies. Proteomics is a science dedicated to the comprehensive understanding of the totality and the dynamics of all of the protein constituents of a sample. Arbitrarily it began in 1975, when O′Farrell developed the 2DE technique combining isoelectric focusing (IEF) and sodium

dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) for large-scale separation of the proteins of biological samples (O′Farrell, 1975). With this technique, denatured proteins migrate to positions termed protein spots on the 2DE gel, as dictated by the relation of their chemical parameters to the applied 2DE parameters (Jungblut et al., 1997). High quality 2DE has a remarkable resolution capacity, being able to separate up to 10,000 sample constituents distinguished by a single amino acid or posttranslational modification (Klose and Kobalz, 1995). The separated proteins in the gel are visualized by staining them with a dye or by blotting them onto membranes and then staining them, resulting in a 2DE protein spot pattern.

The other premier technique employed for large-scale protein and peptide separation in proteomics is liquid chromatography (LC). LC is one of the oldest and best techniques for molecular separation in biochemical analysis, earning Archer Martin and Richard Synge the Nobel Prize for Chemistry in 1952. It is based on the individual behavior of soluble analytes in a mobile phase passed over and interacting differentially with a solid phase or matrix. Various combinations of liquid and solid phases featuring distinct molecular interaction and separation properties have been developed and tried over the years, leading to several popular LC approaches, among them affinity chromatography, ion exchange chromatography, reverse phase chromatography, and size exclusion chromatography. The combination of two or more of these techniques, in conjunction with relatively long durations (several hours) and low flow rates (nano-LC), has remarkable resolution power.

Following separation, it is desirable to identify as many proteins as completely as possible. Originally, purified or separated proteins were mostly analyzed by Edman degradation, a procedure that realizes protein primary structure beginning with a polypeptide’s N-terminus (Edman, 1949). Primary structure information is the most basic and meaningful form of polypeptide characterization and presents the least degree of ambiguities.

A major breakthrough was reached in the late 1980s, when mass spectrometry became available for polypeptide analysis. Originally developed late in the nineteenth century, it is a technique that measures an ionized molecule’s mass and charge. Ionized molecules from an ion source are manipulated by electric fields in an analyzer, where their recorded behavior prior to detection by an ion detector allows calculation of their physical parameters. So-called soft ionization techniques convert polypeptides to gas phase ions without damaging the molecules. The principal soft ionization techniques employed in proteomics are matrixassisted laser desorption/ionization (MALDI) (Karas and Hillenkamp, 1988; Tanaka et al., 1988) and electrospray ionization (ESI) (Fenn et al., 1989). It is common to digest proteins either with enzymes or with chemicals and to

analyze the resulting peptides with mass spectrometry, because mass accuracy, resolution, and charge minimization are all improved in the low mass range, under 3 kd.

The data acquired by mass spectrometry must be converted into meaningful information. A polypeptide’s mass and charge allow the calculation of its primary structure; however, in light of the 20 proteinogenic amino acids and modifications, the possibilities for large peptides and proteins are considerable. In theory, the sequenced genomes of organisms contain the primary structures of all of an organism’s primary translation products. This is an enormous asset for protein identification. Polypeptide masses recorded in mass spectra are used to search databases of conceptually translated genomics data according to primary structure segment masses, in many cases resulting in matches of the mass spectrometric data to amino acid sequences, producing peptide sequence suggestions and conclusive protein identification. This is termed peptide mass fingerprinting or peptide mass mapping (Henzel et al., 1993, Jungblut et al., 1997; Thiede et al., 2005). Modifications can be assessed from mass shifts using the identification as a reference. A number of software suites available commercially or free of charge on the Web expedite this process.

Today’s generation of mass spectrometers deliver compositional and conformational information by fragmenting or promoting dissociation of the analyzed ions and applying a second round of mass spectrometry to the fragment ions. This is termed tandem mass spectrometry, or MS/MS (Senn et al., 1966; Biemann et al., 1966). Metastable dissociation of polypeptides occurs mainly at the peptide bond. If a peptide ion’s charge is retained at the N-terminus following dissociation, the resulting fragment ion is termed b ion, while if the charge is retained at the C-terminus, the fragment ion is termed y ion. These ions allow the additive calculation of mass differences between fragment ion masses and the correlation of the mass differences with amino acid residues. A combination of mass, charge, and MS/MS data—or exclusively MS/MS data, if sufficiently available—can be used to determine protein primary structure, including modifications and modification sites.

Two-dimensional electrophoresis and lens proteomics go together from the outset. Already in 1975 Kibbelaar and Bloemendal applied the water-soluble and water-insoluble proteins of the whole lens, the size exclusion chromatography α, βH, βL, and γ fractions, and the urea-soluble proteins from the calf lens to urea PAGE combined with SDS-PAGE. They produced a first rudimentary protein spot pattern of the major lens proteins, the unmodified crystallins, and suggested α-crystallin interaction with membrane components (Kibbelaar and Bloemendal, 1975). With improvement in 2DE techniques, a definitive protein spot pattern or reference map for the crystallins and other components was achieved in 1982 for the water-soluble proteins of the bovine

lens cortex, with a final definitive nomenclature for β-crystal- lins added in 1984 (Berbers et al., 1982, 1984). Similar investigations in chicken, mouse, rat, and human yielded comparable crystallin reference patterns and α-crystallin characterization with nomenclature and demonstrated the presence of the cytoskeleton proteins vimentin and actin in the urea-soluble fraction, presumably from the anterior epithelial cell layer and outer cortex (Garadi et al., 1983; Garber et al., 1984; Datiles et al., 1992).

Newer investigations have become increasingly comprehensive, often further characterizing crystallin proteins, confirming amino acid sequences predicted from cDNA or detecting discrepancies and annotating sequences and creating higher-quality reference maps, as well as detecting numerous noncrystallin lens proteins (Shih et al., 1998; Lampi et al., 2002; Hoehenwarter et al., 2005). A detailed reference map of the entire adult mouse lens proteome using high-resolution large-scale 2DE separated the urea-soluble proteins into 1,940 spots (Jungblut et al., 1998) and was ultimately followed by an in-depth analysis of the watersoluble and water-insoluble proteins at different ages (Ueda et al., 2002). A 2DE protein spot pattern of the young ureasoluble mouse lens proteome with the characteristic crystallin pattern produced in our laboratory is shown in figure 58.2. An accessible repository for mouse lens and other proteomics data is under preparation in the form of our 2DE database (www.mpiib-berlin.mpg.de/2D-PAGE).

Major LC investigations have characterized the infant to adult water-soluble and the adult water-insoluble human lens proteomes. Following size exclusion chromatography, water-soluble α, βH, βL, and γ fractions were applied to reverse phase chromatography on C4 columns for further fractionation. Whole proteins were then analyzed offline with ESI mass spectrometry. The crystallin proteins αA and αB, βB1, βB2, and βB3, A1, A3, and A4, and γC, γD, and γS were detected and changes in their abundance as well as posttranslational modifications with age were characterized (Ma et al., 1998). Another investigation subjected the water-insoluble monomeric α-crystallins to cation exchange chromatography before reverse phase chromatography and offline ESI mass spectrometry. A high degree of separation was achieved, and it was shown that α-crystallin is the major component of the water-insoluble lens proteins in humans, constituting about half of protein abundance. In addition, numerous α-crystallin protein species were identified and distinguished from the water-soluble fraction (Lund et al., 1996). Large-scale LC-based proteomic investigations of the mouse lens proteome have not been published.

In summary, the investigations described in this section and others not mentioned have proven invaluable for characterizing the proteins of the healthy lens as well as of morphological sections (Garland et al., 1996), and in elucidating some of the complex factors involved in cataract develop-

ment (Garber et al., 1984; David et al., 1994; Calvin et al., 1996; Li et al., 2002) in the mouse and various other species.

Proteomics beyond genomics

The proteins in an organism are not limited to the primary translation products of its genes. Many proteins undergo posttranslational modification. Posttranslational modifications are covalent modifications to a protein’s primary structure that can alter the function of the original unmodified protein. This introduces an additional vast level of functional activity that goes completely beyond the genome and cannot be assayed by genomicsor transciptomics-based research. The taxonomic term protein species was introduced in 1996 (Jungblut et al., 1996) to express this context. A protein species is defined as a polypeptide and possibly one or more other chemical groups that are covalently bonded. As such, it is the most basic term in protein taxonomy. It distinguishes proteins, posttranslationally modified forms of a protein, protein isoforms, different allelic forms of a protein, and any other primary structure variants as mature distinct molecules.

The proteome-wide identification of protein species is one of the major challenges facing proteomics today. A protein species is considered identified only when the entirety of its primary structure is known, so that it can be distinguished from another protein species that differs in only one primary structure element, such as one amino acid or chemical group or, ultimately, one atom. Current techniques, particularly 2DE and mass spectrometry, can achieve this on a small scale (Okkels et al., 2004).

LC procedures are also able to separate the protein species of a proteome but stop short of making them as readily accessible as 2DE. Their primary advantage is the relative speed at which complex protein mixtures can be assayed, thanks to the development of the electrospray ionization technique. Together with a connecting apparatus, it allows polypeptides to be eluted online or semi-online from chromatography into a mass spectrometer (Yates, 1998). To maximize separation performance, multiple steps of different types of chromatography can be combined. This is known as multidimensional protein identification technology, or MudPIT (Washburn et al., 2001). Strategies of this type that analyze peptides from protein digestion or internal cleavage generate between 10,000 and 100,000 distinct mass spectra, making comprehensive evaluation equally as laborious as the complete analysis of spots from 2DE (Swanson and Washburn, 2005; Hoehenwarter et al., 2006a). Although rapid detection of modifications is possible, the assignment of the identified peptides and modifications to individual molecules and thus the identification of the protein species are not.

The lens proteins of the crystallin superfamily undergo extensive posttranslational modification in all species. This process begins before birth and increases as the organism ages, with early-onset modifications becoming more abundant and additional modification types appearing. Indeed, the water-soluble proteins of the nucleus and cortex of the adult human lens were analyzed separately with 2DE, which revealed a nuclear protein spot pattern that is quite distinct from the previously described cortical or whole-lens pattern (Garland et al., 1996). This is evidence of a high degree of

posttranslational modification in the very young lens, as the development of the nucleus is essentially prenatal, and so the nuclear protein species must be derived from posttranslational modification of the primary crystallin proteins. As this report and numerous others have made clear, many types of posttranslationally modified crystallins exist and are abundant in the healthy lens.

The fact that the protein species are mature molecules with individual primary structures implies that they have defined and possibly distinct functions in the supramolecular

order of the transparent mass of proteins in the lens. This is illustrated for some αA-crystallin protein species separated with 2DE (figure 58.3). The protein species were analyzed with mass spectrometry; it was ascertained that they were products of the αA-crystallin gene, and a nomenclature was introduced (Hoehenwarter et al., 2008). One of the protein species, α-A_B, was analyzed in detail (Hoehenwarter et al., 2006a). The protein species’s N-terminus is serine residue 42 of the full-length αA-crystallin protein and very probably the result of truncation by the calcium-depen- dent calpastatin protease Lp82. Its position on calibrated 2DE gels is in accordance with its theoretical molecular weight and pI. Nevertheless, the protein species was not identified, as only 50% sequence coverage was achieved with mass spectrometry. A model of its secondary and tertiary structure was produced with ab initio structure prediction algorithms (Bystroff and Shao, 2002). The insights gained from the model allowed the formulation of a hypothesis for the function of the protein species in the healthy lens. It is involved in the regulation of the size and function of the native protein. Together with other evidence, this discovery suggests that the protein species related to the primary translation products of the α-crystallin genes are also α-crystallin subunits, and that many more subunits than previously suspected influence the properties of the native HMW α- crystallin oligomer.

In contrast to the number of protein species apparently necessary for the development of the healthy lens, some crystallin protein species are known to induce cataract. Their conformation is incompatible with their environment, so

they become insoluble, leading to opacities in the lens and visual impairment. Tryptophan oxidation to kynurenine was observed in γBand γC-crystallins of adult mice ( Jungblut et al., 1998), but a clear connection of this modification to cataract is missing. Certain protein species such as truncated βB1or γ-cystallin (David et al., 1994; Gong et al., 1997; Descamps et al., 2005; Hoehenwarter et al., 2008) are specifically connected to the onset and development of cataract, their appearance in the lens unequivocally leading to the disease. Other protein species may be involved in cataractogenesis; however, they are also found in the healthy tissue. This duality is not fully understood, but it is conceivable that the protein species are deleterious only when their abundance is perpetuated beyond a certain threshold. Also, it has been shown that modifying factors are present in the lens of the mouse, and that the interactions of the crystallin protein species with these protein species affect the onset and development of cataract as well as healthy lens development (Hoehenwarter et al., 2008).

The concept of protein species with individual functions introduces a new level of complexity that is not unrealistic. These functions must be elucidated for a true understanding of the biological processes in the lens and other tissues, organs, and organisms. The first big step will be the complete identification of the proteome at the protein species level, meaning an exact description of native components. One of the techniques that could achieve this in the near future is the combination of multidimensional LC for protein species separation and MS/MS mass spectrometry of whole proteins for protein species identification. This approach,