Ординатура / Офтальмология / Английские материалы / Biochemistry of the Eye 2nd edition_Whikehart_2003

Proteins • 29

Figure 2–14

Graphs of protein concentrations in normal and cataractous lenses. The areas between the lines represent the amounts of soluble and insoluble protein (respectively) while the top line indicates total protein. (Adapted from Spector A, 1984)

a mixture of crystallins as well as a noncrystallin 43,000-dalton protein, a polypeptide located on the inner face of the plasma membranes of lens fiber cells. Spector (1984) has suggested that a 26,000-dalton intrinsic membrane protein may also be part of the HM fractions in cataracts. HM3 has a molecular weight range between 2 and 33 million daltons, a very large molecular species (Harding, Crabbe, 1984). HM3 is held together by disulfide bonds and is associated with cortical cataracts.

Fraction HM4 is composed of crosslinks that are not disulfide bonded and occurs exclusively in nuclear cataracts. HM4 is also insoluble and requires a denaturing agent for solubilization. This fraction is associated with the dark color of nuclear cataracts and, until recently, the source of the color was speculative, (Harding, 1981; Aquilina, et al, 1999). It is an important observation that the cataract formation associated with these fractions cause both light scatter as well as light absorption (Harding, 1991). This twofold component of cataract formation represents a considerable severity for the patient inasmuch as light scattering causes both the blurring of images as well as light glare (Bettelheim, Chylack, 1985). The properties of these HM fractions are summarized in Table 2–4.

It has been hypothesized that senile cataracts take place because of growing HM fractions that form at the inner layer of the lens cell plasma membranes and grow into the cell cytoplasm. The protein complexes continue to grow as the lens ages by forming either disulfide or other

30 • Biochemistry of the Eye

1Disulfide linked.

2Not disulfide linked, but containing one or more light absorbing (i.e., colored) components. 3May also bond to a 26 kD protein.

covalent bonds (Figure 2–15). This may be seen in A of the figure. This is the stage to which nuclear cataracts form, but with nondisulfide bonds. An important difference with cortical cataracts, however, is that the strain produced in the cell membrane by the growing HM aggregates, as seen in B of the figure, may lead to membrane rupture (arrow). The resultant cellular debris for the cortical cataracts would include membrane fragments such as the micellar form in C to which are attached HM crystallin aggregates. These would, at least partially, constitute the fragmented or globular formations, which are described clinically.

The reason why such an aggregation of disulfide bonded and other crosslinked crystallins occurs is not certain. Nor is it certain where the degree of formation must extend to begin membrane disruption. Some older patients are seen who have limited lens fiber cell disruption and yet have clinically clear lenses. In the case of disulfide bonded crystallins, Garner, Spector (1980) have shown that there is a difference in the oxidation state of both methionine and cysteine in the crystallins and the 43,000 dalton protein of normal 60 to 65-year-old human lenses vs. those that are cataractous. These investigators suggested that, in the noncataractous state, the sulfur containing amino acids are buried in normal conformation of crystallins and are prevented from oxidizing (that is, forming sulfoxide and disulfide bonds). Minimal oxidation of exposed groups, however, may cause conformational changes in the crystallins that expose other oxidizable groups, which, in turn, bring about aggregate formation. Glutathione (a tripeptide: γ-Glu-Cys-Gly), is thought to protect crystallins from crosslinking by binding to such exposed groups.

Evidence indicates, however, that its function may be overwhelmed in cataract formation (Spector, 1984). This crosslinking would apply only to cortical aggregates that form cortical cataracts and is indicated in Figure 2–16. The process of progressive oxidation might be introduced by extralenticular hydrogen peroxide in the aqueous (Spector, 1984).

The cause for nuclear cataracts is more cryptic and the nature of the color in brunescent nuclear cataracts, mentioned previously, has remained speculative until very recently. A review of some of the earlier studies is helpful in understanding the problem. The tryptophan amino acid components of crystallins had been suspected to be associated with nuclear cataracts (Harding, Crabbe, 1984). Normally, there is an increase in yellow coloration of the lens, which augments with age and which had been thought to be associated with changes in the tryptophan components of crystallins. Radiation in the range of 300 to 400 nm (including sunlight) has the potential of being cataractogenic since

Proteins • 31

Figure 2–15

The aggregation of lens proteins proposed to occur in senile cataract formation. Both nuclear and cortical cataracts aggregate as at (A). The aggregated crystallins (white blobs) are seen bound to each other by disulfide (or other) bonds. They are also bound to 43K and 26K proteins at the plasma membrane of the lens fiber cells. In cortical cataracts, the aggregation proceeds to cause a rupture in the membrane (arrow at B), which destroys lens fiber cells and produces cellular debris as seen at (C). (Adapted from Spector A, 1984)

the lens absorbs these wavelengths. As a result of this, a possible connection between such ultraviolet light and tryptophan alteration was suggested by Pirie (1971, 1972) who reported that ultraviolet light was able to oxidize tryptophan to N’-formylkynurenine in proteins in vitro (Figure 2–17 A and B). She thought that such altered amino acids in crystallins may lead to the yellow to brown coloration in nuclear cataracts. It was also indicated that this process might eventually lead to the nondisulfide crosslinking that is characteristic of HM4 aggregates found in nuclear cataracts. Some candidate substances, found in lenses, which have been proposed to link tryptophan with nondisulfide crosslinking are anthranilate and β-carboline (Garcia-Castineiras, Dillon, Spector, 1978; Truscott, Faull, Augusteyn, 1977) (see Figure 2–17 C and D).

Subsequently, van Heyningen (1973) demonstrated that hydroxykynurenine instead of kynurenine was the metabolite that could be linked to the interference of light passing through the lens acting as a UV filter.

Although there has been considerable confusion about the role of hydroxykynurenine in subsequent years, Wood and Truscott (1993),

32 • Biochemistry of the Eye

Figure 2–16

The progressive oxidation of lens crystallins. Some investigators believe that the normally folded (i.e., globular) form of crystallins protect the oxidizable amino acids: methionine (-S-CH3) and cysteine (-SH) as seen at

(A). In the course of aggregation, methionine is first oxidized to SO-CH3, possibly by hydrogen peroxide in the adjacent aqueous. This action could partially unfold the protein as seen at (B). Subsequently, the exposed cysteines would oxidize, as -S-S-, forming disulfide bonds with other crystallins and membrane proteins in a series of chain reactions as seen at (C) and (D).

demonstrated that 3-hydroxykynurenine (3OHK) and its glucose bound analogue are produced by the catabolism of the amino acid tryptophan in the lens epithelial cells and, possibly, the most outer lens fiber cells. The metabolic pathway of tryptophan catabolism is shown in Figure 2–18. The normal function of these metabolites is to act as filters for UV light (300 to 400 nm) in order to protect the retina from UV damage (Dillon, 1994). Chen, et al (1997) found that at least the N-terminal domain of α-crystallins was involved in the formation of colored compounds that are associated with nuclear cataracts. Almost simultaneously, Aquilina,

Figure 2–17

The oxidation of the amino acid tryptophan to kynurenine can be obtained in in vitro proteins by ultraviolet light (as in A and B). Anthranilate, a breakdown product of kynurenine (C) has been found in cataractous lenses. However, no relationship has been established between the possible formation of kynurenine itself in the lens and any lens coloration. β-Carboline, an example of one of many forms, is shown in (D). It has also been found in the lens and was suggested to be a fused ring form of tryptophan and another amino acid in crystallins, which produces yellow or brown coloration there. This is no longer thought to be true.

Proteins • 35

Figure 2–20

Partial diagrammatic cross section of a rod outer segment showing the placement of rhodopsin molecules (black eggs with hooks) in the discs and plasma membrane of the rod.

The hooks represent sugar-containing N-termini of the molecules, which prevent molecular flip-flopping and maintain the orientation of the molecules. It is well known that the vast majority of rhodopsin molecules are in the discs. Proteins, such as peripherin/rds (located at each end of the disc [speckled blob]), prevent rhodopsin from migrating around the ends of the discs. (Adapted from Anderson RE: Biochemistry of the eye. San Francisco, 1983, American Academy of Ophthalmology.)

region contains several hydroxy amino acids, which can be phosphorylated. Phosphorylation acts as a mechanism for turning off the sensitivity of the activated protein to light as described in Chapter 7. The portion of the molecule that traverses the membrane consists of 7 α-helices (secondary structure), whose sequences (primary structure) are composed of substantial amounts of hydrophobic amino acids. Having α-helices that traverse membranes is a characteristic common to many intrinsic membrane proteins and these helices impart a strong association of the protein with the lipids that make up the membrane environment.

Rhodopsin has an additional, nonprotein component: vitamin A. This lipid soluble vitamin is contained within the membrane lipidassociated portion of the molecule (see arrow, Figure 2–21). The vitamin A component is bound to the protein at the amino acid lysine, #296 from the N-terminal end of the molecule. As part of the rhodopsin molecule, vitamin A is present as an aldehyde (known also as retinal) and consists of the 11-cis form (Figure 2–22). This form represents the most energetically favorable configuration for its confinement among the helices of the protein. The bond linkage of the aldehyde form with the protein is that of a protonated (H+ added) Schiff base:

retinal – CH = NH+ – protein

36 • Biochemistry of the Eye

Figure 2–21

Representation of a rhodopsin molecule situated in a rod disc membrane. Note the presence of seven alpha helices in the area of the membrane, the 11-cis retinal buried within the helices (arrow), the small sugar chains at the N-terminus (small spheres), and the phosphorylated serine amino acids near the C-terminus (where the phosphate groups are located). (Adapted from Dratz EA, Hargrave PA: Trends Biochem Sci 8:128, 1983.)

A Schiff base has the characteristic of being less “permanent” or stable than many covalent bonds so that vitamin A can be more easily detached upon light stimulation.

Rhodopsin is referred to as a holoprotein when retinal is attached to lysine #296. In 1958, George Wald (1968) demonstrated that light isomerizes the 11-cis retinal group of rhodopsin to all-trans retinal (see Figure 2–22). This creates an energetically unstable molecule that rapidly proceeds through several intermediate forms: bathorhodopsin, lumirhodopsin, metarhodopsin I and II in which the Schiff base linkage is deprotonated and finally broken (see Figure 2–22). The completion of the reaction results in the release of vitamin A from the protein as all-trans retinal. The vitamin-detached protein then is called opsin. When any nonprotein portion of a holoprotein (such as rhodopsin) is released from its protein, the remaining protein portion is labelled an apoprotein. Opsin is, therefore, an apoprotein. The nonprotein portion, such as vitamin A, is termed a prosthetic group. Table 2–5 summarizes the properties of rhodopsin.

Proteins • 37

Figure 2–22

The conversion of rhodopsin to all-trans retinal and opsin. Rhodopsin is shown at the top with 11-cis retinal bound to opsin via a protonated Schiff base. The initial reaction (1), caused by a photon of light striking the molecule, is the conversion of 11-cis retinal to all- trans retinal while bound to the protein producing bathorhodopsin (a form in which the structure of retinal is strained). Three intermediate forms are then rapidly made in sequence (2): lumirhodopsin, metarhodopsin I, and metarhodopsin II (the last form shown). Each of these forms, with its own spectral properties, produce slightly different protein conformations (not shown). In the conversion of metarhodopsin I to metarhodopsin II, the Schiff base is deprotonated. Metarhodopsin II is also known as photoexcited rhodopsin and is further discussed in Chapter 6. Finally, the Schiff base is broken (3) with the release of all- trans retinal and opsin. Rhodopsin is regenerated in two stages (4–5). All- trans retinal is isomerized to 11-cis retinal by the protein enzyme: retinal isomerase (4) and the 11-cis retinal recombines with opsin (5).

It has been known for many years that rhodopsin also has certain spectral or light absorbing properties that are not part of the visual transduction process per se. Figure 2–23 shows the absorption spectrum of frog rhodopsin before and after exposure to light, that is, before and after the conversion of rhodopsin to all-trans retinal and opsin. This process is also called the bleaching of rhodopsin. Three peaks are seen. The α-peak (approximately 515 nm) is the absorption of 11-cis retinal

T A B L E 2 – 5 PROPERTIES OF RHODOPSIN

1Less retinal and the oligosaccharides.

2See Chapter 3 for structures. Adapted from Shichi H, 1983.

38 • Biochemistry of the Eye

Figure 2–23

The absorption spectrum of rhodopsin and opsin. The spectra of intermediate forms are not shown. The γ-peak is characteristic of all proteins, while the β-peak is the absorption peak of unbound all-trans retinal and the α-peak is bound 11-cis retinal.

bound to opsin (as rhodopsin). The β-peak (approximately 375 nm) is the absorption of all-trans retinal, which is not bound to opsin. The γ-peak (280 nm) is the protein absorption peak common to both opsin and rhodopsin. The shifting of these absorption signals led investigators to discover the intermediate forms of rhodopsin and its final products in the bleaching process. Rhodopsin is able to absorb light by virtue of having that light raise the electronic and other energy levels inherent in the rhodopsin molecule. This is only possible at specified wavelengths of light and the relationship of absorption to the protein concentration can be expressed by an equation developed from Beer’s law (Meites, Thomas, 1958):

A = abc

where A is the absorbance, a is the absorptivity, b is the light pathlength, and c is the concentration. The absorbance may be considered as the ratio of the light shone on a sample compared to the light that is transmitted through the sample. The absorptivity is a quantity that mathematically contains the physical-chemical characteristics of the sample and its surroundings. Absorptivity must be determined empirically (i.e., experimentally). For example, the molar absorptivity of rhodopsin = 40,000 cm–1 M–1. The light pathlength is the distance that the light travels in passing through a sample and is usually 1 cm. This light passes through the sample in a special container known as a cuvette. The concentration of the sample is the most useful part of the equation inasmuch as it gives the amount of sample present in a given volume. Accordingly, one can determine the amount of rhodopsin present when the absorptivity is known together with the light path and the absorption. Absorption values are obtained in an instrument known as a spectrophotometer (Figure 2–24).

The conversion of rhodopsin to opsin is only the beginning of the visual transduction process. In Chapter 7 the remainder of the mechanism will be described. Rhodopsin is the visual transduction protein of rod photoreceptors whose role is principally that of visual sensation, particularly at lower light levels. Color vision, however, is realized by light