Mapping of Glutathione and Its Precursor Amino Acids Reveals a Role for GLYT2 in Glycine Uptake in the Lens Core

Abstract

purpose. To correlate the distribution of glutathione (GSH) and its precursor amino acids (cysteine, glycine, and glutamate) with the expression of their respective amino acid transporters in the rat lens.

methods. Whole rat lenses were fixed, cryoprotected, and cryosectioned in either an equatorial or axial orientation. Sections were double labeled with cystine, glycine, glutamate, GSH, GLYT1, or GLYT2 antibodies, and the membrane marker wheat germ agglutinin (WGA). Sections were imaged by confocal laser scanning microscopy. Cystine, glycine, glutamate, and GSH labeling were quantified by using image-analysis software and intensity profiles plotted as a function of distance from the lens periphery. Western blot analysis was used to verify regional differences in amino acid transporter expression.

results. Cystine and glycine labeling in equatorial sections was most intense in the outer cortex, was diminished in the inner cortex, but was increased again in the core relative to the inner cortex. Glutamate and GSH labeling was most intense in the outer cortex and was diminished in the inner cortex to a minimum that was sustained throughout the core. The distribution of cystine and glutamate levels correlated well with the expression patterns observed previously for the cystine/glutamate exchanger (Xc−) and the glutamate transporter (EAAT4/5), respectively. Although high levels of glycine labeling in the outer cortex correlated well with the expression of the glycine transporter GLYT1, the absence of GLYT1 in the core, despite an increase of glycine in this region, suggests an alternative glycine uptake system such as GLYT2 exists in the core. Equatorial sections labeled with GLYT2 antibodies, showed that labeling in the outer cortex was predominantly cytoplasmic, but progressively became more membranous with distance into the lens. In the inner cortex and core, GLYT2 labeling was localized around the entire membrane of fiber cells. Western blot analysis confirmed GLYT2 to be expressed in the outer cortex, inner cortex, and core of the lens. Axial sections labeled for glycine revealed a track of high-intensity glycine labeling that extended from the anterior pole through to the core that was associated with the sutures.

conclusions. The mapping of GSH and its precursor amino acids has shown that an alternative glycine uptake pathway exists in mature fiber cells. Although GLYT1 and -2 are likely to mediate glycine uptake in cortical fiber cells, GLYT2 alone appears responsible for the accumulation of glycine in the center of the lens. Enhancing the delivery of glycine to the core via the sutures may represent a pathway to protect the lens against the protein modifications associated with age-related nuclear cataract.

The principal antioxidant in the lens is glutathione (GSH), a tripeptide synthesized from the amino acids cysteine, glutamate, and glycine by the sequential actions of the enzyme γ-glutamylcysteine synthetase and glutathione synthetase. ¹ Cysteine is the rate-limiting substrate for GSH synthesis, but it is inherently unstable in free solution. However, cystine, the dimeric oxidized form of cysteine, is more stable and abundant than cysteine and on intracellular accumulation is rapidly reduced to cysteine. ^{2 3} Thus, while cysteine synthesis can occur via the transsulfuration pathway, ⁴ the direct uptake of cystine from the aqueous humor is the more likely mechanism for the accumulation of cysteine in the lens. In the lens, we have identified the cystine/glutamate exchanger Xc−, ⁵ a heterodimer composed of a heavy chain (4F2hc) and a light chain (xCT). ⁶ We have shown Xc− to work in combination with different members of the X_AG family, which include the excitatory amino acid transporters (EAAT1 to -5) ⁵ and the alanine serine cysteine transporters (ASCT1 and -2), ⁷ to mediate the exchange of extracellular cystine for intracellular glutamate (Fig. 1) . 

Glycine is the final precursor amino acid necessary for GSH synthesis and is incorporated with γ-glutamlycysteine to form GSH. ¹ Glycine uptake is mediated by a family of Na⁺/Cl⁻-dependent neurotransmitter transporter proteins that include transporters for γ-aminobutyric acid, proline, and monoamines. ⁸ Using RT-PCR, we identified transcripts for both GLYT1 and -2 in the rat lens, but we could only localize the GLYT1 transcript reproducibly. ⁷ Using immunocytochemistry, we showed that GLYT1 expression was restricted to cortical regions of the lens, indicating that GLYT1 may play a role in the uptake of glycine in cortical fiber cells (Fig. 1) . 

Taken together, our previous localization studies have shown that amino acid transporters capable of accumulating the precursor amino acids required for GSH synthesis are differentially expressed in the rat lens ^{5 7} (Fig. 1) . The question now arises as to whether the transporters in the different regions of the lens are actually functional. This question is particularly pertinent in the outer cortex of the lens where many of the transporters we have identified are localized predominately to the cytoplasm of cortical fiber cells. ^{5 7 9 10} We have hypothesized that these cytoplasmic transporters represent a vesicular pool of transporters that are inserted into the plasma membrane at discrete stages of fiber cell differentiation to compensate for the inability of mature anucleate fiber cells to perform de novo protein synthesis. ¹¹ To investigate transport function in different regions of the lens, we used an immunocytochemical approach that allows the distribution of free amino acid levels in the rat lens to be mapped at subcellular resolution, thereby enabling amino acid accumulation to be correlated with the previously determined expression patterns for transporters known to mediate cystine, glutamate, and glycine uptake in other cell types. To validate the utility of this method, we initially correlated free cystine levels with the expression pattern of Xc−. ¹²  

In this report, this metabolite mapping approach is extended to GSH and its other precursor amino acids: glycine and glutamate. We found the levels of GSH and all its precursor amino acids to be high in the outer cortex, a result consistent with the previously observed positive expression patterns for the different amino acid transporters in this region. The levels of these metabolites all dropped to a minimum in the inner cortex, and in the case of GSH and glutamate these low levels were maintained into the core of the lens. In contrast, cystine and glycine levels showed a marked increase in intensity relative to the inner cortex, suggesting active accumulation of these two amino acids in the lens core. In the case of cystine, this accumulation was consistent with the expression of Xc− in this region. However, the observed increase in glycine levels in the core and the apparent restriction of GLYT1 to the outer cortex suggests that an alternative glycine uptake mechanism exists in mature fiber cells. Our results indicate that GLYT2 may act as the transporter responsible for glycine uptake in the lens core. 

Materials and Methods

Reagents

The anti-rabbit antibodies designed to detect free cystine, glutamate, glycine, and GSH were provided by Robert Marc ^{13 14} (John Moran Eye Center, University of Utah, Salt Lake City). The C-terminal tail-specific GLYT1 antiserum was purchased from Chemicon International (Temecula, CA). The C-terminal tail-specific GLYT2 affinity-purified antibody and its corresponding control peptide were purchased from Alpha Diagnostic International (San Antonio, TX). The goat anti-rabbit AlexaFluor 488 secondary antibody, donkey anti-goat-Cy3 secondary antibody, and the membrane marker wheat germ agglutinin conjugated to tetramethyl rhodamine isothiocyanate (WGA-TRITC) or fluorescein isothiocyanate (WGA-FITC) were all obtained from Invitrogen-Molecular Probes (Eugene, OR). Phosphate-buffered saline (PBS) was prepared from PBS tablets (Sigma-Aldrich, St. Louis, MO). Unless otherwise stated, all other chemicals were from Sigma-Aldrich. 

Animals

All animals were treated according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Twenty-one-day-old Wistar rats were killed by CO₂ asphyxiation and the eyes removed. Lenses were extracted from the globe and placed in PBS. Lenses for protein purification were rolled on sterile filter paper to remove any adherent tissues and decapsulated by using sharpened forceps to remove the epithelial cells from the fiber cells. 

Immunocytochemistry

Whole lenses were fixed either in 0.75% paraformaldehyde and 0.01% glutaraldehyde (for cystine, glycine, glutamate, and GSH) or 0.75% paraformaldehyde alone (for GLYT1/2), cryoprotected and cryosectioned in either an equatorial or axial orientation using standard protocols developed in our laboratory. ¹⁵ Sections were washed and incubated in blocking solution (3% bovine serum albumin and 3% normal goat serum) for 1 hour to reduce nonspecific labeling. The sections were then labeled with anti-cystine (1:400), anti-glycine (1:100), anti-glutamate (1:20), anti-GSH (1:100), anti-GLYT1 (1:1000), or anti-GLYT2 (1:50) diluted in blocking solution, followed by secondary goat anti-rabbit AlexaFluor 488 (1:200), or secondary donkey anti-rabbit AlexaFluor 488 (1:800) for 1 hour each. Control sections in which the primary antibodies were omitted or the primary antibody had been incubated with a control peptide (GLYT2 only) were also prepared. To highlight cell morphology, cell membranes were labeled with either WGA-TRITC (1:50) or WGA-FITC (1:50) in PBS. Sections were then washed, mounted in antifading agent (AF100; Citifluor, London, UK) and viewed using a confocal laser scanning microscope (TCS 4D or SP2; Leica, Heidelberg, Germany). A series of overlapping image stacks were acquired along the equatorial radius of the lens and used to generate maximum image projections that were then tiled together to form a seamless image montage. Intensity profiles for cystine, glycine, glutamate, and GSH, were extracted from these montages using methods described by Li et al. ¹²  

Western Blot Analysis

Crude fiber membranes from the outer cortex, inner cortex, and inner core of the lens were prepared from 10 to 15 decapsulated lenses, as previously described. ⁵ The concentration of lens membranes was determined using the bicinchoninic acid (BCA) detection kit (Pierce, Rockford, IL). Proteins (30 μg/lane) were separated on a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane. The membrane was incubated with blocking solution (5% milk in 1× Tris-buffered saline (TBS; 2 mM Tris-HCl, 140 mM NaCl [pH 7.6], 1% Tween-20) at room temperature for 1 hour. The protein blots were incubated overnight with 2 μg/mL primary antibody in 1% BSA-TBS, followed by incubations with biotinylated secondary antibody (1:10,000; GE Healthcare, Piscataway, NJ), and streptavidin-HRP (1:20,000; GE Healthcare) for 1 hour each. Labeled protein was visualized by chemiluminescence detection (ECL plus; GE Healthcare) and exposure onto film (Hyperfilm; GE Healthcare). 

High-Performance Liquid Chromatography

Whole lenses were dissected into three fractions: outer cortex, inner cortex, and core, as previously described. ¹² All three lens fractions were homogenized in 5 mM HCl, 5 mM EDTA, and 5 mM ethylene glycol-bis (β-aminoethyl ether)-N,N,N′N′-tetraacetic acid. The homogenates were centrifuged at 12,000 rpm for 20 minutes and the supernatant retained. For the thiol-containing amino acids GSH and cystine, the supernatant was precipitated with the reducing agent Tris 2-carboxymethyl phosphine (100 mg/mL) and then mixed with 10% trichloroacetic acid. The samples were then derivatized in 0.1% 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate, 0.125 M boric acid, 4 mM EDTA, 1.55 M NaOH. For the non–thiol-containing amino acids glutamate and glycine, the supernatants were mixed with 0.04 M sulfuric acid containing 15 μM of L-nor-Valine (internal standard), and 10% sodium tungstate at 4°C. The samples were then derivatized in 10 mM 6-aminoaquinolyl-N-hydroxysuccinimidyl carbamate (AQC; dissolved in dry acetonitrile and 0.2 M borate buffer [pH 8.8]). All samples were then analyzed by reversed-phase high-performance liquid chromatography (Alliance 2690; Alphatech, Burlington, MA). Standard curves and separations were performed with a 3-μm C-18 column (250 × 4.6-mm Luna; Phenomenex, Torrance, CA) in gradient mode, at a flow rate of 800 μL/min. Absorbance was measured by a fluorescence detector operating at an excitation wavelength of 385 nm and an emission wavelength of 515 nm. The total amount of amino acids in each lens fraction was calculated as previously outlined by Li et al. ¹² The statistical significance of the difference between amino acid amounts in the inner cortex and core were evaluated by Student’s t-test, based on the null hypothesis that the samples had equal means. 

Results

Quantitative Mapping of Cystine, Glycine, Glutamate, and GSH from Labeled Sections

Marc et al. ¹³ have developed an extensive panel of antibodies that specifically recognize low-molecular-weight metabolites. These antibodies should detect only free metabolites fixed in place with glutaraldehyde. To assess the utility of these antibodies to monitor GSH, glutamate, cystine, and glycine levels in the lens, a series of control experiments were performed (Fig. 2) . Equatorial cryosections obtained from lenses fixed either in paraformaldehyde plus 0.01% glutaraldehyde (Figs. 2A 2B 2C 2D , top) or paraformaldehyde only (Figs. 2A 2B 2C 2D , bottom) were double labeled with either the GSH, glutamate, cystine, or glycine antibody (green) and the membrane marker WGA (red). Strong GSH, glutamate, cystine, and glycine labeling was observed only in the presence of glutaraldehyde, indicating that the antibody detects only free GSH, glutamate, cystine, or glycine fixed with glutaraldehyde. Because the glutaraldehyde fixative exhibits some autofluorescence, an additional control was performed to ensure that the signal detected originated solely from the antibody labeling. Sections labeled from lenses fixed in paraformaldehyde plus glutaraldehyde in which the primary antibody was omitted showed only WGA membrane labeling (Figs. 2A 2B 2C 2D , middle) indicating autofluorescence from glutaraldehyde to be negligible. 

Having established our controls in the cortex of the lens, we then extended our investigation of mapping the distribution of GSH and its precursor amino acids in different regions of the rat lens using an imaging approach developed by Li et al. ¹² Cryosections obtained from the lens equator were double labeled with antibodies to either GSH, cystine, glycine, and glutamate together with the membrane marker WGA. Seamless image montages were assembled which captured the maximum signal intensity at every given depth within the section and also with distance across the section (Fig. 3 , top). From the resultant image montages, intensity profiles were extracted to map the average signal intensity along the radius of an equatorial section (Fig. 3 , bottom). Representative intensity profiles collected from each image montage illustrate how labeling varies from the lens periphery (excluding the epithelium) for each amino acid to a distance ∼1300 to 1600 μm into the lens. The levels of GSH and its precursor amino acids were high in the outer cortex and dropped to a minimum in the inner cortex. In the case of GSH and glutamate these low levels were maintained into the core of the lens (Figs. 3A 3B) . In contrast, cystine and glycine levels showed a marked increase in intensity relative to the inner cortex (Figs. 3C 3D) . To check whether the observed elevation in the lens core for cystine and glycine were not an artifact of nonspecific binding intensity, profiles were collected from sections in which either the primary cystine or glycine antibodies were omitted. These profiles showed no such change in signal intensity with distance into the lens, confirming cystine and glycine levels to be consistently and significantly elevated above background levels in the outer cortex and core relative to the inner cortex. 

We compared our quantitative immunocytochemical results to the more traditional biochemical approach, HPLC, for quantifying GSH and its precursor amino acids. Table 1shows the concentrations (in micromolar) of GSH and its precursor amino acids in each fraction and the amount of amino acids (micromoles/gram) in each fraction normalized to wet weight. The amounts of GSH and its precursor amino acids determined by HPLC exhibited similar profiles to those obtained by immunocytochemistry. Thus, results from both methods confirm the bimodal distribution of cyst(e)ine and glycine and suggest that these two amino acids are actively accumulated in the lens core. 

Comparison of Precursor Amino Acid Distribution with Transporter Localization

We next wanted to determine whether the observed distributions of amino acids correlated with the known expression patterns of their transporters. ^{5 7} Lim et al., ⁵ have mapped the expression patterns of the cystine/glutamate exchanger (Xc−), ⁵ the glutamate recycling transporters ^{5 7} (EAAT4/5 and ASCT2), and the glycine transporter ⁷ (GLYT1) throughout the lens with subcellular resolution. A key finding of these earlier studies was that in the outer cortex, amino acid transporters were located both within the cytoplasm (vesicular) and the plasma membrane. Furthermore, for some transporters the subcellular distribution changed in a differentiation-dependent manner. A graphic summary of mean amino acid intensity profiles (n = 3 lenses) and changes in the subcellular localization of amino acid transporters is plotted as a function of relative distance into the lens to facilitate comparisons between the two datasets (Fig. 4) . In this analysis, a positive correlation between the membranous expression of a transporter and the cytoplasmic accumulation of its amino acid is deemed indicative of transporter functionality. Since uncertainty surrounds the molecular identity of GSH transporters in the lens, ¹⁶ we were unable to compare GSH distribution with the localization of its transporter. 

A detailed comparative analysis of cystine levels and Xc− expression has been reported by Li et al. ¹² The profile observed for glycine mirrored the bimodal cystine profile being high in the outer cortex, decreased to a sustained low in the inner cortex, before rising again in the core. However, although high levels of glycine correlated well with the membranous expression of GLYT1 in the outer cortex, the absence of GLYT1 in mature fiber cells did not correlate with the elevated glycine levels in this region. This finding is highlighted in Figure 5which shows images of glycine distribution (Figs. 5A 5B 5C 5D)and the expression of GLYT1 (Figs. 5E 5F 5G)in different regions of the lens. Thus, our metabolite mapping suggests that an alternative glycine uptake mechanism is likely to exist in deeper lying fiber cells. 

Identification and Localization of GLYT2 at the Protein Level

Considering that GLYT2 is the only other known member of the Na⁺-dependent GLYT family, we hypothesized that GLYT2 is the alternative transporter responsible for mediating glycine uptake in the center of the lens. We performed immunocytochemical labeling of equatorial sections labeled with the isoform-specific GLYT2 antibody (green) and the membrane marker WGA (red). Mapping of GLYT2 revealed labeling to be present throughout the whole lens extending from the periphery to the lens core (Figs. 5H 5I 5J) . In peripheral fiber cells, labeling of GLYT2 was predominantly cytoplasmic with minimal labeling of the plasma membrane (Fig. 5H) , a finding similar to that observed for GLYT1. ⁷ However, in contrast to GLYT1, GLYT2 labeling was observed in the inner cortex and core. In these regions, GLYT2 labeling was punctate and localized around the broad and narrow sides of mature fiber cell membranes (Figs. 5I 5J) . Labeling of GLYT2 in all three regions was completely knocked down by either omitting the GLYT2 antibody (data not shown) or by preabsorption of GLYT2 with its corresponding antigenic peptide (Figs. 5K 5L 5M) . Taken together, our immunocytochemical findings suggest regional differences in the expression of GLYT1 and -2. 

To verify this, Western blot analysis was performed on membranes prepared from total fiber (F), outer cortex (OC), inner cortex (IC), and core (C) regions of the lens. Equal amounts of each fraction were loaded (Fig. 6A)and the blots probed with GLYT1 or -2 antibodies (Figs. 6B 6C) . Protein bands for GLYT1 at 46 and 85 kDa were observed in total fiber membranes (Fig. 6B) . The band at 85 kDa is consistent with published reports that have demonstrated the molecular mass of GLYT1 to lie anywhere between 50 and 110 kDa as a result of differential glycosylation. ^{17 18} The band at 46 kDa is likely to represent the nonglycosylated form of GLYT1, since it has been shown that PGNase treatment of GLYT1 yields a band of this molecular mass. ¹⁸ Although protein bands for GLYT2 of 60, 70, 85, 90, 120, and 160 kDa were observed in total fiber membranes, these bands were present only in the cortical fractions and were absent from the lens core (Fig. 6C) . The size of the GLYT2 has been shown to lie anywhere between 70 and 110 kDa as a result of differential glycosylation. ^{17 18} Preabsorption of the GLYT2 antibody with its corresponding antigenic peptide knocked down the majority of this labeling except for a 90-kDa band present only in the outer cortex (Fig. 6D) . In particular, these experiments showed that an 85-kDa band, expressed in all three fractions of the lens, was knocked down by preabsorption of GLYT2, a result that indicates the validity of our immunocytochemical localization of GLYT2 in the lens core. 

Mapping a Sutural Pathway of Glycine Delivery to the Lens Core

The elevated levels of glycine in the lens core, suggest that glycine delivery to the lens center is mediated by the lens sutures similar to previous observations for cystine. ¹² To investigate this, axial sections were doubled labeled with glycine antibodies (green) and the membrane marker WGA (red; Fig. 7 ). A suture that extends from the anterior pole to the core of the lens is evident in Figure 7A . Representative high-power images of glycine labeling in the outer cortex (Fig. 7B)and inner cortex (Fig. 7C)of the lens show strong labeling of glycine along the suture line and the membranes of fiber cells radiating from the suture. In the core, glycine labeling was uniformly spread round the entire membrane (Fig. 7D) . Based on these results, we suggest that glycine may be delivered to the core of the lens via the sutures where it is then accumulated by GLTY2. 

Discussion

By using an immunocytochemical approach to correlate the intracellular accumulation of GSH and its precursor acids with the membranous expression of their respective transporters, we have inferred transporter functionality and identified regional differences in amino acid uptake. We have shown that the outer cortex is a region of active amino acid uptake and GSH synthesis, since cyst(e)ine, glutamate, glycine, and GSH, are all present at high levels, and the amino acid transporters are found in the membrane. In contrast, in the inner cortex, the intracellular levels of all amino acids and GSH decreased toward a minimal level, despite the presence of amino acid transporters in the membrane. We have proposed that the failure to observe accumulation of amino acids in this region is due a restriction of the extracellular space ¹⁹ that creates a diffusion barrier that impairs the delivery of amino acids to their respective transporters expressed in the inner cortex. In the core, the observation that the levels of cystine and glycine increase relative to the inner cortex indicates that for these two amino acids an alternative delivery pathway exists that bypasses the extracellular diffusion barrier formed in the inner cortex. We suggest that this pathway resides in the sutures and that it directly delivers cystine and glycine to the core where they spread via the extracellular space and then are accumulated by their respective transporters. 

In contrast to cystine and glycine, the levels of GSH and glutamate did not increase in the core relative to the inner cortex. This observation suggests that either active transporters for GSH and glutamate uptake do not exist in the lens core, that GSH and glutamate are too large to be delivered to the core via the sutures, or both. With regard to glutamate, we know that a putative glutamate transporter, ASCT2 is expressed in the lens core, but only low levels of glutamate are present in the aqueous humor. ²⁰ Previously, we have proposed that ASCT2 works in conjunction with Xc− in the core to drive the uptake of cystine. ⁷ The observed elevation of cystine levels in the core suggests that ASCT2 is in fact functional. However, the failure to detect an increase of glutamate in the core indicates that the sutural delivery of glutamate to the core is minimal due to the low levels of glutamate present in the aqueous. Thus, we propose that ASCT2 only mediates the local recycling of glutamate to maintain the glutamate concentration gradient necessary for cystine/glutamate exchange. In the case of GSH, high levels are present in the aqueous humor, but no increase in GSH was observed in the core of the lens. These levels suggest that the larger size of GSH restricts its delivery via the sutures and/or that the core lacks a GSH uptake mechanism. Resolving these issues will require knowledge of the molecular identity of GSH transporters in the lens. In the absence of data supporting the direct delivery and/or uptake of GSH in the lens core, we propose that GSH levels in this region are originally established in the outer cortex during fiber cell differentiation and after fiber cell internalization into the inner cortex and ultimately the core are maintained by the local reduction of GSSG to GSH by glutathione reductase. ¹  

Although the accumulation of cystine in the core correlated well with the membranous expression of the Xc−, the increase in glycine levels did not correlate with the localization of GLYT1 only to the outer cortex. This suggests that an alternative glycine transporter exists in deeper lying fiber cells and prompted us to search for an alternative uptake system. Although amino acid transport systems such as ASC and system L are capable of carrying small amounts of glycine, the transport proteins in the GLYT family of which there are two isoforms, GLYT1 and -2, are the most specific for glycine. ²¹ To determine whether GLYT2 was expressed in the lens, we used a commercial antibody raised against the C terminus of rat GLYT2. This antibody was isoform specific, but was not able to distinguish between variants of GLYT2 (GLYT2a and -2b) which differ only in their N-terminal sequences. Western blot analysis and immunocytochemistry demonstrated GLYT2 to be expressed in all regions of the lens extending from the periphery to the center of the lens (Figs. 5 6) . The cortical localization of GLYT1 and -2 was predominantly cytoplasmic, with some labeling of the membrane suggesting both isoforms to be involved in glycine uptake in the lens cortex. However, the shift to membranous expression of GLTY2 in the inner cortex and core, strongly suggests that this isoform mediates glycine uptake in deeper-lying fiber cells. 

The differential expression of GLYTs in the rat lens is consistent with the known functional properties of these high-affinity Na⁺ dependent glycine uptake transporters. GLYT1 has a stoichiometry of 2Na⁺/Cl⁻/glycine, whereas GLYT2 has a stoichiometry of 3Na⁺/Cl⁻/glycine. ^{22 23} A difference of one Na⁺ in ionic coupling implies that the available driving force for glycine uptake for GLYT2 is two orders of magnitude larger than for GLYT1 under physiological conditions. ²⁴ This difference affords GLYT2 the ability to accumulate glycine to concentrations that exceed those found in the extracellular spaces. Furthermore, GLYT1 and -2 are high-affinity transporters for glycine with K _m values in the micromolar range. Although these K _m values vary between cell systems, GLYT2 has a consistently higher affinity for glycine than GLYT1. ²² As a result, the differential expression of the GLYTs in the lens establishes a gradient of transporter affinities, which increases with distance into the lens and allows deeper fiber cells to take up glycine from the extracellular space, where the glycine concentration diminishes with distance into the lens (Fig. 8) . The differential expression of the GLYTs in the lens draws parallels with previous work in our laboratory on the facilitative glucose transporters GLUT1 and -3. ⁹  

The differential expression of GLYT1 and -2 in the lens also exhibits parallels to the observed switch in the glutamate recycling partner for Xc− from EAAT4/5 to ASCT2, a glutamate uptake system that functions in the low-pH environment of the lens core. A histidine residue at position 421 in GLYT1 has been shown to be responsible for the reduced uptake of glycine at low pH (<pH 7.0) in exogenous expression systems. ²⁵ This residue is not conserved in the GLYT2 subtypes, and therefore glycine transport by GLYT2 is unaffected by low-pH conditions. ²⁵ Collectively, our findings suggest that the lens has evolved a very sophisticated system that utilizes the differential expression of nutrient transporters to match their functional properties to the environmental conditions and the nutrient supply that prevails in different regions of the lens. In the core, this differential expression is achieved in the absence of de novo protein synthesis via the insertion of transporters located in cytoplasmic vesicles into the plasma membrane. This process ensures that deeper-lying fiber cells are capable of nutrient uptake under low-pH conditions and when nutrient concentrations become marginal. 

Given that the lens core lacks the capacity to synthesize GSH, the role of glycine in this region is unclear. In Lim et al., ⁵ we proposed that cystine uptake and its subsequent reduction to cysteine in the core may constitute a low-molecular-mass antioxidant defense system. By analogy, we have searched the literature to propose alternative roles for glycine in the lens core. In the lens, glycine was shown to decrease glycation of proteins by efficiently scavenging glucose. ²⁶ The membranous expression of glycine in the core region (Fig. 4D)may be due to its ability to protect against glycation of membrane proteins in this region. Although glycation is associated with cortical diabetic cataracts, it has also been implicated in old age nuclear cataracts. Even if glucose levels are normal, advanced-stage glycation products may accumulate as a result of ageing processes. ²⁷ As such, glycine may be an effective anti-cataract therapy, because it offers significant advantages over other antiglycating agents like aminoguanidine and aspirin, as it is physiologically available and nontoxic to nonneuronal tissues. In summary, our immunocytochemical mapping approaches have enabled us to identify potential anticataract targets and to visualize pathways for the direct delivery of therapeutic agents specifically to the lens core. 

 Supported by the Health Research Council of New Zealand, the Marsden Fund, and the University of Auckland Research Committee.

 Submitted for publication June 1, 2007; revised July 12 and 24, 2007; accepted September 4, 2007.

 Disclosure: J. Lim, None; L. Li, None; M.D. Jacobs, None; J. Kistler, None; P.J. Donaldson, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Schematic of amino acid uptake in the outer cortex and core of the lens. (A) In the outer cortex of the lens, Xc− and EAAT4/5 are coexpressed, indicating that these transporters may work together to accumulate cystine. Xc− uses the high glutamate concentration to drive the exchange of extracellular cystine for intracellular glutamate. This cycle of exchange is maintained by EAAT4/5 which actively removes glutamate from the extracellular space. ⁵ ASCT2 may mediate the uptake of glutamine for conversion to glutamate in the cortex, whereas the transport of glycine is likely to be via the Na⁺-dependent glycine transporter GLYT1. Adapted from Lim J, Lam YC, Kistler J, Donaldson PJ. Molecular characterization of the cystine/glutamate exchanger and the excitatory amino acid transporters in the rat lens. Invest Ophthalmol Vis Sci. 2005;46:2869–2877. (B) In the core, Xc− and ASCT2 are coexpressed. Because the core of the lens is acidic, we hypothesize that Xc− works with ASCT2, which is able to transport glutamate at low pH. As the core of the lens is not capable of GSH synthesis, the purpose of cystine uptake in this region is purported to lie with cysteine itself, acting as a low-molecular-mass antioxidant to protect cells from oxidative damage. Reprinted from Experimental Eye Research, 83, Lim J, Lorentzen KA, Kistler J, Donaldson PJ, Molecular identification and characterisation of the glycine transporter (GLYT1) and the glutamine/glutamate transporter (ASCT2) in the rat lens, 447–455, © 2006, with permission from Elsevier.

Figure 2.

 

Antibody detection of GSH, glutamate, cystine and glycine in the cortex of the rat lens. (A–D) Equatorial cryosections double labeled with the membrane marker WGA (red) and an antibody that recognizes GSH (A), glutamate (B), cystine (C), or glycine (D) conjugated to glutaraldehyde (green). Top: In lenses fixed in paraformaldehyde plus 0.01% glutaraldehyde, strong labeling of GSH and its precursor amino acids is observed in cortical fiber cells. Middle: lenses fixed in paraformaldehyde plus glutaraldehyde, but labeled with WGA alone, showed no evidence of GSH or precursor amino acid labeling, indicating that glutaraldehyde autofluorescence was negligible. Bottom: lenses fixed in paraformaldehyde only and double labeled with the GSH, glutamate, cystine, or glycine antibody and WGA showed no labeling of GSH or its precursor amino acids, indicating that the antibodies detected only GSH, glutamate, cystine, or glycine conjugated to glutaraldehyde.

Figure 3.

 

Quantification of GSH, glutamate, cystine, and glycine in different regions of the lens. Representative seamless image montages (top) of GSH (A), glutamate (B), cystine (C), and glycine (D) taken from equatorial sections were used to extract intensity profiles that were plotted against distance into the lens (bottom). The bottom traces in the cystine (C) and glycine (D) profiles represent intensity profiles collected from a section in which either the primary cystine or glycine antibody was omitted. Scale bar, 100 μm.

Table 1.

 

Concentrations and Amounts of GSH and Its Precursor Amino Acids in Different Regions of the Lens Determined by HPLC

Amino Acids	Outer Cortex			Inner Cortex			Core
		Conc (μmol/L)	Amount (μmol/g)	Conc (μmol/L)	Amount (μmol/g)	Conc (μmol/L)	Amount (μmol/g)
GSH	202.84 ± 48.38	3.02 ± 0.14	20.72 ± 1.31	1.50 ± 0.82	0.81 ± 0.26	0.26 ± 0.22*
Cysteine	18.91 ± 6.86	0.19 ± 0.06	4.9 ± 0.15	0.17 ± 0.04	5.51 ± 0.47	0.40 ± 0.04, †
Glycine	51.35 ± 26.80	0.81 ± 0.40	16.25 ± 3.32	0.78 ± 0.23	17.30 ± 9.62	2.19 ± 1.14, †
Glutamate	152.21 ± 41.86	1.95 ± 0.26	28.35 ± 0.50	1.45 ± 0.61	9.00 ± 0.42	1.0 ± 0.34

*  GSH levels were significantly lower in the core relative to the inner cortex (P < 0.05).

†  Cysteine and glycine levels were shown to be significantly higher in the core relative to the inner cortex (P < 0.05).

Figure 4.

 

Correlation of the distribution of precursor amino acids with the expression of amino acid transporters. (A) Mean intensity profiles for GSH and each amino acid obtained from three lenses were plotted against relative distance (r/a, where r is the radius, ∼1200 μm, and a is the distance from the center of the lens). (B) Published image montages of amino acid transporter labeling ^{5 7} were analyzed to extract the subcellular distribution of transporters as a function of r/a and plotted as bar graphs. DF/MF, differentiating fiber/mature fiber marks the region where loss of fiber cell nuclei occurs (r/a ∼0.7).

Figure 5.

 

Glycine distribution and the expression of GLYT1 and -2 in different regions of the rat lens. Equatorial cryosections were double labeled with glycine, GLYT1, or GLYT2 antibodies (green), and the membrane marker WGA (red). (A) Montage of extended confocal images of a lens section labeled with glycine showing the areas from which high-magnification images (B–M) were recorded. (B–D) Representative images of glycine labeling in the outer cortex (B), inner cortex (C), and core (D) of the lens showed regional differences in the distribution of glycine. (E–G) Representative images of GLYT1 antibody labeling in the outer cortex (E), inner cortex (F), and core (G) of the lens showed restricted cortical localization of GLYT1. (H–J) Representative images of GLYT2 antibody labeling in the outer cortex (H), inner cortex (I), and core (J) of the lens showed GLYT2 to be expressed throughout the lens. (K–M) Labeling of GLYT2 in the outer cortex (K), inner cortex (L), and core (M), respectively, in which labeling was knocked down by preabsorption of GLYT2 with its corresponding antigenic peptide.

Figure 6.

 

Protein expression of GLYT1 and -2 in different regions of the rat lens. Membrane preparations obtained by homogenization of decapsulated whole lenses (F) or dissection of lenses into outer cortex (OC), inner cortex (IC), and core (C) fractions were analyzed by Western blot analysis. Equal amounts of each fraction were loaded onto an SDS-PAGE gel and stained with Coomassie Blue (A), and the subsequent blots probed with GLYT1 (B) or GLYT2 (C) antibodies. Protein bands for GLYT1 (∼85 kDa) were observed only in cortical fractions, whereas bands for GLYT2 (∼80 kDa) were revealed in all three fractions. (D) The 80-kDa band corresponding to GLYT2 was knocked down by preabsorption of GLYT2 antibodies with its corresponding antigenic peptide (CP). Note that since the lens core exhibits a higher protein concentration than the outer regions, loading equal amounts of protein will tend to underestimate the relative expression of low abundant proteins in this region. Thus, it is not possible to quantify the relative expression of GLYTs in the different regions.

Figure 7.

 

A delivery pathway for glycine in the lens core. Axial sections were double labeled with the membrane marker WGA (red) and the glycine antibody (green). (A) Labeling of the membranes clearly shows a suture line (arrows) that extended from the anterior pole to the core of the lens. Labeling with the glycine antibody reveals glycine to be high in the outer cortex and low in the inner cortex, before rising again in the core. (B–D) Representative high-power images of glycine and membrane labeling in the outer cortex (B), inner cortex (C), and core (D) of the lens show strong labeling of glycine along the suture and membranes of fiber cells radiating from the suture. Scale bar: (A) 100 μm; (B–D) 10 μm.

Figure 8.

 

Molecular model of glycine uptake in different regions of the lens. Schematic diagram summarizing the differential expression of GLYT isoforms in the rat lens. GLYT1 and -2 are both expressed in the epithelium and cortical fiber cells, but only GLTY2 is expressed in mature fiber cells of the lens core. In the lens cortex, membranous and cytoplasmic pools of both GLYT isoforms are present. When differentiating fiber cells lose their nuclei and become mature anucleate fiber cells (DF/MF transition), GLYT1 labeling disappears and GLYT2 is inserted into the plasma membrane. Since GLYT2 is a higher affinity transporter (3Na⁺:1Cl⁻:glycine) than GLYT1 (2Na⁺:1Cl⁻:glycine), this process of differential expression establishes an affinity gradient, which increases the ability of deeper fiber cells to extract a diminishing supply of glycine from the extracellular space. The bimodal intracellular accumulation of glycine is due the preferential delivery of glycine to the lens core via the sutures, where it is accumulated by GLTY2. Dashed arrows: Na⁺ uptake; bold arrows: glycine uptake.