12 G. KING THE AND JOURNAL R. HOLMES OF EXPERIMENTAL ZOOLOGY 282:12–17 (1998) Human Ocular Aldehyde Dehydrogenase Isozymes: Distribution and Properties as Major Soluble Proteins in Cornea and Lens GORDON KING AND ROGER HOLMES* Faculty of Science and Technology, Griffith University, Brisbane, Queensland 4111, Australia ABSTRACT Human aldehyde dehydrogenase isozymes (ALDHs; EC 22.214.171.124) exhibit very high levels of activity in anterior eye tissues. Human corneal ALDH1 and ALDH3 isozymes are present as major soluble proteins (3% and 5%, respectively, of corneal soluble protein) and may play major roles in protecting the cornea against ultraviolet radiation (UVR)–induced tissue damage, as well as contributing directly to ultraviolet B (UV-B) photoreception. The human lens exhibits high levels of ALDH1 activity (1–2% of lens-soluble protein) and lower levels of ALDH3 activity. Kinetic analyses support a role for these enzymes in the metabolism of peroxidic aldehydes, which have been reported in ocular tissues. J. Exp. Zool. 282:1217, 1998. © 1998 Wiley-Liss, Inc. Aldehyde dehydrogenases (ALDHs; EC 126.96.36.199) are products of a complex gene family and are differentially distributed between tissues and subcellular organelles of the body (Hsu et al, ’94; Weiner, ’96). The class 1 (ALDH1) and class 2 (ALDH2) isozymes are tetrameric proteins with 54-kDa subunits, which exhibit high levels of activity within liver cytoplasm and mitochondria, respectively (Greenfield and Pietruszko, ’77; Jenkins and Peters, ’83). In contrast, the class 3 enzyme (ALDH3) is present in high levels within human stomach, cornea, esophagus, and hepatocarcinoma tissue and has a 54-kDa dimeric subunit composition (Teng, ’81; Holmes, ’88; Yin et al., ’88). These isozymes are encoded by their respective genes, which are differentially localized on the human genome, namely, ALDH1 (chromosome 9q21), ALDH2 (chromosome 12q24), and ALDH3 (chromosome 17p11.2) (see Hsu et al., ’94). The resulting ALDHs have an amino acid identity of approximately 65% for ALDH1 and ALDH2 and about 40% between ALDH3 and the ALDH1 and ALDH2 subunits (Hempel et al., ’84, ’85; Hsu et al., ’92). ALDHs catalyze the irreversible oxidation of a wide range of biologic aldehydes, including products of the metabolism of ethanol (acetaldehyde) and other alcohols, biogenic amines (“biogenic” aldehydes), diamines and polyamines, retinol (retinaldehyde), steroids, and carbohydrates, as well as products of lipid peroxidation (peroxidic aldehydes) (Pietruszko, ’83; MacKerrell et al., ’86; © 1998 WILEY-LISS, INC. Algar and Holmes, ’89; Sladek et al., ’89). The isozymes exhibit distinct kinetic properties consistent with differential metabolic roles in aldehyde metabolism. Based on comparative kinetic properties, liver cytoplasmic ALDH1 may serve in a broad detoxification role for a range of peroxidic and “biogenic” aldehydes and as a major catalyst of retinaldehyde oxidation (MacKerrell et al., ’86; Sladek et al., ’89). Liver mitochondrial ALDH2 exhibits very low Km values for acetaldehyde as substrate and is predominantly responsible for acetaldehyde metabolism in the body (see Weiner, ’96). In addition, this isozyme is capable of oxidizing a range of “biogenic” and peroxidic aldehydes (MacKerrell et al., ’86). Human ALDH3 exhibits a “very high” Km for acetaldehyde and prefers peroxidic aldehydes (excluding malondialdehyde) as substrates, as well as other medium-chain aliphatic and aromatic aldehydes (Wang et al., ’90; King and Holmes, ’93). This isozyme also exhibits the highest level of catalytic efficiency among these ALDH classes in the biologic oxidation of aldehydes (King and Holmes, ’93, ’97). Grant sponsor: National Health and Medical Research Council of Australia; Grant number: 94/1098. *Correspondence to: Dr. R.S. Holmes, Vice-Chancellor’s Office, The University of Newcastle, Callaghan, NSW 2308, Australia. Received 27 March 1998; Accepted 27 March 1998 HUMAN OCULAR ALDH ISOZYMES This article describes recent studies on the distribution of ALDH1 and ALDH3 in the human cornea and lens and reports on the purification and properties of these isozymes from human cornea. The article also reviews a number of working hypotheses concerning the potential roles for these isozymes in anterior eye tissues, namely, peroxidic aldehyde metabolism, UV-B photoreception, preventing oxidative damage to other corneal proteins, and assisting in corneal transparency. MATERIALS AND METHODS Tissue sources Human corneas and lenses were obtained with appropriate permission from the Queensland Eye Bank and stored frozen at –70°C until used. ALDHs/antibodies Purified human corneal ALDH3 and human lens ALDH1 were obtained using procedures described previously (King and Holmes, ’93, ’97). The partially pure recombinant human ALDH3, human liver ALDH1, rabbit antihuman hepato– carcinoma ALDH3, and rabbit antihuman liver ALDH1 polyclonal antibodies were a gift from Drs. L. Hsu, A. Shibaya, and A. Yoshida of the Beckman Research Institute of the City of Hope, Los Angeles, California. Rabbit antihuman ALDH3 polyclonal antibodies were produced from purified human corneal ALDH3 (King and Holmes, ’93). Sodium dodecyl sulfate (SDS) gel electrophoresis Polyacrylamide gels (12% separating and 4% stacking) containing 1% SDS were prepared in a mini Protean slab gel system according to the procedure of Laemmli (’70), as modified by Ames (’74). After electrophoresis, the gels were stained using a standard Coomassie blue technique. Immunoblotting Western blots of IEF and SDS-polyacrylamide gels were prepared using standard procedures. Reactions of these blots with primary antibodies were performed at room temperature using antibody diluted into 5% w/v Dutch Jug milk powder in TRIS-buffered saline at pH 7.5 for 1 hr. Reaction with the second antibody was performed at room temperature with the peroxidase-linked anti-IgG under similar conditions. Development of the antibody staining was performed using a diaminobenzidine–nickel chloride procedure (Scopsi and Larsson, ’86). 13 Enzyme and protein assays ALDH activity was determined using a spectrophotometric assay procedure, performed in 0.1 M pyrophosphate buffer at pH 8.5 (Holmes and VandeBerg, ’86). ALDH assays at low concentrations were conducted as for the spectrophotometric assays, except that the rate of production of NADH was followed by fluoresence at 458 nm. Protein concentrations were estimated using a bicinchoninic acid–based procedure (Smith et al., ’85). RESULTS Purification of human corneal ALDH1 and ALDH3 Human corneal ALDH1 and ALDH3 were purified from human corneas using a combination of ion-exchange and affinity chromatography in filtered nitrogen-saturated buffers at 4°C. Corneas were homogenized in five volumes of 0.05 M Tricine buffer (pH 7.5; 0.1 mM EDTA, 1 mM DTT, and 0.1% deoxycholate) at 0°C. The centrifuged homogenate was diluted fourfold into loading buffer and adjusted to pH 5.8 prior to loading onto a 10-ml CM-cellulose column. ALDH1 eluted with the wash buffer (10 mM phosphate buffer, pH 5.8, containing 1 mM EDTA and 1 mM DTT). ALDH3 was eluted with 100 mM phosphate buffer (pH 7.4), also containing 1 mM EDTA and 1 mM DTT. The ALDHs in both fractions were further purified using a 5-ml AMP-Sepharose column. For ALDH1, this column was equilibrated with 50 mM phosphate buffer (pH 7.0) containing 0.25 M sodium chloride, 1 mM EDTA, and 1 mM DTT. Following elution of the unbound protein, highly purified ALDH1 was then eluted with loading buffer containing 0.1 mM NAD. For ALDH3, this column was equilibrated with 10 mM phosphate buffer (pH 7.0) containing 1 mM EDTA and 1 mM DTT and then washed with this buffer containing 0.1 mM NAD. ALDH1 containing fractions were pooled and concentrated using Centricon-30 microconcentrators where required. Kinetic and immunochemical properties of human corneal ALDH isozymes Table 1 compares the kinetic and biochemical properties observed for the purified human corneal ALDH3 and ALDH1 isozymes. ALDH3 exhibited a high Km value with acetaldehyde as substrate but showed a lower Km value and high catalytic efficiency with the peroxidic aldehyde trans-2-hexenal. Human corneal ALDH1 and ALDH3 were differentially active with malon- 14 G. KING AND R. HOLMES TABLE 1. Kinetic and biochemical properties of human corneal ALDH1 and ALDH3 isozymes Human corneal ALDH isozyme Property Km Acetaldehyde trans-2-Hexenal Malondialdehyde kcat(s–1) Acetaldehyde trans-2-Hexenal Malondialdehyde Disulfuram inhibition Cross-reactivity Antihuman liver ALDH1 Antihuman corneal ALDH3 Antihuman hepatocarcinoma ALDH ALDH3 ALDH1 67 mM 0.1 mM 0.0 117 µM 0.2 µM 15 µM 36 53 0 Insensitive to 50 µM 0.27 0.20 0.13 Fully inhibited at 4.2 µM – +++ +++ +++ – – dialdehyde as substrate, with the former isozyme exhibiting activity with this peroxidic aldehyde, with a Km value of 15 µM, whereas ALDH3 was inactive with malondialdehyde under the conditions used. The isozymes also were distinguished by their differential sensitivity to disulfuram inhibition and cross-reactivity with antibodies against class 1 (human liver ALDH1) and class 3 (human corneal ALDH3 and hepatocarcinoma ALDH) ALDHs. Figure 1 illustrates a Western blot of an SDS-PAGE gel on which human corneal soluble extracts and a cloned human stomach Fig. 1. Western blot of an SDS-polyacrylamide gel. Lane 1: human corneal homogenate reacted with rabbit antihuman hepatocarcinoma ALDH3; lane 2: a cloned human stomach ALDH3 preparation reacted with rabbit antihuman hepatocarcinoma ALDH3; lane 3: human corneal homogenate reacted with rabbit antihuman corneal ALDH3; and lane 4: a cloned human stomach ALDH3 preparation reacted with rabbit antihuman corneal ALDH3. Electrophoretic migration is from top to bottom. ALDH3 preparation were reacted with antihuman hepatocarcinoma ALDH and antihuman corneal ALDH3 polyclonal antibodies. Major ALDH3 cross-reactivity was observed in both human cornea and the cloned human stomach ALDH3 using both antibodies, which was consistent with immunochemical identity for human ALDH3, whether derived from cornea, stomach, or hepatocarcinoma sources. DISCUSSION Human corneal and lens ALDH isozymes This and other studies (King and Holmes, ’93, ’97) from this laboratory have shown that human corneal and lens extracts exhibited two major forms of ALDH activity, designated as ALDH1 and ALDH3. These enzymes showed kinetic, biochemical, and immunochemical properties similar to human liver ALDH1 and human stomach and hepatocarcinoma ALDH3 isozymes described previously (Greenfield and Pietruszko, ’77; Wang et al., ’90; Hsu et al., ’92; Weiner, ’96). Purified human corneal ALDH1 exhibited properties similar to human liver ALDH1 with respect to its kinetic characteristics with acetal–dehyde and malondialdehyde as substrates and in its sensitivity to disulfuram inhibition (King and Holmes, ’97). In addition, human corneal ALDH1 cross-reacted with rabbit antihuman liver ALDH1 polyclonal antibodies, demonstrating that this enzyme is identical with or highly homologous to human liver ALDH1. Moreover, human corneal ALDH1 shared these properties with those recently reported for human lens ALDH1 (King and Holmes, ’97), confirming that this enzyme is present in both human cornea and lens. HUMAN OCULAR ALDH ISOZYMES ALDHs: Major soluble proteins in human cornea and lens Soluble extracts of the human cornea have been shown to contain very high levels of ALDH3 (Gondhowiardjo et al., ’92; King and Holmes, ’93) and ALDH1 (King and Holmes, ’97) isozymes, representing approximately 5% and 3% of soluble corneal protein, respectively. With the exception of albumin, which is the major soluble protein found in human cornea (Zhu and Crouch, ’92), ALDH3 and ALDH1 are the most prevalent soluble proteins observed in this tissue. In addition, these enzymes are predominantly restricted in their distribution to the epithelial and endothelial cells of the cornea and are therefore likely to be found more highly concentrated within these cells (King, ’97). Figure 2 summarizes the comparative distribution of both ALDH3 and ALDH1 activity and protein levels within human cornea and lens. Human cornea demonstrated the highest levels of ALDH activity, principally as ALDH3 activity, although human lens contained higher levels of ALDH1 soluble protein on a wet-weight basis. Moreover, immunohistochemical studies (King, ’97) have demonstrated that both isozymes are predominantly localized within corneal and lens epithelial cells rather than being evenly distributed throughout the stroma of the cornea or the 15 fiber cell–cortex regions of the human lens. Consequently, the actual concentration of these enzymes within corneal and lens epithelial cells will be very much higher than that reported within crude extracts of whole tissue. Human corneal ALDH3 and ALDH1: Functional aspects The presence of ALDHs as major soluble proteins within human (and other mammalian) corneas has led to a number of hypotheses concerning their function (or functions) within this anterior eye tissue. Abedinia et al. (’90) initially proposed that ALDH3 may serve a dual role, namely, the oxidation of peroxidic aldehydes generated following UV-B absorption by the cornea and as a UV-B photoreceptor, particularly in the form of the binary complex of the enzyme with NADH as coenzyme, which is likely to readily absorb in the UV-B range. Cooper et al. (’93) also have proposed that corneal ALDH3 may serve a structural role (as a corneal “crystallin”), in a manner similar to that of crystallin proteins in the lens, which provide a transparent matrix suitable for visible light transmission. More recently, Uma et al. (’96) have provided evidence that corneal ALDH3 may play a role in preventing oxidative damage by free-radical species generated following UVR absorption. In addition, the presence of ALDH1 in the human cornea may assist in one or more of these functions and will particularly provide a means of oxidizing malondialdehyde, which has been reported in ocular tissues (Bhuyan et al., ’81). Figure 3 provides a diagrammatic illustration for these proposed roles for human corneal ALDHs and represents a summary of the working hypotheses proposed to explain the presence of very high levels for these isozymes in mammalian corneal epithelial cells. Human lens ALDH1 and ALDH3: Functional aspects Fig. 2. ALDH activity and protein composition of the human cornea and lens. The solid bars represent ALDH activity (mIU/mg wet tissue). The cross-hatched bars represent ALDH protein (µg/mg wet tissue). The presence of very high levels of ALDH1, as well as low levels of ALDH3, within the epithelial cell layer of the human lens is consistent with a functional role (or roles) for these isozymes, similar to that described earlier for the human cornea (King, ’97; King and Holmes, ’98). Lens epithelial cells form a single layer through which UVR and visible radiation must pass prior to being absorbed by the lens (UV-A radiation) or passing through to the retina (visible light). These isozymes therefore may serve to detoxify peroxidic aldehydes, which have been reported in the 16 G. KING AND R. HOLMES Fig. 3. Diagrammatic illustration of the proposed roles for corneal ALDH as major soluble proteins to assist in light transfer, catalysts of peroxidic aldehyde metabolism, assist- ing in UV-B photoreception as E-NADH complexes, and in free-radical (OH•) detoxification. mammalian lens (Bhuyan et al., ’81). In addition, these major soluble proteins may serve “crystallin” type roles within these cells and/or directly contribute toward the direct absorption of residual UV-B radiation prior to entry of UVR into the lens. In summary, the presence of very high levels of ALDH3 and ALDH1 isozymes within human corneal and lens epithelial cells has led to a number of hypotheses concerning the functions of these enzymes within human anterior eye tissues: oxidation of peroxidic aldehydes generated following UVR absorption (King and Holmes, ’93); UV-B absorption, particularly as the E-NADH binary complex (Abedinia et al., ’90); serving in a structural role (corneal and lens “crystallins”), providing a clear soluble matrix for the passage of visible light (Cooper et al., ’93); and preventing oxidative damage by free-radical species generated following UVR absorption (Uma et al., ’96). 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