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Human Ocular Aldehyde Dehydrogenase Isozymes:
Distribution and Properties as Major Soluble
Proteins in Cornea and Lens
Faculty of Science and Technology, Griffith University, Brisbane, Queensland
4111, Australia
Human aldehyde dehydrogenase isozymes (ALDHs; EC 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. 282:12–17, 1998. © 1998 Wiley-Liss, Inc.
Aldehyde dehydrogenases (ALDHs; EC
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;
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
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.
Tissue sources
Human corneas and lenses were obtained with
appropriate permission from the Queensland Eye
Bank and stored frozen at –70°C until used.
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.
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).
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).
Purification of human corneal
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-
TABLE 1. Kinetic and biochemical properties of human corneal ALDH1 and ALDH3 isozymes
Human corneal ALDH isozyme
Disulfuram inhibition
Antihuman liver ALDH1
Antihuman corneal ALDH3
Antihuman hepatocarcinoma ALDH
67 mM
0.1 mM
117 µM
0.2 µM
15 µM
Insensitive to 50 µM
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.
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.
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
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
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). The catalytic
properties of these enzymes reported in this
article are consistent with a differential role in
peroxidic metabolism: ALDH1—broad peroxidic
aldehyde specificity, including malondialdehyde
and medium-chain aliphatic aldehydes as substrates, but with lower catalytic efficiency; and
ALDH3—being inactive with malondialdehyde
as substrate but showing high catalytic efficiency with medium-chain-length aliphatic and
aromatic aldehydes.
The human eye samples were kindly supplied
by Professor L. Hirst and Dr. Peter Madden of
the Queensland Eye Bank.
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