(Received for publication, June 22, 1995; and in revised form, August 8, 1995)
From the
Rapidly growing oocytes in the laying hen are, in addition to the liver, targets of the so-called ``reverse cholesterol transport'' (RCT) (Vieira, P. M., Vieira, A. V., Sanders, E. J., Steyrer, E., Nimpf, J., and Schneider, W. J.(1995) J. Lipid Res. 36, 601-610), pointing to the importance of this process in nonplacental reproduction. We have begun to delineate the details of this unique transport pathway branch by molecular characterization of the first nonmammalian lecithin-cholesterol acyltransferase (LCAT), the enzyme that catalyzes an early step in RCT. The biological significance of the enzyme is underscored by the high degree of protein sequence identity (73%) maintained from chicken to man. Interestingly, the conservation extends much less to the cysteine residues; in fact, two of the cysteines thought to be important in mammalian enzymes (residues 31 and 184 in man) are absent from the chicken enzyme, providing proof of their dispensability for enzymatic activity. Antibodies prepared against a chicken LCAT fusion protein cross-react with human LCAT and identify a 64-kDa protein present in enzymatically active fractions obtained by hydrophobic chromatography of chicken serum. The developmental and tissue distribution pattern of LCAT in females is striking; during embryogenesis and adolescence, LCAT expression is extremely high in liver but undetectable in brain. Upon onset of laying, however, brain LCAT mRNA increases suddenly and is maintained at levels 5 times higher than in liver, in stark contrast to most mammals. In adult roosters, the levels of LCAT transcripts in brain are lower than in liver. Together with the molecular characterization of chicken LCAT, these newly discovered developmental changes and gender differences in its expression establish the avian oocyte/liver system as a powerful model to delineate in vivo regulatory elements of RCT.
Reverse cholesterol transport (RCT) ()is the
physiological process that transports excess cholesterol back to the
liver for secretion(1) , thereby counteracting the accumulation
of cholesterol in peripheral cells. The enzyme lecithin-cholesterol
acyltransferase (LCAT, EC 2.3.1.43) catalyzes the initial step in this
cascade, i.e. the esterification of cell-derived free
cholesterol, concomitant with transfer of the esters into the core of
high density lipoprotein (HDL) particles. Apolipoprotein A-I (apoA-I),
the major protein component of plasma HDL, is a potent activator of
LCAT(2) . By a yet poorly understood mechanism, the cholesteryl
esters carried in HDL are then transported via the plasma back to the
liver. The important physiological role of this enzyme has become
evident from studies of mutations in the LCAT gene causing functional
deficiencies of the enzyme(3) . Patients with LCAT deficiency
accumulate unesterified cholesterol in their peripheral tissues,
associated with premature atherosclerosis, kidney disease, and central
nervous system impairment(4, 5) .
In mammals, the liver is the sole physiologically important target for RCT-derived peripheral cholesterol. However, in the course of our studies of lipoprotein metabolism in the chicken, we recently have identified an alternative deposition site, i.e. the developing oocytes of laying hens(6) . In agreement with these findings, laying hens have been reported to possess high RCT activity and plasma HDL levels(7, 8) . Results from Smith and co-workers (9) imply that the majority of the cholesteryl esters in chicken plasma are synthesized by LCAT. High plasma levels of HDL possibly reflect the significant demand for yolk lipids in the growing oocytes, single cells that accumulate as much as 5 g of lipid in the form of plasma-borne lipoproteins. It seems likely that supplementation of the oocytes with lipoproteins cannot be satisfied merely by uptake of very low density lipoprotein (VLDL) and vitellogenin. Indeed, the oocyte appears to compete with the liver for the products of the RCT; recently, we have identified bona fide, cholesteryl ester-containing HDL particles in the yolk of chicken oocytes(6) . Thus, in the chicken, and possibly in other oviparous species, RCT and LCAT are tightly linked to the reproductive effort. Despite this obvious biological significance, knowledge about molecular characteristics of LCAT in any nonmammalian species is lacking.
In this paper we report the molecular characterization of LCAT from Gallus domesticus. Remarkable is the low degree of conservation of cysteine residues in the otherwise highly conserved, catalytically active, avian enzyme. The current studies also have revealed a hitherto unrecognized striking developmental and sex-specific pattern of LCAT gene expression in this avian species, i.e. a sharp rise of LCAT transcript levels in the brain of sexually maturing hens.
Figure 1:
A, nucleotide sequence and deduced
amino acid sequence of the chicken LCAT cDNA. The amino acid sequence
is given in the single-letter code; the residues of the presumed leader
peptide are in lower case letters. The in-frame stop codons
are underlined once, and the polyadenylation signal (AAUAAA)
is underlined in boldface. The tetranucleotide
(GAUG), resembling a motif thought to be responsible for the
destabilization of specific mRNAs after estrogen withdrawal (see text)
is marked with a double underline. B, alignment of
the deduced amino acid sequence of chicken and human LCAT. The amino
acid sequences are given in the single-letter code and are numbered
from the NH terminus of the mature protein (+1).
Negative numbers refer to the presumed leader peptide sequence. The shaded areas indicate identical residues in the chicken and
human sequences. The 6-residue stretch, identical to the interfacial
active binding site of the lipase family, is underlined in boldface. The four potential glycosylation sites are indicated
by brackets. The active serine is indicated with an asterisk, and cysteines in the human sequence, not found in
the chicken enzyme, are marked with dots.
The overall similarity at both the nucleotide and amino acid level of the avian enzyme with mammalian LCATs including those of man, baboon, rat, and mouse (17, 18, 19, 20) is striking. For instance, the chicken LCAT protein sequence is 73% identical with that of the human enzyme, and amino acids unequivocally involved in LCAT function are conserved. This includes Ser-181 of the catalytically active site(21, 22) , contained in the motif GXSXG. This motif is found not only in LCATs, but also in lipases (lipoprotein lipase, hepatic lipase, lingual lipase, and pancreatic lipase);(23, 24, 25, 26, 27, 28, 29, 30) (Table 1). In addition, adjacent to the GXSXG motif the chicken enzyme contains the so-called interfacial binding region(17) , an extended linear hydrophobic amino acid stretch (VFLIGHSMGNLNVLYFLL) common to catalytic factors interacting with lipids(29, 31, 32) .
Based on these extensive similarities between the avian gene product and its mammalian counterparts, it is surprising and interesting that this conservation does not extend to cysteine residues. Chicken LCAT contains 5 cysteines, whereas all mammalian LCAT harbor 6 cysteine residues. The 4 cysteines known to be involved in intramolecular disulfide-bonds in mammalian enzymes (Cys-50/Cys-74 and Cys 313/Cys-356) are unchanged; the fifth cysteine, in position 26 has been replaced by an Ile in the mammalian enzymes. Significantly and surprisingly, Cys-31 (replaced by Phe) and Cys-184 (substituted by Asn) are absent from the chicken protein. Biochemical data obtained with the human enzyme (33, 34) had suggested that these two cysteines may be crucial for acyl-transfer activity. However, chicken LCAT is catalytically active (see below), strongly supporting earlier site-directed mutagenesis studies (35) that showed that these cysteines are not essential for LCAT activity. In addition, there are no amino acid replacements at sites that when mutated cause classical human familial LCAT deficiencies or fish eye disease(3, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45) . Also, all of the four potential asparagine-linked glycosylation sites found in mammalian LCATs are conserved in the chicken sequence (Asn-Xaa-Ser/Thr, at residues 20, 84, 272, and 384).
The 3`-untranslated region of our clone (690 nucleotides) exceeds that of human LCAT mRNA by 536 nucleotides (17) (Fig. 1A). It contains an additional in-frame stop codon and the polyadenylation signal, AAUAAA. There are also five repeats of a tetranucleotide, GAUG, resembling the situation in the 3`-untranslated end of the mRNA for apo-VLDL-II, a strictly estrogen-dependently expressed avian apolipoprotein(46) . This tetranucleotide, among other primary and secondary structures, has been suggested to be involved in destabilization of specific mRNAs after estrogen withdrawal(47, 48) .
Figure 2:
Tissue distribution of LCAT mRNA in laying
hens and mature roosters. Northern blot analysis was performed with 15
µg of total RNA isolated from different tissues of laying hens (25
weeks old) and mature roosters (28 weeks old). The upper panels show the hybridization pattern obtained with a P-labeled LCAT cDNA (1929-bp fragment). The lower
panels show the same blots after rehybridization with a labeled
rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA
probe. The size of the chicken LCAT transcript (approximately 2
kilobase pairs) was determined by comparison with size markers
(ribosomal RNAs and HindIII-cut DNA, not
shown).
Figure 3:
Developmental pattern of LCAT gene
expression in chicken liver (Li) and brain (Br).
Northern blot analysis was performed with RNA pooled from four embryos
each (9, 14, and 16 days old), four hatchlings each (3 days and 2 weeks
old), immature hens (11 and 15 weeks old), and laying hens (25 weeks
old). Total RNAs (15 µg/lane) were applied to a 1.2% agarose gel
and probed with a P-labeled chicken LCAT cDNA fragment (upper panel) and subsequently with a rat
glyceraldehyde-3-phosphate dehydrogenase probe (lower panel)
as described in the legend to Fig. 2.
Figure 4: Column chromatographic purification and characterization of plasma LCAT. Laying hen plasma was subjected to column chromatography on phenyl-Sepharose; fractions were assayed for activity and analyzed by Western blotting as described under ``Materials and Methods.'' A, a 50-µl aliquot of each of the indicated fractions was tested for LCAT activity. B, equal amounts of protein from fractions 2, 8, 14, 20, 26, 32, 38, 44, 50 were separated by 8% SDS-polyacrylamide gel electrophoresis under reducing conditions and analyzed by Western blotting. C, LCAT from laying hen (LH) or human (Hu) plasma was partially purified by phenyl-Sepharose chromatography, and aliquots of the fractions containing the highest LCAT activity were analyzed by Western blotting with our rabbit anti-chicken LCAT antiserum (Imm) or preimmune serum (Con). The broad range molecular weight standards (Bio-Rad) were used as markers.
Molecular characterization of the first premammalian LCAT has revealed novel details about the structure/function relationships and features of expression of this important enzyme in lipoprotein metabolism. The chicken (G. domesticus), situated in evolution before the emergence of mammals, expresses all components identified as essential for the process of reverse cholesterol transport. In fact, in the hen, reverse transport of LCAT-generated cholesteryl esters is not only directed to the liver, but also to the ovary, and is therefore particularly important for normal development of the oocytes, i.e. for reproduction.
The delineation of the primary structure of mature chicken LCAT revealed that it is identical to the most distant known enzyme, that from man, in 284 of its 391 residues (73% identity); at the carboxyl terminus, mammalian enzymes have acquired a proline-rich extension of 23-25 residues. However, all of the residues identified to be important for the catalytic activity of mammalian enzymes are already present in the chicken sequence. LCAT dysfunctions in the human diseases, LCAT deficiency and fish eye disease, have been reported to result from mutations at different sites(3, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 52, 53) . Significantly, all of the residues corresponding to these crucial sites in the human wild-type enzyme are identical to those in chicken LCAT. In addition, all four potential glycosylation sites are conserved in the avian sequence (Asn-Xaa-Thr/Ser, at residues 20, 84, 272, and 384). Mutations introduced by site-directed mutagenesis revealed that substitution of one of these sites (Asn-272) causes diminished LCAT activity(54) . All of these findings are in accordance with the consequences of evolutionary pressure to retain functionally important residues.
Special attention has been paid to the role(s) of the cysteine residues in LCAT structure and function. Overall, the chicken enzyme harbors 5 cysteines; one of these (Cys-26) has been replaced by an Ile in the known mammalian enzymes. Through acquisition of two new cysteines (at positions 31 and 184), mammalian LCATs possess 6 cysteines; however, these 2 additional cysteines are present in reduced form(55, 56) . The remaining 4 cysteines, common to chicken and mammalian enzymes, are known to form disulfide bonds in the human enzyme (between positions 50 and 74 and positions 313 and 356, respectively(56) ), and thus likely in the avian enzyme as well.
Mutations introduced at the paired cysteine sites result in
impaired LCAT secretion and activity(56) . Early biochemical
studies on human LCAT had implied that the free cysteines at position
31 and 184 are also essential for enzymatic activity of
LCAT(33, 34) . Chemical modification of the 2 cysteine
residues in human LCAT suggested their participation in a complex
catalytic mechanism involving an obligatory LCAT-S-acyl intermediate
formed in a reaction with both of the respective free cysteine
residues(33) . These data are not consistent with later
findings of Francone and Fielding (35) who generated LCAT which
carried mutations in either or both of the free cysteine residues. Even
the double mutant (Cys-31 Gly/Cys-184
Gly) was active in
the formation of cholesteryl esters(35) . The ancestral LCAT of
chicken, in which these free cysteines are not yet present, is
catalytically active. This provides proof for the notion that the
cysteine residues in positions 31 and 184 are not required for
cholesteryl ester synthesis. It will be interesting to study at which
point in evolution, and possibly why, these cysteines arose.
Another significant aspect of the present work relates to the developmental and apparently sex-specific pattern of LCAT gene expression ( Fig. 2and Fig. 3). In mature hens, LCAT message is predominantly found in brain, and comparatively low levels are found in liver and adrenals. In contrast, roosters express higher mRNA levels in liver than in brain and very low levels in testes. Thus, in comparison with the rooster and with mammalian systems(20, 57) , the laying hen shows a strikingly different and hitherto unrecognized distribution of LCAT transcripts. Lipoprotein metabolism and transport pathways of the hen must ensure sufficient nutritional supplementation of the embryo, i.e. deposition of lipid-rich yolk into growing oocytes. Besides the two major precursors of the yolk components, VLDL and vitellogenin, a bona fide HDL fraction has been demonstrated to constitute a component of egg yolk(6, 58) . While VLDL is the predominant lipoprotein fraction of laying hen plasma, the presence of considerable amounts of HDL suggests that the cholesteryl esters derived from plasma HDL also contribute to the yolk cholesterol pool(6) . Esterification of cholesterol takes place in the HDL particle by the action of LCAT, underscoring the importance of this enzyme in reproduction.
Upon onset of egg laying, birds show dramatic changes in their lipoprotein profile(59, 60) . Immature hens and roosters have low VLDL levels, and in both animals the HDL fraction dominates over the low density fraction. This distribution reverses upon maturation of the female or following estrogen-treatment of roosters; there is an up to 1000-fold induction of certain apolipoproteins and lipid synthetic enzymes(46, 61) . We thus wondered whether we could observe, as a corollary to active reverse cholesterol transport, a relationship between the developmental stages of the hen and LCAT expression.
In this context, in embryos, female chicks, and young hens, LCAT gene expression occurs predominantly in the liver, likely correlating with their high plasma HDL levels. Recent reports also demonstrated apoA-I expression in the liver of developing chicks(62) . In man, mouse, and rat, the liver is the predominant tissue of LCAT mRNA expression (20, 57) . Interestingly, one primate species, the baboon, has been reported to exhibit 3 times higher LCAT mRNA levels in brain than in liver of female animals(18) ; the authors observed similar results in male baboons. In all other studies so far, extrahepatic tissues were found to express only trace amounts of LCAT(20, 57) .
It is probably not unexpected to find LCAT mRNA expression in brain, which is separated from the plasma compartment by the blood-brain barrier and thus from proteins that cannot freely cross this boundary. However, several lines of evidence indicate that the brain maintains cholesterol homeostasis by both uptake as well as removal. Pitas et al.(63) demonstrated that all known components of proper lipoprotein metabolism are present and functional in the brain. ApoE and apoA-I are the major apolipoproteins in human and canine cerebrospinal fluid(63, 64, 65, 66) . Recently, further apoproteins have been described from human cerebrospinal fluid, such as apoA-IV, D, and J(67) . Since the adult brain does not accumulate cholesterol, it must possess a mechanism(s) to dispose of excess cholesterol. ApoA-I-carrying HDL particles in conjunction with LCAT may well participate in this reverse cholesterol transport process. Notably, LCAT, albeit with low activity, has been demonstrated in human cerebrospinal fluid(68) . In brain of laying hens, we find dramatically increased LCAT mRNA levels compared with liver, possibly reflecting the need for maintenance of cholesterol homeostasis in this partially closed system. LCAT action in brain may be important for function of the central nervous system, since several patients with LCAT deficiency showed signs of hearing loss and sensory impairments(4) . Whether high levels of LCAT mRNA expression correlate with high enzyme activity in the brain of adult chickens and the baboon (18) needs to be addressed in further studies.
The observed tissue-specific expression of LCAT may be subject to tight control. For instance, in addition to known promoter elements(69) , we might find tissue-specific posttranscriptional control of the LCAT gene. We have observed in the 3`-untranslated region of chicken LCAT elements previously characterized as important for estrogen regulation at the level of mRNA stability(47, 48) . Thus, the chicken with its dramatic developmental changes and gender differences in LCAT gene expression provides a powerful system to delineate possibly novel control elements of LCAT regulation in vivo.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X91011[GenBank].