(Received for publication, May 19, 1995)
From the
Rat hepatic asialoglycoprotein receptors (ASGP-Rs) are
hetero-oligomers composed of three homologous glycoprotein subunits,
designated rat hepatic lectins (RHL) 1, 2, and 3. ASGP-Rs mediate the
endocytosis and degradation of circulating glycoconjugates containing
terminal N-acetylgalactosamine or galactose, including
desialylated plasma glycoproteins. We have shown in permeable rat
hepatocytes that the ligand binding activity of one subpopulation of
receptors (designated State 2 ASGP-Rs) can be decreased or increased,
respectively, by ATP and palmitoyl-CoA (Weigel, P. H., and Oka, J.
A.(1993) J. Biol. Chem. 268, 27186-27190). We proposed
that a reversible and cyclic acylation/deacylation process may regulate
ASGP-R activity during endocytosis, receptor-ligand dissociation, and
receptor recycling. In the accompanying paper (Zeng, F-Y., and Weigel,
P. H.(1995) J. Biol. Chem. 270, 21388-21395), we show
that the ligand binding activity of affinity-purified State 2 ASGP-Rs
is decreased by treatment with hydroxylamine under mild conditions
consistent with these ASGP-Rs being fatty acylated in vivo. In
this study, we used a chemical method to determine the presence of
covalently-bound fatty acids in individual ASGP-R subunits. The
affinity-purified ASGP-R preparations were separated by
SDS-polyacrylamide gel electrophoresis under nonreducing conditions,
and the gel slices containing individual RHL subunits were treated with
alkali to release covalently bound fatty acids, which were subsequently
analyzed by gas chromatography and confirmed by gas chromatography-mass
spectrometry. Both stearic and palmitic acids were detected in all
three receptor subunits. Pretreatment of ASGP-Rs with hydroxylamine
before SDS-polyacrylamide gel electrophoresis reduced the content of
both fatty acids by 66-80%, indicating that most of these fatty
acids are attached to cysteine residues via thioester linkages.
Furthermore, when freshly isolated hepatocytes were cultured in the
presence of [H]palmitate, all three RHL subunits
in affinity-purified ASGP-Rs were metabolically labeled. We conclude
that RHL1, RHL2, and RHL3 are modified by fatty acylation in intact
cells.
Covalent binding of long chain saturated fatty acids occurs with a wide variety of membrane proteins post-translationally and significantly influences protein localization and/or function(1) . Two of the most common modifications involve acylation with myristate and palmitate. Myristate is usually attached to an N-terminal glycine via an amide bond in a relatively stable linkage. In contrast, palmitate is attached either to cysteine residues via a thioester bond or to serine residues via an ester bond; both linkages, particularly the thioester, are hydrolyzed by alkali treatment(2) . Palmitoylation of many proteins has been shown to be a dynamic process(3, 4) .
The hepatic
asialoglycoprotein receptor (ASGP-R) ()mediates the
endocytosis of desialylated glycoproteins containing terminal galactose
or N-acetylgalactosamine residues(5, 6) . The
functional rat ASGP-R is a hetero-oligomer composed of three subunits,
RHL1, RHL2, and RHL3, with molecular masses of 41,500, 49,000, and
54,000 Da, respectively(7) . The amino acid sequences of all
three subunits are closely related, and they are the products of two
different genes(8) . RHL1 is the major subunit of the ASGP-R,
while RHL2 and RHL3, encoded by the same gene, are minor subunits and
differ only in the type and amount of post-translational carbohydrate
modification(9) .
We and others have previously demonstrated that the ASGP ligands are endocytosed and intracellularly processed by two functionally different receptor populations via two distinct pathways(6, 10) . We have designated these two receptor populations as the State 1 ASGP-Rs and the State 2 ASGP-Rs. In intact cells, the State 2 ASGP-Rs undergo a transient inactivation/reactivation cycle during receptor recycling (11) . This same cycle has been successfully reconstituted in permeable rat hepatocytes(12, 13, 14) . In permeablized cells the State 2 ASGP-Rs are inactivated by the addition of ATP in a time- and temperature-dependent manner(12) ; these ATP-inactivated receptors can then be quantitatively reactivated by the addition of palmitoyl-CoA(13, 14) .
In the accompanying paper(15) , we also demonstrate that the activity of one population of affinity-purified ASGP-Rs, the State 2 receptors, is selectively inactivated by treatment with hydroxylamine, a chemical frequently used to release thioester-linked fatty acids from proteins. These above results suggest that one or all ASGP-R subunits are modified by fatty acylation in vivo and that a reversible acylation/deacylation process may be involved in regulating the ligand-binding activity of ASGP-Rs as they function during receptor mediated endocytosis.
In this study, we have used gas chromatography-mass spectrometry to examine directly whether total (State 1 plus State 2) ASGP-Rs contain covalently-bound fatty acids. The results demonstrate that all three ASGP-R subunits contain covalently-linked palmitate and stearate. Treatment of each RHL subunit with hydroxylamine under mild conditions released both fatty acids in relatively large amounts, indicating that most fatty acids are attached via thioester linkages.
To release ester-linked fatty acids from the RHL proteins, the minced gel pieces were dried under nitrogen after the final wash and 0.7 ml of 1.5 N NaOH was added to each vial. After shaking at 30 °C for 3 h, the vials were cooled on ice and then neutralized with 0.3 ml of 6 N HCl. To each vial, 4.0 ml of chloroform/methanol (1:2, v/v) was added, and the vials were shaken at room temperature for 10 min. The chloroform/methanol extract was transferred to another new vial, each gel was washed with 1.5 ml of chloroform, and the wash was combined with each extraction. Water (1 ml) was added to each vial to obtain a two-phase solution, and the chloroform phase was transferred to a clean new vial and dried under nitrogen. The gels were also dried under nitrogen.
To hydrolyze amide-linked fatty acids, the dried, minced gel pieces were incubated with 1.0 ml of 6 N HCl at 110 °C for 6 h in Teflon-lined screw-capped vials. After cooling to room temperature, 4.0 ml of methanol/chloroform (1:1) was added and the vials were shaken for 10 min. The extract was transferred to a new vial, the gel pieces were washed with 1.5 ml of chloroform, and this wash was added to the extract solution. Two phases were formed by addition of 1 ml of water and after vigorous mixing followed by phase separation the chloroform layer was transferred to a clean vial and dried under nitrogen.
Fatty acids can be covalently attached to proteins either to
glycine residues via N-linked amide bonds, to cysteine
residues via thioester bonds, or to serine residues via hydroxyester
bonds(1, 2) . The thioester and hydroxyester linkages
are labile to alkaline treatment. To determine if the ASGP-R is
modified by fatty acylation, we used a direct chemical method to assess
whether any of the ASGP-R subunits contain covalently bound fatty
acids. Freshly purified ASGP-R, prepared from isolated rat hepatocytes
by affinity chromatography, was examined immediately, since storage
resulted in activity loss and potential deacylation as noted in the
accompanying paper(15) . To exclude the possibility that
reducing agents such as -mercaptoethanol or dithiothreitol may
release some covalently-bound fatty acids, SDS-PAGE was carried out
under nonreducing conditions and the samples were not boiled. Gel
pieces containing the separated subunits RHL1, RHL2, and RHL3 were cut
out, and subjected to extensive washing, extraction, and alkaline or
acid hydrolysis as described under ``Experimental
Procedures.'' The released fatty acids were derivatized to their
methyl esters and analyzed by GC-MS.
To assess the reliability of
this method, we determined the optimal conditions for the washing
steps, the alkali treatment to release ester-linked fatty acids, and
the derivatization of fatty acids to their methyl esters. This was
necessary in order to remove SDS and other components that could give
contaminating GC peaks. In particular, we found that (i) extensive
washing of the gel pieces with water and then methanol/water and (ii)
performing the methanolysis with BF-methanol at <60
°C were both critical in order to reduce the background of observed
GC peaks. A blank gel of the same size was used as negative control.
Using our final conditions, this negative control gave small, but
detectable peaks at positions corresponding to methyl palmitate and
methyl stearate. The intensities of these peaks were very similar, if
not identical, to those shown in the left panels of Fig. 1. These background peaks were present even in gels on
which no samples had been loaded and could not be eliminated by
changing the experimental conditions. The intensities of these peaks
were also significantly increased by increasing the gel size analyzed
(data not shown). For this reason, we used the same size blank or
subunit-containing gels (verified by weight) to minimize the effect of
differing gel size on the results. To verify that SDS-PAGE and the
washing procedure after SDS-PAGE could effectively remove noncovalently
bound fatty acids, BSA which is known to contain many kinds of
noncovalently bound fatty acids, was subjected to the same procedure.
The result showed no detectable fatty acids in either the
chloroform/methanol wash or the alkaline hydrolyzate in comparison with
a blank gel (not shown), indicating that SDS-PAGE and the subsequent
wash procedures (before alkali treatment) completely removed
noncovalently bound fatty acids.
Figure 1: Gas chromatographic analysis of fatty acids released. Affinity-purified ASGP-Rs were separated by SDS-PAGE under nonreducing conditions, and the bands containing RHL1, RHL2, and RHL3 were excised and subjected to chloroform-methanol wash and subsequent mild alkaline hydrolysis as described under ``Experimental Procedures.'' Derivatized fatty acids released by the chloroform-methanol (left panel) and alkaline hydrolysis (right panel) treatments were analyzed by GC.
The analysis of alkaline
hydrolysates of purified ASGP-R by GC-MS clearly demonstrated the
presence of palmitate and stearate in each of the three RHL subunits ( Fig. 1and Fig. 2). One GC peak in these samples was
identified as the methyl ester of palmitate by two criteria: First, its
retention time (11.65 min) on gas chromatography was identical to that
of standard methyl palmitate and this peak co-chromatographed with the
standard methyl palmitate. Second, mass spectrometric analysis of this
GC peak (retention time = 11.65 min) gave a molecular ion (m/z = 270), which is the expected mass of
methyl palmitate. Furthermore, additional ion mass peaks at 74
(McLafferty ion), 87, and 143, are the expected major molecular
fragment ions from the methyl esters of saturated fatty acids. The mass
spectrum of the 11.65-min peak was identical to that of the standard
methyl palmitate (Fig. 2A). The methyl ester of
stearate was identified by the same criteria. All samples contained a
GC peak with a retention time of 13.86 min, identical to that of
standard methyl stearate, and GC-MS analysis revealed a molecular ion (m/z = 298) and other major ions
characteristic (21, 24) of the methyl ester of
stearate (Fig. 2B). In addition to methyl palmitate and
methyl stearate, small amounts of the methyl esters of myristic, oleic,
and linoleic acids were also detected by GC in the alkaline
hydrolyzates of all three subunits (Fig. 1). Palmitate and
stearate methyl esters were not detected in the chloroform/methanol
wash of the gels containing ASGP-R subunits (Fig. 1, left
panel). These esters were only found after processing the alkaline
hydrolysates. After alkali treatment, the incubation of the gels
containing receptor subunits with strong acid should release
amide-linked fatty acids. The analysis of the acid hydrolysates of RHL1
and RHL2 by GC showed no detectable methyl esters of any fatty acid
above background (data not shown), indicating the absence of
amide-linked fatty acids in these two receptors subunits. RHL3,
however, gave significant amounts of palmitate and stearate (6
times background).
Figure 2:
Mass
spectrometric analysis of the GC peaks identified as palmitate and
stearate methyl esters. The peaks at 11.65 min (A) and 13.85
min (B) from the GC analysis of fatty acids released from
individual RHL1, RHL2, and RHL3 subunits as shown in Fig. 1were
derivatized and analyzed by GC-MS. The mass spectra of both standard
fatty acid methyl esters are also shown. The major ion peaks (such as m/z = 74 (McLafferty ion), 87, and 143) that
are characteristic of fatty acid methyl esters were found in both GC
peaks from all RHL samples. Ion peaks unique to methyl palmitate (m/z = 227, 239, and 270 (M))
or to methyl stearate (m/z = 255, 267, and 298
(M
)) were found in each sample as
indicated.
To determine if palmitate and stearate are
attached to Cys residues of the receptor subunits via thioester
linkages, the affinity-purified ASGP-Rs were treated prior to SDS-PAGE
with hydroxylamine under mild conditions. Total ASGP-R was first
treated with NHOH, and then each RHL subunit was purified
by SDS-PAGE and treated with alkali (Table 1). Quantitation of
the released fatty acids showed that hydroxylamine treatment released
73, 100, and 68% of the methyl palmitate that could be detected in
RHL1, RHL2, and RHL3, respectively. Similarly, for methyl stearate 74,
100, and 79% of this fatty acid detectable in RHL1, RHL2 and RHL3,
respectively, was released by NH
OH treatment. Tris
treatment had no significant effect on any of the RHL subunits (Table 1). These results indicate that most (>70%) of the
covalently bound fatty acids in an alkali-labile linkage are attached
to each RHL subunit via thioester-linkages to Cys.
Taking the molar
ratio of the subunits (RHL1:RHL2:RHL3 4:1:1) into account and
assuming comparable yields, RHL2 and RHL3 contain about 4-5 times
more covalently bound fatty acids than RHL1 (Table 1). Although
the stoichiometries of fatty acids per RHL subunit have not been
determined, these relative results were very similar in three
independent experiments (Table 1).
To confirm the above
biochemical analyses, metabolic labeling experiments were performed to
assess the incorporation of radiolabeled fatty acids into ASGP-R in
intact hepatocytes. After cells were cultured in the presence of
[H]palmitate for 16 h, ASGP-Rs were
affinity-purified and analyzed by SDS-PAGE and fluorography. All three
RHL subunits were labeled with [
H]palmitate,
although RHL1 had significantly more radioactivity than either RHL2 or
RHL3 (Fig. 3). To verify that the
H radioactivity
represents palmitate, RHL1 was isolated, treated with alkali, and the
hydrolysate was analyzed by HPLC (Fig. 4). The majority (81%) of
the released radoactivity comigrated with authentic palmitate
confirming that this fatty acid was covalently incorporated into
ASGP-Rs in vivo. The remaining radioactivity which migrated as
small alkyl fragments (probably C2-C10) could be derived in part from O-acetylated sialic acids on RHL1. Similar metabolic labeling
experiments using [
H]stearate or
[
H]myristate did not show detectable
incorporation of these fatty acids into any of the RHL subunits of
affinity-purified ASGP-R (not shown). Ongoing experiments indicate that
the human ASGP-R is also metabolically labeled when HepG2 or HuH7 cells
are cultured in the presence of [
H]palmitic acid. (
)
Figure 3:
Metabolic labeling of active ASGP-Rs using
[H]palmitate. Hepatocytes were cultured with 400
µCi/ml [
H]palmitate for 16 h, and active
ASGP-Rs were purified as described under ``Experimental
Procedures.'' Nine-tenths of the purified ASGP-Rs (from one 60-mm
dish containing about 2
10
cells) were analyzed by
SDS-PAGE and fluorography (lane 1), and one-tenth (lane
2) was analyzed by Western blotting and detected using a mixture
of subunit-specific affinity-purified antibodies specific for RHL1 or
RHL2/3.
Figure 4:
Metabolically labeled RHL1 contains
authentic [H]palmitate. Hepatocytes were cultured
with [
H]palmitate, and radiolabeled ASGP-Rs were
purified and separated by SDS-PAGE. RHL1-containing bands were excised,
and subjected to extensive wash, alkali treatment, and extraction of
released fatty acids. The hydrolysate was analyzed by reverse-phase
HPLC as described under ``Experimental Procedures.'' The
retention times for co-chromatographed myristic acid (C14), palmitic
acid (C16), and stearic acid (C18) are marked by arrows.
A number of diverse proteins including viral
proteins(25) , the subunits of G proteins(26) ,
the G protein-coupled receptors (27, 28, 29) , and the transferrin receptor (30, 31) have been shown to be modified by palmitate
via thioester linkages. Palmitoylation has been suggested to play an
important functional role in many proteins. For example, the
palmitoylation of subunits of G proteins is involved both in membrane
attachment as well as in modulating signaling capability (32, 33) . Palmitoylation of the neuronal growth cone
protein GAP-43 reduces its ability to catalyze nucleotide exchange on a
G protein (34) . Many investigators have shown that
palmitoylation/depalmitoylation of a variety of proteins is a dynamic
process(3, 4) .
Although the present study shows
for the first time that ASGP-Rs are fatty acylated, Stockert ()found evidence over 10 years ago that
[
H]palmitate might be incorporated into the human
ASGP-R. Our results here clearly demonstrate that all three subunits of
the ASGP-R are modified by fatty acylation. The GC-MS analysis shows
the presence of both palmitate and stearate in each RHL subunit. The
results from the GC-MS analyses were confirmed by the finding that all
three subunits of the ASGP-R are metabolically labeled by
[
H]palmitic acid. Initial metabolic labeling
studies with [
H]stearate did not show
incorporation into ASGP-Rs, although [
H]palmitate
was readily incorporated. In earlier studies, however, stearyl-CoA was
as effective as palmitoyl-CoA in being able to reactivate State 2
ASGP-Rs that had been inactivated by ATP treatment of permeable
cells(13) . Furthermore, as shown in Table 1and the
accompanying paper(15) , the sensitivity of fatty acid release
by hydroxylamine treatment at neutral pH is indicative of a thioester
linkage. Pretreatment with hydroxylamine released approximately
66-100% of the stearate or palmitate in RHL1, RHL2, and RHL3 that
could be released by alkaline hydrolysis. Therefore,
66% of the
covalently bound fatty acids in alkali-labile linkages is probably
present as thioesters. This percentage could actually be significantly
greater if any fatty acyl migration occurs during receptor
purification. Some thioesters could react with nucleophilic -NH
or -OH groups in proximal amino acids to produce amide-linked or
esterified fatty acids. This may explain why virtually all of the
metabolically incorporated [
H]palmitate is
released by mild NH
OH treatment (15) and,
therefore, presumably present exclusively as thioesters.
The GC-MS and metabolic labeling results show apparent differences in the extent of RHL subunit fatty acylation, the fatty acids involved and the proportion of thioester linkages. It is premature to consider these differences significant, since the two methods are so different and each has inherent biases. For example, chemical analysis of purified total receptor should represent a steady-state incorporation of fatty acids mediated by every acylating and deacylation enzyme that recognizes ASGP-Rs from every part of the receptor trafficking pathway. Multiple cellular sites of modification (e.g. cell surface, endosome, or vesicles recycling back to the surface) and multiple fatty acylation sites within the ASGP-R likely make the overall modification pattern and process very complicated. Metabolic labeling studies, which are frequently not at a steady-state, can preferentially identify the fastest fatty acylation step for newly synthesized molecules or a site(s) of the most rapid fatty acyl turnover. More extensive studies will be required to characterize these processes in intact cells.
The cytoplasmic domains of each RHL subunit have a single Cys
residue within 5 amino acids of the transmembrane domain, which is a
likely site for fatty acylation. The same position in the transferrin
receptor is palmitoylated(30, 31, 35) .
Studies are in progress to determine the site(s) of modification in the
three RHL subunits. The three ASGP-R subunits are known to have
different extents of glycosylation(9) , and are also
differentially modified by phosphorylation in their cytoplasmic
domains(36, 37) . Our present results strongly suggest
that the three subunits are also modified by fatty acylation to
different extents. Thus, in the steady-state situation, all RHL
subunits contain palmitate and stearate in similar ratios, but the
amount of these two fatty acids in RHL1 is smaller than in RHL2 and
RHL3. We report in the accompanying paper (15) that treatment
of radiolabeled affinity-purified ASGP-Rs with hydroxylamine completely
removes the [H]palmitic acid from RHL1, RHL2, and
RHL3. The metabolic labeling experiments indicate that, in fact, only
the State 2 ASGP-Rs (one of two receptor subpopulations that we have
previously characterized) are palmitoylated and the vast majority of
fatty acylation is via thioester bonds.
One population of ASGP-Rs (the State 2 ASGP-Rs) undergoes a transient inactivation-reactivation cycle during receptor recycling(6, 11, 12, 13, 14) . Such an inactivation-reactivation cycle may provide a biological basis for the high efficiency of the ligand-receptor segregation step in this and in other recycling receptor systems(6) . That fatty acyl-CoAs can regulate ASGP-R activity in permeable hepatocytes(13, 14) , suggests that a reversible fatty acylation-deacylation process could be the molecular basis of the State 2 receptor inactivation-reactivation cycle. The finding that one population of purified ASGP-Rs is selectively inactivated by hydroxylamine treatment, under mild conditions that releases essentially all of the covalently associated fatty acids,(15) , supports this hypothesis. Thus, we conclude that the three ASGP-R subunits are modified by palmitate and stearate, and that acylation-deacylation of the ASGP-R could directly regulate its activity.
An important point about the fatty acylation results presented here is that we analyzed the total active ASGP-R pool, which contains both State 1 and State 2 receptors. It is likely that the State 1 ASGP-Rs are not fatty acylated; fatty acylation may occur exclusively in the State 2 ASGP-R population(15) . Further studies will address whether fatty acylation in RHL1, RHL2, and/or RHL3 is the molecular basis for the two functionally distinct receptor populations and how fatty acylation/deacylation regulates receptor activity.