(Received for publication, June 12, 1996, and in revised form, December 3, 1996)
From the Bernhard Nocht Institute for Tropical Medicine, A novel apoprotein of an apparent molecular mass
of 86 kDa in its unreduced form was identified in human
triglyceride-rich lipoproteins. This protein was purified and the amino
acid sequence of six proteolytic fragments was found to overlap with
that of the factor H-related proteins. In parallel we identified the
cDNA encoding a new complement factor H-related protein, termed
FHR-4. The sequences of the new apoprotein overlapped with that of the FHR-4 protein. Similar to the previously described factor H-related proteins, FHR-4 contains a hydrophobic signal sequence followed by a
stretch of five repetitive elements termed short consensus repeats.
Recombinant FHR-4 protein was expressed in the baculovirus system and
has an apparent molecular mass of 42 kDa. In addition a 84-kDa dimeric
form of the recombinant FHR-4 was detected. Using an immunoaffinity
column with antibodies raised against the recombinant FHR-4, we
isolated a 86-kDa protein from human plasma. The different molecular
mass of the recombinant FHR-4 and the dimeric FHR-4 in plasma is due to
different carbohydrate moieties. The 86-kDa plasma protein and the
novel apolipoprotein had identical mobility on SDS-polyacrylamide gel
electrophoresis analysis and reacted with antisera raised against the
reFHR-4 and the purified apoprotein. In conclusion, we have identified
a novel factor H-related protein, FHR-4, in human plasma and
demonstrate that this protein is present in triglyceride-rich
lipoproteins in a dimeric form. This observation provides an intriguing
new aspect on possible function(s) of this novel protein and the other
factor H-related proteins.
Several human factor H-related plasma proteins have been
identified recently that represent a family of structurally and
immunologically related proteins and that are termed factor H-related
proteins 1-3 (FHR-1 to FHR-3).1 Similar to
human complement factor H, these proteins are exclusively composed of
repetitive elements termed short consensus repeats (SCRs). Factor
H-related molecules of human and mouse origin have been isolated on the
cDNA, the protein, and the genomic level (1, 2). Three distinct
human factor H-related cDNA clones, termed H36 (or pFH1.4),
DDESK59, and DOWN16 have been isolated, and the corresponding plasma
proteins are termed factor H-related proteins 1-3 (FHR-1 to FHR-3)
(2-6). The H36 cDNA encodes two human plasma proteins of 37 and 42 kDa, which represent the differently glycosylated forms FHR-1 Factor H, FHL-1, and all FHR proteins are structurally related, and
Northern blot analyses confirmed that all identified molecules of human
and mouse origin are synthesized in the liver. Individual SCRs of these
proteins display a significant identity to each other and to SCRs of
factor H. The proteins are also immunologically related: all human
proteins react with antiserum that was raised against human factor H
(2).
Several apoproteins have been described to be important structural and
functional components of human triglyceride-rich lipoproteins (TG-Lp),
as there are chylomicrons (CM) and very low density lipoproteins (VLDL)
(11-13). The main structural apoproteins are apoB-48 in TG-Lp and
apoB-100 in VLDL. Both lipoproteins contain apoE, which serves as
ligand for lipoprotein receptors (14), apoC-II as a cofactor for
lipoprotein lipase activity (15), and apoC-III, known to modulate the
receptor binding affinity (16). ApoA-I and ApoA-IV are further
apoproteins associated with these TG-Lp, and both are known to activate
the enzyme lecithin-cholesteryl acyltransferase (17, 18). However,
detailed studies on further possible human apoproteins of plasma CM or
VLDL have not been performed.
In our experiments with iodinated human TG-Lp, we detected one
additional major apoprotein that has not yet been characterized. This
novel apoprotein was purified and found to be identical to the factor
H-related protein 4, which was cloned and recombinantly expressed in
parallel. The recombinant FHR-4 protein, as expressed in insect cells,
is of amphipathic nature and the native protein is detected as a
homodimer in human plasma. The FHR-4 plasma protein is found free and
associated with TG-Lp and other lipoproteins.
Human TG-Lp were obtained
from plasma of patients with lipoprotein lipase deficiency, a genetic
disorder that leads to massive accumulation of TG-Lp, due to lack of
the hydrolytic activity in plasma (19). The TG-Lp were isolated from 36 ml of plasma by ultracentrifugation. The plasma was adjusted to 10%
sucrose, and 6 ml were layered under 6 ml of PBS. The first separation was performed in the SW 42 rotor (Beckmann for 45 min at 4 °C, 38,000 rpm). The TG-Lp were isolated from the top of the tube and again
adjusted to 10% sucrose. 2 ml were layered under 10 ml of PBS and
recentrifuged under the same conditions. The TG-Lp were isolated from
the top, and the protein concentration was determined by a modification
of the Lowry method (20). The protein content of the isolated TG-Lp
were typically around 0.46 mg/ml. The TG-Lp were iodinated with
Na125I by the iodine-monochloride method (21). The specific
activity reached was 20-80 cpm/ng of protein. For the SDS-PAGE 100 µl of iodinated TG-Lp were delipidated in chloroform/methanol (8:5). The autoradiography was performed for 4 h on a Cronex film. The samples were either applied to the gel in unreduced form or reduced with dithioerythritol (10-min incubation at 95 °C). The apparent molecular mass of the differently prepared proteins was distinct. In
the unreduced form it appeared as an 86-kDa band, while after reduction
with dithioerythritol or For analytical purpose all human lipoproteins were isolated from normal
human plasma by ultracentrifugation. The plasma was adjusted to a
density of 1.21 g/ml with KBr and spun for 16 h at 4 °C and
39,000 rpm in the SW 42 rotor (Beckmann). The total lipoprotein
fraction was taken from the top of the tubes and either used for the
lipoprotein separation in a density gradient ultracentrifugation or
were delipidated (chloroform/methanol, 8:5) and directly applied to an
SDS-PAGE for protein analysis. The bottom (B1) was also delipidated and
analyzed on the SDS-PAGE. For a further separation the lipoproteins
were again adjusted to the density 1.21 g/ml with KBr and underlayered
under a four-step density gradient as described earlier (31). After the
density gradient centrifugation the tubes were punctured at the bottom,
and 0.5-ml fractions were eluted. Cholesterol measurements were
performed in all fractions (enzymatic test kit from Boehringer
Mannheim). The bottom fraction (B2) and the various VLDL, LDL, and HDL
fractions were pooled according to the cholesterol measurements and
were delipidated with chloroform/methanol (8:5) for SDS-PAGE. The B2
fraction contains all proteins dissociated from the lipoproteins during
the second ultracentrifugation step.
Plasma lipoproteins were separated on
a Superose 6 column (10 × 300 mm; Pharmacia Biotech Inc.). The
column was equilibrated with PBS. 200 µl of plasma were applied to
the column, and the run was performed at room temperature with a flow
rate of 0.3 ml/min. Protein was detected with the adsorbance of 280 nm.
Fractions of 0.5 ml were collected and prepared for SDS-PAGE. For the
TG-Lp the whole fractions were delipidated with chloroform/methanol, 1:1 (v/v), while for the protein peak, corresponding to the bottom fraction of the ultracentrifugation step, only 5% of the material was
used for gel electrophoresis.
For the isolation of
water-soluble apolipoproteins the TG-Lp were delipidated as described
above, and the apoproteins were extracted by PBS. Thereby the novel
protein could be separated from the water-insoluble apolipoprotein B. The extraction was repeated twice, and the samples were concentrated by
precipitation in chloroform/methanol (8:5) before SDS-PAGE. A 10%
SDS-PAGE was performed according to Neville (22) with reduced samples.
The proteins were electroblotted onto nitrocellulose and stained by Coomassie on both edges. The 106-kDa band of the novel apoprotein was
cut out and used for immunization.
In order to obtain
sequence information the 106-kDa protein was electroeluted from
SDS-PAGE in a Biotrap elution chamber (Schleicher & Schuell, Dassel,
Germany). The elution buffer was 25 mM Tris, 192 mM glycine, and 0.025% SDS, pH 8.6. The eluted protein was precipitated with chloroform/methanol (8:5) to remove the detergent and
then digested with trypsin or V8 protease (protein sequencing grade,
Boehringer, Mannheim, Germany). Digestions were performed in 100 µl
of the respective standard buffers (0.1 M Tris/HCl, pH 8.5, 2 mM CaCl2 for trypsin; 0.1 M
Tris/HCl, pH 8.0, for protease V8) at 37 °C for 12 h at an
estimated substrate/protease ratio of 5:1 (w:w). The proteolytic
fragments were separated by narrowbore HPLC (130A, Applied Biosystems)
on a reverse phase column (Vydac C4, 300 A pore size, 5 µm particle
size, 2.1 × 250 mm) peptides were eluted with a linear gradient
(0-80% for 50 min; solvent A: water, 0.1% trifluoroacetic acid;
solvent B: 70% acetonitrile, 0.09% trifluoroacetic acid) at a flow
rate of 200 µl/min. Peptide containing fractions detected at 214 nm
were collected manually, concentrated, and further purified by a second
HPLC run (Nucleosil C8, 5 µm, 1.6 × 125 mm, gradient as above).
Protein sequences were determined by standard Edman degradation on an
automatic peptide sequenator (473A, Applied Biosystems).
A human
liver oligo(dT)-primed cDNA library in Several plasmids, which showed a restriction
pattern distinct from the FHR-3 cDNA, were further analyzed. Their
cDNA inserts were sequenced in double-stranded form by the dideoxy
chain termination method (24) using Total cellular
human RNA was extracted with guanidinium thiocyanate and isolated by
centrifugation over CsCl (25). 8 µg of RNA were separated by
electrophoresis in a formaldehyde-agarose gel and subsequently
transferred to a nylon membrane (PALL).
Recombinant FHR-4,
delipidated lipoprotein fractions, and fractions obtained by Nickel
chromatography were separated by SDS-PAGE using either 10 or 12% gels
according to Neville (22) using unstained broad range markers (Bio-Rad)
or a 12% SDS-PAGE according to Laemmli (23, 26) using prestained low
range markers (Bio-Rad) as standards. Proteins were visualized either
by Coomassie staining, silver staining, or were electroblotted.
Proteins separated according to the Neville method were transferred to
nitrocellulose in buffer chambers, while proteins separated by Laemmli
SDS-PAGE were transferred by semidry blotting (27). Membranes were
blocked for 30 min using either 5% (w/v) dried milk in PBS or 5%
bovine serum albumin in Tris/HCl, pH 8.6. Incubations with the specific
antibodies were performed for the indicated times. Dilutions of the
polyclonal rabbit antibodies used in the incubations were 1:500 for the
antibody raised against the novel apolipoprotein and 1:1000 against the recombinant FHR-4. After washing in PBS for five times membranes were
incubated with peroxidase-conjugated goat anti-rabbit antibody (Daco or
Jackson) for 2-3 h. Protein bands were visualized by the addition of
0.3% (w/v) 4-chloro-1-naphthol in 10% (v/v) methanol in PBS.
For library screening a
full-length FHR-3 fragment was used. A specific fragment of FHR-4,
representing mainly SCR 5 and part of the 3 Antiserum raised against reFHR-4 (10 µl)3 was coupled to 200 µl of protein
A-Sepharose (Pharmacia) by agitation for 1 h at room temperature.
After two washing steps in 10-fold volume of 0.2 M sodium
borate, pH 9.0, protein A was resuspended in the same buffer and mixed
with the cross-linker dimethyl pimelimidate (Pierce) (20 mM) for an additional 30 min at room temperature. Subsequent washing steps were performed in 0.2 M
ethanolamine, pH 8.0, under agitation for 2 h at room temperature
followed by PBS. The beads were stored in the same buffer at
4 °C.
Human serum or supernatant of infected insect cells was precleared by
incubation for 1 h at room temperature with protein A-Sepharose.
Sepharose beads were discarded, and the serum was incubated overnight
at 4 °C with 20 µl of FHR-4-protein A-Sepharose and 20 µl of
PBS. After washing three times in PBS and once in 10 mM
potassium phosphate buffer, pH 8.0, the bound protein was eluted with
triethanolamine, pH 11.5. After neutralization with 1 M
sodium phosphate, pH 6.8, the eluted protein was analyzed by SDS-PAGE
and by Western blotting.
In studies designed
to understand the catabolism of human TG-Lp, the apoprotein composition
of these lipoproteins was analyzed after iodination. In addition to the
known apoproteins apoB-100, apoB-48, apoA-I, apoA-IV, apoE, and apoC, a
protein of approximately 106 kDa was found as a major labeled band in
reduced samples separated by SDS-PAGE (Fig.
1A). This protein was found associated with the TG-Lp also after repeated recentrifugation (see below, Fig. 9) and
was therefore considered as a new apoprotein. To confirm the
association of the 106-kDa protein with lipoproteins, we used gel
filtration on Superose 6 (FPLC). As in ultracentrifugation in FPLC the
106-kDa protein was found associated with TG-Lp (Fig. 2). The
distribution was correlated to the lipoprotein levels. In Fig.
2B only the TG-Lp, the LDL, and the plasma
protein fraction ("bottom") are shown, as in HDL the amount of this
apoprotein is rather low. The 106-kDa protein was isolated by SDS-PAGE,
and antibodies were raised in rabbits. Analysis of the enriched
apoprotein with a glycan detection kit revealed that it is highly
glycosylated (Fig. 1B, lane 2). Partial amino acid sequence
analysis of proteolytic fragments obtained from this novel apoprotein
was performed and the sequence of six fragments revealed striking
sequence identity with the FHR-3 and the predicted protein sequence of
a new cDNA (SAC6, see below; Fig. 5).
In order to identify sequences
coding for additional factor H-related proteins an oligo(dT)-primed
human liver cDNA library was screened with a full-length cDNA
clone (DOWN16), which encodes the FHR-3 protein. One clone showed a
restriction pattern distinct from the FHR-3 coding cDNA, and this
cDNA was used to rescreen the same library (data not shown). The
nucleotide sequence of the longest clone isolated termed SAC6 is shown
in Fig. 3. This clone is 1315 nucleotides long and has a poly(A) tail.
The motif TCT AAC ATG (position 80-88) shows a good match
(six out of nine, including the ATG) with the consensus sequence of
initiation sites GCC ACC ATG (28). There is a poly(A) signal
"AATAAA" at position 1283-1288.
The nucleotide sequence of clone
FHR-4 displays an open reading frame of 331 amino acids encoding a
protein of 37.3 kDa (Fig. 3). Within the predicted amino
acid sequence four potential N-linked glycosylation sites of
the type Asn-X-Ser/Thr were found at positions 127-129
(Asn-Ser-Ser), 186-188 (Asn-Thr-Thr), 206-208 (Asn-Ser-Ser), and
310-312 (Asn-Thr-Ser), respectively. Given the homology to the FHR-3
protein, the corresponding protein was designated FHR-4. The
hydrophobicity analyses of the NH2-terminal amino acid
residues predicted a potential signal peptide, indicating that the
FHR-4 protein is expressed via the secretory pathway (29). According to
the criteria common for signal peptide cleavage sites, we suggest that
the leader sequence is cleaved at position 19. The molecular mass of
the secreted, nonglycosylated product was calculated to 35.5 kDa. A
striking similarity was observed in the amino acid sequence of the
predicted FHR-4 protein and that obtained from five of the six
proteolytic fragments of the novel apolipoprotein. Among 80 amino acids
that were identified by amino acid sequence analysis, 60 residues were
identical to the protein predicted from the cDNA sequence, 10 residues could not be exactly determined and 10 residues showed a
mismatch to the predicted FHR-4 sequence. Only fragment I shows a major
discrepancy to the predicted cDNA sequence. Among the 22 residues
of this fragment 12 residues were identical and 3 could not be
determined. Thus this fragment shows a match of only 68.2%. The
remaining five fragments (fragments II-VI) showed 94.9% match to the
predicted sequence, and this identity suggests that the two proteins
are highly related or may even be identical.
Structural alignment of the protein encoded by the SAC6
cDNA indicated a protein composed of a NH2-terminal
signal peptide followed by five SCRs (Fig. 4). Each of
the five SCRs includes the essential four Cys (C) residues
(boxed with double lines in Fig. 4) and
additional conserved amino acids such as a Pro (P), an Asn
(N), a Gly (G), a Leu (L), 2 Tyr
(Y), 2 Gly (G), a Trp (W), and a Pro
(P) residue (Fig. 4). The signal peptide and the individual
SCRs display identity to SCRs of FHR-3 (DOWN16 cDNA), FHR-2
(DDESK59 cDNA), FHR-1 (H36 cDNA), and factor H, respectively (Fig. 5 and Table I). SCRs 1, 2, and 3 of
FHR-4 are homologous to SCRs 6, 8, and 9 of factor H, having an overall
amino acid identity of 70.3, 62.9, and 64.4%, respectively. SCRs 4, 5 of FHR-4 are related to SCRs 19 and 20 of factor H, displaying an identity of 63.9 and 39.1%. These two SCRs, are conserved in position and sequence in factor H and all identified factor H-related proteins (Fig. 5 and Table I). This comparison demonstrates that the
COOH-terminal ends of FHR-4 and FHR-3 are highly related with a
identity of 98.4% for SCR 4 and 93.8% for SCR 5, respectively. The
relatedness between FHR-4 and FHR-3 is underlined by the
identical amino acids of their signal peptides.
Amino acid homology of individual short consensus repeats of the FHR-4
protein with members of the factor H family
Medical
Clinic,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
and
FHR-1
(7). FHR-1
has two carbohydrate side chains attached, and
the FHR-1
protein has one carbohydrate side chain attached.
Similarly the DDESK59 cDNA encodes a glycosylated 29-kDa (FHR-2
)
and a nonglycosylated 24-kDa plasma protein (FHR-2). The product of the
DOWN16 cDNA, the FHR-3 protein, has been described as a 55-kDa
plasma protein.2 Similarly, four factor
H-related cDNAs, termed 13G1, 23L1, 3A4, and 9C4, have been
isolated from a mouse liver cDNA library (8). Genomic analysis has
demonstrated that the human FHR-2 gene and the mouse FHR
transcripts are derived from loci that are distinct from the
factor H gene (9, 10).
Preparation of Human Lipoproteins
-mercaptoethanol a 106-kDa band was
detected.
ZAP (Stratagene) was
screened according to standard procedures (23) with a cDNA fragment
(DOWN16) representing the previously described FHR-3 protein (5).
-35S-dATP and
Sequenase II (U. S. Biochemical Corp.). Various oligonucleotide primers were synthesized, and the sequence of the cDNA was
determined in both orientations.
-untranslated region was
used as a probe for Northern blot analysis. The insert was excised,
purified on low melt agarose gels, and after labeling with
32P by random priming (Amersham Corp.) was used for
hybridization at 47 °C (10 × Denhardt's, 5 × SET (1 × SET: 150 mM NaCl, 30 mM Tris, 2 mM
EDTA, pH 8.0), 0.1% SDS, 0.1% sodium pyrogenphosphate, and 250 mg/ml
denatured salmon sperm DNA). Following hybridization for 14-18 h the
filters were washed at a final stringency of 0.1 × SSC, 0.1% SDS
at 47 °C. The filters were exposed at
70 °C using intensifying
screens (Quanta III, DuPont).
Identification of a Novel Apolipoprotein
Fig. 1.
Characterization of a novel apoprotein in
human TG-Lp. A, SDS-PAGE of iodinated TG-Lp. The identified
apolipoproteins are indicated by their name, and the novel
TG-Lp-associated protein of 106 kDa is indicated by the
arrow. Proteins were reduced with -mercaptoethanol prior
to electrophoresis. The mobility of the size markers are indicated on
the left. B, apoproteins (50 µg) obtained from human TG-Lp
were analyzed by SDS-PAGE after reduction with dithioerythritol and
transferred to membranes. Filters representing individual lanes were
analyzed by Coomassie staining (lane 1) by treatment with a
glycan detection kit (lane 2) and immunodetection with a
polyclonal antiserum raised against the purified 106-kDa protein
(lane 3). Peroxidase labeled goat anti-rabbit was used for
visualization.
[View Larger Version of this Image (40K GIF file)]
Fig. 9.
The dimeric form of FHR-4 is present in
plasma and in the lipid-fraction of plasma. A, Detection of
the FHR-4 protein in lipoprotein fractions. Lipoproteins were isolated
from human plasma by ultracentrifugation and equal amounts (80 µg) of
the bottom (B1) and the lipoprotein fraction were separated
by SDS-PAGE and analyzed by Western blotting using anti-FHR-4
antiserum. B, identification of FHR-4 and factor H in
lipoprotein particles of different density. The individual fractions,
obtained after a second ultracentrifugation of the isolated
lipoproteins (as shown in A) are indicated. The fraction
designated B2 shows the bottom fraction obtained after the
second ultracentrifugation and represents proteins that were
dissociated from the isolated lipoproteins. High density lipoproteins
(HDL), low density lipoproteins (LDL),
intermediate density lipoproteins (IDL), and very low
density lipoproteins (VLDL) were delipidated and 50 µg of
each applied to a 10% SDS-PAGE (Neville system). Proteins were
transferred to a nylon membrane and subjected to immunoblotting using
antiserum against FHR-4 (lanes 1, 3, 5, 7, and 9)
or against the factor H-like protein 1 (FHL-1) (lanes 2, 4, 6, 8, and 10). The band detected with FHL-1 antiserum in
lanes 2, 6, and 10 might represent the 150-kDa
factor H-like protein. The mobility of the size markers is indicated on
the left.
[View Larger Version of this Image (44K GIF file)]
Fig. 2.
Column chromatography of human lipoproteins.
A, column profile of a Superose 6 run (FPLC) of 200 µl of
a postprandial plasma sample of a patient with hyperlipidemia type I. The absorbance was measured at 280 nm. B, SDS-PAGE (10%) of
column fractions. Total TG-Lp and LDL fractions were delipidated, while
only 5% of the bottom fractions was applied to the gel. Fractions
representing HDL are not shown, since its relative low amount of the
106-kDa protein in these fractions. The 106-kDa protein needs further enrichment to demonstrate its presence in these fractions (data not
shown). The additional band detected in the TG-Lp fraction has also
been detected in human chylomicrons and has not been further
characterized.
[View Larger Version of this Image (32K GIF file)]
Fig. 5.
Comparison between individual SCRs of FHR-4,
factor H, and the factor H-related proteins FHR-1 to FHR-3. Amino
acid comparison of the homologous SCRs using the single letter code. Identical amino acids are shown by dots. The
individual lines represent the leader sequence, and SCRs
1-5 of FHR-4. The SCRs of the individual proteins which are used for
alignment are indicated on the left. Sequences obtained by
amino acid sequence analysis of proteolytic fragments of the human
apolipoprotein purified from human TG-Lp are aligned and shown above
the predicted protein sequences. Matching residues are shown by
"periods," unidentified residues by "X," and
conflicting residues are indicated by the single letter amino acid
code.
[View Larger Version of this Image (44K GIF file)]
Fig. 3.
Nucleotide sequence of cDNA clone SAC6
and derived protein sequence. The nucleotide sequence and the
amino acid sequence of the predicted FHR-4 protein is shown below in
the single letter amino acids code. The numbers referring to the
nucleotide acid sequence are indicated on the left and that
of the amino acid are shown below. The polyadenylation signal is
underlined, and the poly(A) tail is also shown.
[View Larger Version of this Image (33K GIF file)]
Fig. 4.
Amino acid alignment of the SCRs predicted
for the FHR-4 protein. The sequences were aligned based on their
conserved amino acids according to the SCR structure. The essential Cys residues are boxed with double lines and
conserved residues are aligned. The N-linked glycosylation
sites are underlined.
[View Larger Version of this Image (10K GIF file)]
FH
FHR-1
FHR-2
FHR-3
Leader
40%
42.1%
42.1%
100%
SCR 1
70.3%
44.3%
42.6%
70.8%
SCR 6
SCR
1
SCR 1
SCR 1
SCR 2
62.9%
31.4%
31.2%
98.4%
SCR 6
SCR 3
SCR 2
SCR 3
SCR
3
64.4%
34.5%
34.5%
35.3%
SCR 9
SCR 2
SCR
2
SCR 3
SCR 4
63.9%
63.9%
68.3%
98.4%
SCR 19
SCR 4
SCR 3
SCR 4
SCR
5
39.1%
39.1%
47.7%
93.8%
SCR 20
SCR
5
SCR 4
SCR 5
Expression of the FHR-4 mRNA in human
liver was demonstrated by Northern blot analyses. A fragment specific
for FHR-4 cDNA hybridized to several mRNA species with an
estimated size of 1.4, 2.2, and 3.5 kilobases (Fig. 6).
As the SAC6 cDNA sequence represents an almost full-length clone,
the 1.4-kilobase mRNA transcript encodes the FHR-4 protein. The
identification of several transcripts suggests the existence of
additional closely related, but yet unidentified, cDNAs and
proteins. The new apoprotein described above also seems to be of
hepatic origin, as demonstrated by metabolic labeling with
[35S]methionine in human hepatoma cells (data not
shown).
Immunopurification of FHR-4 from Human Serum
Having
demonstrated the existence of a hydrophobic NH2-terminal
region and expression of the FHR-4 mRNA in human liver, we asked
for the existence of this novel factor H-related protein in human
plasma. By immunopurification and SDS-PAGE analysis according to
Laemmli a single protein of approximately 86 kDa was isolated from
human serum with the specific FHR-4 antiserum (Fig. 7A, lane 1). The mobility of the human plasma protein
isolated by immunopurification with FHR-4 antiserum is distinct from
that of the monomeric and the dimeric recombinant FHR-4 (Fig.
7A, compare lanes 1 and 2), but
identical to the apoprotein (Fig. 8). The specificity of
the immunopurification procedure was demonstrated by immunopurification of the reFHR-4 protein from the culture medium of infected insect cells. Both the 42- and 84-kDa form of the reFHR-4 protein were isolated (Fig. 7A, lane 3). To confirm that the purified
human plasma protein represents a dimeric form of native FHR-4, the isolated plasma protein was reduced by extensive boiling in the presence of dithiothreitol and analyzed by SDS-PAGE. This treatment resulted in a predominant band with an apparent molecular mass of 63 kDa, which appears to represent the reduced monomeric form (Fig.
7B, lane 3). The additional band of weaker intensity was not
further characterized. The mobility of the reduced native protein is
distinct from that of the reduced reFHR-4 (Fig. 7B, compare
lanes 2 and 3), this difference is due to
different types of attached carbohydrates.4
Identity of the New Apoprotein and the FHR-4
Given the overlap in sequence between the proteolytic fragments obtained from the newly identified apolipoprotein and the predicted FHR-4 protein, we asked whether the two proteins might even be identical. To this end the mobility and immunoreactivity of the novel human apoprotein, the immunopurified human plasma protein and the recombinant FHR-4 protein were compared. Both human proteins had identical mobility, when separated on SDS-PAGE in their nonreduced and reduced forms (Fig. 8A, lanes 1 and 2, and B, lanes 1 and 2). In unreduced gels both proteins of human origin migrate at a molecular mass of 86 kDa. However, the mobility of the human proteins differ from that of the recombinantly expressed protein (Fig. 8A, lanes 3 and 6). Upon reduction with dithioerythritol a decrease in mobility (increase of apparent molecular mass) was observed, both human proteins migrate with an apparent molecular mass of 106 kDa. This treatment seems to cause an unfolding of the SCRs, while the two subunits remain attached to each other. The two monomeric forms can be separated by extensive boiling in the presence of dithiothreitol, resulting in a protein of 63 kDa, which represents the reduced unfolded monomeric form (Fig. 7B, lane 3). In addition immunological analysis revealed that all three proteins react with specific antiserum raised either against the reduced purified apolipoprotein or with antiserum raised against the nonreduced purified reFHR-4 protein (Fig. 8). The apoprotein can be isolated with the immunoaffinity column from the soluble apoprotein fraction of TG-Lp. This direct comparison of the mobility and the immunoreactivity with specific antisera revealed that the novel human apolipoprotein and the native form of the plasma FHR-4 protein are highly related and may even be identical.
Plasma Distribution of the FHR-4 DimerThe observed association of the FHR-4 protein with TG-Lp led us to study the plasma distribution of this protein in more detail. To this end lipoproteins were separated from plasma proteins by ultracentrifugation and the distribution of the FHR-4 protein in plasma, and the lipoprotein fractions was analyzed. We found that a relatively large fraction of the FHR-4 was associated with lipoproteins (Fig. 9A, lane 2). Due to the delipidation of the lipoproteins and the lack of a quantification method, we were unable to precisely determine the exact distribution of FHR-4. Separation of the different lipoprotein fractions by a second density gradient ultracentrifugation showed that most of the protein was associated with VLDL, and relatively smaller amounts were detected in the LDL and HDL fractions (Fig. 9B). The more TG-Lp were present in the plasma the more FHR-4 could be detected in this fraction. The appearance of FHR-4 protein in the bottom fraction (B2) after the second centrifugation is due to a partial loss of the protein from the lipoproteins during separation. This phenomenon is also observed for other apoproteins. A very similar distribution was observed in lipoproteins isolated from a series of normal probands and hyperlipidemic patients.5 From these results we conclude that the FHR-4 dimer is present in human plasma as free protein and also in TG-Lp, such as CM and VLDL, as well as other lipoproteins. The high molecular mass band of about 150 kDa detected by FHL-1 antiserum in the bottom fraction, LDL, and VLDL might represent the factor H protein.
We describe the isolation and characterization of a novel member of the family of factor H-related plasma proteins, FHR-4, and demonstrate that this protein is associated with human TG-Lp and VLDL and with other lipoproteins as well. The newly identified molecule is synthesized in human liver and is closely related to the previously described FHR-3 protein (5). While recombinant FHR-4 protein, expressed in the baculovirus system, exists mainly as a monomer, the native protein present in plasma and in TG-Lp exists predominantly in a dimeric form. The immunopurified plasma form of FHR-4 and the protein isolated from TG-Lp have identical mobility and cross-react with the specific antisera. The apparent differences between the plasma forms and the recombinant FHR-4 protein are explained by differences in glycosylation.
The FHR-4 molecule is a member of the factor H gene family. Similar to factor H and to the other factor H-related molecules, the processed FHR-4 protein is exclusively composed of SCRs (2). The FHR-4 protein of human plasma exists as a 86-kDa dimer and is glycosylated. In addition to its presence in plasma as free FHR-4 protein, it is also identified as a constituent of lipoprotein particles. It can be detected in all lipoproteins, namely HDL, LDL, and TG-Lp. The recombinant FHR-4 protein, expressed in the baculovirus system, has a molecular mass of 42 kDa, is glycosylated and can form a homodimer of 84 kDa. The different molecular mass of the dimeric native protein (86 kDa) and the dimeric recombinant protein (84 kDa) is due to attachment of different carbohydrate moieties.4 The reduced protein has an apparent molecular mass of 106 kDa in SDS-PAGE, indicating unfolding of the SCRs before monomerization.
The amino acid sequences of the predicted FHR-4 protein and of the proteolytic fragments obtained from the purified novel apoprotein are highly related but not identical. Five of the protein fragments (fragments II-VI) display a match of 94.9%; however, fragment I shows only an identity of 68.2%. Although polymorphic variants have been described for other members of the FHR gene family (9, 30, 31), the reason(s) for the observed differences are yet unclear. However, the identical mobility of the protein isolated from human TG-Lp and of the native plasma protein immunopurified using antiserum raised against the recombinant FHR-4 protein in SDS-PAGE and the cross-reactivity of the two proteins with the corresponding specific antisera indicates that both proteins are highly related or even identical.
The two factor H-related proteins FHR-4 and FHR-3 show a similar structural organization, both are organized in five SCRs and show a striking identity on the amino acid level. However the two proteins are distinct, as only the FHR-4 protein, but not the FHR-3 protein, has a domain related to SCR 9 of factor H. This domain is also represented by peptides II and III obtained from the 106-kDa protein isolated from TG-Lp (Fig. 5), again highlighting an identical feature of the two proteins. The individual SCRs of FHR-4 show a high degree of identity to SCRs of factor H and to the FHR-3 protein (Table I). In particular SCRs 2, 4, and 5 of FHR-4 have over 93% identity to the corresponding SCRs of FHR-3. The relatedness of the two proteins is also indicated by the amino acid identity of their signal peptides. SCRs 4 and 5 of FHR-4 show also significant identity to SCRs 19 and 20 of factor H. The characterized biological functions of factor H (inactivation of the alternative pathway convertases and cofactor activity for the cleavage of C3b, as well as polyanion/heparin binding) have been mapped to the NH2-terminal SCRs 1-4 and to SCR13, respectively (2, 32-34). The SCRs present in FHR-4, FHR-3, FHR-2, FHR-1, and homologous SCRs of factor H exclude these functionally characterized protein domains. Using recombinant FHR-4 protein for functional analysis demonstrates that this protein lacks the complement regulatory functions of factor H.6 The function of the newly identified FHR-4 protein seems, therefore, distinct from the complement regulatory role of factor H. A distinct biological function has, however, not yet been described for these members of the family of factor H-related proteins. Here we demonstrate that the FHR-4 protein is associated with lipoproteins and TG-Lp, thus indicating a potential function of this protein in lipid metabolism. Although FHR-4 is identified in all analyzed lipoprotein particles, the distribution of this protein is not uniform. Semiquantitative analyses suggest that in relation to other apoproteins the majority of lipid-associated FHR-4 was found in TG-Lp, such as CMs and VLDL. The amount of FHR-4 in these lipoproteins in relation to the free form is dependent on the level of these lipoproteins in plasma.
Similar as described here for the FHR-4 protein, an association with lipoprotein particles has recently been demonstrated for two additional members of this protein family, i.e. FHR-1 and FHR-2 (35). Thus suggesting for the FHR proteins a general role as constituents of lipoproteins.
At present also three complement regulatory proteins have been found
associated with lipoproteins. C4-binding protein (C4BP), the
glycophosphatidylinositol lipid-anchored membrane protein CD59
(protectin), and the soluble human complement lysis inhibitor (clusterin) are also associated with lipid particles (36-40). While C4BP is associated mainly with VLDL and LDL proteins, the other two
complement regulatory proteins, which inhibit formation or insertion of
the membrane attack complex are part of human HDL particles. Clusterin
is incorporated into lipoproteins due to amphipathic -helical
structures, and the similar biochemical properties qualify FHR-4 as an
apolipoprotein. Lipid-free proteins and highly concentrated fractions
of the two proteins show a tendency to aggregate and are poorly soluble
in water.
The association of FHR-4, a new member of the FHR family with lipoproteins, suggests either a role for these proteins in lipid transport, a functional interaction of lipids or lipoproteins with the complement system, or the use of lipoproteins as transport vehicles for these amphipathic proteins.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) X98337[GenBank].
We thank Dr. Ulrich Harmel for human liver samples. For performing the lipoprotein separations we thank the medical student Anne Kölln and Nicolette Meyer for performing the experiments for the revised form of the manuscript.