The Human Factor H-related Protein 4 (FHR-4)
A NOVEL SHORT CONSENSUS REPEAT-CONTAINING PROTEIN IS ASSOCIATED WITH HUMAN TRIGLYCERIDE-RICH LIPOPROTEINS*

(Received for publication, June 12, 1996, and in revised form, December 3, 1996)

Christine Skerka , Jens Hellwage , Wilfried Weber Dagger , Anne Tilkorn Dagger , Friedrich Buck §, Thomas Marti , Eva Kampen , Ulrike Beisiegel Dagger and Peter F. Zipfel

From the Bernhard Nocht Institute for Tropical Medicine, Bernhard-Nocht-Strasse 74, 20359 Hamburg, the Dagger  Medical Clinic, University Hospital Eppendorf, Martinistrasse 52, 20246 Hamburg, and the § Institute for Cell Biology and Clinical Neurobiology, University of Hamburg, Süderfeldstrasse 24, 20246 Hamburg, Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

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.


INTRODUCTION

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-1beta and FHR-1alpha (7). FHR-1beta has two carbohydrate side chains attached, and the FHR-1alpha protein has one carbohydrate side chain attached. Similarly the DDESK59 cDNA encodes a glycosylated 29-kDa (FHR-2alpha ) 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).

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.


EXPERIMENTAL PROCEDURES

Preparation of Human 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 beta -mercaptoethanol a 106-kDa band was detected.

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.

Separation with FPLC

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.

Isolation of a Novel Apolipoprotein

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.

Protein Digest and Sequence Analysis

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).

Labeling of Oligonucleotide Probes and Screening

A human liver oligo(dT)-primed cDNA library in lambda ZAP (Stratagene) was screened according to standard procedures (23) with a cDNA fragment (DOWN16) representing the previously described FHR-3 protein (5).

Characterization of Isolated Plasmids and Sequence Analysis of cDNA Clones

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 alpha -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.

RNA Isolation and Northern Blot Analysis

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).

SDS-PAGE and Western Blot Analysis

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.

Labeling and Hybridization

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'-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).

Immunopurification of the Native FHR-4 Protein from Human Plasma

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 M sodium phosphate, pH 6.8, the eluted protein was analyzed by SDS-PAGE and by Western blotting.


RESULTS

Identification of a Novel Apolipoprotein

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).


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 beta -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.
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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.
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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.
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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.
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A New Factor H-related Protein

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.


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.
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Protein Structure of FHR-4

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 Analysis of FHR-4 and Homologies in the FHR Family

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.


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.
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Table I.

Amino acid homology of individual short consensus repeats of the FHR-4 protein with members of the factor H family

Homology of individual SCR elements of the FHR-4 protein to SCRs of related members of the factor H family. The SCR displaying the highest degree of identity is shown. (FH, factor H; FHR-1-3, factor H-related proteins 1-3)
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 Analysis

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).


Fig. 6. Northern blot analysis with human liver RNA. Total cellular RNA was extracted, and 8 µg of RNA were separated on denaturing agarose gel electrophoresis and blotted onto nylon membranes. A 32P-labeled probe representing a region specific for the SAC6 cDNA was used.
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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


Fig. 7. Characterization of the native FHR-4 protein isolated by immunopurification from human plasma. A, native FHR-4 protein, immunopurified from human plasma using antiserum that was raised against the purified recombinant FHR-4 protein, was separated by SDS-PAGE and detected by Western blotting (lane 1). As a comparison recombinant FHR-4 protein either purified by nickel-chelate chromatography (lane 2) or immunopurified with the same specific antiserum (lane 3) was separated and visualized under identical conditions. B, the mobility of the native FHR-4 protein, isolated from human plasma by immunopurification, is changed by treatment with reducing agents (lane 3). As a comparison purified reFHR-4 protein is separated and detected under identical conditions as described in A in nonreduced (lane 1) and reduced form (lane 2).
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Fig. 8. Identical mobility and reactivity of the novel apolipoprotein purified from human TG-Lp and the FHR-4 protein isolated from human plasma. The human proteins isolated from TG-Lp (A, lanes 1, 4; B, lane 1), proteins isolated by immunopurification with anti-FHR-4 antiserum from human plasma (A, lanes 2 and 5; B, lane 2) and purified, recombinant FHR-4 protein (A, lanes 3 and 6; B, lane 3) were separated by SDS-PAGE under nonreducing conditions by SDS-PAGE according to Laemmli (A) or under reducing conditions in a gel system described by Neville (B). Identical blots were treated with antiserum either raised against the purified 106-kDa TG-Lp (lanes 1-3: anti-apoprotein) or the recombinant FHR-4 protein (lanes 4-6: anti-FHR-4). The blots were extensively stained in order to detect the dimeric form of the recombinant FHR-4 protein. B, after reduction with dithioerythritol the mobility of the protein shifts due to breakage of the intramolecular disulfide bonds, while the interaction of the two dimeric forms is unaffected by this treatment. The reduced proteins are visualized by Western blotting using anti FHR-4 antiserum. The mobility of the markers is indicated on the left.
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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 Dimer

The 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.


DISCUSSION

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 alpha -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.


FOOTNOTES

*   This work was supported by the Deutsche Forschungsgemeinschaft (DFG) in Projects Zi432/1-2 and Klinische Forschergruppe Gr258/10-1. This work is part of the doctoral thesis of J. H. at the Department of Biology at the University of Hamburg. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) X98337[GenBank].


   To whom correspondence and reprint requests should be addressed: Bernhard Nocht Inst. for Tropical Medicine, Bernhard-Nocht-Strasse 74, D-20359 Hamburg, Germany. Tel.: 49-40-31182-472; Fax: 49-40-31182-400.
1    The abbreviations used are: FHR, factor H-related; SCR, short consensus repeat; VLDL, very low density lipoproteins; IDL, intermediate density lipoproteins; LDL, low density lipoproteins; HDL, high density lipoproteins; ChR, chylomicron remnants; CM, chylomicrons; TG-Lp, triglyceride-rich lipoproteins; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; FPLC, fast protein liquid chromatography; HPLC, high performance liquid chromatography.
2    S. Kühn, J. Hellwage, and P. F. Zipfel, unpublished data.
3    C. Skerka, J. Hellwage and P. F. Zipfel, manuscript in preparation.
4    P. F. Zipfel et al., manuscript in preparation.
5    U. Beisiegel, manuscript in preparation.
6    S. Kühn and P. F. Zipfel, unpublished data.

Acknowledgments

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.


REFERENCES

  1. Vik, D. P., Munoz-Canovez, P., Chaplin, D. D., and Tack, B. F. (1989) Curr. Top. Microbiol. Immunol. 153, 148-162
  2. Zipfel, P. F., and Skerka, C. (1994) Immunol. Today 15, 121-126 [CrossRef][Medline] [Order article via Infotrieve]
  3. Skerka, C., Horstmann, R. D., and Zipfel, P. F. (1991) J. Biol. Chem. 266, 12015-12020 [Abstract/Free Full Text]
  4. Estaller, C., Koistinen, V., Schwäble, W., Dierich, M. P., and Weiss, E. (1991) J. Immunol. 146, 3190-3196 [Abstract/Free Full Text]
  5. Skerka, C., Kühn, S., Günther, K., Lingelbach, K., and Zipfel, P. F. (1993) J. Biol. Chem. 268, 2904-2908 [Abstract/Free Full Text]
  6. Skerka, C., Timmann, C., Horstmann, R. D., and Zipfel, P. F. (1992) J. Immunol. 148, 3313-3318 [Abstract/Free Full Text]
  7. Timmann, C., Leippe, M., and Horstmann, R. D. (1991) J. Immunol. 146, 1265-1270 [Abstract/Free Full Text]
  8. Vik, D. P., Munoz-Canoves, P., Kozono, H., Martin, L. G., Tack, B. F., and Chaplin, D. D. (1990) J. Biol. Chem. 265, 3193-3201 [Abstract/Free Full Text]
  9. Skerka, C., Moulds, J., Taillon-Miller, P., Hourcade, D., and Zipfel, P. F. (1995) Immunogenetics 42, 268-274 [Medline] [Order article via Infotrieve]
  10. Vik, D. P., Keeney, J. B., Munoz-Canoves, P., Chaplin, D. D., and Tack, B. F. (1988) J. Biol. Chem. 263, 16720-16724 [Abstract/Free Full Text]
  11. Schaefer, E. J., Jenkins, L. L., and Brewer, H. B. (1978) Biochim. Biophys. Acta 80, 405-412
  12. Schaefer, E. J., Eisenberg, S., and Levy, R. I. (1978) J. Lipid Res. 19, 667-680 [Medline] [Order article via Infotrieve]
  13. Innerarity, T. L. (1991) Encycl. Hum. Biol. 6, 23-35
  14. Sherrill, B. C., Innerarity, T. L., and Mahley, R. W. (1980) J. Biol. Chem. 255, 1804-1807 [Abstract/Free Full Text]
  15. Groot, P. H. E., Oerlemans, M. C., and Scheek, L. M. (1978) Biochim. Biophys Acta 530, 91-98 [Medline] [Order article via Infotrieve]
  16. Sehayek, E., and Eisenberg, S. (1991) J. Biol. Chem. 266, 18259-18267 [Abstract/Free Full Text]
  17. Chen, C. H., and Albers, J. J. (1983) Biochim. Biophys. Acta 753, 40-46 [Medline] [Order article via Infotrieve]
  18. Steinmetz, A., and Utermann, G. (1985) J. Biol. Chem. 260, 2258-2264 [Abstract]
  19. Breckenridge, W. C., Little, J. A., Steiner, G., Chow, A., and Poapst, M. (1978) N. Engl. J. Med. 298, 1265-1273 [Abstract]
  20. Lowry, O. H., Rosebrough, N. R., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 [Free Full Text]
  21. McFarlane, A. S. (1958) Nature 182, 53-57
  22. Neville, D. M., Jr. (1971) J. Biol. Chem. 246, 6328-6334 [Abstract/Free Full Text]
  23. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  24. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467 [Abstract]
  25. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J. (1979) Biochemistry 18, 5294-5299 [Medline] [Order article via Infotrieve]
  26. Heukeshoven, J., and Dernick, R. (1986) Electrophoresis 6, 103-112
  27. Kyhse-Andersen, J (1984) J. Biochem. Biophys. Methods 10, 203-209 [CrossRef][Medline] [Order article via Infotrieve]
  28. Kozak, M. (1984) Nucleic Acids Res. 12, 857-872 [Abstract]
  29. von Heijne, G. (1986) Nucleic Acids Res. 14, 4683-4692 [Abstract]
  30. Feifel, E., Prodinger, W. M., Mölgg, M., Schwaeble, W., Schönitzer, D., Koistinen, V., Misasis, R., and Dierich, M. P. (1992) Immunogenetics 36, 104-109 [Medline] [Order article via Infotrieve]
  31. Meyer, C. G., Skerka, C., and Zipfel, P. F. (1995) Immunogenetics 41, 335 [Medline] [Order article via Infotrieve]
  32. Kühn, S., Skerka, C., and Zipfel, P. F. (1995) J. Immunol. 155, 5663-5670 [Abstract]
  33. Gordon, D. L., Kaufman, R. M., Blackmore, T. K., Kwong, J., and Lubin, D. M. (1995) J. Immunol. 155, 348-356 [Abstract]
  34. Pangburn, M. K., Atkinson, M. A. L., and Meri, S. (1991) J. Biol. Chem. 266, 16847-16853 [Abstract/Free Full Text]
  35. Park, C. T., and Wright, S. D. (1996) J. Biol. Chem. 271, 18054-18060 [Abstract/Free Full Text]
  36. Väkeva, A., Jauhiainen, M., Ehnholm, C., Lehto, T., and Meri, S. (1994) Immunology 82, 28-33 [Medline] [Order article via Infotrieve]
  37. Meri, S. (1994) Immunologist 2, 149-155
  38. Jenne, D. E., Lowin, B., Peitsch, M. C., Bottcher, A., Schmitz, G., and Tschopp, J. (1991) J. Biol. Chem. 266, 11030-11036 [Abstract/Free Full Text]
  39. Jenne, D. E., and Tschopp, J. (1992) Trends Biochem. Sci. 17, 154-159 [CrossRef][Medline] [Order article via Infotrieve]
  40. Funakoshi, M., Sasaki, J., and Arakawa, K. (1988) Biochim. Biophys. Acta 963, 98-108 [Medline] [Order article via Infotrieve]

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