From the New York University Medical Center, Department of Pathology, New York, New York 10016
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ABSTRACT |
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Haptoglobin-related protein (HPR) is a serum protein that is >90% homologous to the acute-phase reactant haptoglobin (Hp). Haptoglobin binds and removes free hemoglobin (Hb) from the circulation. Hpr levels are elevated with tumor progression in the serum of some cancer patients, but the relevance of this observation is not understood. HPR is an integral part of two distinct high molecular weight complexes (trypanosome lytic factor 1 (TLF1) and TLF2) that are lytic for the African parasite Trypanosoma brucei brucei. Previous data indicate that HPR represents the toxic component of both trypanosome lytic factors. It has been proposed that after uptake by the parasite, Hb bound to HPR causes lysis in a peroxidase-dependent process. We report that the molecular architecture of HPR in normal human serum is different from that of Hp and that HPR does not bind Hb in normal human serum. Immunodepletion of all detectable Hb from TLF1 does not deplete TLF1 of HPR or trypanolytic activity, suggesting that the mechanism of parasite lysis is Hb-independent.
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INTRODUCTION |
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Haptoglobin (Hp)1 is an
acute-phase plasma glycoprotein present in normal human serum (NHS) at
concentrations of between 0.2 and 2 mg/ml. The major known function of
Hp is to bind and remove potentially toxic free hemoglobin (Hb) from
the circulation. Haptoglobin binds Hb with 1:1 stoichiometry with an
extremely high affinity (Ka > 1015
M) (1), and Hp·Hb complexes are rapidly removed from the
circulation via receptors in the liver. Haptoglobin is a heterodimer,
and due to structural variation in the
chain of Hp
-
dimers,
there are three Hp types in humans, namely Hp1-1, Hp2-1, and Hp2-2
(2). Types 2-1 and 2-2 exist as a series of disulfide-linked polymers (2).
Haptoglobin-related protein (HPR) displays more than 90% identity to
Hp and is found at a much lower concentration in serum (3). The HPR
gene arose by duplication of the Hp gene and is located 2.2 kilobase
pairs downstream of the Hp gene on chromosome 16. There are 28-amino
acid changes in HPR, 16 of which occur in the chain. In addition,
intron 1 of Hp is only 1.3 kilobase pairs, whereas that of HPR is 9.5 kilobase pairs and contains a retrovirus-like element (4). There is an
Alu sequence in the 5'-flanking region of HPR (3, 4). The HPR gene is
also present in apes and Old World monkeys and is a product of a gene triplication event that occurred early in primate evolution (5).
In NHS, HPR appears to be associated exclusively with either a small HDL subpopulation or a high molecular weight protein complex (6), both of which possess lytic activity against the African cattle parasite Trypanosoma brucei brucei. The HDL trypanolytic factor is termed TLF1 and is composed of phospholipid, apoAI, apoAII, paraoxonase and HPR (7). The second factor, TLF2, is a protein complex containing apoA1 and HPR (6, 8). The natural immunity of humans to T. brucei brucei but not to the morphologically indistinguishable human pathogens T. brucei gambiense and T. brucei rhodesiense that cause sleeping sickness is due to the selective killing of T. brucei brucei by TLF1 and/or TLF2. Consistent with an important role for HPR in trypanosome killing, sera from nonhuman primates that contain the HPR gene (see above) are trypanolytic (9, 10). An exception is Chimpanzee serum, which is not trypanolytic. However, the HPR gene sequence in Chimpanzees was found to contain a frameshift mutation, resulting in premature termination of translation (5). Data indicate that trypanosome lysis is peroxide-dependent (7). It has been proposed that like Hp, HPR binds Hb, and that TLF1-associated HPR·Hb complexes possess peroxidase activity at acid pH. After uptake and delivery to parasite lysosomes, the HPR·Hb complexes would kill trypanosomes by oxidative damage (7).
Other than its role in trypanosome lysis, there is no known function of HPR. A form of HPR apparently distinct from that found in NHS is found in the sera of cancer patients. Hpr levels appear to be useful as a clinical diagnostic marker, since they are elevated in progression and decreased during regression of various carcinomas (11-14). Here we characterize this minor serum protein and further define its role as the toxin in the trypanocidal factors.
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EXPERIMENTAL PROCEDURES |
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Sera--
NHS from a single healthy donor of Hp haplotype 1-1
was stored at 70 °C in aliquots for up to 6 months. Sera from
patients with paroxysmal nocturnal hemoglobinuria and Tangier disease
were the kind gifts of Dr. W. Rosse (Duke University, NC) and Dr.
Sachiya Ohtaki (Miyazaki Medical College, Fukuoka, Japan),
respectively.
Parasites-- Swiss Webster mice were inoculated intraperitoneally with T. brucei brucei TREU 667 stock (15), and the trypanosomes were harvested 2 days later from infected mouse blood and prepared for trypanolytic assay as described previously (16).
Purification of TLF1--
Total lipoproteins were isolated from
NHS by density gradient centrifugation, essentially as described (17).
Solid KBr was added to serum to give a density of 1.25 g/ml, and after
ultracentrifugation (Beckman NVTi 60 rotor, 16 h, 49,000 × g, 10 °C), the top 25% of the gradient containing
lipoprotein was collected. The density of the lipoprotein fraction was
then adjusted to 1.3 g/ml with KBr, and aliquots (4 ml) were layered
under 0.9% NaCl (8 ml). After centrifugation for 3 h at
49,000 × g/10 °C (NVTi 60, Beckman), the HDL band
was isolated. The crude TLF1 preparation was dialyzed against TBS at
4 °C with frequent buffer changes and then concentrated by
ultrafiltration using Amicon (Beverly, MA) XM300 membranes. The TLF1
preparation (aliquots of 0.5 ml at about 50 mg protein/ml) was then
fractionated by fast protein liquid chromatography (Pharmacia Biotech
Inc.) on Superose 6 high resolution 10/30 and Superose 12 high
resolution 10/30 columns connected in tandem (0.2 ml/min in TBS, pH
7.0). Fractions were analyzed by absorbance at 280 nm, and TLF1
fractions were identified by measurement of trypanosome lytic activity
in each fraction (8, 18). Fractions with peak trypanolytic activity
were pooled and concentrated using Centricon 100 devices (Amicon), and
aliquots were stored at 70 °C.
Anti-Hb Immunodepletion of TLF1-- Anti-Hb monoclonal (Genzyme, Cambridge, MA) or polyclonal (Sigma) antibody was incubated with protein G-Sepharose (Pharmacia) for 2 h at 4 °C. Ten µg of anti-Hb antibody was conjugated/µl of protein G-Sepharose. The conjugated beads were washed by centrifugation (phosphate-buffered saline, 0.1% bovine serum albumin) and resuspended to 50% (phosphate-buffered saline, 0.1% bovine serum albumin). After incubation for 1 h at 4 °C, the beads were used to deplete TLF1 preparations of Hb. Fifty µl of TLF1 was treated with 4 µl of anti-Hb-Sepharose (or protein G-Sepharose as control) for 2 h at 4 °C, and the immunoprecipitate was isolated by centrifugation. Immunoprecipitates were washed 5 × with phosphate-buffered saline containing 0.5 M NaCl and 1% Nonidet P-40, followed by three washes with phosphate-buffered saline. Pellets and supernatants were analyzed as described below. Hp·Hb complexes were used in control immunoprecipitation experiments and were prepared as described (8).
SDS-Polyacrylamide Gel Electrophoresis and Immunoblot Analysis-- All samples were separated in SDS-polyacrylamide gel electrophoresis 4-15% acrylamide gradient gels (Bio-Rad) under reducing or nonreducing conditions by standard procedures (19). Separated proteins were transferred to a nitrocellulose membrane, and prestained broad range markers (Bio-Rad) were used as an indicator of complete transfer. Membranes with transferred proteins were developed by either chemiluminescence (Amersham Life Science, Inc.) (as described by the manufacturer) or by standard alkaline phosphatase detection. For Hp detection, membranes were probed with rabbit anti-human Hp (Sigma) at either 1,000 × dilution (for alkaline phosphatase detection) or at 10,000 × dilution (for chemiluminescent detection). For Hb detection, membranes were probed with goat anti-human Hb (Chemicon, Temecula, CA) at 2,500 × dilution, and bound antibody was revealed by means of protein A-HPR antibody at 2,500 × dilution (Cappel, Durham, NC) and ECL reagents (Amersham).
Assay Procedures-- Quantitation of Hp, HPR, and Hb was performed using a Western blotting procedure as described previously (6). For assaying serum Hp and HPR concentrations, various dilutions of serum corresponding to 2-0.02 µl of serum were separated by SDS-polyacrylamide gel electrophoresis before transfer to nitrocellulose membranes (see above). Each gel run for quantitation purposes contained Hp and/or Hb standards of between 100 and 2.5 ng. Immunoblots were scanned, and immunostained bands were quantitated using NIH image 5.7 software. The distinction between Hp and HPR is based on molecular weight and has been previously described (6). Hemoglobin used for standards was purified as described (18).
Parasite Lysis Assays-- Assays were performed as described previously (18). Briefly, 2 × 106 trypanosomes were incubated with the sample under test in a total volume of 200 µl in high glucose Dulbecco's modified essential medium (Life Technologies, Inc.) containing 0.2% bovine serum albumin. Lysis was assessed by means of the fluorescent probe Calcein-AM (Molecular Probes, Eugene, OR) after a 150-min incubation at 37 °C (18).
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RESULTS |
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Structure of HPR--
Haptoglobin is a disulfide-linked
heterodimer, and Western blot analysis of reduced purified Hp1-1 using
an anti-Hp polyclonal antibody revealed the expected 15-kDa and
40-kDa
chains of the protein (Fig. 1,
lane 2). The corresponding subunits for HPR are 12 and 35 kDa, respectively (8, 20) and are shown in Fig. 1, lane 1.
Purified TLF1 was the source of HPR. Native Hp1-1 exists as a
disulfide-linked tetramer (
2-
2) (21), and as expected, an anti-Hp
immunoblot of nonreduced Hp1-1 revealed a single 110-kDa protein (Fig.
1, lane 4). In contrast, immunoblot analysis of nonreduced
HPR revealed a major band of about 45 kDa and additional much fainter
bands of about 90, 120, and 135 kDa, probably representing HPR
multimers (Fig. 1, lane 3). Native Hp2-1 and Hp2-2 exist
as disulfide-linked oligomeric complexes of between 200-700 kDa (21). Thus, the molecular architecture of native HPR is different to that of
all Hp allotypes in that it appears to exist predominantly as a single
-
dimer.
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Haptoglobin-related Protein Is Found in Blood of Patients with Intravascular Hemolysis-- Hp binds free plasma Hb with high affinity, and Hp·Hb complexes are rapidly cleared from the circulation via hepatocyte receptors. We determined Hp and HPR concentrations in NHS and in sera from patients with paroxysmal nocturnal hemoglobinuria (PNH) or Tangier disease. These conditions lead to extensive intravascular hemolysis, resulting in excess free Hb in the circulation and subsequent Hp depletion. As expected, the pathological sera contained very low levels of Hp; between 500-5,000-fold less than in NHS (Table I). In contrast, the concentration of Hpr in both NHS and pathological sera was similar. Therefore, unlike Hp, HPR serum concentration does not decrease in the presence of excess circulating plasma Hb. The simplest interpretation of these data is that either HPR does not bind Hb or that the hepatocyte ligand within HPR·Hb complexes is absent or obscured.
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Hpr Does Not Contain Bound Hb-- In NHS, most plasma HPR is associated with the TLF1 complex, a small HDL subpopulation that possesses trypanosome lytic activity (6, 7). Partially purified TLF1 was fractionated by gel filtration, and two lytic TLF1 fractions eluting at slightly different molecular weights were selected. One contained a high HPR and a low contaminating Hp concentration (Fig. 2, lane 1), and one contained low HPR and high Hp concentration (Fig. 2, lane 2). Each of these fractions was treated with anti-Hb monoclonal antibody bound to protein G beads, and the immunoprecipitate and supernatant were analyzed by anti-Hp Western blot. A quantitative determination of Hp, HPR, and Hb in the various anti-Hb immunodepleted fractions is shown in Table II. Fig. 2 and Table II shows that an anti-Hb antibody coimmunoprecipitated contaminating Hp from TLF1 preparations, but failed to coimmunoprecipitate HPR (Fig. 2, lanes 5 and 6, and Table II). Analysis of supernatants after anti-Hb immunoprecipitation shows that HPR remains in the supernatant (Fig. 2, lanes 3 and 4, and Table II). Not all Hp present is expected to contain bound Hb, so not all Hp was immunoprecipitated. Anti-Hb antibody was added in excess, as indicated by the quantity of control Hp·Hb complexes that were immunoprecipitated (Fig. 2, lane 7 and Table II). Further analysis of anti-Hb-treated fractions by anti-Hb Western blot revealed the removal of all detectable Hb from TLF1 (Fig. 3, Table II).
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Immunodepletion of Hb from TLF1 Does Not Deplete Trypanolytic Activity-- It has been proposed that Hb bound to HPR kills T. brucei in a peroxidase-dependent mechanism (7). However, the data presented above indicate that HPR does not contain bound Hb in NHS. Furthermore, Fig. 4 shows that immunoprecipitation of TLF1 with either monoclonal or polyclonal antibodies to Hb did not remove trypanolytic activity from a TLF1 preparation.
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DISCUSSION |
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We have studied certain properties of HPR in NHS and report several new findings. The molecular architecture of native HPR is distinct from that of Hp. Hpr levels in Hp-depleted patient sera are comparable to levels found in NHS, indicating either that HPR does not bind Hb or that HPR·Hb complexes (unlike Hp·Hb complexes) are not cleared from the circulation. We have not measured the turnover of HPR, but it is unlikely that the maintenance of HPR levels in serum of PNH patients reflects compensatory synthesis after clearance of HPR·Hb complexes. In fact, HPR in NHS does not contain any detectable bound Hb. Therefore, HPR does not share the principal function of Hp, which is to bind and remove free Hb from the circulation. This is consistent with earlier studies reporting no detectable peroxidase activity in HPR identified on native polyacrylide gels (22).2 Finally, the present experiments show that the concentration of HPR in NHS is about 50-fold less than that of Hp, contrasting with the 1,000-fold difference previously predicted from mRNA quantitation (3).
The sequence of HPR is more than 90% identical with that of Hp, and
the predicted Hb binding region of Hp is conserved. The inability of
HPR to bind Hb may be due to a different pattern of disufide bonding
that results in structural differences. Although both Hp and HPR
contain the same total number of cysteines, the cysteine at position 15 of the Hp chain that stabilizes the tetramer through cross-linking
the monomers is replaced by phenylalanine in HPR (4). This is
consistent with our observation that HPR exists predominantly as an
-
dimer. Though HPR can polymerize into higher order complexes,
these are minor components (based on the faint staining with anti-Hp).
It is also possible that the cysteines in HPR are conformationally
shielded from one another, thus preventing oligomer formation.
In NHS, total serum HPR appears to be distributed between two particles termed TLF1 and TLF2, both of which have trypanosome lytic activity. Susceptibility of trypanosomes to these lytic factors determine their human infectivity and defines their host range. There is compelling evidence that HPR represents the toxic component of these factors (6, 7). The unusual hydrophobic N terminus of HPR in NHS (7) may be involved in its targeting to TLF1 (an HDL particle) and to TLF2 (a protein complex that contains the amphipathic apolipoprotein AI) (6). However, the current data does not support the hypothesis that HPR-mediated trypanolysis is due to peroxidase activity of bound Hb (see the introduction). Although Smith et al. (7) detected Hb in their TLF1 preparation (as do we in the TLF1 preparations used in this study), the current data indicate that Hb is a contaminant and not a component of TLF1.
So how does HPR mediate trypanosome lysis? The previous measurement of peroxidase activity in TLF1 may have been due to contaminating Hb, but the inhibition of parasite lysis by catalase indicates that endocytosed TLF1 causes parasite lysis via a peroxide-dependent mechanism (7). In addition to Hb, heme/hemin, Fe3+, and Cu2+ can participate in peroxidation of lipids and proteins and cause cell damage through reactive oxygen intermediates. Hp is known to bind Cu2+ (24) and heme (2), and it is possible that HPR may similarly bind one or more of these molecules, resulting in a parasite lysosome-inducible peroxidase activity. An alternative and intriguing possibility is that the HPR in TLF may induce a parasite-specific peroxidase activity. Further characterization of HPR, in particular identification of a putative peroxidase-conferring moiety, may provide a better understanding of its precise role in trypanosome lysis. Such knowledge may be useful in creating drug delivery systems for African trypanosomes such as trypanolytic recombinant fusion proteins incorporating HPR and parasite receptor ligands.
Other than its role in trypanolysis, there is no known function of HPR. Increased levels of HPR have been reported in sera from patients with various cancers, and HPR levels have been reported to correlate with tumor progression (11-14, 25).2 However, the analysis of cancer-related HPR revealed that it contains an N-terminal extension not found in HPR present in NHS (23). This N-terminal extension appears to be due to an alternative processing of the HPR pre-mRNA, resulting in transcription of intron 1 and translation from the first AUG in intron 1 with the loss of exon 1. The relationship between the two forms of HPR is not known.
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ACKNOWLEDGEMENTS |
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We thank Drs. Jayne Raper and Mary Lee for critical discussions, Dr. Channa Shalitin for critical reading of the manuscript and sharing unpublished data, and Dr. Ronald Nagel for critical reading of the manuscript.
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FOOTNOTES |
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* This investigation received financial support from the United Nations Developmental Program/World Bank/World Health Organization Special Program for Research and Training in Tropical Diseases and from National Institutes of Health Grant AI40206.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.
To whom correspondence should be addressed: New York University
Medical Center, Dept. of Pathology, MSB 127, 550 First Ave., New York,
NY 10016. Tel.: 212-263-8514; Fax: 212-263-8179; E-mail: tomlis01{at}popmail.med.nyu.edu.
1 The abbreviations used are: Hp, haptoglobin; Hb, hemoglobin; HPR, haptoglobin-related protein; NHS, normal human serum; TLF, trypanosome lytic factor; HDL, high density lipoprotein; PNH, paroxysmal nocturnal hemoglobinuria.
2 C. Shalitin, C. Valansi, A. Admon, B. Moskovitz, R. Segal, R. Epelbaum, and O. Nativ, submitted for publication.
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REFERENCES |
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