Characterization of the Human Serum Trypanosome Toxin, Haptoglobin-related Protein

Abstract

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. 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. Haptoglobin (Hp) 1The 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. 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 > 10−15m) (1Hwang P.K. Greer J. J. Biol. Chem. 1980; 255: 3038-3041Abstract Full Text PDF PubMed Google Scholar), 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 α chain of Hp α-β dimers, there are three Hp types in humans, namely Hp1–1, Hp2–1, and Hp2–2 (2Bowman B.H. Kurosky A. Harris H. Hirschhorn K. Advances in Human Genetics. Plenum Publishing Corp., New York1982: 189-261Crossref Scopus (305) Google Scholar). Types 2–1 and 2–2 exist as a series of disulfide-linked polymers (2Bowman B.H. Kurosky A. Harris H. Hirschhorn K. Advances in Human Genetics. Plenum Publishing Corp., New York1982: 189-261Crossref Scopus (305) Google Scholar). Haptoglobin-related protein (HPR) displays more than 90% identity to Hp and is found at a much lower concentration in serum (3Bensi G. Raugei G. Klefenz H. Cortese R. EMBO J. 1985; 4: 119-126Crossref PubMed Scopus (115) Google Scholar). 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 β 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 (4Maeda N. J. Biol. Chem. 1985; 260: 6698-6709Abstract Full Text PDF PubMed Google Scholar). There is an Alu sequence in the 5′-flanking region of HPR (3Bensi G. Raugei G. Klefenz H. Cortese R. EMBO J. 1985; 4: 119-126Crossref PubMed Scopus (115) Google Scholar, 4Maeda N. J. Biol. Chem. 1985; 260: 6698-6709Abstract Full Text PDF PubMed Google Scholar). 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 (5McEvoy S.M. Maeda N. J. Biol. Chem. 1988; 263: 15740-15747Abstract Full Text PDF PubMed Google Scholar). In NHS, HPR appears to be associated exclusively with either a small HDL subpopulation or a high molecular weight protein complex (6Tomlinson S. Muranjan M. Nussenzweig V. Raper J. Mol. Biochem. Parasitol. 1997; 86: 117-120PubMed Google Scholar), both of which possess lytic activity against the African cattle parasiteTrypanosoma brucei brucei. The HDL trypanolytic factor is termed TLF1 and is composed of phospholipid, apoAI, apoAII, paraoxonase and HPR (7Smith A.B. Esko J.D. Hajduk S.L. Science. 1995; 268: 284-286Crossref PubMed Scopus (146) Google Scholar). The second factor, TLF2, is a protein complex containing apoA1 and HPR (6Tomlinson S. Muranjan M. Nussenzweig V. Raper J. Mol. Biochem. Parasitol. 1997; 86: 117-120PubMed Google Scholar, 8Raper J. Nussenzweig V. Tomlinson S. J. Exp. Med. 1996; 183: 1023-1029Crossref PubMed Scopus (61) Google Scholar). 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 (9Seed J.R. Sechelski J.B. Loomis M.R. J. Protozool. 1990; 37: 393-400Crossref PubMed Scopus (46) Google Scholar, 10Hawking F. Ramsden D.B. Whytock S. Trans. R. Soc. Trop. Med. Hyg. 1973; 67: 501-517Abstract Full Text PDF PubMed Scopus (34) Google Scholar). 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 (5McEvoy S.M. Maeda N. J. Biol. Chem. 1988; 263: 15740-15747Abstract Full Text PDF PubMed Google Scholar). Data indicate that trypanosome lysis is peroxide-dependent (7Smith A.B. Esko J.D. Hajduk S.L. Science. 1995; 268: 284-286Crossref PubMed Scopus (146) Google Scholar). 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 (7Smith A.B. Esko J.D. Hajduk S.L. Science. 1995; 268: 284-286Crossref PubMed Scopus (146) Google Scholar). 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 (11Oh S.K. Kim S.H. Abbruzzi G. Adler W.H. Tauber A.I. Hybridoma. 1992; 11: 1-12Crossref PubMed Scopus (4) Google Scholar, 12Kuhajda F.P. Katumuluwa A.J. Pasternack G.R. Proc. Natl. Acad. Sci. U. S. A. 1986; 86: 1188-1192Crossref Scopus (62) Google Scholar, 13Grundner-Culemann E. Bokemeyer C. Neubauer V. Shalitin C. Schmoll H.J. Poliwoda H. Bentner T. J. Cancer Res. Clin. Oncol. 1993; 119: 685-688Crossref PubMed Scopus (3) Google Scholar, 14Shalitin C. Epelbaum R. Valansi C. Segal R. Mekori T. Lover B. Robinson E. Int. J. Cancer. 1991; 49: 861-866Crossref PubMed Scopus (7) Google Scholar). Here we characterize this minor serum protein and further define its role as the toxin in the trypanocidal factors. 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. Swiss Webster mice were inoculated intraperitoneally with T. brucei brucei TREU 667 stock (15Jennings F.W. Urquhart G.M. Murray P.K. Miller P.M. Trans. R. Soc. Trop. Med. Hyg. 1983; 77: 693-695Abstract Full Text PDF PubMed Scopus (23) Google Scholar), and the trypanosomes were harvested 2 days later from infected mouse blood and prepared for trypanolytic assay as described previously (16Lanham S.M. Godfrey D.G. Exp. Parasitol. 1970; 28: 521-531Crossref PubMed Scopus (1124) Google Scholar). Total lipoproteins were isolated from NHS by density gradient centrifugation, essentially as described (17Havel R.J. Eder H.A. Bragden J.H. J. Clin. Invest. 1955; 34: 1345-1353Crossref PubMed Scopus (6498) Google Scholar). 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 (8Raper J. Nussenzweig V. Tomlinson S. J. Exp. Med. 1996; 183: 1023-1029Crossref PubMed Scopus (61) Google Scholar, 18Tomlinson S. Jansen A.-M. Koudinov A. Ghiso J.A. Choi-Miura N.-H. Rifkin M.R. Ohtaki S. Nussenzweig V. Mol. Biochem. Parasitol. 1995; 70: 131-138Crossref PubMed Scopus (44) Google Scholar). Fractions with peak trypanolytic activity were pooled and concentrated using Centricon 100 devices (Amicon), and aliquots were stored at −70 °C. 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 (8Raper J. Nussenzweig V. Tomlinson S. J. Exp. Med. 1996; 183: 1023-1029Crossref PubMed Scopus (61) Google Scholar). All samples were separated in SDS-polyacrylamide gel electrophoresis 4–15% acrylamide gradient gels (Bio-Rad) under reducing or nonreducing conditions by standard procedures (19Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). 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). Quantitation of Hp, HPR, and Hb was performed using a Western blotting procedure as described previously (6Tomlinson S. Muranjan M. Nussenzweig V. Raper J. Mol. Biochem. Parasitol. 1997; 86: 117-120PubMed Google Scholar). 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 (6Tomlinson S. Muranjan M. Nussenzweig V. Raper J. Mol. Biochem. Parasitol. 1997; 86: 117-120PubMed Google Scholar). Hemoglobin used for standards was purified as described (18Tomlinson S. Jansen A.-M. Koudinov A. Ghiso J.A. Choi-Miura N.-H. Rifkin M.R. Ohtaki S. Nussenzweig V. Mol. Biochem. Parasitol. 1995; 70: 131-138Crossref PubMed Scopus (44) Google Scholar). Assays were performed as described previously (18Tomlinson S. Jansen A.-M. Koudinov A. Ghiso J.A. Choi-Miura N.-H. Rifkin M.R. Ohtaki S. Nussenzweig V. Mol. Biochem. Parasitol. 1995; 70: 131-138Crossref PubMed Scopus (44) Google Scholar). 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 (18Tomlinson S. Jansen A.-M. Koudinov A. Ghiso J.A. Choi-Miura N.-H. Rifkin M.R. Ohtaki S. Nussenzweig V. Mol. Biochem. Parasitol. 1995; 70: 131-138Crossref PubMed Scopus (44) Google Scholar). Haptoglobin is a disulfide-linked heterodimer, and Western blot analysis of reduced purified using an polyclonal antibody revealed the α and β of the protein The corresponding for HPR are 12 and (8Raper J. Nussenzweig V. Tomlinson S. J. Exp. Med. 1996; 183: 1023-1029Crossref PubMed Scopus (61) Google Scholar, A.B. Hajduk S.L. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google and are in TLF1 was the of HPR. as a disulfide-linked J. The and of Scholar), and as an of revealed a single protein In analysis of HPR revealed a major band of about and much bands of about and HPR and Hp2–2 exist as disulfide-linked complexes of between J. The and of Scholar). the molecular architecture of HPR is different to that of all Hp in that appears to exist as a single α-β Hp binds free plasma Hb with high and Hp·Hb complexes are rapidly from the circulation via We Hp and HPR concentrations in NHS and in sera from patients with paroxysmal nocturnal hemoglobinuria or Tangier conditions to resulting in free Hb in the circulation and Hp the sera contained levels of between than in NHS In the concentration of Hpr in both NHS and sera was Hp, HPR serum concentration does not in the of plasma Hb. The of data is that either HPR does not bind Hb or that the HPR·Hb complexes is or of Hp and HPR in sera from normal and from patients with in a also that the concentration of HPR in NHS is about lower than that of Hp is much than the previously that was based on a of levels of Hp and HPR (3Bensi G. Raugei G. Klefenz H. Cortese R. EMBO J. 1985; 4: 119-126Crossref PubMed Scopus (115) Google Scholar). In NHS, plasma HPR is associated with the TLF1 a small HDL subpopulation that trypanosome lytic activity (6Tomlinson S. Muranjan M. Nussenzweig V. Raper J. Mol. Biochem. Parasitol. 1997; 86: 117-120PubMed Google Scholar, A.B. Esko J.D. Hajduk S.L. Science. 1995; 268: 284-286Crossref PubMed Scopus (146) Google Scholar). purified TLF1 was fractionated by gel and two lytic TLF1 fractions at different molecular were contained a high HPR and a Hp concentration and contained HPR and high Hp concentration Each of fractions was treated with anti-Hb monoclonal antibody bound to protein and the immunoprecipitate and were analyzed by Western A of Hp, HPR, and Hb in the various anti-Hb fractions is in 2 and that an anti-Hb antibody Hp from TLF1 but to HPR 5 and and of supernatants after anti-Hb immunoprecipitation that HPR in the 3 and and all Hp present is to contain bound Hb, not all Hp was Anti-Hb antibody was added in as by the of control Hp·Hb complexes that were and analysis of fractions by anti-Hb Western blot revealed the of all detectable Hb from TLF1 of Hp, HPR, and Hb in TLF1 preparations after with anti-Hb monoclonal TLF1 TLF1 TLF1 TLF1 Hp Hb Hp Hb Hp Hb Hp Hb in a blot analysis under reducing conditions of TLF1 and revealed with anti-Hb 1 and 2 the from TLF1 after incubation with either protein G-Sepharose or anti-Hb monoclonal antibody bound to protein G-Sepharose respectively. 3 and the from control Hp·Hb complexes after incubation with protein beads or bands were by means of anti-Hb polyclonal antibody and antibody and It has been proposed that Hb bound to HPR T. brucei in a peroxidase-dependent mechanism (7Smith A.B. Esko J.D. Hajduk S.L. Science. 1995; 268: 284-286Crossref PubMed Scopus (146) Google Scholar). However, the data indicate that HPR does not contain bound Hb in that immunoprecipitation of TLF1 with either monoclonal or polyclonal to Hb not remove trypanolytic activity from a TLF1 that a lytic TLF1 preparation that has been of all detectable Hb contains about of HPR. levels of Hb are bound to HPR in there is at a a of HPR Hb molecular of HPR and Hb are In a Hb was added to and its trypanolytic activity was Hb bound to HPR and the lytic of Hb added to TLF1 would be to complex with the HPR and in an in trypanolytic The of Hb to TLF1 not TLF1 trypanolytic activity We that Hb does not a role in We of HPR in NHS and report The molecular architecture of HPR is distinct from that of Hpr levels in sera are to levels found in NHS, either that HPR does not bind Hb or that HPR·Hb complexes Hp·Hb are not from the circulation. We not the of HPR, but is that the of HPR levels in serum of patients after of HPR·Hb In HPR in NHS does not contain detectable bound Hb. HPR does not the function of Hp, which is to bind and remove free Hb from the circulation. is with no detectable peroxidase activity in HPR identified on gels H. Biochem. 1990; Google Scholar). C. A. B. R. R. and for the present experiments that the concentration of HPR in NHS is about than that of Hp, with the previously from quantitation (3Bensi G. Raugei G. Klefenz H. Cortese R. EMBO J. 1985; 4: 119-126Crossref PubMed Scopus (115) Google Scholar). The sequence of HPR is more than 90% with that of Hp, and the Hb region of Hp is The of HPR to bind Hb be due to a different of that in structural both Hp and HPR contain the total of the at of the Hp α chain that the the is by in HPR (4Maeda N. J. Biol. Chem. 1985; 260: 6698-6709Abstract Full Text PDF PubMed Google Scholar). is with observation that HPR as an α-β HPR are minor on the with It is also that the in HPR are from In NHS, total serum HPR appears to be between two termed TLF1 and TLF2, both of which trypanosome lytic of trypanosomes to lytic human and There is that HPR represents the toxic component of (6Tomlinson S. Muranjan M. Nussenzweig V. Raper J. Mol. Biochem. Parasitol. 1997; 86: 117-120PubMed Google A.B. Esko J.D. Hajduk S.L. Science. 1995; 268: 284-286Crossref PubMed Scopus (146) Google Scholar). The of HPR in NHS (7Smith A.B. Esko J.D. Hajduk S.L. Science. 1995; 268: 284-286Crossref PubMed Scopus (146) Google be in its to TLF1 HDL and to protein complex that contains the (6Tomlinson S. Muranjan M. Nussenzweig V. Raper J. Mol. Biochem. Parasitol. 1997; 86: 117-120PubMed Google Scholar). However, the data does not the that is due to peroxidase activity of bound Hb (see the (7Smith A.B. Esko J.D. Hajduk S.L. Science. 1995; 268: 284-286Crossref PubMed Scopus (146) Google Hb in TLF1 preparation (as we in the TLF1 preparations used in this the data indicate that Hb is a and not a component of does HPR trypanosome The measurement of peroxidase activity in TLF1 been due to Hb, but the of parasite lysis by that TLF1 causes parasite lysis via a peroxide-dependent mechanism (7Smith A.B. Esko J.D. Hajduk S.L. Science. 1995; 268: 284-286Crossref PubMed Scopus (146) Google Scholar). In to Hb, and in of and proteins and cause damage Hp is known to bind J. J. G. Nature. PubMed Scopus Google and (2Bowman B.H. Kurosky A. Harris H. Hirschhorn K. Advances in Human Genetics. Plenum Publishing Corp., New York1982: 189-261Crossref Scopus (305) Google Scholar), and is that HPR bind or more of resulting in a parasite peroxidase An and is that the HPR in a peroxidase of HPR, in of a a of its role in trypanosome be useful in delivery for African trypanosomes as trypanolytic proteins HPR and parasite Other than its role in there is no known function of HPR. levels of HPR been in sera from patients with various and HPR levels been to with tumor progression (11Oh S.K. Kim S.H. Abbruzzi G. Adler W.H. Tauber A.I. Hybridoma. 1992; 11: 1-12Crossref PubMed Scopus (4) Google Scholar, 12Kuhajda F.P. Katumuluwa A.J. Pasternack G.R. Proc. Natl. Acad. Sci. U. S. A. 1986; 86: 1188-1192Crossref Scopus (62) Google Scholar, 13Grundner-Culemann E. Bokemeyer C. Neubauer V. Shalitin C. Schmoll H.J. Poliwoda H. Bentner T. J. Cancer Res. Clin. Oncol. 1993; 119: 685-688Crossref PubMed Scopus (3) Google Scholar, 14Shalitin C. Epelbaum R. Valansi C. Segal R. Mekori T. Lover B. Robinson E. Int. J. Cancer. 1991; 49: 861-866Crossref PubMed Scopus (7) Google Scholar, F.P. S. Pasternack G.R. N. J. Med. PubMed Scopus Google However, the analysis of HPR revealed that contains an not found in HPR present in NHS S. A. Valansi C. Shalitin C. Biol. 1996; PubMed Scopus Google Scholar). appears to be due to an of the HPR resulting in of intron 1 and translation from the in intron 1 with the of The between the two of HPR is not We Raper and for Dr. Shalitin for of the and and Dr. for of the