From the William B. Castle Hematology Research Laboratory and the Departments ..... Tsai R, Yu CA, Gunsalus I, Peisach J, Blumberg W, Orme-. Johnson WH ...
Structural Characterization of the Isoenzymatic Forms of Human Myeloperoxidase: Evaluation of the Iron-Containing Prosthetic Group By J. Wright, N. Bastian, T.A. Davis, C. Zuo, S. Yoshimoto, W.H. Orme-Johnson, and A.I. Tauber Myeloperoxidase (MPO) from human neutrophils has been purified and found to exist in three isoenzymatic forms, resolved by ion exchange chromatography. In addition to differences in subunit size and cellular compartmentalization of the isoenzymes, differences have been reported in their activity and susceptibility to inhibition. The structural basis of these isoenzymes is unclear; we attempted to further define their functional characteristics and structural identity. First, we measured respective enzymatic activity using a panel of substrates: MPO I was found to have lower activity with some substrates (pyrogallol, guaiacol, potassium iodide [KI]), but similar activity to the other isoenzymes with Caminoantipyrine. These studies confirm
M
YELOPEROXIDASE (MPO) is a lysosomal enzyme found in the primary granule of the human neutrophi1 and is an important component of the oxygen-dependent antimicrobial activity of the cell.' Hydrogen peroxide derived from the dismutation of superoxide, in turn generated by the neutrophil NADPH-oxidase, is used by MPO to oxidize halide, creating potent oxidative species. These then may attack and degrade a variety of biologic compounds. Controversy regarding the structure of MPO has surrounded its investigation since its first description, but it is now generally agreed that it is a heme-containing glycoprotein existing as a single molecular species composed of two heavy and two light polypeptide subunit^.^-^ Recently, this model has been contested and the possibility of similar light subunits but dissimilar heavy subunits has been raised.' Three isoenzymatic forms of MPO exist as determined by their resolution on ion-exchange chromatography using a linear salt gradient.4 Functional differences exist between these isoenzymes including enzymatic activity, substrate preference, subcellular localization, and subunit size. However, the basis for these differences in structure and function has not been determined. We sought to characterize the iron-containing prosthetic group present in each isoenzyme to determine if differences in function could be attributed to a detectable structural variation in this site. From the William B. Castle Hematology Research Laboratory and the Departments of Medicine and Pathology, Boston City Hospital and Boston University School of Medicine: and the Department of Chemistry, Massachusetts Institute of Technology, Cambridge. Submitted February 6 , 1989; accepted August 25,1989. Supported by research grantsfrom the Whitaker Health Sciences Fund, Cambridge, M A and the National Institutes of Health P60 AR20613. A120064 and GM28358. Address reprint requests to Jonathan Wright. MD. William B. Castle Hematology Research Laboratory, Boston City Hospital, 818 Harrison Ave. Boston. M A 021 18. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C.section 1734 solely to indicate this fact. o I990 by The American Society of Hematology. 0006-4971/90/7501-0027$3.00/0 238
that MPO I is enzymatically distinct from MPO II and MPO 111. Next, we examined the structural basis of these differences by evaluating the iron-containing prosthetic group in each form using electron paramagnetic resonance (EPR) and determination of the pyridine hemochrome. No significant difference between the isoenzymes was noted in these parameters, suggesting that the prosthetic group is the same in each protein. The cause for any difference in enzymatic activity must lie then in variations extrinsic to the heme, and based on previous studies of the gene and protein analysis, the posttranslationalmodification of MPO must account for these isoenzymatic species. 0 1990 b y The American Society of Hematology.
MATERIALS AND METHODS
Materials. Phenylmethylsulfonyl fluoride (PMSF), 4-aminoantipyrine, guaiacol, hexadecyltrimethylammonium bromide (CETAB), and diisopropyl Auorophosphate (DFP) were obtained from Sigma (St Louis, MO); Sephacryl S-200, Percoll, and CMSepharose CL-6B chromatography gel columns were obtained from Pharmacia (Piscataway, NJ). Myeloperoxidase isolation. Azurophil granule-rich fractions from DFP-treated human neutrophils were prepared by nitrogen cavitation and Percoll gradient subfracti~nation,",~ The three isoenzymes of MPO were purified from these fractions as described previously.6 Granules were resuspended in 0.1 mol/L ammonium bicarbonate (pH 8.0) (buffer) and then solubilized in 3% CETAB at 22°C for 30 minutes. After centrifugation at 100,000 x g for 45 minutes, the myeloperoxidase-rich supernatant was applied to a CM-Sepharose CL-6B column (1.5 x 16 cm) equilibrated in 0.1 mol/L buffer. Elution from the column was done by a linear gradient (0.2 to 0.6 mol/L buffer, 300 mL total) that allowed a sharp resolution of the isoenzymatic forms! Column fractions from each of the three peaks in which the Reinheitszahl (RZ), determined by the ration of A,,,/A,8,, was greater than 0.45, were pooled, concentrated by ultrafiltration (MicroProDicon; Bio-Molecular Dynamics, Beaverton, OR) to 1.0 mL, and chromatographed on Sephacryl S-200 (1.0 x 65 cm) using 0.6 mol/L buffer to elute!.' Sephacryl S-200 column fractions in which the R Z value was greater than 0.75 were pooled and lyophilized. These resolved isoenzymes then underwent polyacrylamide gel electrophoresis before other studies were performed to ensure that the purified proteins were free of contaminank6 MPO activity was assayed using several substrates. 4-aminoantipyrine was used as the electron donor as previously described'; one unit of activity was defined as causing a change in absorbance of 1.0 U/min at 510 nm. Methods described previously4 were used for assays with guaiacol, pyrogallol, or potassium iodide. For guaiacol, one unit of activity formed 1 pmol tetraguaiacol per minute (extinction coefficient 26.6 mmol/L-' cm-'). For pyrogallol, one unit equaled the change of 1.0 absorbance U/min at 515 nm. For potassium iodide, one unit was defined as that amount of MPO oxidizing 1 pmol ididelmin. Electron paramagnetic resonance (EPR). EPR spectra were recorded on each of the three isoenzymatic proteins using a Varian Model E-9 spectrometer.' Samples were concentrated to approximately 1.5 mg/mL and 0.5-mL amounts were analyzed in 4 mm (internal diameter [i.d.]) quartz tubes. Temperature was controlled with a stream of helium boil-off gas.' Heterogeneity of the spectra was observed on occasion; addition of sodium chloride (0.1 mol/L) at pH 8.0 allowed conversion of low-spin ferric heme species to a Blood, Vol 75, No 1 (January 1 ), 1990: pp 238-24 1
239
STRUCTURAL CHARACTERIZATION OF HUMAN MPO
high-spin form with a sharply defined lineshape, for comparison of the isoenzyme forms."," Pyridine hemochrome. The pyridine hemochrome spectrum of each isoenzyme was determined as previously described." Briefly, 100 pg of protein was dissolved in 1 mL of 50 mmol/L NH,HCO, pH 8.0; 1 mL IN NaOH was added to the solution followed by 1.2 mL of redistilled pyridine. A small amount of sodium dithionite was added as a reducing agent, and absorbance spectroscopy was performed immediately and at 10 minutes using a Perkin Elmer UV/VIS 590A spectrophotometer.
4
g = 6.88
RESULTS AND DISCUSSION
Isoenzyme activity. The isoenzymatic character of MPO is still controversial. Felberg and Schultz13 reported that MPO consisted of six isoenzymatic forms; Bakkenist et ai' reported the presence of three, based on bands present on polyacrylamide gel electrophoresis of solubilized MPO. Pember et all4 first described the reproducible resolution of three forms by ion exchange chromatography and characterized these in both mouse and human preparations. These isoenzymes were found to vary in substrate preference, susceptibility to inhibition by aminotriazole, and cellular compartment a l i ~ a t i o n .Further '~ investigations by Wright et aI6 disclosed no difference in N-terminal sequence or amino acid analysis between the isoenzymes. MPO is a glycoprotein, and carbohydrate has been demonstrated to make up from 2.5%to 4% of the weight of the holoenzyme.' While initial studied did not disclose differences between the isoenzymes after treatment with neuraminidase or endoglycosidases, a recent report" strongly suggests a variation based on carbohydrate content as determined in degraded protein. This difference in glycosylation may account for the ready separation on ion exchange chromatography. Reports on the kinetics of the three forms have differed in their conclusions,d.6,'6perhaps because of the use of a variety of substrates; in some reports enzymatic activity was similar in all three,6.I6 while others supported a difference between the various forms.4 In our investigations, we studied enzymatic activity with different assays (Table 1 ) and again found no detectable difference in activity with 4-aminoantipyrine as the electron donor. However, with other substrates we confirm the results of Pember et a14showing a difference of activity exhibited by the isoenzymes with pyrogallol, guaiacol, and potassium iodide. By these assays, MPO 1appears to be distinct in its activity from MPO I1 and 111. Prosthetic group. Peroxidase enzymes generally contain protoheme IX; however, uncertainty still exists regarding the prosthetic group of MPO. Efforts to characterize the group have been impeded, initially due to chemical instability on cleavage from the pr~tein.".'~Structural identification has Table 1. Enzymatic Activity of Myeloperoxidase Isoenzymes lsmnzvmes 4-Aminoantipvrine ..
MPOl MPOII MPOIII
KI
Pwoaallol . .
Guaiacol
138.6 10.2 102.5 f 7.5 9.4 & 3.9 16.2 i: 0.5 149.6 f 16.2 203.3 f 14.3 48.1 f 16.9 24.0 i: 1.8 141.6 f 6.3 205.0 f 8.2 37.4 ? 8.6 23.0 3.5
*
In all experiments n 2 3; results are recorded as mean f SEM. Activity is expressed as U/mg protein; units for each substrate are defined in Materials and Methods.
g = 6.90
\I
Fig 1. EPR spectra for MPO resolved into its three isoenzymatic forms. Proteins were dissolved in 0.1 mol/L NH,HCO,, pH 8.0, to which 0.1 mol/L NaCl was added.
necessarily been inferred from absorbance spectros~opy,'~ resonance Raman,20.21 and magnetic circular dichroism studies.22 Although initial reports postulated the prosthetic group as derivative of an ir~n-formyl-porphyrin,'~,~~ recent studies support the presence of a saturated pyrrole, consistent with an iron chlorin chromophore.2@22Uncertainty also existed regarding the equivalence of the two iron chromophores, bound to each of the heavier subunits of MP023; however, data now support equivalence. The enzyme has been resolved into identical dimeric forms,24suggesting there is no difference in heme-subunit interactions; further, resonance Raman spectroscopy provide no convincing support for inequivalence,20.2'but the spectra seen with this modality are more complex than usually observed for heme proteins and require cautious interpretation. EPR studies provide more compelling evidence for equivalence,".2z as resting and C1substituted forms of the enzyme show no obvious evidence of magnetic interaction between different iron centers. To ascertain if the differences in enzymatic activity were due to differences in the prosthetic group not disclosed in studies of whole MPO, we examined each isoenzyme using two methods: EPR and pyridine hemochrome determination. The EPR spectra in Fig 1 show the characteristic high-spin species in each isoenzymatic form, similar to that previously reported with MPO in a pooled p r e p a r a t i ~ n . " , ~The ~ , ~ ~g values differ slightly from some other reports"*2Z.23;in some cases this is clearly due to differences in pH of the buffer.".22 In others, the buffer solutes themselves may have affected the
240
WRIGHT ET AL
spectra or contaminating heterogeneous species may have been presentz3 In comparing the isoenzymes (Fig l ) , no unexpected peak was found in any of the specimens, and no significant g value difference between isoenzymes was noted. Overall, the similarity of the active site environment, according to this criterion of EPR, is quite evident. Blumberg and Peisach26discussed the range of EPR g values in known heme proteins. Clearly the isoenzymes of MPO all fall within the larger class of “peroxidase” spectra; ie, the axial ligands are unlikely to be different from those, for example, in horseradish peroxidase.26 In addition to the similarity noted in EPR spectra, the pyridine hemochrome of each isoenzyme (Fig 2) was identical as no shift was noted at all in the three detectable bands; these spectra were consistent with that previously reported for unresolved MPO.I9 Both of these modalities of investigation indicate that the prosthetic group does not differ between isoenzymes and thus cannot account for the reported differences in activity. We note that differences in the peptide sequence and/or differences in glycosylation could be responsible for the different reactivities of the isoenzymes, without affecting the immediate heme environment as reported by EPR on hemochromogen spectra. With the cloning of the myeloperoxidase gene, another level of investigation is now possible to determine the basis for the isoenzymes. In a recent report,27 three clones of cDNA coding for MPO were isolated from an HL-60 cell cDNA library; further analysis showed that these three
436 436
436
I
I
1\, 550
MPO I Fig 2.
MPO II
MPO Ill
Pyridine hemochrome spectra for MPO isoenzymesfrom
400 to 600 nm immediately after addition of dithionite.
clones came from messenger RNAs (mRNAs) derived by alternative splicing of a transcript from a single gene. At this point it is not clear that these multiple forms of mRNAs account for the multiple forms of MPO, but it may explain a posttranslational modification in glycosylation recently reported in human neutrophil MPO isoenzyme^.'^ ACKNOWLEDGMENT
We thank Dorothy Nuttall for her expert secretarial assistance.
REFERENCES
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and EPR properties of myeloperoxidase. J Biol Chem 260:8301, 1985 12. Furhop J-H, Smith KM: Laboratory methods, in Smith KM (ed): Porphyrins and Metalloporphyrins. Amsterdam, The Netherlands, Elsevier, 1976, p 757 13. Felberg NT, Schultz J: Evidence that myeloperoxidase is composed of isoenzymes. Arch Biochem Biophys 148:407, 1972 14. Pember SO, Fuhrer-Krusi SM, Barnes KC, Kinkade JM Jr: Isolation of three native forms of myeloperoxidase from human polymorphonuclear leukocytes. FEBS Lett 140:103, 1982 15. Selsted ME, Novotny MJ: The isoenzyme forms of human myeloperoxidase result from differential N-glycosylation. Blood 72:152a, 1988 (abstr, suppl 1) 16. Suzuki K, Yamada M, Akashi K, Fugikura T: Similarity of kinetics of three types of myeloperoxidase from human leukocytes and four types from HL-60 cells. Arch Biochem Biophys 245:167, 1986 17. Wu NC, Schultz J: The prosthetic group of myeloperoxidase. FEBS Lett 60:141, 1975 18. Nichol AW, Morel1 DB, Thomson J: Porphyrins derived from the prosthetic group of myeloperoxidase. Biochem Biophys Res Comm 36576,1969 19. Schultz J, Shmukler HW: Myeloperoxidase of the leucocyte of normal human blood. 11. Isolation, spectrophotometry and amino acid analysis. Biochemistry 3:1234, 1964 20. Sibbett SS, Hurst J K Structural analysis of myeloperoxidase by resonance Raman spectroscopy. Biochemistry 23:3007,1984 21. Babcock GT, Ingle RT, Oertling WA, Davis JG, Averill BA, Hulse CL, Stufkens DJ, Bolscher BGJM, Wever R: Raman characterization of human leukocyte myeloperoxidase and bovine spleen green haemoprotein. Insight into chromophore structure and evidence that the chromophores of myeloperoxidase are equivalent. Biochem Biophys Acta 828:58, 1985
STRUCTURAL CHARACTERIZATION OF HUMAN MPO
22. Eglinton DG, Barber D, Thomson AJ, Greenwood C, Segal AW: Studies of cyanide binding to myeloperoxidase by electron paramagnetic resonance and magnetic circular dichroism spectroscopics. Biochim Biophys Acta 703:187, 1982 23. Odajima T: Myeloperoxidase of the leukocyte of normal blood. J Biochem (Tokyo) 87:379, 1980 24. Andrews PC, Parnes C, Krinsky NJ: Comparison of myeloperoxidase and human myeloperoxidase with respect to catalysis, regulation and bactericidal activity. Arch Biochem Biophys 228:439, 1984
24 1
25. Wever R, Roos D, Weening RS, Vulsma T, Van Gelder B F An EPR study of myeloperoxidase in human granulocytes. Biochim Biophys Acta 421:328, 1976 26. Blumberg WE, Peisach J: Bioinorganic chemistry. Adv in ChemSer 100:271, 1971 27. Hashinaka K, Nishio C, Hur S-J, Sakiyama F, Tsunasawa S, Yamada M: Multiple species of myeloperoxidase messenger RNAs produced by alternative splicing and differential polyadenylation. Biochemistry 275906, 1988
