The type 5, acid phosphatase from spleen of humans with hairy cell leukemia. Purification, properties, immunological characterization, and comparison with porcine uteroferrin

The spleens of patients with hairy cell leukemia contain high levels of a tartrate-insensitive, cationic, acid phosphatase (the human Type 5 isozyme). This phosphatase has been purified by a procedure which involves only two chromatographic steps: CM-cellulose chromatography and immunoaffinity chromatography on sheep antibodies generated against porcine uteroferrin. Uteroferrin is an abundant iron-containing acid phosphatase that can be recovered readily from porcine uterine secretions. Like uteroferrin, the purified human Type 5 phosphatase is a glycoprotein of molecular weight about 34,000. It contains two atoms of iron/molecule. The human phosphatase and uteroferrin also resemble each other closely in electrophoretic mobility, substrate specificity, and response to a variety of activators and inhibitors. Mouse monoclonal antibodies have been raised to uteroferrin and to the human Type 5 phosphatase. Three monoclonal antibodies which bind with high affinities to distinct sites on the uteroferrin molecule also recognize the human spleen enzyme, but bind to it with much lower affinity. These antibodies also recognize cationic acid phosphatases purified from bovine and rat spleens. A monoclonal antibody raised against the human enzyme, but selected for binding to uteroferrin, appears to recognize a relatively conserved site on all four phosphatases. We conclude that the human Type 5 isozyme belongs to a growing class of structurally related, iron-containing acid phosphatases which includes the iron-transport protein, uteroferrin.     

Possible function of carbohydrate on glycoproteins secreted by the pig uterus during pregnancy

Uteroferrin is a purple iron-containing acid phosphatase secreted by the porcine uterus under the influence of the hormone, progesterone. It is synthesized by the glandular epithelial cells of the uterine endometrium and during pregnancy is taken up by specialized structures (areolae) opposite each uterine gland. Uteroferrin is then released into the fetal circulation and cleared by the liver or fetal kidney. A major role in iron transport to the fetus has been proposed. Uteroferrin, as purified from uterine secretions of pigs, possesses mainly high mannose (predominately Man₅ and Man₆ chains. These oligosaccharide chains of uteroferrin appear to be responsible for its binding and uptake by reticuloendothelial cells of the fetal liver which is the major site of erythropoiesis of the fetus. Uteroferrin, although implicated in transplantal iron transport, also possesses many of the properties of a lysosomal enzyme and, when newly synthesized, carries the so-called lysosomal recognition marker, mannose 6-phosphate. The phosphate group is masked by a covering N-acetylglucosamine residue, a feature which may account for its secretion rather than retention within lysosomes. Evidence is also presented that the oligosaccharide chains of newly synthesized uteroferrin are larger than those of the mature form and are trimmed after secretion. The phosphate group is also removed. It is not clear whether uteroferrin carbohydrate is implicated in the movement of the glycoprotein across the placenta as well as its uptake by the fetal liver.     

The carbohydrate structure of porcine uteroferrin and the role of the high mannose chains in promoting uptake by the reticuloendothelial cells of the fetal liver

Uteroferrin, the iron-containing, progesterone-induced phosphatase of the porcine uterus, is a glycoprotein carrying a single oligosaccharide chain. Most of the uteroferrin isolated from either uterine secretions or allantoic fluid has endoglycosidase H-sensitive carbohydrate chains with either five or six mannose residues. As determined by 1H-NMR spectroscopy, the Man6 oligosaccharide has the following structure. (Formula: see text) The Man5 species lacks the terminal alpha 1,2-linked residue. Uteroferrin is transported across the pig placenta and has been proposed to be involved in iron transfer to the fetus (see Buhi, W. C., Ducsay, C. A., Bazer, F. W., and Roberts, R. M. (1982) J. Biol. Chem. 257, 1712-1721). Injection of 125I-labeled uteroferrin into the umbilical vein of midpregnant fetuses resulted in incorporation of label into the liver, the major site of fetal erythropoiesis. Light and electron microscope autoradiography revealed that the primary sites of uteroferrin uptake were the reticuloendothelial cells lining the liver sinusoids. Reticuloendothelial cells isolated from either fetal pig or adult rat livers were shown to accumulate uteroferrin when cultured in vitro. Uptake was inhibited by yeast mannan and by glycopeptides isolated from either ovalbumin or uteroferrin. Rat cells did not accumulate uteroferrin whose high mannose chains had been removed using endoglycosidase H. Moreover, the K uptake values (3 X 10(-7) M), specific competition by D-mannose and L-fucose bovine serum albumin, and inhibition by EDTA are consistent with an uptake mechanism involving a receptor for high-mannose oligosaccharides on the liver sinusoidal cells. It is suggested that one function of this receptor in the fetal pig is to remove maternally derived uterine glycoproteins from the fetal circulation. In the case of uteroferrin this process provides iron to the fetal liver.     

Role of uteroferrin in transplacental iron transport in the pig

The pig possesses a noninvasive, diffuse type of epitheliochorial placentation in which the blood supply of the mother is well separated from the absorptive surface of the chorion, a feature that must complicate the movement of nutrient molecules across the placenta. Evidence is presented that a protein synthesized and secreted by the glandular epithelial cells of the maternal uterus of the pig is involved in iron transport to the fetus. This protein, uteroferrin, is induced by progesterone; is purple, which results from an unusual iron center; and possesses acid phosphatase activity. Secreted uteroferrin is taken up by specialized chorionic epithelial cells located in domed structures, called areolae, overlying the mouth of each uterine gland. Uteroferrin then enters the placental venous drainage and its iron is efficiently incorporated into fetal hemoglobin. It is taken up by the fetal liver or cleared by the kidney. The liver is the main site of erythropoiesis in the fetus. From the kidney uteroferrin enters the allantoic sac where it exchanges its iron with fetal transferrin. The rate of uteroferrin biosynthesis in the uterus and its rate of metabolism in the fetus can theoretically provide sufficient iron for the needs of pregnancy, at least until around day 70 of the 115-day gestation. Uteroferrin and transferrin, the iron transport protein of plasma, appear to be unrelated proteins.     

Resonance Raman scattering from uteroferrin, the purple glycoprotein of the porcine uterus.

Uteroferrin, a purple-colored, iron-containing glycoprotein, purified from the uterine fluid of progesterone-stimulated pigs, owes its natural purple color to a broad absorption band centered at 545 nm. Laser excitation within the visible absorption band of uteroferrin results in an intense resonance Raman spectrum which bears a striking resemblance to that reported for Fe(III)-transferrin, the iron transport protein of serum. Excitation profiles for the four resonance-enhanced bands of uteroferrin were obtained from 4579 A to 6741 A, using lines from Ar+ and Kr+ lasers. Each of the profiles have maxima near 545 nm. The spectral similarities of uteroferrin and Fe(III)-transferrin lead to the belief that the Fe(III) binding sites of the two proteins must be, at least in some respects, quite similar. In particular, it is concluded that, as in Fe(III)-transferrin, the metal binding site of uteroferrin contains a tyrosine ligand and that the visible absorption spectrum of uteroferrin results from a phenolate to Fe(III) charge transfer.     

Type 5 acid phosphatase. Sequence, expression and chromosomal localization of a differentiation-associated protein of the human macrophage

The purple acid phosphatases and uteroferrin belong to a diverse multifunctional class of binuclear iron-containing proteins that includes haemerythrin and ribonucleotide reductase. In the pig, uteroferrin has been implicated in the delivery of iron to the foetus, but the role of the related human type 5 acid phosphatase that is principally found in resident tissue macrophages is not yet clear. To define further the function of this metalloenzyme, we have isolated and sequenced a cDNA clone for type 5 acid phosphatase and investigated expression of its gene in human tissues. The phosphatase clone contains an open reading frame of 975 bp and encodes a protein of 325 amino acids, including a signal peptide of 19 residues and two potential sites for N-glycosylation. The type 5 acid phosphatase gene mapped to the short arm of human chromosome 19 and was found to have a restriction fragment length polymorphism on digestion with XbaI. Expression of phosphatase mRNA was restricted to mononuclear phagocytes and the enzyme was induced greater than 20-fold on transformation of normal human monocytes to macrophages by culture in serum-supplemented medium. Type 5 acid phosphatase thus represents a tightly regulated system for the study of molecular events in the differentiation programme of the normal macrophage.     

Porcine purple acid phosphatase: heterologous expression, characterization, and proteolytic analysis

Uteroferrin is an iron-binding glycoprotein, which is abundantly synthesized in porcine uterine glandular endometrium and believed to be involved in maternal/fetal iron transport. In the present study, uteroferrin has been cloned and functionally expressed using baculovirus-infected insect host cells Spodoptera frugiperda. The work also addresses the possible role of proteolytic cleavage to facilitate the release of uteroferrin-bound iron. The enzyme secreted in culture medium exhibits a molecular mass and catalytic properties similar to native porcine uteroferrin. The specific activity was estimated at 233 U/mg using p-nitrophenyl phosphate as substrate. Partial cleavage of the enzyme with trypsin resulted in a 1.7-fold enhancement in specific activity and a two-subunit polypeptide as observed in preparations of most mammalian purple acid phosphatases. Digestion with the aspartic protease pepsin resulted in a 2.5-fold enzyme inactivation correlated with the appearance of low molecular weight polypeptide fragments and the release of enzyme-bound iron.     

The functions of uterine secretions

The likely functions of uterine secretions, often termed histotroph, in the nurture of the early conceptus are reviewed. Particular emphasis has been placed on the pig in which the uterus synthesizes and secretes large amounts of protein in response to progesterone. In this species, which possesses a non-invasive, diffuse type of epitheliochorial placentation, the secretions provide a sustained embryotrophic environment which is distinct from that of serum. A group of basic proteins dominates these uterine secretions after Day 11 of pregnancy and its best characterized member is uteroferrin, an iron-containing acid phosphatase with a deep purple colour. Evidence has accumulated to suggest that uteroferrin, rather than functioning as an acid phosphatase, is involved in transporting iron to the conceptus. Three basic polypeptides which are found noncovalently associated with uteroferrin have been shown to be antigenically closely related to one another and to have arisen by post-translational processing from a common precursor molecule. Their function is unknown. A group of basic protease inhibitors has been identified which bear considerable sequence homology to bovine pancreatic trypsin inhibitor (aprotinin) and may control intrauterine proteolytic events initiated by the conceptuses. The last basic protein so far characterized is lysozyme which is presumed to have an antibacterial role. Finally, two low molecular weight (Mr approximately 18,000) acidic polypeptides have been purified and have sequence homology to a plasma retinol binding protein. Like uteroferrin, these proteins may be responsible for transport of an essential nutrient to the conceptus.     

Interaction of porcine uterine fluid purple acid phosphatase with vanadate and vanadyl cation.

Uteroferrin, the purple acid phosphatase from porcine uterine fluid, is noncompetitively inhibited by vanadate in a time-dependent manner under both aerobic and anaerobic conditions. This time-dependent inhibition is observed only with the diiron enzyme and is absent when the FeZn enzyme is used. The observations are attributed to the sequential formation of two uteroferrin-vanadium complexes. The first complex forms rapidly and reversibly, while the second complex forms slowly and results in the production of catalytically inactive oxidized uteroferrin and V(IV), which is observed by EPR. The redox reaction can be reversed by treatment of the oxidized enzyme first with (V(IV)) and then EDTA to generate a catalytically active uteroferrin. Multiple inhibition kinetics suggests that vanadate is mutually exclusive with molybdate, tungstate, and vanadyl cation. The binding site for each of these anions is distinct from the site to which the competitive inhibitors phosphate and arsenate bind. The time-dependent inhibition by vanadate of uteroferrin containing the diiron core represents a new type of mechanism by which vanadium can interact with proteins and gives additional insight into the binding of anions to uteroferrin.     

Linear electric field effect and electron spin-echo studies of uteroferrin. Evidence for iron coordination by a nitrogen-containing ligand

Uteroferrin and semimethemerythrin, proteins possessing spin-coupled binuclear iron centers, exhibit large linear electric field effects in their mixed-valence, EPR-active states. This indicates that the paramagnetic center of each protein is noncentrosymmetric and suggests that charge may be localized on one of the iron atoms. The magnetic field dependence of the linear electric field effects for both proteins demonstrates that the direction of most facile polarization of the binuclear iron centers is near the orientation giving rise to gmin. Electron spin-echo studies of uteroferrin reveal that its magnetic electron interacts with at least one and possibly two classes of nitrogen nuclei. Furthermore, comparison of echo envelope spectra for uteroferrin with that of ferric bleomycin suggests that one of these nuclei is from a histidine ligand.     

Inhibition of osteoclastic acid phosphatase abolishes bone resorption

Osteoclastic acid phosphatase is a member of a widely-distributed class of iron-containing proteins with acid phosphatase activity. Antibodies raised against one member of this class cross-react with other members from the same or different species, but not with acid phosphatase isoenzymes of different types. When antibodies to one such protein, porcine uteroferrin, are added to medium in which rat osteoclasts are incubated on devitalised cortical bone, both bone resorption and acid phosphatase activity are markedly inhibited. Furthermore, addition of molybdate (an inhibitor of this class of acid phosphatases) also inhibits both bone resorption and enzyme activity. These observations strongly suggest a functional role for osteoclastic acid phosphatase in bone resorption.     

The binding of molybdate to uteroferrin. Hyperfine interactions of the binuclear center with 95Mo, 1H, and 2H

Uteroferrin, an acid phosphatase with a spin-coupled and redox-active binuclear iron center, is paramagnetic in its pink, enzymatically active, mixed-valence (S = 1/2) state. Phosphate, a product and inhibitor of the enzymatic activity of uteroferrin, converts the pink, EPR-active form of the protein to a purple, EPR-silent species. In contrast, molybdate, a tetrahedral oxyanion analog of phosphate, transforms the EPR spectrum of uteroferrin from a rhombic to an axial form. With both electron spin echo envelope modulation (ESEEM) and electron nuclear double resonance (ENDOR) spectroscopies, we observe a hyperfine interaction of [95Mo]molybdate with the S = 1/2, Fe(II)-Fe(III) center of the protein. A pair of 95Mo resonances centered at the 95Mo Larmor frequency at the applied magnetic field and separated by a hyperfine coupling constant of 1.2 MHz is evident. Therefore, a single monomeric species of molybdate is close to, and likely a ligand of, the binuclear cluster. 1H ENDOR studies on uteroferrin reveal at least six sets of lines mirrored about the 1H Larmor frequency. Two pairs of these lines become reduced in intensity when the protein is exchanged against D2O. Moreover, ESEEM and 2H ENDOR spectra display resonances at the 2H Larmor frequency. Therefore, the metal-binding region of the protein is accessible to solvent. Additional deuterium lines observable by ESEEM spectroscopy provide evidence for a population of strongly coupled, readily exchangeable protons associated with the binuclear center. The measured hyperfine coupling constants for these deuterons are orientation-dependent with splittings of nearly 4 MHz at g3 = 1.59 and less than 1 MHz at g1 = 1.94. In the presence of molybdate, ESEEM spectra of D2O-exchanged samples reveal a resonance at the 2H Larmor frequency, with no evidence of spectral components due to strongly coupled deuterons. 1H ENDOR studies of the uteroferrin-molybdate complex show at least seven pairs of lines, mirrored about the 1H Larmor frequency, of which one pair becomes attenuated in amplitude upon deuteration. The active site thus remains accessible to solvent in the presence of molybdate.     

Transfer of iron from uteroferrin (purple acid phosphatase) to transferrin related to acid phosphatase activity

There is continuing controversy as to whether iron can be exchanged from the purple phosphatase, uteroferrin (Uf), to fetal transferrin (Tf) and whether this process might be of physiological relevance during pregnancy in the pig. Here, iron transfer from Uf to apoTf at pH 7.1 was followed by measuring the loss of acid phosphatase activity from native Uf as a function of incubation conditions and time. In the presence of apoTf and 1 mM ascorbate (but not in the presence of either agent alone), 50% of enzyme activity was lost in about 12 h. Loss of activity was accompanied by bleaching of Uf purple color and the appearance of the characteristic visual absorption spectrum of Fe-Tf. Citrate could replace ascorbate in the reaction. Loss of Uf iron did not occur at pH 5.3, at which pH Tf cannot bind Fe. [59Fe]Uf was prepared and shown to be identical in its enzymatic and physical properties with unmodified Uf. Transfer of 59Fe from Uf to apo-Tf was promoted by conditions identical to those which led to loss of purple color and acid phosphatase activity. However, the results suggested that only one of the two iron atoms at the bi-iron center on Uf was readily lost, and that exchange of the second iron occurred more slowly. Loss of iron made Uf more susceptible to denaturation. A third technique, quantitation of the g' = 4.3 signal of iron specifically bound to Tf by EPR, was also tested as a means assaying accumulation of Fe-Tf, but the method was too insensitive to measure the kinetics of iron transfer at physiological protein concentrations. We conclude that iron can be transferred directly from Uf to apoTf in the presence of low molecular weight chelators, and that the process is likely to be of physiological significance.     

Purification and characterization of purple acid phosphatase from developing rat bone

Tartrate-resistant acid phosphatase active on nucleoside di- and triphosphate substrates was isolated from developing rat bone and purified 2500-fold. The enzyme concentration had a purple coloration and activity that was sensitive to reducing agents. Mild reducing agents such as ferrous ion and ascorbic acid caused loss of purple color and increased activity toward substrates severalfold; however, a strong reductant such as dithionite caused loss of both color and activity which were partially restored by addition of ferrous ion and ascorbic acid. Enzyme activity was homogeneous with protein during the final gel permeation steps of chromatography and gave an apparent molecular size of about 40,000 Da. Determination of iron in the most pure preparation revealed the presence of 1.3 atoms of iron per molecule of the tartrate-resistant enzyme E2. Other properties of the purified enzyme include a pI of approximately 9.5 and sensitivity to inhibition by ions of copper, zinc, fluoride, and molybdate. Antibody prepared to the pre-concanavalin A (Con A)-Sepharose purified enzyme reacted with all protein from the Con A step, but it did not react with tartrate-sensitive acid phosphatase from rat bone or with potato acid phosphatase. Purple acid phosphatase from rat bone has many properties that parallel the iron-containing purple acid phosphatases from rat spleen, bovine spleen, and pig uterine secretions.     

Phosphorylation of the oligosaccharide of uteroferrin by UDP-GlcNAc:glycoprotein N-acetylglucosamine-1-phosphotransferases from rat liver, Acanthamoeba castellani, and Dictyostelium discoideum requires alpha 1,2-linked mannose residues

We have investigated the oligosaccharide requirements of the UDP-GlcNAc:glycoprotein N-acetylglucosamine-1-phosphotransferases from rat liver, Acanthamoeba castellani, and Dictyostelium discoideum. Uteroferrin, an acid hydrolase, was phosphorylated by the three N-acetylglucosaminylphosphotransferases, and the phosphorylated oligosaccharides were isolated and analyzed by ion suppression high performance liquid chromatography. In all three cases, the phosphorylated species contained 6 or more mannose residues. Phosphorylation of the Man5GlcNAc2 oligosaccharide could not be detected even though this was the major species on the native uteroferrin. The Man5GlcNAc2 oligosaccharides lack alpha 1,2-linked mannose residues, whereas the larger oligosaccharides contain 1 or more mannose residues in this linkage. Treatment of intact uteroferrin with an alpha 1,2-specific mannosidase-generated molecules whose oligosaccharides consisted almost entirely of species with 5 mannose residues. The N-acetylglucosaminylphosphotransferases could no longer phosphorylate such molecules. These data indicate that at least 1 alpha 1,2-linked mannose residue must be present on uteroferrin's oligosaccharide for phosphorylation to occur.     

The "manganese(III)-containing" purple acid phosphatase from sweet potatoes is an iron enzyme

An improved purification of the purple acid phosphatase from sweet potatoes has been developed, and the properties of the enzyme have been reexamined. Contrary to previous reports, (e.g., Y. Sugiura, et al., J. Biol. Chem., 256, 10664-10670 (1981) ), the enzyme contains two moles of iron and insignificant amounts of manganese. The specific activity of the iron-containing preparations is ca. 14 times higher than that reported previously for the purported "Mn(III)" enzyme. The sweet potato purple acid phosphatase does indeed bind manganese, but it can be removed by dialysis with no changes in specific activity or spectral properties.     

Immunolocalisation of the uterine secretory proteins uterocalin, uteroferrin and uteroglobin in the mare's uterus and placenta throughout pregnancy

Previous studies have shown that the equine uterus produces many progesterone-dependent proteins throughout gestation. In particular, uterocalin and uteroferrin are detectable using electrophoresis or blot analyses but information regarding the immunohistochemical placental distribution of these two proteins is rare and information regarding uteroglobin is still lacking. The aim of the present study was to co-immunolocalise these three secretory proteins in the mare's uterus throughout gestation in an effort to understand their functional role in the maintenance of pregnancy. Therefore, endometrial biopsy samples were obtained from 20 pregnant mares between 16 and 309 days of gestation and labelled immunohistochemically for uteroglobin, uteroferrin and uterocalin. Uteroferrin remained detectable in almost every endometrial gland at all stages but with an increase in staining intensity as gestation advanced. The most progesterone-dependent protein, uterocalin, showed variable staining throughout gestation with the most intense labelling in early pregnancy and during the period of endometrial cup reaction. Uteroglobin secretion was only detectable in traces and only in individual glands throughout gestation. The results indicate that uterocalin and uteroferrin, but not uteroglobin, may play important roles in supplying nutrients for the conceptus, thereby contributing to the maintenance of pregnancy. However, further investigations are necessary to understand the role of uteroglobin during gestation.     

Absence of iron transfer from uteroferrin to transferrin

Transfer of iron from native porcine uteroferrin to apotransferrin was investigated using EPR spectroscopy. Purple (oxidized) or pink (reduced) forms of uteroferrin were incubated with porcine or human apotransferrin under conditions of temperature (37 degrees C) and pH (6.8) approximating those found in the allantoic fluid of the pregnant sow. Studies were also performed in the presence of mediators such as ascorbate, citrate, and ATP in concentrations previously claimed to be effective in promoting large-scale transfer of iron (Buhi, W. C., Ducsay, C. A., Bazer, F. W., and Roberts, R. M. (1982) J. Biol. Chem. 257, 1712-1723). Our experiments indicate that even in the presence of mediators, less than 20% of the iron in uteroferrin is transferred to apotransferrin at the end of 24 h and such transfer may be accompanied by denaturation of uteroferrin. We therefore conclude that the direct transfer of iron to apotransferrin is unlikely to be a physiological role of uteroferrin.