Common epitopes in human isoferritins characterized by murine monoclonal antibodies

Three monoclonal antibodies against human liver ferritin were selected to study antigenic determinants (epitopes) of human isoferritins. These monoclonal antibodies were found to form immunoprecipitin lines with ferritin in double diffusion tests (Ouchterlony), indicating multiple epitopes on a single ferritin molecule. The antibodies revealed high species specificity as well. Monoclonal antibodies MA301 and MA311 appeared to recognize different epitopes, since they did not inhibit each other in competitive enzyme-linked immunosorbent assay (ELISA). However, MA309 recognized both epitopes for MA301 and MA311 with similar competitive inhibition. These epitopes were not detectable when ferritin was treated with 8M urea (pH 2.5) and were detectable upon reconstruction by dialysis against 2 M urea (pH 7.2), suggesting that these monoclonals recognize epitopes in the tertiary structure of the ferritin molecule. As a matter of fact, these monoclonals react preferentially with intact ferritin molecule and only negligibly with subunits. Isoelectric focusing patterns of human ferritins demonstrated that liver, spleen, placenta, and hepatoma cells (Li-7) transplanted in nude mice contained basic isoferritins, whereas HeLa cells (carcinoma), Wa cells (EB virus-transformed B cells), and Raji cells (Burkitt's lymphoma) contained acidic isoferritins. Human heart ferritin displayed a somewhat intermediate pattern between liver and HeLa ferritins. In spite of the heterogeneous population of human isoferritins, the dissociation constants (Kd) of the three monoclonal antibodies to liver, HeLa, and heart isoferritins were quite similar.     

Properties of human tissue isoferritins.

1. Human liver ferritin was separated by preparative isoelectric focusing into six fractions. 2. Except for the least acidic fraction the reactivity with antibody against spleen ferritin increased with rising pI, but with antibody against heart ferritin the reactivity decreased. 3. The highest iron content was found in the most acidic isoferritins and progressively decreased with rising pI. 4. Iron uptake was studied in apoferritin prepared from heart and liver ferritin fractions separated by ion-exchange chromatography. There was good correlation between the rate of iron uptake and pI. The most acidic fractions took up iron more rapidly than did the more basic ones. 5. Ferritin was prepared from heart, liver, spleen and kidney. There was little difference on isoelectric focusing between ferritin obtained from normal tissues and the corresponding iron-loaded tissues from patients who had received multiple blood transfusions. The iron-loaked heart ferritin invariably contained relatively more of the basic isoferritins. Normal and iron-overloaded heart ferritins were separated into isoferritin fractions by ion-exchange chromatography, and in each case there was a fall in iron content as the pI increased. The iron content of ferritin from the iron-overloaded heart was higher throughout than that from normal heart. 6. There is a relationship between the rate of iron uptake by apoferritin and pI, and this probably accounts for the variation in iron content of the isoferritins found in human liver and heart.     

Possibly unique subunit(s) of human ferritins responsible for tissue specificity

The biochemical characteristics of human placental and hepatic ferritins were compared. By DEAE cellulose column chromatography, placental ferritin was found to contain more acidic isoferritins, while hepatic ferritin was richer in basic isoferritins. The electrofocusing patterns of subunits revealed that placental ferritin contained at least one unique subunit on the acidic side and might lack one subunit on the basic side as compared with hepatic ferritin. It is hypothesized that microheterogeneities, immunological as well as biochemical, existing between placental and hepatic ferritins cannot be explained merely in terms of different proportions of known acidic and basic subunits, but appear to have resulted from the existence of the unique acidic subunit of placental ferritin and possibly also a unique basic subunit of hepatic ferritin.     

Heterogeneity in tissue ferritins displayed by gel electrofocusing

1. Horse spleen ferritin and human liver ferritin were examined by gel electrofocusing under conditions that demonstrated equilibrium focusing. Both ferritins were resolved into multiple isoferritins. Both families of isoferritins were separable from one another. 2. Horse spleen ferritin was also resolved into five components by ion-exchange chromatography on DEAE-Sephadex A-50. Each of the major chromatographic fractions contained only a few of the isoferritins seen on gel electrofocusing. Each chromatographic fraction corresponded to different portions of the isoferritin profile. 3. These results indicate that the heterogeneity seen in many ferritins by gel electrofocusing represents structural heterogeneity in the ferritin population as isolated from the tissues.     

A biochemical comparison of normal human liver and hepatocellular carcinoma ferritins.

1. The iron contents, gel migration rates and isoelectric-focusing patterns of normal liver and hepatocellular carcinoma ferritins from the same patients were compared. 2. Sucrose-density-gradient centrifugation showed that the number of iron atoms per ferritin molecule was decreased to approximately half in carcinoma tissue when compared with normal liver. 3. On electrophoresis, hepatocellular carcinoma ferritin migrates faster and is therefore more negatively charged than normal liver ferritin, thus refuting the general view that the more negatively charged a ferritin molecule the greater its iron content. 4. Comparison of tumour and normal liver ferritin subunit compositions on acid/urea/polyacrylamide gels showed hepatocellular carcinoma ferritin to contain an additional, more negatively charged, subunit to normal liver ferritin. 5. Isoelectric focusing showed that hepatocellular carcinoma tissue contains isoferritins with isoelectric points intermediate between the ranges of normal liver and normal heart isoferritins.     

Variation in the distribution of two human heart ferritin species. Isoferritin profile and subunit composition in normal and iron-overloaded subjects.

On polyacrylamide slab electrophoresis two ferritin species, termed fast and slow, were present in three control and four iron-loaded human hearts. The ratio of the fast to slow species varied between hearts and correlated with the distribution of isoferritins, which had pI values ranging from 4.8 to 5.6. The acidic isoferritins were prominent in all hearts, but one control and the four iron-loaded hearts contained, in addition, basic isoferritins. All ferritins were composed of two subunits, an acidic H type and a more basic HL type, which was the main subunit in normal liver. Hearts with the highest proportions of acidic isoferritin contained the highest proportion of the H subunit.     

Isolation of ferritin from human mammary and pancreatic carcinomas by means of antibody immunoadsorbents

In the course of a study of tumor antigens we prepared an absorbed antiserum to a breast tumor that reacted strongly with breast tumor but not with normal breast. The antigen was purified by adsorption to an antibody immunoadsorbent prepared from this antiserum, elution with 2 m potassium thiocyanate at neutral pH, and passage through an immunoadsorbent containing antibodies to human serum. The purified antigen was identified as ferritin by electrophoretic, chemical, and immunological criteria. Isoelectric focusing in acrylamide gels revealed that tumor ferritin contained six bands seen in normal liver ferritin plus a variable number of acidic components not detected in normal liver ferritin. The acidic components were concentrated by chromatography on a DEAE-cellulose column. Similar acidic components described previously in ferritins isolated from cultured human tumor cells, hepatomas, and fetal liver have been designated as “carcino-fetal” ferritins.     

Mouse hepatoma and liver ferritins. Comparative structural studies.

Pure ferritin from male mouse liver produces a single band of monomers (RF = 0.199) with electrophoresis in polyacrylamide gels at pH 9.0. The five sub-bands within this monomeric band appear to represent charge isomers having the same molecular size. Ferritin from BH3 transplantable mouse hepatoma shows two overlapping bands of monomers (RFA = 0.208 and RFB = 0.240); further electrophoretic studies show that these bands represent two subpopulations of molecules differing both in charge and size. Sub-bands are not found in this hepatoma ferritin. The larger tumor ferritin reaches the same end migration position as all liver isoferritins on gradient gels, signifying a very similar or identical molecular size; however, the absence of sub-bands indicates that this hepatoma ferritin differs in charge from the homologous liver proteins. Liver and hepatoma ferritins both produce a single prominent subunit band corresponding to nominal molecular weights of 22 250 and 21 700, with polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and dithiothreitol. With electrophoresis on polyacrylamide gradient slabs containing sodium dodecyl sulfate and dithiothreitol, both liver and hepatoma ferritins now reveal two subunits bands situated at identical positions. The polypeptides of these two closely spaced bands have a nominal molecular weight difference of less than 1000. Neither the hepatoma nor the liver seems to produce the ferritins found in the other tissue. Nevertheless, all these ferritins are composed of the same two types of subunits, albeit in different relative amounts. Observed distinctions in the ferritins from these normal or neoplastic cells must reflect differences in assembly and processing, as well as in the regulated expression of the same ferritin genes.     

Characterization of the binding of ferritin to the rat liver ferritin receptor

The binding characteristics and specificity of the rat hepatic ferritin receptor were investigated using ferritins prepared from rat liver, heart, spleen, kidney and serum, human liver and serum, guinea pig liver and horse spleen as well as ferritins enriched with respect to either H- or L-type subunit composition, prepared by chromatofocusing of rat liver ferritin on Mono-P or by reverse-phase chromatography of ferritin subunits on ProRPC 5/10. No significant difference was apparent in the binding of any of the tissue ferritins, or of ferritins of predominantly acidic or basic subunit composition. However, serum ferritin bound with a lower affinity. The effect of carbohydrate on the ferritin-receptor binding was examined by glycosidase treatment of tissue and serum ferritins. Tissue ferritin binding was unaffected, while serum ferritin binding affinity was increased to that of the tissue ferritins. Inhibition of ferritin binding by lactoferrin was not due to common carbohydrate moieties as previously suggested but was due to direct binding of lactoferrin to ferritin. Therefore, carbohydrate residues do not appear to facilitate receptor-ferritin binding, and sialic acid residues present on serum ferritin may in fact interfere with binding. The results indicate that the hepatic ferritin receptor acts preferentially to remove tissue ferritins from the circulation. The lower binding affinity of serum ferritin for the ferritin receptor explains its slower in vivo clearance relative to tissue ferritins.     

The distribution of ferritin subunit types in porcine tissues

Ferritin was isolated from porcine heart, liver and spleen. Reversed phase high performance liquid chromatography of the ferritin subunits yielded three chromatographic fractions. The relative proportions of the three chromatographic fractions were different for each tissue ferritin. These results support the model which proposes a combination of (at least) two subunit types as the basis for the existence of isoferritins.     

Characterization of the binding of ferritin to the rat liver ferritin receptor

The binding characteristics and specificity of the rat hepatic ferritin receptor were investigated using ferritins prepared from rat liver, heart, spleen, kidney and serum, human liver and serum, guinea pig liver and horse spleen as well as ferritins enriched with respect to either H- or L-type subunit composition, prepared by chromatofocusing of rat liver ferritin on Mono-P or by reverse-phase chromatography of ferritin subunits on ProRPC 5/10. No significant difference was apparent in the binding of any of the tissue ferritins, or of ferritins of predominantly acidic or basic subunit composition. However, serum ferritin bound with a lower affinity. The effect of carbohydrate on the ferritin-receptor binding was examined by glycosidase treatment of tissue and serum ferritins. Tissue ferritin binding was unaffected, while serum ferritin binding affinity was increased to that of the tissue ferritins. Inhibition of ferritin binding by lactoferrin was not due to common carbohydrate moieties as previously suggested but was due to direct binding of lactoferrin to ferritin. Therefore, carbohydrate residues do not appear to facilitate receptor-ferritin binding, and sialic acid residues present on serum ferritin may in fact interfere with binding. The results indicate that the hepatic ferritin receptor acts preferentially to remove tissue ferritins from the circulation. The lower binding affinity of serum ferritin for the ferritin receptor explains its slower in vivo clearance relative to tissue ferritins.     

Immunochemical characterization of human liver and heart ferritins with monoclonal antibodies

A library of 27 murine monoclonal antibodies was obtained by using human liver and heart ferritins as immunogens. The specificity of the antibodies for the two ferritins and their subunits was studied with five different methods. The antibodies elicited by the liver ferritin bound preferentially the immunogen and were specific for the L subunit. Some antibodies elicited by the heart ferritin had characteristics similar to the anti-liver antibodies, other ones bound preferentially the heart over the liver ferritin and were specific for the H subunit. Only two antibodies were able to bind both ferritins and subunits. Some anti-H and anti-L chain antibodies were used to develop and compare four types of immunoassay to quantitate isoferritins. The results indicate that heart ferritin is immunologically more heterogeneous than liver, the H and L subunits having large immunological differences with few, if any, identical epitopes; and that that the architecture of the immunoassays have a strong influence on the crossreactivity of the antibodies with the two isoferritins, probably because H and L chains are not arranged randomly in the assembled protein.     

Structural differences in ferritins from normal and malignant rat tissues.

Ferritins purified from several normal and malignant rat tissues were examined for amino acid composition, content of tryptic peptides, available sulfhydryl groups and subunit sizes and proportion. Ferritin extracted from adult kidney, neonatal liver and hepatic and renal tumors differed from the ferritin of adult rat liver in migration on electrophoretic gels and in antibody affinity, but did not differ among themselves. Nevertheless, they showed distinctive differences in amino acid composition and tryptic peptide content. All of them and also adult liver ferritin contained two major species of subunits differing in molecular weight. The proportions of subunits, and the available sulfhydryl groups of the intact ferritin molecules, differed among these tissue ferritins. On the basis of amino acid and peptide content, the ferritins of hepatomas and the renal tumor analyzed showec the greatest similarity but not identity. The ferritin of neonatal liver was next most similar. Kidney ferritin differed considerably in composition from tumor and neonatal ferritins, while adult liver ferritin was the most extremely divergent of the series examined. A similar progressive difference was found on examining the proportions of subunits and sulfhydryl groups in these ferritins. However, changes in subunit proportion cannot explain the amino acid and peptide compositional changes.     

Heart tissue contains small and large aggregates of ferritin subunits

Ferritin purified from horse heart and applied to nondenaturing polyacrylamide gel electrophoresis migrated as a single band that stained for both iron and protein. This ferritin contained almost equal amounts of fast- and slow-sedimenting components of 58 S and 3-7 S, which could be separated on sucrose density gradients. Iron removal reduced the sedimentation coefficient of the fast-sedimenting ferritin to 18 S, and sedimentation equilibrium gave a molecular weight 650,000, with some preparations containing ferritin of 500,000 molecular weight as well. Sedimentation rates of the 3 S and 7 S ferritins were not affected by iron removal, and sedimentation equilibrium data were consistent with Mr's 40,000 and 180,000, respectively. Preparations of ferritin extracted from horse spleen contained only 67 S (holo) or 16 S (apo) ferritin and no slow-sedimenting species. When examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, all of the ferritins contained the usual H and L subunits (23 and 20 kDa, respectively), but the slow-sedimenting (3 S and 7 S) heart apoferritins also contained appreciable quantities (ca 25%) of three larger subunits of 42, 55, and 65 kDa. All the subunits reacted positively in Western blots to polyclonal antibodies made against specially purified large heart or spleen ferritins containing only 20- and 23-kDa subunits. Similar results were obtained for ferritins from rat heart. The results indicate that mammalian heart tissue is peculiar not just in having an abnormally large iron-rich ferritin but also in having iron-poor ferritins of much lower molecular weight, partly composed of larger subunits.     

The iron content of human liver and spleen isoferritins correlates with their isoelectric point and subunit composition.

The six isoferritins in normal human liver and spleen with pIs between 5.7 and 5.2 were fractionated according to iron content by sucrose density gradient centrifugation. The pIs and subunit composition of the isoferritins in fractions from the gradients were determined by gel electrofocussing and electrophoresis in acid/urea polyacrylamide gels respectively. Fractions with the lowest iron content contained the two basic isoferritins, which were homopolymers of the L subunit and had pIs of 5.7 and 5.6. Increasing iron content correlated with decreasing isoelectric point and an increasing substitution of the H subunit in the isoferritin shell. The two isoferritins with highest iron content consisted of 92% and 8% respectively of the L and H subunits and had pIs of 5.3 and 5.2.     

Conformational studies of a synthetic peptide corresponding to the repeat motif of C hordein.

Cytosolic glutathione S-transferases were purified from the epithelial cells of human small and large intestine. These preparations were characterized with regard to specific activities, subunit and isoenzyme composition. Isoenzyme composition and specific activity showed little variation from proximal to distal small intestine. Specific activities of hepatic and intestinal enzymes from the same patient were comparable. Hepatic enzymes were mainly composed of 25 kDa subunits. Transferases from small intestine contained 24 and 25 kDa subunits, in variable amounts. Colon enzymes were composed of 24 kDa subunits. In most preparations, however, minor amounts of 27 and 27.5 kDa subunits were detectable. Separation into isoforms by isoelectric focusing revealed striking differences: glutathione S-transferases from liver were mainly basic or neutral, enzymes from small intestine were basic, neutral and acidic, whereas large intestine contained acidic isoforms only. The intestinal acidic transferase most probably was identical with glutathione S-transferase Pi, isolated from human placenta. In the hepatic preparation, this isoform was hardly detectable. The specific activity of glutathione S-transferase showed a sharp fall from small to large intestine. In proximal and distal colon, activity seemed to be about equal. In the ascending colon there might be a relationship between specific activity of glutathione S-transferases and age of the patient, activity decreasing with increasing age.     

Structural and functional relationships of human ferritin H and L chains deduced from cDNA clones

We have isolated essentially full-length cDNA clones for human ferritin H and L chains from a human liver cDNA library. This allows the first comparison of H and L nucleotide and amino acid sequences from the same species as well as ferritin L cDNA sequences from different species. We conclude that human H and L ferritins are related proteins which diverged about the time of evolution of birds and mammals. We also deduce the secondary structure of the H and L subunits and compare this with the known structure of horse spleen ferritin. We find that residues involved in subunit interaction in shell assembly are highly conserved in H and L sequences. However, we find several interesting differences in H subunits at the amino acid residues involved in iron transport and deposition. These substitutions could account for known differences in the uptake, storage, and release of iron from isoferritins of different subunit composition.     

Differences between newly formed and recycled free small ribosome subunits in liver cytoplasm.

1. Ferritin has been isolated from the serum of four patients with iron overload by using two methods. 2. In method A, the serum was adjusted to pH 4.8 and heated to 70 degrees C. After removal of denatured protein, ferritin was concentrated and further purified by ion-exchange chromatography and gel filtration. In most cases, only a partial purification was achieved. 3. In method B, ferritin was extracted from the serum with a column of immuno-adsorbent [anti-(human ferritin)] and released from the column with 3M-KSCN. Further purification was achieved by anion-exchange chromatography followed by the removal of remaining contaminating serum proteins by means of a second immunoadsorbent. Purifications of up to 31 000-fold were achieved, and the homogeneity of the final preparations was demonstrated by polyacrylamide-gel electrophoresis. 4. Serum ferritin purified by either method has the same elution volume as human spleen ferritin on gel filtration on Sephadex G-200. Serum ferritin has a relatively low iron content and iron/protein ratios of 0.023 and 0.067 (mug of Fe/mug of protein) were found in two pure preparations. On anion-exchange chromatography serum ferritin has a low affinity for the column when compared with various tissue ferritins. Isoelectric focusing has demonstrated the presence of a high proportion of isoferritins of relatively high pI. 5. Possible mechanisms for the release of ferritin into the circulation are briefly discussed.     

Assembly of intra- and interspecies hybrid apoferritins

An intraspecies hybrid apoferritin was assembled by mixing subunits of horse heart ferritin, which consists mainly of H-type subunits, and horse spleen ferritin, in which L-type subunits predominate. Interspecies hybrid apoferritins were reconstituted from subunits of human liver-horse spleen ferritins and from rat liver-horse spleen ferritins. All the hybrid ferritins migrated as single zones with electrophoretic mobilities intermediate between those of the parent ferritins. Isoelectric focusing data and immunological patterns were consistent with the view that the reassembled apoferritins were composite molecules that contained subunits from each of the interacting forms. Reconstitution occurred in a random manner, as there was no apparent preference for assembly of homologous subunits. These results suggest that intersubunit interaction domains and recognition mechanisms that dictate formation of the highly specific quaternary structure assumed by this protein are common for different species of ferritins.     

Purification and characterization of a small dermatan sulphate proteoglycan implicated in the dilatation of the rat uterine cervix.

Ferritin was purified from chicken liver by two different methods: gel filtration on controlled-pore glass beads, and immunoaffinity chromatography employing a chicken ferritin-specific monoclonal antibody that did not cross-react with horse spleen ferritin. This antibody recognizes intact ferritin and an oligomeric 240 kDa form of the molecule after protein transfer to nitrocellulose, but not the 22 kDa chicken ferritin subunit. Chicken liver ferritin purified by these methods exhibited reduced migration on non-denaturing polyacrylamide gels compared with horse spleen ferritin. These results were consistent with the difference in calculated isoelectric points of chicken and horse ferritin subunits. By two-dimensional gel electrophoresis, chicken ferritin 22 kDa subunits exhibited isoelectric points from 6.1 to 6.6 whereas horse spleen ferritin subunits exhibited isoelectric points of 5.8-6.3. The 240 kDa form of the chicken ferritin molecule had an isoelectric point of 6.6 whereas the 210 kDa form of the horse ferritin molecule had isoelectric points of 5.1 and 4.9. Intact chicken liver ferritin particles were 13.4 +/- 0.8 nm (controlled-pore glass-purified) and 12.5 +/- 0.9 nm (affinity-purified) in diameter when viewed by electron microscopy. Horse spleen ferritin consisted of slightly smaller particles with an average diameter of 11.0 +/- 0.7 nm. However, ferritin from chicken liver and horse spleen co-migrated with an apparent molecular mass of 470 kDa when analysed by Sepharose 4B gel filtration chromatography. These results indicate that, consistent with results from other published purification methods, the chicken ferritin purified by the methods reported here exhibits both structural similarities to, and differences from, horse spleen ferritin.