Effect of tunicamycin on the secretion of serum proteins by primary cultures of rat and chick hepatocytes. Studies on transferrin, very low density lipoprotein, and serum albumin.

Using rat or chick hepatocyte monolayers, we have studied the effect of tunicamycin, a specific inhibitor of protein glycosylation, on the synthesis and secretion of serum proteins. Tunicamycin inhibited glucosamine incorporation into rat liver transferrin and the apoprotein B chain of chick liver very low density lipoprotein (VLDL) by 75 to 90%. In contrasts, amino acid incorporation into these two glycoproteins, as well as into the normally unglycosylated proteins, rat serum albumin and apoprotein A of chick liver VLDL, was decreased by only 10 to 25% in the presence of the antibiotic. Despite the inhibitory effect of tunicamycin on glycosylation, secretion of all four proteins was virtually unimpaired. Thus, the carbohydrate moieties of rat liver transferrin or apoprotein B of chick liver VLDL do not appear to play an essential role in the secretion process.     

Isolation and characterization of a transferrin binding protein from rat plasma

A transferrin binding protein was isolated from normal rat placenta and from iron-deficient rat plasma using a human transferrin affinity column. The yield of the isolated pure protein from iron-deficient rat plasma was about 0.5 micrograms/ml plasma. The major protein had a molecular mass of 85 kDa and contained carbohydrate. Reduction with mercaptoethanol did not change the molecular mass of the plasma transferrin binding protein whereas the native placental transferrin receptor of 180 kDa was reduced to 90 kDa. The transferrin binding protein reacted with both monoclonal and polyclonal antibodies raised against rat transferrin receptor. Immunoblotting of both normal and iron deficient rat plasma showed that the transferrin binding protein had a molecular mass of 85 kDa. In vitro digestion of purified rat placental transferrin receptor and red blood cells with trypsin provided an identical peptide profile, suggesting that the transferrin binding protein in rat plasma is derived from proteolysis of the extracellular portion of the transferrin receptor of the erythroid tissues.     

Polymorphism of transferrin found in the laboratory rat and wild rats in Japan

Polymorphism of the transferrin locus (Tf) was found in the laboratory rat and wild rats in Japan by polyacrylamide gel electrophoresis. Two phenotypes, “a” and “b,” were distinguished in homozygotes. It is suggested that these are controlled by autosomal codominant alleles. In 10 laboratory strains, only the IS strain showed the a type. This allele found in the IS strain was broadly distributed in Japanese wild rats. It is considered to be derived from a wild rat in Japan. Linkage relationship betweenTf andAlp-1 was not established.     

Isolation of rat serum alpha 1-microglobulin. Identification of a complex with alpha 1-inhibitor-3, a rat alpha 2-macroglobulin homologue.

Alpha 1-Microglobulin (alpha 1-m), or protein HC, a low molecular weight plasma protein with immunoregulatory properties, was isolated from rat serum by affinity chromatography using Sepharose-coupled monoclonal anti-alpha 1-m antibodies. High molecular weight forms of alpha 1-m were then separated from the low molecular weight alpha 1-m by gel chromatography of the eluted proteins. The apparent Mr (28,000), the charge heterogeneity, the N-linked carbohydrate, and yellow-brown chromophore suggest that the low molecular weight alpha 1-m is the serum counterpart to urinary alpha 1-m, which was purified previously. A high molecular weight complex of alpha 1-m was also isolated by the gel chromatography. It was homogeneous as judged by nondenaturing polyacrylamide gel electrophoresis. The molecule was bound by antibodies against human alpha 2-macroglobulin, and experiments with antisera against the three alpha-macroglobulin variants in rat serum, alpha 1-macroglobulin, alpha 2-macroglobulin, and alpha 1-inhibitor-3 (alpha 1I3) suggested that alpha 1I3 was the complex-partner of alpha 1-m. An antiserum raised against high molecular weight alpha 1-m was then used to isolate the complex-partner of alpha 1-m from rat serum with affinity chromatography, and this molecule was positively identified as alpha 1I3 by its physicochemical properties. Gel chromatography of the alpha 1I3.alpha 1-m complex suggested a molecule with an Mr of 266,000. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, however, it migrated as three major molecular species with apparent molecular weights of 224,000, 205,000, and 194,000 and several minor species of both higher and lower molecular weights, suggesting a complex subunit structure. alpha 1-m and alpha 1I3 could be detected in all three major species by Western blotting, and NH2-terminal amino acid sequencing suggested a molar ratio of 1:1 of alpha 1-m and alpha 1I3 in all three species. alpha 1I3.alpha 1-m was colorless, did not show light absorbance beyond 300 nm which is typical of low molecular weight alpha 1-m and was electrophoretically homogeneous, suggesting that it lacks the chromophore. Finally, the serum concentrations of the alpha 1I3.alpha 1-m complex and free alpha 1-m were determined as 0.16 and 0.010 g/liter, respectively. Thus, alpha 1I3.alpha 1-m constitutes 1-3% of the total alpha 1I3 in rat serum (w/w) and approximately 60% of the total alpha 1-m.     

The effects of an antibody to the rat transferrin receptor and of rat serum albumin on the uptake of diferric transferrin by rat hepatocytes

The role of high-affinity specific transferrin receptors and low-affinity, non-saturable processes in the uptake of transferrin and iron by hepatocytes was investigated using fetal and adult rat hepatocytes in primary monolayer culture, rat transferrin, rat serum albumin and a rabbit anti-rat transferrin receptor antibody. The intracellular uptake of transferrin and iron occurred by saturable and non-saturable mechanisms. Treatment of the cells with the antibody almost completely eliminated the saturable uptake of iron but had little effect on the non-saturable process. Addition of albumin to the incubation medium reduced the endocytosis of transferrin by the cells but had no significant effect on the intracellular accumulation of iron. The maximum effect of rat serum albumin was observed at concentrations of 3 mg/ml and above. At a low incubation concentration of transferrin (0.5 microM), the presence of both rat albumin and the antibody decreased the rate of iron uptake by the cells to about 15% of the value found in their absence, but to only 40% when the diferric transferrin concentration was 5 microM. These results confirm that the uptake of transferrin-bound iron by both fetal and adult rat hepatocytes in culture occurs by a specific, receptor-mediated process and a low-affinity, non-saturable process. The low-affinity process increases in relative importance as the iron-transferrin concentration is raised.     

Evidence for a novel circulating alpha 1 protein in tumour-bearing rats. Differentiation from acute phase alpha 1 proteins and from alpha 1 fetoprotein

It has been found in this study that the serum from rats bearing a transplanted dibenzanthracene-induced tumour ($\text{RD}\text{3}$), has a high concentration of alpha₁ proteins compared with normal rat serum. These alpha₁ proteins have been isolated by an immunoabsorption method and have been compared by immunological methods with the acute phase alpha₁ proteins isolated by the same method from the serum of rats presenting an inflammatory reaction. It has been found that the isolated $\text{RD}\text{3}$ alpha₁ proteins were composed of two major proteins: one of these corresponded to an inflammatory protein, the alpha₁-AP-globulin. The other may be a new protein, as it is absent from the serum of rats with an acute phase inflammatory reaction and nor does it correspond to alpha₁ feto-protein, a carcino-embryonic protein presenting the same electrophoretic mobility.     

Comparison of the interactions of transferrin receptor and transferrin receptor 2 with transferrin and the hereditary hemochromatosis protein HFE

The transferrin receptor (TfR) interacts with two proteins important for iron metabolism, transferrin (Tf) and HFE, the protein mutated in hereditary hemochromatosis. A second receptor for Tf, TfR2, was recently identified and found to be functional for iron uptake in transfected cells (Kawabata, H., Germain, R. S., Vuong, P. T., Nakamaki, T., Said, J. W., and Koeffler, H. P. (2000) J. Biol. Chem. 275, 16618-16625). TfR2 has a pattern of expression and regulation that is distinct from TfR, and mutations in TfR2 have been recognized as the cause of a non-HFE linked form of hemochromatosis (Camaschella, C., Roetto, A., Cali, A., De Gobbi, M., Garozzo, G., Carella, M., Majorano, N., Totaro, A., and Gasparini, P. (2000) Nat. Genet. 25, 14-15). To investigate the relationship between TfR, TfR2, Tf, and HFE, we performed a series of binding experiments using soluble forms of these proteins. We find no detectable binding between TfR2 and HFE by co-immunoprecipitation or using a surface plasmon resonance-based assay. The affinity of TfR2 for iron-loaded Tf was determined to be 27 nm, 25-fold lower than the affinity of TfR for Tf. These results imply that HFE regulates Tf-mediated iron uptake only from the classical TfR and that TfR2 does not compete for HFE binding in cells expressing both forms of TfR.     

Energy transfer as a probe of protein dynamics in the proteins transferrin and calmodulin.

We have initiated an investigation into the usefulness of fluorescence energy transfer in probing protein dynamics. Our analysis involves measuring the energy transfer efficiency while perturbing the protein conformational equilibrium with heat. As the temperature increases, the amplitudes of vibrations increase, and fluorescence energy transfer should also increase if the donor and acceptor are in a flexible region of the protein. A theoretical analysis developed by Somogyi and co-workers for the temperature dependence of dipole-dipole energy transfer (Somogyi, B., J. Matko, S. Papp, J. Hevessey, G. R. Welch, and S. Damjanovich. 1984. Biochemistry. 23:3403-3411) was tested by the authors in one protein system. Energy transfer from tryptophan to a pyridoxamine derivatized side group in RNase increased 40% over 25 degrees C. Here we report further testing of this model in two additional protein systems: calmodulin, a calcium activated regulatory protein, and transferrin, a blood serum iron shuttle. Our studies show a slight differential sensitivity of the transfer efficiency to heat for the two systems. Normalized energy transfer over 6.5 A in calmodulin from a tyrosine donor to a Tb(III) acceptor increases 40% from 295 to 320 K. Normalized energy transfer over 42 A in transferrin from a Tb(III) donor to an Fe(III) acceptor increases 35% over the same temperature range. Whereas these results demonstrate that thermally induced fluctuations do increase energy transfer as predicted by Somogyi, they also appear rather insensitive to the nature of the protein host environment. In contrast to the F?rster processes examined above, energy transfer over very short distances has shown an anomalously high temperature dependence.     

Receptor-mediated endocytosis of alpha 2-macroglobulin and transferrin in rat caput epididymal epithelial cells in vitro

The endocytic activity of epithelial cells from the rat epididymis in vitro has been examined by following the uptake of tracer compounds conjugated to proteins. Transferrin-gold and alpha 2-macroglobulin-gold were taken up initially in coated pits, internalized and sequestered into tubular-vesicular structures, multivesicular bodies and, in the case of alpha 2-macroglobulin, into lysosomes. Uptake could be prevented by an excess of unlabeled protein. Studies using 125I-alpha 2-macroglobulin and 125I-transferrin also showed that the uptake of these proteins was specific and could be displaced with increasing amounts of unlabeled protein. In addition, binding of 125I-transferrin to cells was saturable at 4 degrees C. These studies indicate that transferrin and alpha 2-macroglobulin are taken up by receptor-mediated endocytosis. In contrast, a fluid phase marker, bovine serum albumin-gold (BSA-gold), was initially taken up predominantly in uncoated caveolae rather than coated pits, and could not be displaced with excess BSA. By virtue of their charge, polycationized ferritin and unlabeled colloidal gold were taken up and internalized by adsorptive endocytosis, a pathway which is similar to fluid phase endocytosis. The uptake and internalization of alpha 2-macroglobulin and transferrin differed in a number of respects. Uptake and internalization of alpha 2-macroglobulin but not of transferrin was dependent on extracellular calcium. Only alpha 2-macroglobulin was transferred into lysosomes, whereas transferrin was recycled to the cell surface. Although the proton ionophore, monensin, and the transglutaminase inhibitor, dansylcadaverine, did not stop uptake and internalization of either alpha 2-macroglobulin or transferrin, they did prevent the transfer of alpha 2-macroglobulin to lysosomes.     

Role of the transferrin receptor in lymphocyte growth: a rat IgG monoclonal antibody against the murine transferrin receptor produces highly selective inhibition of T and B cell activation protocols

We have produced a new rat IgG monoclonal antibody against the murine transferrin receptor (TR). This antibody (C2F2) exhibits a surprisingly selective pattern of inhibition of murine lymphocyte activation protocols. C2F2 inhibits the mixed lymphocyte reaction (MLR) and the generation of cytotoxic T cells. Interestingly, although interleukin 1 (IL 1)-dependent thymocyte co-stimulatory activity is strongly inhibited by C2F2, interleukin 2 (IL 2)-dependent thymocyte co-stimulation is only marginally reduced. IL 2-dependent growth of CTLL cells is also not inhibited by C2F2. These data suggest that IL 1-dependent helper T cell activation is very sensitive to C2F2-mediated inhibition. Studies with phytohemagglutinin, Concanavalin A, and lipopolysaccharide induced activation also indicate that the inhibitory effects of C2F2 are selective, and T cell activation may be more sensitive to inhibition than B cell activation. Although there is little published information about the functional effects of other rat anti-mouse TR antibodies, the available data suggest that the patterns of inhibition produced by anti-TR antibodies may be individually distinct. Anti-TR antibodies may constitute a new set of highly selective probes for the study of lymphocyte activation.     

Microheterogeneity of serum transferrin, haptoglobin and alpha 2 HS glycoprotein examined by high resolution two-dimensional electrophoresis.

A cell-free system of the cephalosporin C fungus, $\text{Cephalosporium}\text{acremonium}$, has been described which conversts penicillin N to a cephalosporin-like material which was biologically-active upon treatment with penicillinase with subsequent loss of activity when treated with cephalosporinase. We have confirmed this conversion independently in our laboratories and have shown that the activity was lost upon boiling, indicating the reaction is enzymatic in nature. Furthermore, we have shown that the production of the cephalosporin-like material was stimulated by the addition of ascorbic acid (3.8 μM) and ferrous ions (0.075 μM) to the reaction mixture, components which are co-factors in monooxygenase and dioxygenase reactions.     

The C2 variant of human serum transferrin retains the iron binding properties of the native protein

The tryptic digests of blood samples obtained from transferrin C1 and C2 (TfC 1 and TfC2 hereafter) genotypes were analysed by Liquid Chromatography coupled to Electrospray Mass Spectrometry (LC/ESI--MS/MS). The analytical results confirmed the single base change in exon 15 of the Tf gene. The solution behaviour and the iron binding properties of the two Tf variants were studied by UV-visible spectrophotometry and by circular dichroism. It appears that TfC2 globally manifests the same spectral features as the native protein. The local conformation of the two iron binding sites is conserved in the two Tf variants as evidenced by the visible absorption and CD spectra. Also, the iron binding capacities and their pH-dependent profiles are essentially the same. Overall, our investigation points out that the single amino acid substitution in TfC2 (Pro 570 Ser) does not affect the general conformation of the protein nor the local structure of the iron binding sites. The implications of these results for the etiopathogenesis of Alzheimer's disease are discussed.     

Phosphorylation of maize and Arabidopsis HMGB proteins by protein kinase CK2alpha

In plants, a variety of chromatin-associated high mobility group (HMG) proteins belonging to the HMGB family have been identified. We have examined the phosphorylation of the HMGB proteins from the monocotyledonous plant maize and the dicotyledonous plant Arabidopsis by protein kinase CK2alpha. Maize CK2alpha phosphorylates the maize HMGB1 and HMGB2/3 proteins and the Arabidopsis HMGB1, HMGB2/3, and HMGB4 proteins. Maize HMGB4 and HMGB5 and Arabidopsis HMGB5 are not phosphorylated by CK2alpha. Depending on the HMGB protein up to five amino acid residues are phosphorylated in the course of the phosphorylation reaction. The HMGB1 proteins from both plants are markedly more slowly phosphorylated by CK2alpha than the other HMGB substrate proteins, indicating that certain HMGB proteins are clearly preferred substrates for CK2alpha. The rate of the phosphorylation reaction appears to be related to the ease of interaction between CK2alpha and the HMGB proteins, as indicated by chemical cross-linking experiments. MALDI/TOF mass spectrometry analyses demonstrate that the HMGB1 and HMGB2/3 proteins occur in various phosphorylation states in immature maize kernels. Thus, HMGB1 exists as monophosphorylated, double-phosphorylated, triple-phosphorylated, and tetraphosphorylated protein in kernel tissue, and the tetraphosphorylated form is the most abundant version. The observed in vivo phosphorylation states indicate that protein kinase(s) other than CK2alpha contribute(s) to the modification of the plant HMGB proteins. The fact that the HMGB proteins are phosphorylated to various extents reveals that the existence of differentially modified forms increases the number of distinct HMGB protein variants in plant chromatin that may be adapted to certain functions.