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Glycosylation is the most complex post-translational modification. Thus, it contributes to versatile chemical compositions of proteins, leading to high amounts of protein species. The structural heterogeneity of glycoproteins was also described by the definition of glycoforms. We therefore introduced a new term called "glycoprotein species" to join the two concepts from different fields of biology. In this study, we further determined the theoretical numbers of glycoprotein species of two recombinant glycoproteins - a therapeutical antibody and the human protease inhibitor alpha-1-antitrypsin (A1AT) - based on structural analysis of their N-glycans. Moreover, we showed that variations in the used cell lines and their cultivation conditions strongly influence the number of glycoprotein species in case of recombinant A1AT production., Biological Significance: Protein glycosylation is a major source for the huge amount of protein species. This study extends the sight of protein species by the following contributions: 1) The new term "glycoprotein species" was defined to introduce the concept of glycoforms into the field. 2) An estimation of the number of potential glycoprotein species of two particular glycoproteins was given. 3) The influence of production conditions for recombinant glycoproteins on glycoprotein species generation was displayed., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Congenital disorders of glycosylation (CDG) comprise a family of inherited multisystemic disorders resulting from the deficiency of glycosylation pathways. N-glycosylation defects are classified as two biochemical and genetic established types, of which CDG-Ia is the most frequent. We performed 2-DE proteomic analysis on serum from two functional hemizygous CDG-Ia patients bearing T237M and D65Y missense changes. Comparative analysis of control/patient serum proteome allowed us to identify differential expression of 14 proteins. The most remarkable groups included proteins involved in immune response, coagulation mechanism and tissue protection against oxidative stress. The patient bearing D65Y mutation had less favourable clinical outcome and showed more abnormalities in the spot patterns, suggesting that the proteomic results might also be correlated with the phenotype of CDG patients. This study describes for the first time the differential expression of alpha(2)-macroglobulin, afamin, fibrin and fibrinogen in CDG disorder and shows how the proteomic approach might be useful for understanding its physiopathology.

Tumor cells synthesize and release a variety of substances, including proteases and protease inhibitors involved in cell growth and proliferation. alpha1-Antitrypsin (AAT) is a serine proteinase inhibitor synthesized primarily in the liver, but also in extra-hepatic tissues and cells, including tumor cells. AAT exists not only in a native, active inhibitory form, but also in several, non-inhibitory forms, such as cleaved and/or degraded. This study was designed to investigate the synthesis of AAT by melanoma cells, ME 1477, and the effects of native, cleaved and C-terminal fragment of AAT (C-36) on cell functional activity. We found that ME 1477 cells synthesize and secrete AAT with the same apparent molecular mass as described for AAT purified from plasma, but with no measurable inhibitory activity. As determined by Western blot after immunoprecipitation of [32S]-labeled AAT, exogenous native or modified forms of AAT added to the cells at a concentration of 10 microM did not change AAT synthesis. Moreover, cells exposed to native AAT show decreased [3H]-thymidine incorporation by 53% and tissue inhibitor of metalloproteinases (TIMP)-1 levels by 36%. In contrast, cells treated with C-36 peptide significantly increased metalloproteinase activity, and [3H]-thymidine incorporation by 35%. Specifically, pro-collagenase-1 levels were found to be increased by 1.4-fold and decreased by 1.5-fold in cells treated with C-36 peptide and native AAT, respectively. Cleaved form of AAT had no significant effects on parameters measured. Data obtained from this study suggest that specific forms of AAT have multiple effects on tumor cell viability and play diverse roles in tumorogenesis.

Atherosclerotic plaques contain a significant number of macrophage foam cells and are associated with an inflammatory state. Inflammation induces the secretion from monocytes and other cells of cytokines, reactive oxygen species, proteinases and proteinase inhibitors among many other molecular species. AAT is prominent among the serine proteinase inhibitors and is an important regulator of leukocyte elastase and proteinase-3. It has been shown that the stable AAT-proteinase complex can upregulate AAT biosynthesis, and we have shown that the shorter, carboxyl terminal peptide (C-36) resulting from proteinase cleavage of AAT polymerizes, and in its fibrillar form alters cellular metabolism. To test for a possible link between the inflammation-generated C-36 peptide and cellular processes associated with atherogenesis, we have studied the effects of the fibrillar form of this peptide at varying concentrations on human monocytes in culture. We have found that fibrillar C-36 at concentrations of greater than or equal to 5 micromol/l in monocyte cultures for 24 h significantly increases LDL binding and uptake, upregulates LDL receptors, induces cytokine production and glutathione reductase activity, and upregulates AAT synthesis. The expression of CD36 protein, LDL Scavenger receptor, is also upregulated by fibrillar C-36 and native LDL in the presence of C-36-activated monocytes is more oxidized than with unactivated control monocytes. The majority of monocytes cultured for 24 h in the presence of C-36 fibrils were transformed morphologically into macrophages. These data establish a direct molecular link, mediated by C-36 peptide of AAT, between inflammation and the oxidation and accumulation of lipid in monocyte-derived macrophages. This may be important for an understanding of the events conducive to atherogenesis.

The idea of generating transgenic livestock which secrete into their milk large quantities of proteins for therapeutic use, was pioneered in the late 1980s with the disclosure of the production of a number of transgenic sheep. One particular animal, a sheep called Tracy, produced milk where over 50% of the protein consisted of human alpha 1 anti-trypsin. Sheep-derived protein has now entered clinical trials for cystic fibrosis (UK, USA) and congenital emphysema (UK). There are many other examples where this technology is making inroads into more traditional ways of making biopharmaceuticals. However, although robust, this technology has several limitations, including an inability to allow targeted insertion/modification of the animal genome, long timelines to production flocks/herds, and the rather unpredictable expression levels seen when different transgenic founders are compared. We believe that there is now a technical solution to all of these problems. Dolly is a high profile example of a new technology comprising the generation of identical animals from cultured somatic cells. This work has many implications. In the commercial context, the real benefits of this advance will be seen when genetically engineered somatic cells are shown to be suitable nuclear donors, and particularly when the manipulations are targeted to pre-determined sites in the host cell genome. The first objective has now been achieved with the birth of Polly, a cloned sheep which contains the human gene encoding Factor IX, a protein involved in preventing haemophilia.

Background/aims: Some of the mutant forms of cellular proteins not only lose their function, but also cause diseases by their toxic effects. One of the challenging tasks in the field of gene therapy will be "gene replacement" accomplished by inhibiting mutant gene expression and providing normal function of the same gene, simultaneously. Although lung involvement in alpha1-antrypsin (alpha1-AT) deficiency is caused by the lack of alpha1-AT function, the liver involvement is due to the accumulation of the mutated alpha1-AT protein. Therefore, one possible approach to prevent and treat the disease manifestations of alpha1-AT deficiency is to inhibit the expression of the mutated gene and replace it with normally functioning alpha1-AT protein in the liver., Methods: For the inhibition of alpha1-AT gene expression, panels of alpha1-AT-specific hammerhead ribozymes designed to target different GUC sites in the alpha1-AT mRNA were evaluated in a human hepatoma cell-line, transduced with retroviral vectors which express ribozymes under the control of a human tRNA promoter. A bi-functional vector was also constructed, which contained a functional alpha1-AT ribozyme and was combined with a modified alpha1-AT gene, whose product was engineered to be resistant to the specific alpha1-AT ribozyme. This construct was transduced into target hepatoma cells., Results: The transduced hepatoma cells showed the effective expression of modified alpha1-AT, under the conditions where the endogenous alpha1-AT gene expression was inhibited., Conclusion: This ribozyme-mediated, specific gene replacement is a first step in the gene therapy of alpha1-AT deficiency.

alpha 1-Proteinase inhibitor (alpha 1-PI) augmentation therapy has been licensed for treatment of alpha 1-PI-deficient individuals with pulmonary emphysema. The currently available product is purified from pooled human plasma. To obtain larger amounts of protein free from possible unknown plasma contaminants, human alpha 1-PI has been produced by recombinant DNA. Since wild-type alpha 1-PI is susceptible to oxidative impairment, several alpha 1-PI variants in which the active site oxidation-sensitive residue is replaced by inert residues have been constructed. This article is aimed at reviewing the history, biological efficacy, advantages, disadvantages, and concerns linked to alpha 1-PI recombinant DNA and site-specific mutagenesis technology.

Lung transplantation is an option for appropriately selected patients with end-stage emphysema caused by alpha 1-antitrypsin deficiency. The functional results have been excellent after single or bilateral lung transplantation, and the medium-term survival results have been good. However, the role of alpha 1-antitrypsin replacement therapy after lung transplantation remains uncertain, and further investigation is needed.

The Mus musculus alpha 1-protease inhibitor gene cluster encodes five highly related proteins. The most significant amino acid polymorphisms lie within the reactive-site loop which is important in determining serpin substrate specificity. All five genes are transcribed in M. musculus adult liver and presumably secreted into plasma. In an attempt to characterize their protein products all five cDNAs were expressed in recombinant mammalian cells and the protease inhibition activity of each determined. Only two of the proteins were efficient inhibitors of neutrophil elastase, the major physiological target of the sole human alpha 1-protease inhibitor (antitrypsin). Four of the proteins were active against chymotrypsin, while no substrate could be identified for the fifth.

In this study we provide evidence for downregulation of alpha 1-Proteinase Inhibitor (alpha 1-PI) synthesis in cells of monocytic origin by the glucocorticoid analog, dexamethasone. This factor significantly reduced the basal level of alpha 1-PI expression as well as antagonized the effect of lipopolysaccharide and interleukin-6, stimulators of alpha 1-PI synthesis in cells of monocyte/macrophage lineage. Since increased levels of all of these mediators are observed in both acute infections and inflammatory reactions, their combination may affect the contribution of monocytes to the synthesis of alpha 1-PI.

Rat hepatoma cells H-35 cultured in serum-free medium were exposed to interleukin-6 (IL-6), interleukin-1 (IL-1), hepatocyte growth factor (HGF), retinoic acid (RA), or a mixture of these factors. Production of acute phase proteins, responding to IL-6 alone (type 2) or to the mixture of IL-6 and IL-1, was assessed by electroimmunoassay and the corresponding mRNAs were compared by Northern blot analysis. HGF enhanced IL-6-induced synthesis of alpha-2-macroglobulin but reduced synthesis of C3 complement and alpha-1-acid glycoprotein. Retinoic acid reduced the response to IL-6 of alpha-2-macroglobulin but enhanced that of alpha-1-acid glycoprotein and especially of C3 complement. In general, changes in protein secretion were correlated with the contents of their corresponding cellular mRNAs. These results indicate that hepatocyte growth factor can enhance basal or IL-6-induced gene expression of type 2 and reduce the expression of type 1 acute phase proteins, whereas the action of retinoic acid is opposite. The modulation of acute phase response by HGF and RA likely involves transcriptional factors and regulatory sequences in the genes coding for these two types of acute phase proteins.

Hepatic gene therapy could be used to treat a number of inherited blood diseases such as hemophilia or thrombophilia. Although liver-directed retroviral transduction can result in long-term gene expression in vivo, the low level of protein production has limited its clinical application. We reasoned that the insertion of liver-specific promoters into retroviral vectors would increase gene expression in vivo. The 347-bp human alpha 1-antitrypsin (hAAT), the 810-bp murine albumin (mAIb), the 490-bp rat phosphoenolpyruvate carboxykinase (rPECK), and the 596-bp rat liver fatty acid binding protein promoters were inserted into a Moloney murine leukemia retroviral backbone containing the hAAT reporter gene. Vectors that produced appropriately sized RNA and hAAT protein in vitro were tested in vivo by transducing regenerating rat livers. Long-term serum expression of the hAAT reporter gene was normalized to retroviral transduction efficiency as determined by using a polymerase chain reaction-based assay of genomic DNA from transduced rat livers. The hAAT, mAIb, and rPEPCK promoters were, respectively, 35-, 8-, and 0.02-fold as strong as the previously studied constitutive Pol-II promoter. We conclude that the hAAT promoter resulted in the highest expression from a retroviral vector and may result in therapeutically significant expression of other clinically significant blood proteins.

A plasmid (pTG7101) containing the full-length human alpha 1-antitrypsin gene was encapsulated in small liposomes and used for "in vivo" gene transfer to mouse hepatocytes, by i.v. injection (100 ng DNA/mouse and dose). The expression of human protein was evaluated by microspectrophotometry after human alpha 1-antitrypsin immunoperoxidase reaction on liver cryosections and the presence in mouse plasma of de novo synthesized protein was detected by ELISA analysis. Our results indicate that a single dose of encapsulated plasmid induces the expression of human alpha 1-antitrypsin in mouse hepatocytes and a large effect (70%) remains two weeks after treatment. However, no effect was observed when mice were treated with buffer or free plasmid (100 ng/mouse) plus an equivalent lipid dose of empty liposomes. In addition, whereas no additive effect was observed after repetitive treatment-doses, the partial hepatectomy three hours after a single treatment-dose, significantly increased the presence of human alpha 1-antitrypsin in mice plasma.

Recombinant human leukemia inhibitory factor (rhLIF) produced by Escherichia coli was administered subcutaneously (sc) to rhesus monkeys at doses of 2, 10, and 50 micrograms/kg body weight/d for 14 days to assess its biologic activities in vivo. Serum levels of positively regulated acute phase proteins (APP) (C-reactive protein, alpha 1-antitrypsin, haptoglobin, and ceruloplasmin) were increased, whereas the negatively regulated APP prealbumin decreased in response to rhLIF treatment. During the second week of treatment, blood platelet counts began to increase, resulting in a maximum of a twofold increase above normal levels a week after termination of the rhLIF treatment. No changes were seen in total and differential white blood cell counts in blood progenitor levels and in red blood cell numbers. The low- and medium-dose rhLIF treatments were tolerated without significant side effects. The animals treated with the high dose showed a reduction in body weight of approximately 10%. In conclusion, rhLIF was shown to stimulate APP and to increase the number of platelets in circulation in nonhuman primates.

Hepatocytes respond to inflammatory stimuli by changing the synthesis and N-glycosylation of acute phase plasma proteins (APP). So far, interleukin (IL) 6, transforming growth factor beta (TGF beta), tumor necrosis factor alpha (TNF) and IL-1 have been found to control N-glycosylation patterns of APP. Cytokines either increased (type I) or decreased (type II) the ratio of bi-relative to more branched N-glycans on APP. In this study, we describe the effect of leukemia inhibitory factor (LIF), interferon gamma (INF gamma) and dexamethasone (dex) on production of alpha 1-protease inhibitor (PI) and alpha 1-antichymotrypsin (ACT) and on glycosylation of PI in the human hepatoma cell line HepG2. Cytokines and dex were used separately and in various combinations including also IL-6 and TGF beta. Production of the antiproteases was quantitated by immunoelectrophoresis of the proteins accumulated in the culture medium. Glycosylation pattern of PI was assessed by crossed immunoaffinity electrophoresis (CIAE) with Concanavalin A (Con A) as a ligand. The production of ACT and PI was increased by LIF, decreased by INF gamma and unaffected by dex. LIF and INF gamma each like IL-6, decreased PI-Con A reactivity while dex like TGF beta enhanced PI-Con A reactivity. Combination of dex with LIF yielded additive effects while combination of dex with either INF gamma, L-6 or TGF beta acted synergistically on PI-Con A reactivity. Combinations of multiple cytokines and dex produced additive, inhibitory or synergistic effects. The type of glycosylation profile of PI secreted by HepG2 cells depended on the composition and amounts of interacting cytokines and dex.

The potent serine protease, neutrophil elastase (NE), is stored in neutrophil azurophilic granules, where it is available to degrade phagocytosed material and can be released by the cell to assist in tissue migration and help clear tissue debris. While neutrophils carry NE, they cannot produce it; the NE gene is expressed only in bone marrow granulocyte precursor cells. Protection of normal tissues from the destructive capacity of NE is provided by alpha 1-antitrypsin (alpha 1 AT), a 52-Kd serine antiprotease produced by hepatocytes and mononuclear phagocytes. In the context of the broad destructive capacity of NE, we evaluated the concept that human neutrophils may be able to modulate the extracellular activity of NE by synthesizing and secreting alpha 1AT. Immunocytochemical analysis demonstrated that the neutrophil contains alpha 1AT. Northern analysis and in situ hybridization with alpha 1AT-specific probes demonstrated the presence of alpha 1AT messenger RNA transcripts within neutrophils. [35S]methionine-labeling of neutrophils followed by immunoprecipitation of the supernatant with an anti-alpha 1AT antibody and sodium dodecyl sulfate-acrylamide gel analysis demonstrated that neutrophils can synthesize alpha 1AT de novo and secrete the synthesized molecule. In the presence of major neutrophil degranulation, the antiprotease effect of neutrophil alpha 1AT is overwhelmed, allowing the NE to act unopposed in the extracellular microenvironment. However, in conditions where small amounts of NE are released by neutrophils, at least some of the secreted newly synthesized alpha 1AT was capable of complexing with NE. Thus, despite the fact that the neutrophil cannot synthesize NE, it can synthesize and secrete alpha 1AT, the inhibitor of NE, ie, the neutrophil is capable, to some extent, of modulating NE activity in the local milieu without the help of antiproteases produced by other cells.

This report compares the relative levels of messenger RNA species coding for plasma proteins in rat visceral yolk sac and fetal liver from 12.5 days to 21.5 days gestation. Transthyretin, retinol-binding protein, transferrin and alpha 1-fetoprotein mRNAs were detected in both tissues, although relative levels were much higher in the yolk sac compared to fetal liver, in early gestation. Messenger RNA coding for the positive acute phase proteins thiostatin, fibrinogen, alpha 2-macroglobulin and alpha 1-antitrypsin were detected at a low but significant level in yolk sac, while the levels in fetal liver steadily increased from 16.5 days gestation and, with the exception of alpha 1-antitrypsin, reached levels higher than those found in adult liver just prior to birth. Albumin, inter-alpha 1-trypsin inhibitor, alpha 1-acid glycoprotein, haptoglobin, vitamin D-binding protein and ceruloplasmin messenger RNA levels were either very low or undetectable in yolk sac and fetal liver. Secretion of proteins by yolk sac endoderm occurred largely across the basolateral surface, i.e. towards the fetal compartment. These data support the hypothesis that one function of the yolk sac in the rat is the synthesis and secretion of a select group of plasma proteins to maintain homeostasis in the fetal compartment in the period before the fetal liver has matured sufficiently to carry out this function.

Molecular analysis of alpha 1-antitrypsin (alpha 1AT) Wbethesda revealed that it differs from the normal M1 (Ala213) allele by a single base mutation causing an amino acid substitution Ala336 GCT----Thr ACT. Evaluation of alpha 1AT biosynthesis directed by the Wbethesda allele showed that although Wbethesda alpha 1AT mRNA was translated normally in vitro, transfection of the Wbethesda cDNA into COS-I cells was associated with human alpha 1AT secretion of 50% that of cells transfected with a normal alpha 1AT cDNA. The pattern of alpha 1AT biosynthesis was not intracellular accumulation as observed with the common Z alpha 1AT deficiency allele, but reduced intracellular alpha 1AT, suggesting intracellular degradation of the newly synthesized Wbethesda molecule. Together these observations suggest that in heterozygous combination with a Z or Null alpha 1AT allele, the Wbethesda variant causes "alpha 1AT deficiency", thus classifying it as an alpha 1AT "at risk" allele for emphysema.

Alpha 1 protease inhibitor antigen was identified in the culture medium of the human ascites hepatoma cell line SK-HEP-1. Trypsin inhibitory activity and alpha 1 Pl antigen accumulated in serum-free medium concomitantly over a period of several days. Radioactive alpha 1 Pl antigen was detected in conditioned medium from cultures supplemented with 35S-L-methionine, indicating a synthesis and release of the protein. Alpha 1 Pl antigen in conditioned medium appeared to be antigenically identical to that in human plasma, and the newly synthesized (radiolabeled) antigen co-migrated with plasma, alpha 1 Pl after immunoelectrophoresis or SDS-polyacrylamide gel electrophoresis. Moreover, evidence is presented that the synthesized inhibitor exhibits functional activity, since the 35S-labeled alpha 1 Pl in conditioned medium complexes with trypsin. We conclude that SK-HEP-1 cells in culture produce functionally active alpha 1 Pl which may be identical to that in plasma.

The synthesis of plasma proteins directed by mRNA from human brain tissues was studied by combining in vitro or in ovo translation of mRNAs with crossed immunoelectrophoresis of the mRNA-directed labeled polypeptides, followed by autoradiography of the washed plates. Poly(A)-containing mRNA was prepared from different developmental stages of fetal and postnatal human brain and also from primary glioblastomas and meningiomas. Several plasma protein-like polypeptides were identified in the autoradiographs by their migration coordinates in the two-dimensional gels, compared with immunoprecipitates formed by mature, unlabeled, stainable proteins. These included polypeptides migrating like Gc globulin, haptoglobin, fibrinogen, alpha-fetoprotein, transferrin, cholinesterase, and alpha 2-macroglobulin; other, yet unidentified plasma proteins, were also observed. In general, the synthesis of these plasma proteins appeared to be more pronounced in fetal and neoplastic brain tissues than in postnatal tissues. However, clear immunoprecipitates for some of these plasma proteins could also be detected in products directed by mRNA from particular regions of mature, normal brains, indicating that some synthesis of plasma proteins takes place in the human brain even as late as 40 years of age. mRNAs for several proteins were also identified in samples of neoplastic brain. mRNA for transferrin was identified in normal fetal and adult brain but not in either the glioblastomas or meningiomas studied. Microinjected Xenopus oocytes, in which post-translational processing occurs as well, were also used to translate fetal brain mRNA. Several plasma proteins could be detected in the translation products which were induced and stored in the oocytes. These included hemopexin, which could not be detected in the in vitro system. Others, such as cholinesterase, were found to be secreted by the oocytes. These findings indicate that different cell types in the human brain may produce and either store or secrete particular plasma proteins at defined stages in their development.

Invented 20 years age, crossed immunoelectrophoresis (X-IEP) today is a technique of unusual power and myriad application. It combines very high resolution with exquisite specificity by alloying 2-dimensional electrophoresis with immunoprecipitation for symbiotic new potentialities. The consequent matchless quantitative/qualitative capabilities of X-IEP for analyzing antigens in complex mixtures, particularly by their idiomatic internal comparison, are still not widely recognized. Because of this and the supposed complications of its use and interpretation, X-IEP is more rarely used than it should be. This essay discusses contemporary X-IEP with the particular aims of demonstrating that it is not difficult to use and of explaining with selected examples why it is peculiarly powerful for analyzing antigen mixtures like the body fluids, tissue and cell extracts, and microbial homogenates.

The major serum antiprotease is alpha 1-antitrypsin (A1AT). Deficiency of A1AT can result in infantile cirrhosis and premature emphysema, both of which have a high degree of morbidity and significant mortality. Although synthesized primarily by the liver, A1AT has been histochemically localized in monocytes and macrophages in vitro and has been shown to be produced in tissue culture of monocyte-macrophage origin. This study was planned to quantitatively and qualitatively assess the in vivo monocyte-macrophage system contribution to serum A1AT. We used bone marrow transplantation (BMT) as an experimental method because there is commanding evidence that after engraftment, the monocyte-macrophage system of the recipient is replaced by that of donor origin. Protease inhibitor (Pi) typing was done on 150 potential BMT recipients and on their potential donors before transplantation. From these initial recipients, 92 eventually underwent transplantation, and 11 recipient-donor pairs, in which each donor's Pi type contained a band not in the recipient's Pi type, were chosen for the study. Six recipients survived beyond 100 days after BMT, and in these cases the donor contained either an S or an M2 band in his or her Pi type not present in the recipient. Using a silver stain method on diluted serum of known M1M2 and MS types, we were able to detect a 2% dilution of the S band and a 25% dilution of the M2 band. When the same method was applied to gels used in typing recipient Pi after BMT, we were unable to detect any contribution to serum A1AT by the donor monocyte-macrophage system.

A series of subclones of the H4II line of the Reuber H35 rat hepatoma produce substantial amounts of three plasma proteins, transferrin, alpha 1-antitrypsin and fibrinogen. Immunocytochemical staining demonstrated that each of these proteins is synthesized by essentially every cell of these cell populations. Cells of dedifferentiated variant clones either cease to produce the proteins, or exhibit a substantial reduction that is accompanied by variability in the synthetic activity of individual cells of the population. As previously observed with regard to angiotensinogen production, the variant clones clearly divide into two categories: those that show only a reduction in synthesis are able to give rise to revertants, whereas the negative clones fail to do so. Revertant cells exhibit a dramatic restoration of the synthesis of plasma proteins, which in some cases, exceeds by severalfold the rates seen in the differentiated clones of origin. In addition, the revertant cells synthesize alpha-fetoprotein, a function that is not expressed by H4II cells or its daughter subclones. Immunocytochemical staining revealed that, with regard to several plasma proteins including albumin, fibrinogen and alpha-fetoprotein, the cell populations of revertant clones are very heterogeneous, for only a fraction of the cells synthesizes each protein. Hybrid cells resulting from several types of crosses, exhibited extinction of the plasma proteins, the exception being transferrin, whose production was maintained, but at a reduced level and in only a fraction of the cells. Taken together, our results show that the expression of albumin and transferrin can be dissociated from one to another, and from that of fibrinogen, alpha 1-antitrypsin and angiotensinogen.

A two-step procedure to incorporate 125I-iodotyrosine into protein synthesized in a reticulocyte lysate is described. In the first step, the iodination of tyrosyl tRNA was catalyzed by a solid-state glycouril compound. More than one-third of 200 microCi of radioiodine became bound to 70 micrograms of aminoacyl tRNA after 15 min at 0 degrees C. The isotope was distributed in a three-to-one ratio of monoiodotyrosine to di-iodotyrosine. In the second step, the soluble product of the radioiodination was transferred directly into a nuclease-treated reticulocyte lysate coded with RNA isolated from the human hepatoma cell line Hep G2. Fractional recovery of radioiodine in nascent protein was maximally 7.6%. Reaction of the product of translation with antibody against alpha-antitrypsin separated an 125I-containing protein having a molecular weight estimated as 47,000. The synthesis of unprocessed alpha-antitrypsin was confirmed by cleavage of the labeled protein with leader peptidase and by its displacement from immunocomplex formation with purified alpha-antitrypsin. The amount of 125I incorporated into alpha-antitrypsin was proportionate to iodinated tRNA additions up to a concentration of 70 micrograms/ml. The synthesis of alpha-antitrypsin as detected in radioautograms after gel electrophoresis was more than twice as sensitive using radioiodinated aminoacyl tRNA as compared with [35S]methionine. Iodine labeling of thyroxine-binding globulin was also demonstrated in the translation product of Hep G2 RNA. Since the specific activity of the radioiodine is high and the means for detection of the isotope efficient, the method described can facilitate the demonstration of quantitatively minor translation products.

Various biosynthetic forms of rat alpha 1-antitrypsin (alpha 1AT) have been isolated by immunoprecipitation of in vitro and in vivo synthesized products. Rat alpha 1AT is synthesized in a rabbit reticulocyte system as a 45,000-Da preprotein with a 23-amino acid signal sequence. The majority of the amino acids in the signal sequence have been identified and resemble the signal peptides of other secretory proteins with respect to the abundance and positions of hydrophobic amino acids. Evidence from the translation of rat liver RNA in the presence of dog pancreas microsomes, from the translation of rat liver polysomes, and from tunicamycin-treated rat hepatocytes established that cleavage of the signal peptide of pre-alpha 1AT results in the formation of a 42,000-Da protein, the polypeptide backbone of mature alpha 1AT. A 50,000-Da glycoprotein is immunoprecipitated from translations programmed with rat liver microsomes or with rat liver mRNA and dog pancreas microsomes. Cotranslational glycosylation of alpha 1AT appears to occur in a stepwise fashion since three glycosylated forms of alpha 1AT (approximately 45,000, 47,000, and 50,000 Da) can be detected in polysome translations. These proteins are susceptible to cleavage by endo-beta-N-acetylglucosaminidase H and are digested to the same product, indicating that they have identical polypeptide chains. Two intracellular forms of alpha 1AT were detected in cultured rat hepatocytes, a 50,000- and a 52,000-Da protein; only the larger protein was immunoprecipitated from the medium of these cells. Digestion with endo-beta-N-acetylglucosaminidase H indicated that the 50,000-Da protein is a core glycosylated processing intermediate, whereas the 52,000-Da protein, which comigrated with purified serum alpha 1AT, appears to contain complex carbohydrate sidechains. When glycosylation was inhibited by incubation of hepatocytes with tunicamycin, a nonglycosylated 42,000-Da protein was immunoprecipitated from the cells and the culture medium, indicating that glycosylation of alpha 1AT is not essential for its secretion.

Utilizing a recently developed method (Boradeption) for transferring water-insoluble hydroxyorganoborane compounds into the cells, we observed inhibition of protein synthesis by three of these compounds and inhibition of secretion of plasma proteins by four of them in human hepatoma HepG2 cells. These effects were specific in that the cell viability was not affected and an increase in protein catabolism was not observed. Three compounds caused a compound-specific alterations in the electrophoretic mobility of secreted glycoproteins due to underlying changes in the N-linked carbohydrate moieties. Results presented suggest a potential new source of cellular probes.