Molecular structure of human fibroblast and leukocyte interferons: probe by lectin and hydrophobic chromatography.

Structural differences between human leukocyte virus-induced interferon and human fibroblast polyinosinic-polycytidylic acid (rIn-rCn)-induced interferon have been noted in previous studies. This study reports the behavior of human leukocyte and fibroblast interferon, induced by virus and by rIn-rCn, in several lectin and hydrophobic chromatographic systems. Differences in both glycosylation and in hydrophobicity of human leukocyte and fibroblast interferons are documented. Human fibroblast interferon is a glycoprotein, whereas our evidence suggests that human leukocyte interferon probably is not. Also, fibroblast interferon is more hydrophobic than leukocyte interferon, as probed on several hydrophobic adsorbents. The possible relationships of these differences to each other and to antigenic variations are discussed. Generally, the differences appear to be attributable to the cell type in which the interferon was induced. However, our results suggest that at least subtle differences in the processing of the induction signal (virus or rIn-rCn) within the same cell type may occur, slightly altering some structural features.     

Enzymatic modifications of human fibroblast and leukocyte interferons

The sensitivity of highly purified human fibroblast interferon and partially purified human leukocyte interferon to several proteolytic and glycolytic enzymes was determined with respect to antiviral activity, isoelectric point, molecular weight, and thermal stability. Leucine aminopeptidase altered the distribution of isoelectric points for both interferons but produced little change in molecular weights; this enzyme somewhat reduced the activity of only leukocyte interferon. Treatment of fibroblast interferon with carboxypeptidases A and B did not greatly decrease antiviral activity, but it did slightly reduce the molecular weight of the interferon and substantially altered the distribution of isoelectric point values; similar treatment of leukocyte interferon caused some loss in activity, especially of the 17,000-molecular-weight species. Both interferons were inactivated rapidly by treatment with the endoproteases trypsin, pepsin, bromelain, and subtilisin. Chymotrypsin shifted the isoelectric points of both interferons, but only leukocyte interferon was significantly inactivated. Treatment with neuraminidase and beta-galactosidase changed the isoelectric point distribution but did not affect the activity or thermal stability of either interferon; such a treatment reduced the molecular weight of fibroblast interferon and the size heterogeneity of leukocyte interferon. Treatment with neuraminidase and then leucine aminopeptidase greatly reduced the activity of both interferons, especially leukocyte interferon. The data indicate that biologically active forms of fibroblast and leukocyte interferons can be distinguished by their relative sensitivity to certain proteases.     

Chemical characterization of recombinant human leukocyte interferon A using fast atom bombardment mass spectrometry

Proteolytic digests of biologically active fractions of recombinant human leukocyte interferon A expressed in large quantities in Escherichia coli were analyzed by fast atom bombardment mass spectrometry and high-performance liquid chromatography. The values observed in the mass spectra of digests of the major fraction of recombinant human leukocyte interferon A with trypsin and Staphylococcus aureus protease V8 accounted for 93% of the amino acid sequences of human leukocyte interferon A predicted from the nucleotide sequence of the gene encoding the protein, indicating that the major fraction of recombinant human leukocyte interferon A was expressed with the same amino acid sequence as that translated from the nucleotide sequence of the gene encoding the protein. Mass spectrometry of proteolytic digests of two minor fractions of recombinant human leukocyte interferon A and mass and amino acid analyses of their high-performance liquid chromatography fractions showed that the amino group of the N-terminal amino acid residue of interferon was in part acetylated, and the Cys-1 and Cys-98 residues were oxidized to cysteic acid or linked to glutathione. These findings suggest that amino acid residues in recombinant proteins prepared in large quantities in E. coli are modified post-translationally.     

Human leukocyte interferon: Isolation and characterization of several molecular forms

Purification of human leukocyte interferon by high-performance liquid chromatography yielded eight major interferon species. These were characterized by their molecular weight, amino acid composition, and antiviral activity on human and bovine cells. The molecular weights of the purified species ranged from 16,000 to 21,000. The tryptic peptide profiles of all the pure species were compared. Both amino acid compositions and tryptic peptide profiles revealed a structural similarity among the various species.     

The use of multifunctional adsorbents to purify membrane-bound phosphatases from Zymomonas mobilis. Purification of acid phosphatase, alkaline phosphatase and ATPase.

The purification of detergent-solubilized membrane-bound phosphatases from Zymomonas mobilis using novel adsorbents is described. The prepared adsorbents have a hydrophobic core with functional groups attached. These functional groups may either increase or decrease the hydrophobicity of the adsorbent, or participate in other forms of interactions. Adsorption of acid phosphatase (ACP), alkaline phosphatase (ALP) and ATPase to these adsorbents was salt-promoted. Desorption was achieved by decreasing the salt concentration or by displacement with increasing concentration of Triton X-100. The results indicate that chromatography on multifunctional adsorbents that are predominantly hydrophobic in character is a procedure that can have a general applicability in purification of membrane proteins.     

Purification and characterization of human leukocyte interferon components.

Human leukocyte interferon, prepurified either by acid ethanol extraction or by affinity chromatography with antibodies, was further purified by gel filtration in the presence of sodium dodecyl sulfate. Interferon was eluted from gel filtration columns as an apparently homogeneous entity with a molecular weight of 26,600, resulting in an up to 50-fold additional purification during a single step. The antiviral activity could be further resolved into two components by hydroxylapatite adsorption chromatography. The isolated components (A and B) were distinguishable by isoelectric focusing and polyacrylamide gel electrophoresis. The apparent molecular weights were 20,000 to 16,000 and 16,000, respectively. No differences were detected in their susceptibility toward reduction of disulfide bonds by beta-mercaptoethanol. Both could be obtained on a preparative scale with minimal losses in biological activity.     

Binding of human fibroblast interferon to concanavalin A-agarose. Involvement of carbohydrate recognition and hydrophobic interaction.

Human fibroblast interferon binds to a concanavalin A-agarose (Con A-Sepharose) equilibrated with methyl alpha-D-mannopyranoside, or levan; in contrast, it is only partially retarded on a similar column equilibrated with ethylene glycol. Interferon does not bind, however, to a lectin column equilibrated with both methyl alpha-D-mannopyranoside and ethylene glycol. Thus, a hydrophobic interaction between fibroblast interferon and the immobilized lectin seems to account for a large portion of the binding forces involved. Other hydrophobic solutes, such as dioxane, 1, 2-propanediol, and tetraethylammonium chloride, were found equally or more efficient than ethylene glycol in displacing interferon from the lectin column. The elution pattern of interferon from a concanavalin A-agarose (Con A-Sepharose) column, at a constant ehtylene glycol concentration and with an increasing mannoside concentration, reveals the existence of four distinct interferon components. The selective adsorption to and elution from a concanavalin A-agarose (Con A-Sepharose) column resulted in about a 3000-fold purification of human fibroblast interferon and complete recovery of activity. The specific activity of the partially purified interferon preparation is about 5 X 10(7) units per mg of protein. The chromatographic behavior of human leukocyte interferon is remarkable in that it does not bind to concanavalin A-agarose at all indicating the absence of carbohydrate moieties recognizable by the lectin, or if present, their masked status. When concanavalin A was coupled to an agarose matrix (cyanogen bromide activated) at pH 8.0 and 6.0 human fibroblast interferon bound to both lectin-agarose adsorbents and could be recovered with methyl alpha-D-mannopyranoside. Concanavalin A, immobilized directly on agarose matrix at pH 8.0 and 6.0, thus displays only carbohydrate recognition toward interferon. By contrast, unless a hydrophobic solute was included in the solvent containing methyl mannoside, human fibroblast interferon could not be recovered from concanavalin A-agarose coupled at pH 9.0. When concanavalin A was immobilized via molecular arms, in tetrameric as well as dimeric forms, the binding of interferon again occurred exclusively through carbohydrate recognition. Thus, the hydrophobic interaction can be eliminated by appropriate immobilization of the lectin, and then adsorbed glycoproteins, as exemplified here by interferon, can be recovered readily with methyl mannoside alone.     

Separation of human leukocyte interferon components by concanavalin A-agarose affinity chromatography and their characterization.

Human leukocyte interferon (HL-IF), produced by mixed leukocytes infected with Newcastle disease virus, was resolved into three distinct fractions when chromatographed on concanavalin A-agarose. The major portion (70--75%) of interferon appeared in the breakthrough (BT fraction). The bound interferon (25--30%) was displaced from the column as two peaks: the first was eluted with 0.01 M methyl alpha-D-mannoside, yielding 15-20% of the interferon activity (alpha-MM fraction), and the second by including ethylene glycol (70%) in the eluant, yielding the remaining 5--15% of the interferon (EG fraction). No interferon was retained when HL-IF produced in the presence of glycosylation inhibitors (tunicamycin or 2-deoxy-D-glucose) was chromatographed on concanavalin A-agarose, suggesting that the fraction of interferon retained by this lectin is glycosylated. The three fractions of interferon (BT, alpha-MM, and EG) were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, cross-species antiviral activity, and neutralization by specific antisera. The BT fraction contains exclusively the 16 000 molecular weight component of human leukocyte interferon. The majority of the alpha-MM fraction (90%) is the 21 000 molecular weight component. However, the EG fraction contains the 16 000 and 21 000--23 000 molecular weight components in essentially equal proportions. On the basis of cross-species antiviral activity and neutralization by specific antisera, the BT and alpha-MM fractions are leukocyte-type interferon and the EG fraction seems to be primarily of fibroblast type.     

Some species of human leukocyte interferon are glycosylated

The carbohydrate content of all of the species of human leukocyte interferon (IFN-alpha) which have been derived from patients with chronic myelogeneous leukemia (CML) and purified to homogeneity has now been determined. Amino sugar content was measured by high-performance liquid chromatography and fluorescamine detection of acid hydrolysates of each sample. Two species showed significant amounts of glucosamine. Most of the purified species of leukocyte interferon from a myeloblast cell line were also tested, and two species were found to contain sugar residues. These forms also differed from the CML interferons in that they revealed the presence of greater amounts of galactosamine. The apparent lack of carbohydrate in some of the higher-molecular-weight species of interferon implicated factors other than glycosylation in the molecular weight differences. The results indicate that some species of IFN-alpha are glycosylated to various degrees.     

Molecular size heterogeneity of human leukocyte interferon

Molecular sieving of human leukocyte interferon revealed an apparent molecular weight of 26,000. However, after denaturation by guanidine hydrochloride in the presence of a reducing agent and reactivation by extensive dialysis, a molecular weight of only 21,000 was observed. The reactivated human leukocyte interferon (mol wt 21,000) gave a single peak of activity when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, confirming that a single molecular weight species was generated by the denaturation and reactivation procedure. A partial unfolding of the molecule was evident when the interferon preparation was heated to 50 degrees C in the absence or presence of an unfolding agent and then sieved on Sephadex G-100 Superfine. These results suggest that the interferon molecule undergoes a proteolytic cleavage probably by a protease present in extracellular fluid. Thus, a peptide fragment dissociates from the parent molecule when human leukocyte interferon is denatured in the presence of a reducing agent, resulting in a drop of 5,000 in molecular weight; interestingly, the resultant 21,000 molecular weight form still retains its antiviral activity.     

Purification and characterization of interferons from a continuous myeloblastic cell line

Several leukocyte interferon species have been purified from a continuous human myeloblast cell line. The purification procedure involving selective precipitations, gel chromatography, and several steps of high performance liquid chromatography results in interferons with specific activities of 1 to 4 X 10(8) units/mg on bovine MDBK cells. The total yield of interferon is 23%, with the yield of the individual fractions ranging from 0.2 to 11.4%. Five fractions are homogeneous as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Molecular weights of the interferons were estimated by mobility on the sodium dodecyl sulfate gels and range from 17,600 to 26,200. The species differ in their relative antiviral activities on two cell lines, bovine MDBK and human AG-1732. In addition, the pure species have similar, but distinct, amino acid compositions and tryptic peptide profiles. These result support the conclusion that leukocyte interferon consists of several homologous proteins.     

Human interferon and cell growth inhibition. III. Separation of activities by treatment with sodium dodecyl sulphate

When human leukocyte interferon was treated with boiling sodium dodecyl sulphate antiviral activity without detectable effect on the growth of human amnion cells could be separated from the growth inhibitory activity by a single gel filtration. Similar results were obtained with mouse L-cell interferon. It is concluded that the two effects of interferon can be separated in distinct molecular entities.     

Interference of a human leukocyte interferon preparation with stimulatory activities in leukocyte-conditioned medium for human hematopoietic stem cells

Medium conditioned by leukocytes in the presence of phytohemagglutinin (PHA-LCM) promotes the growth of multilineage hemopoietic progenitors derived from human bone marrow. However, PHA-LCM prepared in the presence of a human leukocyte interferon preparation does not support mixed colony formation. Crude PHA-LCM preparations were characterized by gel filtration, affinity chromatography, and gel electrophoresis. The elution profile on Sephacryl S-300 of PHA-LCM prepared without interferon showed a distinct peak that stimulated the growth of pluripotent stem cells (CFU-gemm) and committed precursors (CFU-c, BFU-e). Gel filtration of PHA-LCM, prepared with 1000 U/ml of interferon, revealed a change in the elution profile. The eluted material demonstrated no growth-promoting activities. We conclude that the abolished stimulatory activity of PHA-LCM, prepared with human leukocyte interferon, might be due to a reduced production of stimulatory molecules, suggesting that interferon interferes with the molecular events required for colony formation of committed and noncommitted hemopoietic progenitors.     

Optimizing dye-ligand density with molecular analysis for affinity chromatography of rabbit muscle L-lactate dehydrogenase

Ligand density is an important factor in determining the binding capacity and separation efficiency for affinity chromatography. A molecular analysis method based on the three-dimensional structure of protein and protein-ligand interactions was introduced to optimize the dye-ligand density for target protein separation. Expanded-bed adsorption (EBA) of L-lactate dehydrogenase (LDH) from rabbit muscle crude extract with Procion Red HE-3B as the dye-ligand was used as the model. After the analysis of LDH three-dimensional molecular structure and dye-protein interaction modes, the rational dye-ligand distance was predicted at about 20 A for efficiently binding LDH. A series of dye-ligand adsorbents with different ligand densities were prepared, and the isotherm adsorption equilibria of LDH were measured. High adsorption capacity of LDH was achieved at about 1600 U/mL adsorbent. Packed-bed chromatography was performed, and the elution effects were investigated. Finally, an EBA process was achieved to capture the LDH directly from rabbit muscle crude extract. The method established in the present work could be expanded to guide the screening of ligand density for other affinity chromatographic processes.     

Differential inactivation and separation of homologous and heterologous antiviral activity of human leukocyte interferon by a proteolytic enzyme.

Human leukocyte interferon (HuLeIF) can express its antiviral activity on both human and bovine cells. The rates of inactivation of HuLeIF by α-chymotrypsin, as expressed on human and bovine cells, are not the same: the ability to induce activity on human cells is lost significantly more rapidly than the activity detected on bovine cells; usually a margin of greater than one hundred-fold exists after α-chymotrypsin treatment. HuLeIF, when subjected to analysis on 10% SDS-PAGE, can be separated into two molecular weight species, one having apparent molecular weight of approximately 21,000 daltons, the other 18,000 daltons. A more rapidly migrating form (molecular weight 16,500 daltons) can also be isolated, which is considerably more active on bovine cells than on human cells. α-chymotrypsin-treated samples analyzed by SDS-PAGE show a clear separation of the activities expressed on human and bovine cells. The residual activity detected on human cells is isolated only in the 21,000 component while the activity found on bovine cells is recovered only as the 16,500 dalton species.     

Solubilization and hydrophobic immobilization assay of a cAMP binding protein from Dictyostelium discoideum plasma membranes

A cAMP binding site present on isolated plasma membranes of aggregation-competent $\text{D.}\text{discoideum}$ cells has been solubilized with the nonionic detergent Emulphogene BC-720. An assay has been developed based on the principle of hydrophobic chromatography, in which the detergent solubilized cAMP binding protein is immobilized on alkyl-agarose beads at low detergent concentration. This allows the necessary rapid separation of bound and free [³H]-cAMP by filtration of the beads. The kinetics and nucleotide specificity of the detergent solubilized cAMP binding protein are comparable to those of the cAMP chemotactic receptor on intact cells and plasma membranes. The alkyl-agarose bead assay may have general utility for the assay of detergent solubilized membrane receptors.     

Inhibition of macrophage migration by virus-induced interferon preparations.

It was found that a preparation of mouse L cell interferon induced by Newcastle disease virus (NDV) possessed not only interferon activity but also inhibitory activity upon migration of guinea pig peritoneal macrophages (MIF activity). These activities were also observed in a preparation of human leukocyte interferon induced by NDV. The interferon and MIF activities shared common characteristics in the dose response, time course of in vitro production, thermal stability, sensitivity to trypsin and periodate, and elution pattern in CM-Sephadex column chromatography. However, gel filtration pattern with Sephadex G-100 showed two separate peaks. Fractions collected from the first peak, corresponding to a molecular weight of about 45 000, had only the MIF activity, while those collected from the second peak, corresponding to a molecular weight of about 30 000, had both the interferon and MIF activities. A preparation of mouse brain interferon induced by Japanese encephalitis virus had a much weaker MIF activity than the L cell interferon, although these preparations were equal in interferon activity (5000 units/ml).     

Proteome wide evaluation of the separation ability of hydrophobic interaction chromatography by fluorescent dye binding analysis

Hydrophobic interaction chromatography (HIC) is an important tool in the industrial purification of proteins from various sources. The HIC separation behavior of individual (or model) proteins has been widely researched by others. On the contrary, this study focused on the fractionation ability of HIC when it is challenged with whole proteomes. The impact of the nature of three different proteomes, that is, yeast, soybean, and Chinese hamster ovary cells, on HIC separation was investigated. In doing so, chromatography fractions obtained under standardized conditions were evaluated in terms of their overall hydrophobicity—as measured by fluorescence dye binding. This technique allowed for the calculation of an average protein surface hydrophobicity (S₀) for each fraction; a unique correlation between S₀ and the observed chromatographic behavior was established in each case. Following a similar strategy, the effect of three different ligands (polypropylene glycol, phenyl, and butyl) and two adsorbent particle sizes (65 and 100 µm) on the chromatographic behavior of the yeast proteome was evaluated. As expected, the superficial hydrophobicity of the proteins eluted is correlated with the salt concentration of its corresponding elution step. The findings reveled how—and in which extent—the type of ligand and the size of the beads actually influenced the fractionation of the complex biological mixture. Summarizing, the approach presented here can be instrumental to the study of the performance of chromatography adsorbents under conditions close to industrial practice and to the development of downstream processing strategies. Copyright © 2013 John Wiley & Sons, Ltd.     

Inhibition of Macrophage Migration by Virus-Induced Interferon Preparations1

It was found that a preparation of mouse L cell interferon induced by Newcastle disease virus (NDV) possessed not only interferon activity but also inhibitory activity upon migration of guinea pig peritoneal macrophages (MIF activity). These activities were also observed in a preparation of human leukocyte interferon induced by NDV. The interferon and MIF activities shared common characteristics in the dose response, time course of in vitro production, thermal stability, sensitivity to trypsin and periodate, and elution pattern in CM-Sephadex column chromatography. However, gel filtration pattern with Sephadex G-100 showed two separate peaks. Fractions collected from the first peak, corresponding to a molecular weight of about 45 000, had only the MIF activity, while those collected from the second peak, corresponding to a molecular weight of about 30 000, had both the interferon and MIF activities. A preparation of mouse brain interferon induced by Japanese encephalitis virus had a much weaker MIF activity than the L cell interferon, although these preparations were equal in interferon activity (5000 units/ml).     

Molecular weight of the functional unit of human leukocyte, fibroblast, and immune interferons

There is good agreement between the target molecular weight and the known molecular weight of human leukocyte interferons (about 20,000). The target molecular weight of fibroblast interferon, 31,000 to 42,000, is significantly larger than the monomer molecular weight of 21,000 to 24,000, suggesting that the dimer may be the predominant active functional unit in solution. A range from 63,000 to 73,000 for the target molecular weight of several different fractions of immune interferon (including natural crude as well as the recombinant form) indicates that the functional form of the immune interferon may be a trimer or tetramer. Thus, these studies indicate that the functional unit of leukocyte interferon is the monomer, that of fibroblast interferon is a dimer, and that of immune interferon is probably a tetramer (or trimer).