Natural murine interferon-gamma. Evidence for post-translational proteolytic processing

Murine interferon-gamma (MuIFN-gamma), produced by the T-cell lymphoma, L12-R4, and stimulated with phorbol myristic acetate, was purified by rat monoclonal antibodies AN-18.17.24. The purified product retained its biologic activity and consisted of two proteins (Mr = 17,500 and 16,500), as determined by sodium dodecyl sulfate-gel electrophoresis. Both species were found to be glycosylated, since their Mr values decreased to 14,500 and 13,500 when the producing cells were stimulated in the presence of tunicamycin. Analysis of intracellular and secreted forms in the presence of [35S] methionine revealed that MuIFN-gamma is synthetized as a single peptide of Mr = 17,500 and undergoes proteolytic cleavage during or after secretion. Peptide mapping by reverse phase high pressure liquid chromatography showed indeed that the peptide profile of both species is very similar, suggesting a high degree of homology in the primary structure. These results demonstrate that the molecular heterogeneity of the MuIFN-gamma is probably the outcome of proteolytic processing during or after secretion.     

Human macrophage colony-stimulating factor heterogeneity results from alternative mRNA splicing, differential glycosylation, and proteolytic processing

The single gene for human macrophage colony-stimulating factor (M-CSF, or CSF-1) generates multiple mRNA species that diverge within the coding region. We have characterized translation products of these mRNA species from native and recombinant sources. Immunoblots of reduced native M-CSF indicate that multiple glycosylated species ranging from 25 kd to 200 kd are secreted by human monocytes and cell lines. In contrast, CV-1 cells expressing a short M-CSF clone secrete only 24 kd recombinant M-CSF. Synthetic peptide antibodies were developed to distinguish between secreted recombinant M-CSF from long and short mRNA splicing variants. Immunoblot analysis indicates that alternative mRNA splicing generates some M-CSF protein heterogeneity. Most secreted MIA PaCa-2 M-CSF reacts with long-clone-specific antibody. Lectin affinity chromatography shows that variable glycosylation contributes significantly to MIA PaCa-2 M-CSF size heterogeneity. In addition, cell lysates also contain larger M-CSF species that apparently undergo proteolytic processing before secretion. The data indicate that M-CSF protein heterogeneity results from both pre- and post-translational processing.     

Monitoring recombinant human interferon-gamma N-glycosylation during perfused fluidized-bed and stirred-tank batch culture of CHO cells

Chinese hamster ovary cells producing recombinant human interferon-gamma were cultivated for 500 h attached to macroporous microcarriers in a perfused, fluidized-bed bioreactor, reaching a maximum cell density in excess of 3 x 10(7) cells (mL microcarrier)-1 at a specific growth rate (mu) of 0.010 h-1. During establishment of the culture, the N-glycosylation of secreted recombinant IFN-gamma was monitored by capillary electrophoresis of intact IFN-gamma proteins and by HPLC analysis of released N-glycans. Rapid analysis of IFN-gamma by micellar electrokinetic capillary chromatography resolved the three glycosylation site occupancy variants of recombinant IFN-gamma (two Asn sites occupied, one Asn site occupied and nonglycosylated) in under 10 min per sample; the relative proportions of these variants remained constant during culture. Analysis of IFN-gamma by capillary isoelectric focusing resolved at least 11 differently sialylated glycoforms over a pI range of 3.4 to 6.4, enabling rapid quantitation of this important source of microheterogeneity. During perfusion culture the relative proportion of acidic IFN-gamma proteins increased after 210 h of culture, indicative of an increase in N-glycan sialylation. This was confirmed by cation-exchange HPLC analysis of released, fluorophore-labeled N-glycans, which showed an increase in the proportion of tri- and tetrasialylated N-glycans associated with IFN-gamma during culture, with a concomitant decrease in the proportion of monosialylated and neutral N-glycans. Comparative analyses of IFN-gamma produced by CHO cells in stirred-tank culture showed that N-glycan sialylation was stable until late in culture, when a decline in sialylation coincided with the onset of cell death and lysis. This study demonstrates that different modes of capillary electrophoresis can be employed to rapidly and quantitatively monitor the main sources of glycoprotein variation, and that the culture system and operation may influence the glycosylation of a recombinant glycoprotein.     

N-glycans of recombinant human interferon-gamma change during batch culture of chinese hamster ovary cells

A recombinant Chinese hamster ovary (CHO) cell line making human interfron-gamma (IFN-gamma) was grown in 12-L stirred tank fermentors in three batch fermentations under conditions of constant temperature, pH, and dissolved oxygen tension. In addition to cell growth, metabolite, and productivity data, a detailed analysis of the carbohydrate structures attached to each glycosylation site of IFN-gamma was achieved using matrix-assisted laser desorption mass spectrometry (MALDI-MS) in combination with exoglycosidase array sequencing. Complex biantennary oligosaccharides (particularly Gal(2)GlcNAc(4)Man(3) which was core alephl-6 fucosylated at Asn(25) but not at Asng(97)) were most prevalent at both glycosylation sites. However, considerable microheterogeneity arising from the presence of triantennary and truncated glycan structures was also observed. The proportion of the dominant core glycan structure (Gal(2)GlcNAc(4)Man(3) +/- Fuc(1)) decreased by 15-26% during batch culture, with increases in the proportion of oligomannose and truncated glycans over the same time period. Prolonged culture resulting from an extended lag phase led to further accumulation of oligomannose and truncated structures, reaching up to 52% of total glycans attached to Asng(97) by 240 h of culture. The implications of these glycosylation changes for optimizing the time for harvesting cell cultures, and for the clearance of recombinant therapeutic products in vivo are discussed. (c) 1995 John Wiley & Sons, Inc.     

Differences in glycosylation pattern of human secretory ribonucleases.

The major secretory ribonuclease (RNase) of human urine (RNase HUA) was isolated and sequenced by automatic Edman degradation and analysis of peptides and glycopeptides. The isolated enzyme was shown to be free of other urine RNase activities by SDS/polyacrylamide-gel electrophoresis and activity staining. It is a glycoprotein 128 amino acids long, differing from human pancreatic RNase in the presence of an additional threonine residue at the C-terminus. It differs from the pancreatic enzyme in its glycosylation pattern as well, and contains about 45 sugar residues. Each of the three Asn-Xaa-Ser/Thr sequences (Asn-34, Asn-76, Asn-88) is glycosylated with a complex-type oligosaccharide chain. Glycosylation at Asn-88 has not been observed previously in mammalian secretory RNases. Preliminary sequence data on the major RNase of human seminal plasma have revealed no difference between it and the major urinary enzyme; their similarities include the presence of threonine at the C-terminus. The glycosylation pattern of human seminal RNase is very similar to that of the pancreatic enzyme. The structural differences between the secretory RNases from human pancreas, urine and seminal plasma must originate from organ-specific post-translational modifications of the one primary gene product. Detailed characterization of peptides and the results of gel filtration of tryptic and tryptic/chymotryptic digests of performic acid-oxidized RNase have been deposited as Supplementary Publication SUP 50146 (4 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1988) 249, 5.     

Natural human interferon-gamma. Complete amino acid sequence and determination of sites of glycosylation

Fresh human peripheral blood lymphocytes were induced with desacetylthymosin -alpha 1 and staphylococcal enterotoxin B. The induced gamma interferon (or IFN-gamma, immune interferon, type II interferon) was purified to homogeneity utilizing controlled-pore glass, concanavalin A-Sepharose, Bio-Gel P100, or Sephacryl S-200, and reversed phase high performance liquid chromatography. This procedure resulted in two active species with apparent Mr = 20,000 and 25,000 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both species were found to have identical amino acid sequences with a pyroglutamate residue as NH2-terminus. In both cases six different COOH termini were found. They are, at least qualitatively, identical in both species. There are two possible Asn-X-Ser/Thr glycosylation sites. Both carry carbohydrates in the Mr = 25,000 species whereas in the Mr = 20,000 species only one site is glycosylated. This likely explains the difference in apparent molecular weight between the two species and the expected molecular weight based upon the amino acid sequence.     

Biosynthetic analysis of the human interferon-gamma receptor. Identification of N-linked glycosylation intermediates

Biosynthesis of the human IFN gamma receptor was studied using metabolic labeling techniques and immunoprecipitation with receptor-specific monoclonal antibodies. Colo-205 and HepG2 cells labeled with [35S]methionine gave rise to two components with molecular mass 75 and 90 kDa following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. No bands were detected when immunoprecipitation was performed using irrelevant monoclonal IgG or in the presence of excess ligand, a condition known to block antibody-receptor interaction. When Colo-205 were labeled for increasing periods of time, the 75-kDa form was detected after 5 min, whereas the 90-kDa form appeared only after 60 min. Pulse-chase analysis established that the 75-kDa form was the precursor of the 90-kDa component. Only the 90-kDa form was detected on extrinsically radioiodinated Colo-205 cell surfaces. This observation was confirmed by Western blot analysis of isolated Colo-205 membranes. Digestion of labeled precipitates with peptide:N-glycosidase F caused a 22% reduction in the apparent molecular weight of the IFN gamma receptor. Receptor derived from tunicamycin-treated Colo-205 labeled for 5 min displayed a single molecular mass of 65 kDa and expressed ligand binding activity. Longer labeling periods in the presence of tunicamycin revealed the appearance of a second ligand-binding form of 70 kDa. Thus, Colo-205 IFN gamma receptors carry asparagine (N)-linked oligosaccharides and possibly some other form of post-translational modification.     

Posttranslational processing of recombinant human interferon-gamma in animal expression systems.

We have characterized the heterogeneity of recombinant human interferon-gamma (IFN-gamma) produced by three expression systems: Chinese hamster ovary cells, the mammary gland of transgenic mice, and baculovirus-infected Spodopera frugiperda (Sf9) insect cells. Analyses of whole IFN-gamma proteins by electrospray ionization-mass spectrometry (ESI-MS) from each recombinant source revealed heterogeneous populations of IFN-gamma molecules resulting from variations in N-glycosylation and C-terminal polypeptide cleavages. A series of more specific analyses assisted interpretation of maximum entropy deconvoluted ESI-mass spectra of whole IFN-gamma proteins; MALDI-MS analyses of released, desialylated N-glycans and of deglycosylated IFN-gamma polypeptides were combined with analyses of 2-aminobenzamide labeled sialylated N-glycans by cation-exchange high-performance liquid chromatography. These analyses enabled identification of specific polypeptide cleavage sites and characterization of associated N-glycans. Production of recombinant IFN-gamma in the mammalian expression systems yielded polypeptides C-terminally truncated at dibasic amino acid sites. Mammalian cell derived IFN-gamma molecules displayed oligosaccharides with monosaccharide compositions equivalent to complex, sialylated, or high-mannose type N-glycans. In contrast, IFN-gamma derived from baculovirus-infected Sf9 insect cells was truncated further toward the C-terminus and was associated with neutral (nonsialylated) N-glycans. These data demonstrate the profound influence of host cell type on posttranslational processing of recombinant proteins produced in eukaryotic systems.     

Metabolic effects on recombinant interferon-gamma glycosylation in continuous culture of Chinese hamster ovary cells

Asparagine linked (N-linked) glycosylation is an important modification of recombinant proteins, because the attached oligosaccharide chains can significantly alter protein properties. Potential glycosylation sites are not always occupied with oligosaccharide, and site occupancy can change with the culture environment. To investigate the relationship between metabolism and glycosylation site occupancy, we studied the glycosylation of recombinant human interferon-gamma (IFN-gamma) produced in continuous culture of Chinese hamster ovary cells. Intracellular nucleotide sugar levels and IFN-gamma glycosylation were measured at different steady states which were characterized by central carbon metabolic fluxes estimated by material balances and extracellular metabolite rate measurements. Although site occupancy varied over a rather narrow range, we found that differences correlated with the intracellular pool of UDP-N-acetylglucosamine + UDP-N-acetylgalactosamine (UDP-GNAc). Measured nucleotide levels and estimates of central carbon metabolic fluxes point to UTP depletion as the cause of decreased UDP-GNAc during glucose limitation. Glucose limited cells preferentially utilized available carbon for energy production, causing reduced nucleotide biosynthesis. Lower nucleoside triphosphate pools in turn led to lower nucleotide sugar pools and reduced glycosylation site occupancy. Subsequent experiments in batch and fed-batch culture have confirmed that UDP-sugar concentrations are correlated with UTP levels in the absence of glutamine limitation. Glutamine limitation appears to influence glycosylation by reducing amino sugar formation and hence UDP-GNAc concentration. The influence of nucleotide sugars on site occupancy may only be important during periods of extreme starvation, since relatively large changes in nucleotide sugar pools led to only minor changes in glycosylation.     

Biosynthesis of the v-sis gene product: signal sequence cleavage, glycosylation, and proteolytic processing.

The v-sis oncogene and its cellular homolog c-sis encode chain B of platelet-derived growth factor. Cells transformed by v-sis produce a platelet-derived growth factor-related molecule which is able to stimulate the platelet-derived growth factor receptor in an autocrine fashion. Site-directed mutagenesis was used to construct several mutations which substitute charged residues for hydrophobic residues in the proposed signal sequence of the v-sis gene product. Two of these mutations resulted in the synthesis of altered v-sis gene products with an unexpected nuclear location and a loss of biological activity. We also report here the intracellular localization of the v-sis gene product to the endoplasmic reticulum-Golgi compartment, where signal sequence cleavage and N-linked glycosylation occur. The v-sis gene product contains no transmembrane regions, as it is completely protected within isolated microsomes from trypsin proteolysis. Site-directed mutagenesis was also used to alter a proposed proteolytic processing site in the v-sis gene product. This mutant v-sis gene, which encodes Asn-Ser in place of Lys-Arg at residues 110 to 111, was found to retain full biological activity.     

Effects of weakly basic amines on proteolytic processing and terminal glycosylation of secretory proteins in cultured rat hepatocytes.

We examined the effects of weakly basic amines on the secretion and post-translational modifications of secretory proteins in cultured rat hepatocytes. Weakly basic amines such as methylamine, chloroquine and NH4Cl strongly inhibited not only protein secretion, but also the proteolytic conversion of a proform of complement C3, allowing the precursor to be released into the medium. The amines, however, had no effect on the proteolytic conversion of prohaptoglobin into its subunits. Since available evidence indicates that the conversion of pro-C3 occurs at the Golgi complex while that of prohaptoglobin takes place in the endoplasmic reticulum, it is most likely that the weak bases specifically affect the proteolytic event occurring at the Golgi complex. Electron microscopic observations confirmed that the amines caused morphological changes of the Golgi complex, consisting of dilated cisternae and swollen vacuoles. When the glycosylation of alpha 1-protease inhibitor and haptoglobin was examined, it was found that the amines caused a marked accumulation in the cells of both glycoproteins corresponding to the mature secreted forms. Neuraminidase digestion demonstrated that the glycoproteins accumulating in response to the amines had acquired terminal sialic acid. The results indicate that the amines do not significantly affect terminal glycosylation, in contrast with their definite effect on proteolytic processing, despite the fact that both modifications take place in the Golgi complex.     

Components and proteolytic processing sites of arylsulfatase B from human placenta.

Previous studies have shown that mature arylsulfatase B purified from human sources is composed of two non-identical chains with apparent molecular masses of 43 kDa and 8 kDa. Arylsulfatase B purified from human placenta in the present study, however, included another 7 kDa component that could be detected only by carbohydrate staining on reducing SDS-PAGE employing the Tris-Tricine system. The 43 kDa and 7 kDa components contained a carbohydrate moiety, but the 8 kDa one did not, as demonstrated by periodic acid-Schiff staining, Con-A lectin blotting, endo-glycosidase treatment and in vitro phosphorylation by UDP-N-acetylglucosamine: lysosomal enzyme N-acetylglucosamine 1-phosphotransferase. The purified arylsulfatase B migrated as a single polypeptide of 58 kDa on non-reducing SDS-PAGE, indicating that the three chains are linked by disulfide bonds. In order to determine the origin of the components, N-terminal sequencing of the isolated polypeptides was performed. As a result, the 43, 7 and 8 kDa components were found to commence with Ala-41, Ala-424 and Asp-466, respectively. These results suggest that after removal of the signal peptide, human arylsulfatase B undergoes proteolytic processing on at least two sites during maturation.     

Recombinant human laminin-5 domains. Effects of heterotrimerization, proteolytic processing, and N-glycosylation on alpha3beta1 integrin binding

Human laminin-5 fragments, comprising the heterotrimeric C-terminal part of the coiled-coil (CC) domain and the globular (G) domain with defined numbers of LG subdomains, were produced recombinantly. The alpha3' chain with all five LG subdomains was processed proteolytically in a manner similar to the wild-type alpha3 chain. Conditions were established under which the proteolytic cleavage was either inhibited in cell culture or was brought to completion in vitro. The shorter chains of the laminin-5CCG molecule, beta3'and gamma2', produced in a bacterial expression system associated into heterodimers, which then combined spontaneously with the alpha3' chains in vitro to form heterotrimeric laminin-5CCG molecules. Only heterotrimeric laminin-5CCG with at least subdomains LG1-3, but not the single chains, supported binding of soluble alpha3beta1 integrin, proving the coiled-coil domain of laminin-5 to be essential for its interaction with alpha3beta1 integrin. The N-glycosylation sites in wild-type alpha3 chain were mapped by mass spectrometry. Their location in a structural model of the LG domain suggested that large regions on both faces of the LG1 and LG2 domains are inaccessible by other proteins. However, neither heterotrimerization nor alpha3beta1 integrin binding was affected by the loss of N-linked glycoconjugates. After the proteolytic cleavage between the subdomains LG3 and LG4, the LG4-5 tandem domain dissociated from the rest of the G domain. Further, the laminin-5CCG molecule with the alpha3'LG1-3 chain showed an increased binding affinity for alpha3beta1 integrin, indicating that proteolytic processing of laminin-5 influences its interaction with alpha3beta1 integrin.     

Biosynthesis of rice seed alpha-amylase: proteolytic processing and glycosylation of precursor polypeptides by microsomes

Microsomes prepared from the rice seed scutellum were incubated in wheat germ extracts (S-100 fraction) to direct the synthesis of alpha- amylase, a secretory protein subject to proteolytic processing (cleavage of the N-terminal signal sequence) as well as glycosylation during its biosynthesis. The characterization and identification of the immunoprecipitable products synthesized were performed by SDS gel electrophoresis and subsequent fluorography. The molecular weight of the alpha-amylase synthesized by the microsomes was found to be identical with that of the mature secretory form of the enzyme on the basis of electrophoretic mobilities. A significant portion of the enzyme molecules synthesized was shown to be segregated into the microsomal vesicles and protected against digestion by endo-beta-N- acetylglucosaminidase, indicating that both proteolytic processing and glycosylation of the precursor polypeptide chains take place in the microsomes. The modification of the polypeptide chains was further examined by disrupting the microsomal membranes with Triton X-100. Detergent treatment of the microsomes prior to protein synthesis caused an inhibition of both proteolytic processing and glycosylation of the polypeptide chains, leading to the synthesis of the unprocessed nascent (precursor I), processed but nonglycosylated nascent (precursor II) forms, in addition to the mature form of alpha-amylase. Furthermore, the results of time-sequence analysis of the inhibitory effect of Triton X-100 on the modification of the polypeptide chains have led us to conclude that both proteolytic processing and subsequent glycosylation occur in the microsomes during the biosynthesis of alpha- amylase.     

Oligosaccharide processing at individual glycosylation sites on MOPC 104E immunoglobulin M. Differences in alpha 1,2-linked mannose processing

Processing of the asparagine-linked oligosaccharides at the known glycosylation sites on the mu-chain of IgM secreted by MOPC 104E murine plasmacytoma cells was investigated. Oligosaccharides present on intracellular mu-chain precursors were of the high mannose type, remaining susceptible to endo-beta-N-acetylglucosaminidase H. However, only 26% of the radioactivity was released from [3H]mannose-labeled secreted IgM glycopeptides, consistent with the presence of high mannose-type and complex-type oligosaccharides on the mature mu-chain. [3H]Mannose-labeled cyanogen bromide glycopeptides derived from mu-chains of secreted IgM were isolated and analyzed to identify the glycopeptide containing the high mannose-type oligosaccharide from those containing complex-type structures. [3H]Mannose-labeled intracellular mu-chain cyanogen bromide glycopeptides corresponding to those from secreted IgM were isolated also, and the time courses of oligosaccharide processing at the individual glycosylation sites were determined. The major oligosaccharides on all intracellular mu-chain glycopeptides after 20 min of pulse labeling with [3H]mannose were identified as Man8GlcNAc2, Man9GlcNAc2, and Glc1Man9GlcNAc2. Processing of the oligosaccharide destined to become the high mannose-type structure on the mature protein was rapid. After 30 min of chase incubation the predominant structures of this oligosaccharide were Man5GlcNAc2 and Man6GlcNAc2 which were also identified on the high mannose-type oligosaccharide of the secreted mu-chain. In contrast, processing of oligosaccharides destined to become complex type was considerably slower. Even after 180 min of chase incubation, Man7GlcNAc2 and Man8GlcNAc2 were the predominant structures at some of these glycosylation sites. The isomeric structures of Man8GlcNAc2 obtained from all of the glycosylation sites were identical. Thus, the different rates of processing were not the result of a different sequence of alpha 1,2-mannose removal.     

Glycation and post-translational processing of human interferon-gamma expressed in Escherichia coli

Until recently, nonenzymatic glycosylation (glycation) was thought to affect the proteins of long living eukaryotes only. However, in a recent study (Mironova, R., Niwa, T., Hayashi, H., Dimitrova, R., and Ivanov, I. (2001) Mol. Microbiol. 39, 1061-1068), we have shown that glycation takes place in Escherichia coli as well. In the present study, we demonstrate that the post-translational processing (proteolysis and covalent dimerization) observed with cysteineless recombinant human interferon-gamma (rhIFN-gamma) is tightly associated with its in vivo glycation. Our results show that, at the time of isolation, rhIFN-gamma contained early (but not advanced) glycation products. Using reverse phase high performance liquid chromatography in conjunction with fluorescence measurements, enzyme-linked immunosorbent assay, and mass spectrometry, we found that advanced glycation end products arose in rhIFN-gamma during storage. The latter were identified mainly in the Arg/Lys-rich C terminus of the protein, which was also the main target of proteolysis. Mass spectral analysis and N-terminal sequencing revealed four major (Arg140/Arg141, Phe137/Arg138, Met135/Leu136, and Lys131/Arg132) and two minor (Lys109/Ala110 and Arg90/Asp91) cleavage sites in this region. Tryptic peptide mapping indicated that the covalent dimers of rhIFN-gamma originating during storage were formed mainly by lateral cross-linking of the monomer subunits. Antiviral assay showed that proteolysis lowered the antiviral activity of rhIFN-gamma, whereas covalent dimerization completely abolished it.     

Influence of intracellular nucleotide and nucleotide sugar contents on recombinant interferon-gamma glycosylation during batch and fed-batch cultures of CHO cells

Both the macroheterogeneity of recombinant human IFN-gamma produced by CHO cells and intracellular levels of nucleotides and sugar nucleotides, have been characterized during batch and fed-batch cultures carried out in different media. Whereas PF-BDM medium was capable to maintain a high percentage of the doubly- glycosylated glycoforms all over the process, mono-glycosylated and non-glycosylated forms increased during the batch culture using SF-RPMI medium. Intracellular level of UTP was higher in PF-BDM all over the batch culture compared to the SF-RPMI process. UDP-Gal accumulated only during the culture performed in PF-BDM medium, probably as a consequence of the reduced UDP-Glc synthesis flux in SF-RPMI medium. When the recombinant CHO cells were cultivated in fed-batch mode, the UTP level remained at a relatively high value in serum-containing RPMI and its titer increased during the fed-phase indicating an excess of biosynthesis. Besides, an accumulation of UDP-Gal occurred as well. Those results all together indicate that UTP and UDP-Glc syntheses in CHO cells cultivated in SF-RPMI medium in batch process, could be limiting during the glycosylation processes of the recombinant IFN-gamma. At last, the determination of the energetic status of the cells over the three studied processes suggested that a relationship between the adenylate energy charge and the glycosylation macroheterogeneity of the recombinant IFN-gamma may exist.