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INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA

The Function of the Human Interferon-β1a Glycan Determined in Vivo

Medical Biotechnology Center, University of Southern Denmark, Odense C, Denmark (L.D.-O., M.M., B.F.); and Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark (M.T.-A., P.H.)

Received February 20, 2008; accepted April 28, 2008.

	Abstract

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Recombinant human interferon-β (rhIFN-β) is the leading therapeutic intervention shown to change the cause of relapsing-remitting multiple sclerosis, and both a nonglycosylated and a significantly more active glycosylated variant of rhIFN-β are used in treatment. This study investigates the function of the rhIFN-β1a glycan moiety and its individual carbohydrate residues, using the myxovirus resistance (Mx) mRNA as a biomarker in Mx-congenic mice. We showed that the Mx mRNA level in blood leukocytes peaked 3 h after s.c. administration of rhIFN-β1a. In addition, a clear dose-response relationship was confirmed, and the Mx response was shown to be receptor-mediated. Using specific glycosidases, different glycosylation analogs of rhIFN-β1a were obtained, and their activities were determined. The glycosylated rhIFN-β1a showed significantly higher activity than its deglycosylated counterpart, due to a protein stabilization/solubilization effect of the glycan. It is interesting to note that the terminating sialic acids were essential for these effects. Conclusively, the structure/bioactivity relationship of rhIFN-β1a was determined in vivo, and it provided a novel insight into the role of the rhIFN-β1a glycan and its carbohydrate residues. The possibilities of improving the pharmacological properties of rhIFN-β1a using glycoengineering are discussed.

Human IFN-β1a is a glycoprotein comprising 166 amino acid residues in its matured form (Derynck et al., 1980). It contains a single N-glycosylation site (Asn80), and the glycan structures of the protein from different sources have been determined. For example, low heterogeneity of the glycans of Chinese hamster ovary cell-derived rhIFN-β1a has been reported, and 95% of the structures were found to be biantennary complex glycans containing terminating galactose sialylation and core fucosylation with the overall carbohydrate residue composition NeuAc₂Gal₂Man₃GlcNAc₄Fuc₁ (Conradt et al., 1987). In addition, the glycans of Chinese hamster ovary cell-derived IFN-β show high similarity to the naturally occurring human (Kagawa et al., 1988) and murine (Civas et al., 1988) counterpart. Protein-conjugated carbohydrates are increasingly being recognized as functional modulators of gene products or by exhibiting independent functions affecting the stability, solubility, clearance, and receptor-binding properties of proteins (Oh-eda et al., 1990; Kodama et al., 1993; Logsdon et al., 2004). Although, the IFN-β1a glycan has been hypothesized to be involved in receptor binding (Civas et al., 1988), it is generally accepted that the carbohydrate moiety is involved in solubility and stability of the protein. This was supported by a 10 times higher antiviral activity of the glycosylated rhIFN-β1a compared with the nonglycosylated variant as well as altered physicochemical properties, e.g., increased propensity of aggregation of the deglycosylated rhIFN-β1a (Watanabe and Kawade, 1983; Conradt et al., 1987; Runkel et al., 1998).

Based on the fact that the bioactivity of rhIFN-β1a is a critical parameter in the treatment of RRMS and to a large extent seems to depend on the presence of a conjugated glycan moiety, we determined the structure/bioactivity relationship to further investigate the carbohydrate role of rhIFN-β1a. After a careful validation of the Mx biomarker, native rhIFN-β1a and glycosylated analogs were monitored for activity. We present in vivo data confirming that the glycan moiety of rhIFN-β1a is crucial for its bioactivity by exerting protein stabilization/solubilization. The major contributing factor for these effects could be pinpointed to the terminating sialic acid residues.

	Materials and Methods

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Experimental Animals. This study was performed using adult Mx-congenic mice on a C57BL/6 background (breeding couples were kindly donated by Drs. Otter Haller and Dr. Peter Staeheli, University of Freiburg, Freiburg, Germany) and adult Mx-congenic IFNAR-knockout (ko) mice (provided by Dr. Peter Staeheli). The C57BL/6 Mx⁺ mice were bred at the Biomedical Laboratory at the University of Southern Denmark, and the Mx⁺IFNAR-ko mice were bred at the department of Virology and Microbiology at the University of Freiburg. The mice were cared for in accordance with guidelines of the Danish National Animal Care Committee, thereby meeting international guidelines on the ethical use of animals. Commonly used inbred mouse strains such as C57BL/6 and SJL carry a variant allele of the Mx1 gene, and these strains are phenotypically Mx^– (Staeheli et al., 1988). Wild mice and mice of the strain A2G have a functional Mx1 locus (Haller et al., 1987). The functional Mx gene was introduced into the C57BL/6 mice by crossing C57BL/6 with A2G mice (genotype Mx⁺/Mx⁺) and continuing backcrossing the Mx⁺ offspring with C57BL/6 until the 11th backcross generation was reached (Horisberger et al., 1983).

IFN-β. Rebif (rhIFN-β1a, sponsored by Serono Nordic/Merck Serono, Hellerup, Denmark) was used to validate the Mx marker. Several protein chemical approaches were performed to purify rhIFN-β1a from the rhIFN-β1a formulation containing high concentrations of mannitol and human serum albumin (HSA), e.g., size exclusion chromatography, and HSA-affinity depletion. However, this was not accomplished due to the high concentration difference (the rhIFN-β1a/HSA concentration ratio was 1:100) and the adhesive nature of HSA. Hence, commercially available rhIFN-β1a (98% pure; R&D Systems, Minneapolis, MN) was used for the determination of the glycostructure/bioactivity relationship. In addition, rhIFN-β1a, in a new stabilizing formulation containing a high concentration of the surfactant poloxamer 188 (polyethylene-polypropylene glycol), was used to study the kinetics of the desialylated analog.

IFN-β Administration. Both rhIFN-β1a and the commercially available rhIFN-β1a were diluted in sterile phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 1.4 mM KH₂PO₄, and 8 mM Na₂HPO₄, pH 7.4) and injected s.c. (0.5 ml). HSA (Sigma-Aldrich, St. Louis, MO) was added to the glycosidase-trimmed rhIFN-β1a variants (4 µg/ml) before injection. Several controls were performed: 1) a vehicle control, where vehicle solution was administrated exactly as rhIFN-β1a in a dose corresponding to 100,000 IU; 2) a control for endogenous production of type I and III IFNs, where mice were treated with activity-depleted (denatured) rhIFN-β1a; and 3) a control using IFNAR-ko mice to show that the response was receptor mediated.

Blood Sampling. Screening for IFN-β-induced transcription of the Mx gene was performed by collecting blood before injection and every 3 h within the first 24 h posttreatment (hpt) using 100,000 IU rhIFN-β1a (n = 2). Three blood samples were collected by eye vein puncture (using EDTA-coded pipettes; Danotherm Electric, Rødovre, Denmark) from each mouse, the last with no postsurvival. The eye vein puncture was performed by a well trained animal-technician, and the blood volume collected from each mouse was below 10% of the total blood volume of the mouse. Based on the results of this initial screen, the Mx level was determined for each hour within the first 6 hpt with 100,000 IU rhIFN-β1a.

Real-Time PCR. RNA purification from whole blood was performed using a Versagene RNA blood kit (Gentra Systems, Inc., Minneapolis, MN). The purification was initiated within 10 min after collections and performed according to the manufacturer's instructions. The RNA was eluted in diethylpyrocarbonate-treated water, which was used throughout the experiments. As determined by means of optic density using a GeneQuant Pro (GE Healthcare, Chalfont St. Giles, UK), 250 ng of RNA was mixed with 300 ng of random hexamer primers (Roche, Basel, Switzerland), and 0.5 mM/nucleotide was added (final concentration). After incubation for 5 min at 65°C, the sample was cooled on ice. Subsequently, 50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl₂, and 10 nM dithiothreitol were added (final concentrations), and the mixture was incubated for 10 min at 25°C. Next, 200 units of Moloney murine leukemia virus were added, and the sample was incubated for 50 min at 37°C followed by termination for 15 min at 70°C. Microtiter plates (96 wells) were used, and the reactions were detected real-time using SYBR Green dye-based detection on an iCycler (Bio-Rad, Milpitas, CA). The primers were designed using Beacon Designer (BioSoft International, Palo Alto, CA) so that one primer for each transcript spanned over an exon-exon junction. The concentration of Mg²⁺ was adjusted for each primer design [2 mM for the Mx system and 2.5 mM for the hypoxanthine phosphoribosyltransferase 1 (HPRT1) system]. Specifically, the following primers were used: Mx sense, 5'-TCA GTT TCC TCA AAA GGG GTT GAC-3'; Mx antisense, 5'-AAT ATT CCG TCT GCA CTC CTG GTA-3'; HPRT1 sense, 5'-GTT AAG CAG TAC AGC CCC AAA ATG-3'; and HPRT1 antisense, 5'-AAA TCC AAC AAA GTC TGG CCT GTA-3'. cDNA was mixed with 50% RealQ RT-PCR Master Mix (Ampliqon/Bie and Berntsen, Herlev, Denmark) and 0.3 µM of each primer and SYBR Green (diluted according to the manufacturer) (Lonza Copenhagen ApS, Vallensbaek Strand, Denmark), and 10 nM fluorescein (Bio-Rad) were added to a final volume of 25 µl in each well. The RealQ RT-PCR Master Mix contained hot start DNA polymerase, deoxynucleotide triphosphates, and 1.5 mM MgCl₂, requiring additional MgCl₂ to be added. A three-step protocol was used for the reactions, initiated by a heating step at 95°C for 15 min followed by 35 cycles of a 95°C step for 10 s, a 60°C step for 20 s, and finally a 72°C step for 30 s. Standard series were included on all plates, and all samples where run as triplets. The starting quantity (SQ), based on a standard curve (4 points), for each sample was calculated by the average of each triplet. Data are presented as the ratio between the SQ of the target transcript (Mx) and the SQ of the reference transcript HPRT1. To verify that the designed primers reacted specifically with the target transcript, melting point detection and gel electrophoresis were used to show that PCR gave rise to a single product. The melting point was established by raising the temperature of the PCR products to 95°C for 1 min and then cooling it down to 55°C followed by 80 cycles of 10 s each in which the temperature was raised 0.5°C per cycle. In addition, the PCR products were run on a 3% NuSieve (Cambrex Bio Science) agarose gel with 0.3x Tris-acetate EDTA buffer together with a 50 base pair (bp) and a 100-bp DNA ladder. Furthermore, real-time PCR of 18S was used to validate the consistency of HPRT1 transcription. A sample with a higher concentration of target mRNA than the experimental samples was chosen for generating the standard curve, and it was diluted to a concentration lower than any of the experimental samples (5x dilution series).

Denaturation of rhIFN-β1a. Two micrograms of rhIFN-β1a was dissolved in 20 µl of alkylation buffer containing 6 M guanidine HCl, 30 mM Tris, and 1 mM EDTA. 1,4-Dithiothreitol was added to a final concentration of 50 mM. This mixture was incubated at 56°C for 45 min. Iodoacetamide was added to a final concentration of 100 mM, and the mixture was placed in the dark for 45 min. Salts and other reagents were removed by washing the samples three times in H₂O using a spin column with a 3-kDa molecular mass cutoff (Vivascience, Hannover, Germany). After the final washing step, a minor fraction was kept for protein mass determination, and the remainder was used immediately for bioactivity determination.

Carbohydrate Trimming. In a set of parallel experiments, different glycosidases were used to trim the carbohydrate moiety of rhIFN-β1a, generating different glycosylation analogs. In particular, rhIFN-β1a was treated with the following: 1) fucosidase, 2) sialidase, and 3) PNGase F. To perform the defucosylation experiment (1), 3 µg of rhIFN-β1a was dissolved in 30 µl of 20 mM sodium citrate phosphate buffer, pH 6.0, containing 20 mU of -fucosidase (bovine kidney) (Prozyme, San Leandro, CA). This mixture was incubated for 48 h at 37°C. For the desialylation (2), 3 µg of rhIFN-β was dissolved in 30 µl of 50 mM Na₂HPO₄, pH 6.0, and 8 mU sialidase A (Arthrobacter ureafaciens) (Prozyme) was added. This mixture was incubated for 2 or 48 h (individual experiments) at 37°C. Finally, for the deglycosylation experiment (3), 4 µg of rhIFN-β was dissolved in 40 µl of 20 mM Na₂HPO₄, pH 7.2. Subsequently, 4 U PNGase F (Flavobacterium meningosepticum) (Roche, Mannheim, Germany) was added, and the mixture was incubated at 37°C for 18, 48, or 72 h. An additional 4 U PNGase F was added for every 24 h. For all experiments, 10 µl of the digested sample was mixed with SDS-sample buffer and used for gel electrophoresis, whereas another 10 µl was used for protein mass determination. The rest of the samples were immediately used for bioactivity determination.

Structural Characterization. Samples were mixed with reducing SDS-sample buffer, heated to 100°C for 5 min, and analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) (4–20% gradient gel). The proteins were visualized using Coomassie Brilliant Blue staining. Selected gel bands were excised, and in-gel was digested overnight at 37°C using trypsin as described previously (Shevchenko et al., 1996). The resulting peptide mixtures were used for protein identification and glycan analysis on the peptide level. To perform peptide mass fingerprinting, 0.5 µl of the peptide mixture was deposited directly onto the target, 0.5 µl of matrix [-cyano-4-hydroxycinnamic acid, 10 mg/ml in 70% acetonitrile (MeCN), 5% formic acid (FA)] was added, and the mixture was dried. For the glycan analysis, glycopeptides were enriched before the mass analysis using hydrophilic chromatography. To do this, the peptide mixture was dried and redissolved in 10 µl of 80% MeCN, 2% FA, and subsequently loaded onto a custom-made microcolumn packed with hydrophilic material (ZIC-HILIC, 200 Å, 10 µm; kindly provided by Sequant, Umea, Sweden) into GELoader tips (Eppendorf GmbH, Hamburg, Germany) as described previously (Thaysen-Andersen et al., 2007). After a thorough washing step using the same buffer, the glycopeptides were eluted directly onto the target using 0.5 µl of 2% FA, mixed with 0.5 µl of matrix (2,5-dihydroxybenzoic acid), and dried. In contrast, hydrophobic chromatography was used as sample clean-up for intact protein mass measurements. Hence, samples were loaded on microcolumns packed with Poros R1 (Applied Biosystems, Framingham, MA) as described previously (Gobom et al., 1999). The column was equilibrated and washed using 10 µl of 5% FA, and the glycoprotein was coeluted with the matrix onto the target using 0.8 µl of sinapic acid (20 mg/ml) dissolved in 70% MeCN in 5% FA. Peptide mass fingerprinting and glycan analyses were performed using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) on a Bruker Ultraflex (Bruker Daltonics, Bremen, Germany). Reflector mode was activated, and all samples were analyzed in positive polarity mode. A few species were verified based on their fragmentation pattern using the tandem mass spectrometry mode. Internal two-point calibrations were made when possible using known masses of tryptic peptides or other components. Alternatively, external calibrations using tryptic lactoglobulin were used. Data were viewed with the programs MoverZ (Genomic Solutions, Ann Arbor, MI) or FlexAnalysis version 2.4 (Bruker Daltonics). The mass accuracy was generally below 30 ppm for all MS analyses on peptide levels.

Statistics. Results are presented in bar graphs as means ± S.D., and statistics were performed using Prism, version 4 (GraphPad Software Inc., San Diego, CA), applying a one-way analysis of variance (ANOVA) followed by a Tukey's multiple comparison test to analyze the result in each graphic presentation. Changes in the Mx level 3 h posttreatment compared with no treatment (NT) were additionally analyzed using a paired t test. Differences were considered statistically significant for P values <0.05.

	Results

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In this study, the type I IFN-inducible Mx gene was used as an in vivo marker for monitoring the treatment of rhIFN-β1a and selected glycosylation analogs. Real-time PCR was applied to detect the level of Mx transcripts in blood leukocytes. The constitutive Mx mRNA level (no treatment) was calibrated to equal one in all graphic presentations, meaning that the values for Mx (SQ)/HPRT1 (SQ) represent a fold increase. For each individual experiment, the same batch of rhIFN-β1a was used, and the HPRT1 levels were shown to be constant.

Validation of the Mx Biomarker. Initial screening for induction of Mx at every 3 h within the first 24 hpt of a single injection of 100,000 IU rhIFN-β1a indicated an increase of the Mx mRNA level 3 h posttreatment (data not shown). Based on this observation, a time course study was performed analyzing the Mx response every hour within the first 6 hpt. This experiment showed that the Mx level in leukocytes peaked 3 hpt, showing Mx levels approximately 20-fold above the constitutive level (Fig. 1A). Consequently, 3 hpt was used as a fixed time point in all experiments. The dose-response study showed a clear relationship between the rhIFN-β1a dose and the Mx response (Fig. 1B). Because 100,000 IU generated an appropriate response, this dose was used throughout the experiments. As expected, administration of vehicle alone did not affect the Mx level, demonstrating a specific Mx response. To further rule out an endogenous production of type I/III IFN, which would cause an unspecific Mx response, the activity of denatured (activity depleted) rhIFN-β1a was determined and showed no Mx response. To verify that the Mx response was receptor-mediated, Mx⁺IFNAR-ko mice were treated with rhIFN-β1a, and no induction of Mx was observed (Fig. 1C). Note that the lack of response demonstrated that the IFN-induced Mx response was receptor-mediated. In this and the subsequent experiments, newer batches of rhIFN-β1a were used, generating a higher Mx/HPRT1 ratio than observed in the initial experiments using the same dose. Each of the PCR products of the Mx and HPRT1 genes appeared as a single band on a gel, and both migrated to a position that matched the theoretical size. This proved specific amplifications of the target sequences, which was additionally documented by melting-point analysis, showing a single melting point for all products of each primer system (data not shown).

View larger version (9K): [in this window] [in a new window]   Fig. 1. Mx response in leukocytes after rhIFN-β1a treatment. Real-time PCR for Mx mRNA. A, time profile of the Mx response in blood leukocytes after treatment with 100,000 IU of rhIFN-β (Rebif) (time points for each group of mice; NT, 1 and 4 hpt; NT, 2 and 5 hpt; NT, 3 and 6 hpt; n = 5). Peak levels of Mx mRNA were achieved 3 hpt. B, response of Mx 3 hpt as a result of different doses of rhIFN-β1a. The graphic presentation shows a concentration-dependent effect, 100,000 IU is the same data as presented 3 hpt in A. Vehicle and denatured rhIFN-β1a (dIFN) were used as controls. C, Mx+IFNAR-ko and Mx+ controls treated with 100,000 IU. Data are presented as SQ of Mx mRNA/SQ of mRNA of the reference gene HPRT1, calibrated so that NT equals one, and analyzed using parametric statistic (A, paired t test and C, one-way ANOVA) and presented as means ± S.D., n = 5. ***, P < 0.001. Only discussed P values are shown in the graphics.

Structural Characterization of Native rhIFN-β1a and Glycosylation Analogs. To allow reliable conclusions to be drawn from the bioactivity measurements, the native rhIFN-β1a and its glycosylation analogs were characterized. The primary structure and the glycan moiety of native rhIFN-β1a were determined using MALDI-TOF MS. Peptide mass fingerprinting showed that the amino acid sequence was in agreement with the primary sequence obtained from nucleotide sequencing (data not shown) (Derynck et al., 1980). The glycan was investigated both on the glycoprotein (Fig. 2, A and B) and the glycopeptide (Fig. 2C) level. Upon glycopeptide enrichment, a single peak (m/z 5561.6) appeared in the spectrum (Fig. 2C, top spectrum). Based on this mass and fragmentation analysis (data not shown), the peak was determined to correspond to the rhIFN-β1a peptide Gln72-Lys99 conjugated to a complex biantennary carbohydrate structure containing a core fucosylation and two terminal sialic acid residues. On the glycoprotein level (Fig. 2A, top spectrum), the most abundant peak (22,379 Da, average mass) matched the protein mass of native rhIFN-β1a conjugated with the same glycan structure as determined on the peptide level. In addition, two minor peaks appeared at lower mass values corresponding to structures containing fewer sialic acid residues. However, based on the glycopeptide data, these structures were fragmentation products artificially generated in the MS exclusively at the glycoprotein level.

View larger version (32K): [in this window] [in a new window]   Fig. 2. Characterization of native rhIFN-β1a and its modified variants. A, intact protein mass determination of rhIFN-β1a and its variants using MALDI-TOF MS. The intact mass of rhIFN-β1a (IFN-β, top spectrum) was given by the most abundant peak (22,379 Da, average mass). Two minor peaks were observed at lower m/z values corresponding to the MS-induced loss of one and two labile sialic acids residues, respectively. The denatured IFN (dIFN) (second top spectrum) was achieved using carbamidomethylation derivatization, and its mass was determined to be 22,553 Da. Three types of glycosidase incubations were carried out (three bottom spectra), all giving rise to trimmed rhIFN-β1a variants, where specific parts of the carbohydrate moiety was removed. rhIFN-β1a was incubated with sialidase for 2 h (and 48 h; data not shown), whereas the other glycosidases (fucosidase and PNGase F) were incubated for 48 h. The heterogeneity generated from the sialic acid loss was removed using sialidase and PNGase F (two bottom spectra), and the two minor peaks observed at higher m/z values than the intact protein were adduct ions normally associated with the sinapic acid matrix. The structure of rhIFN-β1a and its variants are illustrated in the left of the spectra. B, SDS-PAGE of rhIFN-β1a and glycosylation analogs. High purity of rhIFN-β1a (band just below the 25-kDa marker) and low glycosidase amounts were observed, with the exception of PNGase F-incubated rhIFN-β1a, where the enzyme was visible as a band above the 35-kDa marker. A minor part of rhIFN-β1a remained glycosylated after the 48-h incubation with PNGase F. C, investigation of the glycan structure on the glycopeptide level. To ensure that the heterogeneity of sialylation observed on the protein level was a consequence of MS-induced fragmentations, IFN-β and sialidase-incubated IFN-β were characterized on the peptide level after trypsin digestion of the gel bands in B. IFN-β (top spectrum) appeared exclusively with two terminating sialic acid residues on the peptide level, confirming the predicted glycan structure. A complete removal of the sialic acid residues could be performed using 2 h of sialidase incubation (lower spectrum).

Several glycosidases, i.e., fucosidase, sialidase, and PNGase F, were used to trim the carbohydrate moiety of rhIFN-β1a, generating a set of glycosylation analogs. The reaction products were characterized using intact protein mass determination (Fig. 2A, three bottom spectra) and gel electrophoresis (Fig. 2B) before the bioactivity determination. Incubation of rhIFN-β1a with fucosidase for 48 h resulted in a mass decrease corresponding to the loss of a single fucose residue (146 Da). The small mass change was not sufficient to generate an observable shift in the migration distance on the gel, and the gel band appeared in the same region as native rhIFN-β1a (approximately 23 kDa). Sialidase incubation of rhIFN-β1a for 2 h was sufficient to produce a single peak corresponding to the loss of two sialic acid residues (2 x 291 Da) (Fig. 2C, bottom spectrum). For this reaction product, a small shift in the migration was observed on the gel. Proteolytical digestion of the gel bands allowed extraction and analysis of the protein and provided a source of sample on the peptide level. In addition, the purity of the sample could be estimated based on the gel experiments. Contaminating components were not observed for the native rhIFN-β1a or for the defucosylated and desialylated analogs, indicating low enzyme amounts. The desialylated analog incubated for 48 h was characterized using the same procedures, and the product, which was similar to the 2-h variant, showed no sign of degradation (data not shown).

Finally, the entire glycan moiety was removed from rhIFN-β1a by incubation with PNGase F for 48 h. This was shown by the decrease in protein mass matching the loss of the carbohydrate structure. The deglycosylated product migrated significantly further on the gel to a position at approximately 20 kDa. However, a minor gel band (5–10% of the major band) appeared in the intact rhIFN-β1a region illustrating incomplete glycosidase reaction. This glycosylated compound was not observed in the MS analysis as a likely consequence of the high deglycosylated/glycosylated ratio and the ion suppression effect, which is known to suppress glycosylated species in MS compared with their nonglycosylated counterparts (Nielsen and Roepstorff, 1989). In contrast to the highly potent sialidase, which completed the reaction in 2 h at low enzyme concentrations, a long reaction time (48 h) at a higher enzyme concentration was necessary to obtain the observed degree of deglycosylation using PNGase F. As a consequence, PNGase F was observed at its molecular mass at approximately 35 kDa on the gel. Incomplete reaction (deglycosylated/glycosylated ratio of approximately 1) was observed after 18 h of incubation (data not shown). In contrast, an extended incubation (72 h) and higher PNGase F concentrations resulted in a visible precipitate, which was probably caused by aggregation of deglycosylated rhIFN-β1a. Hence, 48 h was chosen as the common incubation period for all three glycosidases because this produced a high ratio of deglycosylated/glycosylated rhIFN-β1a for all reactions, and no precipitation was observed at this point.

View larger version (11K): [in this window] [in a new window]   Fig. 3. Mx response to treatment with rhIFN-β1a after 48-h incubation with and without specific glycosidases. Real-time PCR for Mx mRNA in leukocytes 3 hpt with rhIFN-β1a and a set of trimmed rhIFN-β1a variants generated by incubation with specific glycosidases for 48 h at 37°C. Treatment with deglycosylated rhIFN-β1a lead to a 10-fold reduction in the Mx response compared with the native rhIFN-β1a. A similar reduction was observed for the desialylated rhIFN-β1a, and the Mx response for both the deglycosylated and the desialylated rhIFN-β1a was not significantly different from the response to HSA. However, treatment with defucosylated rhIFN-β1a resulted in an Mx response that was not significantly different from treatment with the native rhIFN-β1a. The dose of treatment corresponds to the protein amount for 100,000 IU of native rhIFN-β1a. Data are presented as in Fig. 1 and analyzed using a one-way ANOVA. n = 3, with the exception of IFN 48 h and HSA, where n = 6. ***, P < 0.001. ns, not significant.

Next, to investigate the function of the sialic acid residues, e.g., receptor binding or protein stabilization, we took advantage of the highly potent sialidase and performed the experiment using a short, 2-h incubation. Treatment with this desialylated rhIFN-β1a analog gave rise to an Mx response similar to the response caused by administration of native rhIFN-β1a incubated under the same conditions (Fig. 4). Hence, the 2-h incubation of the desialylated analog did not cause a significant reduction of the bioactivity as opposed to the 48-h incubation of the same variant.

View larger version (10K): [in this window] [in a new window]   Fig. 4. Mx response to treatment with desialylated rhIFN-β1a incubated for 2 h. Real-time PCR for Mx mRNA in leukocytes 3 hpt with desialylated rhIFN-β1a generated by incubation with sialidase for 2 h at 37°C. The rapid desialylation of rhIFN-β1a gave rise to an Mx response similar to the response to native IFN-β1a incubated under the same conditions. As a means of control, the Mx responses to sialidase + rhIFN-β1a (incubated individually) and sialidase itself were measured. Sialidase did not affect the Mx response to rhIFNβ-1a significantly, and treatment with sialidase did not have a significant effect on the Mx level compared with treatment with HSA. Data are presented and analyzed as in Fig. 3, n = 3. ***, P < 0.001. ns, not significant.

View larger version (10K): [in this window] [in a new window]   Fig. 5. Effect of incubation and specific glycosidases on the Mx-response rhIFN-β1a. Real-time PCR for Mx mRNA in leukocytes 3 hpt with incubated rhIFN-β1a and different glycosidases. A, the Mx response to treatment with rhIFN-β1a incubated at 37°C for 2 or 48 h compared with the response to native rhIFN-β1a kept at 4°C (storage temperature according to the manufacturer) and HSA. The 48-h incubation of rhIFN-β1a at 37°C gave rise to a 3.5-fold reduction in the bioactivity of rhIFN-β1a compared with native rhIFN-β1a kept at 4°C, whereas the short, 2-h incubation of rhIFN-β1a at 37°C did not cause a significant reduction in the bioactivity. B, the Mx response to treatment of PNGase F and fucosidase both incubated at 37°C for 48 h. The two glycosidases showed no significant induction of the Mx level compared with HSA, and neither of them showed a significant effect on the Mx response to rhIFN-β1a when treated in combination (not incubated together). C, the Mx response to treatment with desialylated rhIFN-β1a generated by incubation for 48 h at 37°C in a stabilizing buffer containing a polymer. The stabilizing effect was shown by a conserved bioactivity of rhIFN-β1a incubated for 48 h at 37°C. The desialylated variants showed a significant reduction of activity compared with the native rhIFN-β1a, but due to the stabilizing buffer, this analog retained a measurable dose-dependent bioactivity. Data are presented and analyzed as in Fig. 3. n = 3. *, P < 0.05; **, P < 0.01; ***, P < 0.001. ns, not significant.

	Discussion

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Compared with the constitutive level, the increase of the Mx mRNA response observed 3 hpt with rhIFN-β1a showed that rhIFN-β1a administration could be monitored by the downstream Mx gene transcript. It also showed that the mRNA level peaked sharply at this time point. The Mx mRNA synthesis rate has previously been shown to peak 3 hpt with type 1 IFN in embryonic cells from Mx⁺ mice (Staeheli et al., 1986), whereas the Mx response in humans has been shown to peak 9 to 12 hpt upon rhIFN-β1a administration (Pachner et al., 2003). This verifies the Mx gene transcript as a sensitive biomarker, but it also stresses the importance of posttreatment time standardization, highlighting one concern for its clinical use as a biomarker for rhIFN-β1a treatment. The lack of Mx responses upon injection of vehicle and denatured (activity depleted) rhIFN-β1a demonstrated high specificity of the Mx response to functional protein. This confirmed that no significant endogenous production of type I or type III IFN occurred and, consequently, showed that the response could be ascribed to the bioactivity of the administered rhIFN-β1a. The lack of Mx response upon rhIFN-β1a treatment of the IFNAR-ko mice documented that the effect was mediated through the IFNAR. Our result provided evidence of a cross-species activity as observed by others (Kasama et al., 1995); however, high doses were needed for appropriate responses. The cross-species activity agreed well with the high similarity of the human and murine IFN-β on the three-dimensional structural level (Senda et al., 1992; or Karpusas et al., 1997). Taken together, the observed Mx responses could be ascribed to the bioactivity of the administered rhIFN-β1a, demonstrating Mx as a dynamic marker for rhIFN-β1a treatment in Mx-congenic mice.

The native rhIFN-β1a and its glycosylation analogs were characterized using MS and SDS-PAGE before the activity determinations to ensure that the desired structures were obtained. The significantly lower bioactivity of the deglycosylated rhIFN-β1a compared with the native protein was in good agreement with earlier observations, where a 10-fold activity difference of the two variants was shown using in vitro assays (Runkel et al., 1998). Using the Mx biomarker, we similarly observed a 10-fold activity difference of the glycosylated rhIFN-β1a and the deglycosylated variant. However, because the deglycosylated rhIFN-β1a generated an Mx response similar to the constitutive level, the difference could de facto be much higher. Incubation of the deglycosylated rhIFN-β1a for less than 48 h could potentially determine the cause of the activity differences, e.g., glycan involvement in protein stability or receptor binding. However, the low glycosidase activity ruled out a shorter incubation due to incomplete glycosidase reactions. Longer incubation was also excluded as a consequence of protein precipitation as previously observed (Runkel et al., 1998). In addition to complete deglycosylation, the fucose and sialic acids were specifically removed in individual experiments because these residues are known to be involved in numerous biological functions (Angata and Varki, 2002; Becker and Lowe, 2003).

Incubation of the desialylated rhIFN-β1a analog for 48 h depleted the bioactivity of the protein, as observed for the deglycosylated rhIFN-β1a, and was shown not to degradate the protein. This demonstrated a crucial role of the sialic acid residues for activity retention of the protein. Unlike the deglycosylation, complete desialylation could rapidly be achieved. Hence, the activity of the completely desialylated rhIFN-β1a was also measured after 2 h of incubation. The short incubation did not generate a significant reduction of activity, thereby illustrating a retained functional activity (receptor binding properties) of the desialylated analog as previously observed in vitro by only a small antiviral loss of activity of IFN-β upon sialic acid removal (Bocci et al., 1977). In agreement, systematic mutational mapping of sites on IFN-β, important for receptor binding, demonstrated a protein rather than a glycan involvement (Runkel et al., 2000). The distinct activity differences measured among the desialylated analogs (incubated 2 and 48 h) demonstrated a stability promoting effect of the sialic acid residues of rhIFN-β1a. In agreement with our findings, the sialic acids have previously been shown to stabilize proteins as illustrated by an abolishment of in vivo activity of erythropoietin upon desialylation (Tsuda et al., 1990). Here, thermal protection of the sialic acid residues was also suggested by an in vitro activity reduction of the asialoprotein after short heat exposure. Likewise, the terminating sialic acid residues have been shown to play a key role for biological activity of rhIFN-β1a in vitro (Utsumi et al., 1995). Based on our results, which show a stabilizing role of the sialic acids, it can be suggested that this lower activity might be a consequence of reduced protein stability of the desialylated variant. The same investigators have reported that asialo-rhIFN-β1a is cleared rapidly from the blood circulation by asialoglycoprotein receptors in the liver (Kasama et al., 1995). Our data indicate that the hepatic clearance of desialylated rhIFN-β1a by such receptors was negligible. Thus, we assume that the administered rhIFN-β1a is rapidly distributed in the organism and quickly interacts with the IFNARs in various tissues. In support of this assumption, the Mx mRNA synthesis peaked 3 hpt in vitro (Staeheli et al., 1986), as observed in vivo in our experiments.

The results presented here are in agreement with the conclusion drawn by other researchers (Runkel et al., 1998), suggesting that the overall function of the glycan moiety of rhIFN-β1a is to solubilize and stabilize the protein. This was supported by data obtained using biophysical approaches, i.e., size exclusion chromatography, SDS-PAGE, and thermal denaturation as well as structural data (Radhakrishnan et al., 1996; Karpusas et al., 1997; Klaus et al., 1997; Runkel et al., 1998). Hence, our data add in vivo support to these conclusions and, very importantly, pinpoint the stability promoting region of the carbohydrate moiety to the sialic acid residues. This solubilizing and stabilizing role of the sialic acids fits well with their charged (negative) character as well as the terminating location in the glycan, thereby allowing substantial contact with the surrounding solvent. The results also showed that the fucose residue was not essential for retaining the activity of rhIFN-β1a. This agrees well with the uncharged nature and buried position of the fucose residue in the reducing end of the glycan with limited solvent contact.

The native rhIFN-β1a was heat-labile as determined by the significant decrease of bioactivity after the 48-h incubation at 37°C. Because comparative bioactivity measurements were performed for all individual experiments, using samples incubated under the same conditions, this activity reduction did not affect the presented conclusions. However, the data showed that although the native rhIFN-β1a was somewhat stabilized by the terminating sialic acids compared with the extremely heat-labile deglycosylated and desialylated analogs, it remained fairly sensitive to heat degradation. Finally, the bioactivity of the desialylated analog after 48 h of incubation was investigated using a protein stabilizing buffer. The fact that the bioactivity was severely reduced, although not completely lost, in combination with a preserved bioactivity of native rhIFN-β1a incubated for 48 h further emphasized the stabilizing role of the sialic acid residues. The investigation of the kinetics of the desialylated analog showed that the bioactivity followed a similar pattern as that observed in the initial time course study, illustrating similar kinetics of the desialylated and native rhIFN-β1a.

Because the glycan, and the sialic acids in particular, have been shown to stabilize rhIFN-β1a, it can be hypothesized that specific modulations of the glycan moiety (glycoengineering) may contribute to increased stability of the native protein by, i.e., hypersialylation of the galactose residues of the biantennary structure or increased branching (tri- or tetraantennary structures) with more terminating sialic acid residues as a result. Because rhIFN-β1a is a pharmaceutical product, where the limited stability of the protein necessitates careful handling with respect to storage temperature, buffer conditions, and time, this glycoengineering might improve its applications. Other protein modulations have been attempted to improve the pharmacological properties of IFNs, e.g., both IFN- and β have been conjugated to a single polyethylene glycol moiety (10–40 kDa) that led to a 10-fold increase in elimination half-life (Pepinsky et al., 2001). Furthermore, the plasma clearance of IFNs have been lowered 140-fold by fusing IFNs to HSA (Osborn et al., 2002). Although these IFN modulations hold the advantage of a low blood clearance, it can be speculated that the dramatic size increment limits the permeability of the protein to the target tissue, e.g., the central nervous system (Holliday and Benfield, 1997), thereby reducing the overall in vivo bioactivity. Glycoengineering, in contrast, has the advantage of potentially adding significant stability to rhIFN-β1a while preserving its natural size and consequently its distribution features.

In conclusion, we have determined the structure/bioactivity relationship of rhIFN-β1a and generated solid support for a stability promoting role of the glycan. It is noteworthy that the study brought evidence that the sialic acid residues were crucial for the stabilization and consequently the activity of the protein in vivo. This approach demonstrates that information of the glycan and its individual carbohydrate residues of glycoprotein can be generated in vivo and hold the potential to be applied to other glycosylated therapeutics in various diseases.

	Acknowledgements

 
We thank Line Bjørndal Graversen, Anne Mette Durand, and Ket Hansen (Biomedical Laboratory, University of Southern Denmark) for help with breeding the mice and collecting blood. Finally, we acknowledge Drs. Otter Haller and Peter Staeheli for their support.

	Footnotes

 
This work was supported by grants from Warwara Larsen Foundation, Novo Nordisk/Novozymes, Lundbeck Foundation, and the Danish Multiple Sclerosis Society.

L.D.-O. and M.T.-A. contributed equally to this work.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.108.138263.

ABBREVIATIONS: IFN, interferon(s); IFNAR, IFN- receptor; Mx, myxovirus resistance; rh, recombinant human; RRMS, relapsing-remitting multiple sclerosis; ko, knockout; HSA, human serum albumin; hpt, hours posttreatment; HPRT1, hypoxanthine phosphoribosyltransferase 1; SQ, starting quantity; bp, base pair; PAGE, polyacrylamide gel electrophoresis; MeCN, acetonitrile; FA, formic acid; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MS, mass spectrometry; ANOVA, one-way analysis of variance; NT, no treatment.

Address correspondence to: Lasse Dissing-Olesen, Medical Biotechnology Center, University of Southern Denmark, Winsløwparken 25, 2, DK-5000 Odense C, Denmark. E-mail: ldolesen{at}health.sdu.dk

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