N-糖基化对毕赤酵母表达的DSPAα1分泌和活性的影响

李剑凤，阎岩，王庆民，孙丽霞，王晶翼

齐鲁制药有限公司药物研究院，济南 250100

Introduction

DSPAs (Desmodus rotundus salivary plasminogen activators) are a group of serine proteases with plasminogen activator (PA) activity, and are comprised of four distinct variants: α1, α2, β and γ[1]. Comparedto the currently commonly available PAs, DSPA proteins show remarkably effective thrombolytic activity, especially in the case of DSPAα1. DSPAα1 consists of several distinct peptide regions: a finger domain, an epidermal growth factor region, a kringle domain and a serine protease domain[1-2]. The peptide backbone of DSPAα1 includes two N-glycosylation sites at residues Asn-117 and Asn-362, as predicted by the consensus sequence Asn-X-Ser/Thr[3]. In contrast to tPA (tissue-type plasminogen activator), DSPAα1 has exceptionally high fibrin specificity, and in animal models has shown remarkable efficacy for thrombolysis, low incidence of systemic bleeding, low reocclusion rate, an effective time window extending up to nine hours, and the absence of potential neurotoxic effects[4-7]. These superior properties imply that DSPAα1 presents a potentially safer and more efficacious thrombolytic agent than tPA. The clinical trials of DSPAα1 in acute ischemic stroke studies also demonstrated that it is a promising thrombolytic agent[8].

Originally, natural DSPAα1 was purified directly from vampire bat salivary gland, which clearly limited its wider application. Subsequently, recombinant DSPAα1 has been produced in a number of different host cell systems, including baby hamster kidney (BHK)[9], Chinese hamster ovary (CHO)[10], insect (Sf 9)[10]and plant (tobacco)[11]. Pichia pastoris, a methylotropic yeast, has been developed as a host for heterologous protein expression because of its low costs, high expression levels, efficient secretion, posttranslation modification, proper protein folding and the potential for very high cell density fermentation[12-14]. However, some groups have reported that a few proteins expressed in yeast contain non-human classes of N-linked glycans of the high-mannose type, which can be immunogenic in humans and can also lead to accelerated clearance, thus limiting their therapeutic value[15-16]. The direct solution of this problem is to eliminate the N-linked glycan in the synthesized protein through appropriate site-directed mutagenesis of the coding gene, but sometimes such mutations can result in major effects on the folding, activity and secretion of the enzyme[17-20].

In this study, an efficient and cost-effective Pichia pastoris expression system was developed to produce recombinant DSPAα1, and the two glycosylation sites (Asn-117 and Asn-362) were eliminated by site-specific mutation to study the influence of the N-linked glycans on protein expression and enzyme activity.

1 Materials and methods

1.1 Strains, reagents and media

The Pichia pastoris strains GS115, KM71 and the expression vectors pPIC9, pPIC9K were obtained from Invitrogen (California, USA). All enzymes for DNA applications were purchased from Promega. The media used, buffered minimal methanol (BMMY), buffered minimal glycerol (BMGY) and minimal dextrose (MD), were prepared following the manual for the Pichia pastoris expression kit (Invitrogen). The GeneEditor™ in vitro Site-Directed Mutagenesis System kit was purchased from Promega.

1.2 Synthesis of DSPAα1 and site-directed mutagenesis to produce non-glycosylated DSPAα1

The codon-optimized DNA sequence of DSPAα1 was synthesized by Shanghai Sangon Biological Engineering Technology and Service Co., Ltd. (China), and contained Xho I and EcoR I sites at the 5′ and 3′termini, respectively. In addition, the 5′ terminus also contained a site for kex2 cleavage (GenBank accession GQ412732).

The GeneEditor™ in vitro Site-Directed Mutagenesis System was used to create the Asn117 and Asn362 to Gln mutations, individually or together. The mutant primers included pM117: 5′-pGTATCAACTGG CAGAGCAGCCTTCTG-3′ and pM362: 5′-pCAAGT TTCTGTTTCAGAAAACCGTC-3′. Briefly, the oligonucleotide primers (representing the mRNA strand), containing the desired mutation (Asn117 to Gln117, or Asn362 to Gln362), were annealed to the synthesized DSPAα1 DNA and extended by temperature cycling using the very high fidelity enzyme T4 DNA polymerase, then ligated by using T4 DNA ligase. The mutant plasmids were transformed into the E. coli strain BMH71-18 and positive transformants were cultured in LB broth containing GeneEditor™Antibiotic Selection Mix. The plasmids were purified and transformed into JM109 competent cells, and clones were selected and their DNA sequences were determined to confirm the desired mutation.

1.3 Construction of expression plasmids

The wild-type DSPAα1, or the three mutant genes, were digested with Xho I and EcoR I, and then inserted into the vector pPIC9 cut at the Xho I and EcoR I sites, to yield plasmids pP9DSPAα1 (s). Then the four genes were cloned into pPIC9K to yield pP9KDSPAα1 (s). The plasmid pPIC9K contains an AOX1 promoter that allows methanol-inducible high-level expression in Pichia pastoris, an α-factor signal sequence for secretion, and a kanamycin resistance gene and a histidinol dehydrogenase gene (his4) for positive clone selection.

1.4 Construction and selection of recombinant yeast

The four recombinant plasmids pP9KDSPAα1 (s), linearized with Sal I, were transformed into competent Pichia pastoris cells by electroporation. 1 mL of ice-cold 1 mol/L sorbitol was added to the electroporation cuvette immediately after pulsing. His+transformants were selected on histidine-deficient MD agar plates. Several His+clones were randomly picked and inoculated into YPD (1% yeast extract, 2% tryptone, 2% dextrose). The transformants were verified by PCR using gene-specific primers.

1.5 Expression of recombinant yeast

Several recombinant clones from YPD were grown in 20 mL of BMGY medium in 100 mL Erlenmeyer flasks and incubated at 30°C with shaking at 200 r/min until the OD600was 10−20. Cells were collected by centrifugation at 4 000 r/min for 5 min and resuspended in BMMY medium (pH 6.0), then transferred to 50 mL BMMY media in 500 mL Erlenmeyer flasks. The final OD600was approximately 2.0. The cultures were grown at 20°C with shaking at 250 r/min for a week. Methanol was added once a day at a concentration of 1:200 (V/V).

1.6 SDS-PAGE and fibrin degradation assay of DSPAα1

The culture supernatants were collected by centrifugation at 4 000 r/min for 10 min. The supernatant and cell lysate were analyzed by reducing sodium dodecyl sulfate (SDS), 12% polyacrylamide gel electrophoresis (PAGE). The concentration of recombinant DSPAα1 protein was assayed by SDS-PAGE based on a standard curve generated by the purified recombinant DSPAα1, which was quantified by the Bradford protein quantitation method. Enzyme activities were detected by a modified fibrin assay as described[21]. A fibrin-agarose gel plate was prepared by mixing 0.125 mg agarose in 25 mL saline, adding 13.2 mg fibrinogen, 14 μL 100 IU/mL of thrombin and 280 μL 0.5 mg/mL plasminogen. About 10 μL of supernatant from cultures of transformants was applied to each well on the plate and incubated at 37°C for six hours.

2 Results

2.1 Heterologous expression of DSPAα1 (wild-type) in Pichia pastoris

The DSPAα1 gene DNA sequence, which incorporated yeast-preferred codons, was 1 434 bp in length, encoding a 477 amino acid mature protein. Insertion of the cDNA sequence into the vector pPIC9K between the Xho I and EcoR I sites produced a 10.8 kb plasmid. Since the constructed plasmid contained an α-factor signal peptide sequence in front of the DSPAα1 gene, the expressed recombinant DSPAα1 was secreted out of the host cells.

The plasmids pP9KDSPAα1 (wild-type) and pPIC9K were linearized with Sal I and transformed into the Pichia pastoris cells GS115 and KM71. Selection on MD plates yielded thousands of His+transformants. Thirty colonies from GS115 and KM71, respectively, plus the control strain, were randomly picked into YPD medium, and transferred to BMMY for further evaluation in submerged fermentation analysis. The transformants’ broth supernatants were analyzed by SDS-PAGE where a protein band (62 kDa) was clearly observed and absent in the control strain (Fig. 1A). Little difference in DSPAα1 expression levels was observed among the GS115 or the KM71 cultures (data not shown), although the DSPAα1 expression levels for KM71 were about 1.5 times more than for GS115 (70 mg/L), up to 105 mg/L (Fig. 1B). Three colonies from the thirty KM71 or GS115 tested, named KM71-DSPAα1-1~3 and GS115-DSPAα1-1~3, respectively, were further analyzed in detail.

Fig. 1 SDS-PAGE analysis of the recombinant protein DSPAα1. (A) M: protein marker; 1: concentrated supernatant from the negative control after cultured 5 days; 2: concentrated broth supernatant from the GS115-DSPAα1-1 after cultured 5 days. (B) M: protein marker; 1: supernatant from the GS115-DSPAα1-1 from a 5-day culture; 2: supernatant from KM71-DSPAα1-1 from a 5-day culture.

Since the recombinant DSPAα1 was able to cleave plasminogen into plasmin to degrade fibrin, the PA (plasminogen activator) activity of the recombinant protein in transformants’ broth supernatant was analyzed by the fibrin assay. The expression levels of three positive transformants (from GS115 and KM71) were monitored and showed that the protein expression levels continued to increase from day 1 to day 7 following methanol induction (Fig. 2). By day 8, the pH of the supernatant had increased and the recombinant DSPAα1 yield began to decrease.

Fig. 2 Fibrin assay of recombinant DSPAα1 in broth supernatant of transformants. 1: the negative control; 2: the positive control tPA; 3-6: GS115-DSPAα1-1 (24 h, 72 h, 120 h, 168 h); 7-10: GS115-DSPAα1-2 (24 h, 72 h, 120 h, 168 h); 11-14: GS115-DSPAα1-3 (24 h, 72 h, 120 h, 168 h).

2.2 Analysis of the DSPAα1 mutants

The DSPAα1 mutant transformants were selected in the same way as the wild-type DSPAα1. The expression from Pichia pastoris of glycosylated DSPAα1 and the DSPAα1 non-glycosylated at one or two sites (Fig. 3A) was studied and the PA activity of the DSPAα1 mutants was also evaluated. The three DSPAα1 mutant transformants were assayed by flask fermentation. As shown in Fig. 3B, the apparent molecular weight of the DSPAα1 mutant N117Q in culture was decreased to 58 kDa comparing to the wild-type DSPAα1, which was taken as proof that the sugar chain of Asn117 had been eliminated. The results of N362Q and N117Q/N362Q showed that the expression levels for these two mutants were dramatically decreased such that they couldn’t be detected by SDS-PAGE. However, compared to wild DSPAα1, the cell lysate of three mutants all had an extra concentrated protein band with an apparent size of 70 kDa corresponding to DSPAα1 with a signal peptide at the N terminus. The enzyme activites were also detected, and the results showed that the mutants enzyme activites dramatically decreased (Fig. 3C).

After purification and detection, the specific activity of the mutants N117Q and N362Q was less than 25% of the wild-type DSPAα1. The yield for the third mutant N117Q/N362Q was so low that enough protein couldn’t be obtained to assay.

Fig. 3 Analysis of the DSPAα1 mutants. (A) Schematic representation of N-glycosylation mutants. (B) SDS-PAGE analysis of the recombinant protein DSPAα1 and mutants. M: protein marker; 1: supernatant from the wild DSPAα1; 2: cell lysate of the wild DSPAα1; 3: supernatant from the DSPAα1 mutant N117Q; 4: cell lysate of the DSPAα1 mutant N117Q; 5: supernatant from the DSPAα1 mutant N362Q; 6: cell lysate of the DSPAα1 mutant N362Q; 7: supernatant from the DSPAα1 mutant N117Q/N362Q; 8: cell lysate of the DSPAα1 mutant N117Q/N362Q. (C) Fibrin assay of DSPAα1 mutants in broth supernatant of transformants. 1: wild DSPAα1; 2: DSPAα1 mutant N117Q; 3: DSPAα1 mutant N362Q; 4: DSPAα1 mutant N117Q/N362Q.

3 Discussion

It has been reported in the literature that DSPAα1 has been expressed in various hosts, namely insect, tobacco and mammalian cells. The expression in insect and tobacco cells is problematic because of low expression levels and, for tobacco, also high levels of proteolysis. On the other hand, the mammalian cell expression systems are high in cost and require stringent control procedures to detect possible infectious agents. The ability of the yeast Pichia pastoris to be grown rapidly on simple media to high cell density, with secretion of expressed foreign proteins gives it advantages over other expression systems[22]. Therefore, in this study Pichia pastoris was chosen to express DSPAα1. To our knowledge, this is the first time that DSPAα1 has been expressed in Pichia pastoris.

Pichia pastoris is a widely used industrial methylotrophic yeast which has been developed as a host for expression of foreign proteins[23]. In this study, the results indicated that functional DSPAα1 was successfully expressed in two Pichia pastoris strains and the yield of the recombinant DSPAα1 in KM71 was up to 105 mg/L, which is higher than those obtained in other expression systems (the highest reported yield in CHO cells is 60 mg/L)[9-11]. The Pichia pastoris strain KM71, with its slow methanol utilization (MutS) genotype could be predicted to be better for high-level expression of DSPAα1 than is GS115 which has wild-type methanol utilization (Mut+) genotypic expression system. These well known effects are related to the AOX promoter and its repression. In the case of DSPAα1, the 14 disulfide bonds of this protein may require extended times to permit proper post-translational processing[1]. Our results do show that Pichia pastoris is the most efficient host among those systems reported to express functional DSPAα1. Given the good yields of recombinant DSPAα1 produced under the shaking flask conditions, the prospects look promising for optimizing this expression system to produce recombinant DSPAα1 on a large scale[24]. In our experiments the expressed recombinant DSPAα1 produced by the two strains was the dominant protein found in the medium supernatant which should simplify purification of the protein in industrial settings.

As shown in SDS-PAGE, the recombinant DSPAα1 produced in Pichia pastoris is a hyper-glycoenzyme (apparent size 62 kDa), which contains about 24% carbohydrate content and these glycan maybe limited DSPAα1 therapeutic value[20]. To attempt to overcome these drawbacks, three glycosylation minus DSPAα1 mutants were constructed to evaluate the role of the sugar chains in the DSPAα1 protein. The results showed that the apparent molecular weights of mutants were clearly decreased. Although the intracellular protein concentrations were essentially normal, the greatly reduced or largely absent quantities of protein recoverable from the broth supernatant strongly suggests that the secretion of the non-glycosylated proteins was impaired as a result of the glycan deletion. The extracellular protein levels for mutant N362Q was less than for the mutant N117Q, thus implying that the glycan at Asn362 is more important for secretion of DSPAα1 than that at Asn117. The double mutant N117Q/N362Q could not be detected in the broth supernatant because of the very low yield. These results indicated that both glycosylation sites (Asn117 and Asn362) played important roles in the secretion of DSPAα1 from Pichia pastoris. The specific activities of each were also assayed and both mutants (N117Q and N362Q) showed less than 25% of the activity of wild-type DSPAα1. This demonstrated that the N-linked glycans of the secreted DSPAα1 are also critical for DSPAα1’s enzyme activity which has also been reported in other studies[19,25].

Unfortunately, these study results are discouraging because the deletion of the two N-glycans had a dramatic influence on both the secretion and the enzymatic activity of DSPAα1. This demonstrated that recombinant, non-glycosylated DSPAα1 is of no practical use. However, there have been encouraging recent advances involving the glycoengineering of Pichia pastoris that have resulted in the humanizing of the protein glycosylation pathways in this cell[26-27]. Glycosylated recombinant proteins synthesized in these humanized yeast cells can be predicted to show better overall characteristics because of their native glycosylation properties. Improving the specific activity of non-glycosylated DSPAα1 through protein engineering is also a realistic and practical approach. These alternative solutions will be pursued in the future.

In conclusion, we report here that DSPAα1 has been expressed successfully in Pichia pastoris. The expression levels of DSPAα1 could be enhanced further by optimizing culture fermentation conditions or by enlarging the scale of culture. N-glycosylation of this protein is critical in order to maintain enzyme activity and for efficient secretion of the DSPAα1 protein from Pichia pastoris.

ACKNOWLEDGEMENT

The authors sincerely acknowledge Dr. David Spencer and Dr. Kaisong Zhou for proofreading.

[1] Krätzschmar J, Haendler B, Langer G, et al. The plasminogen activator family from the salivary gland of the vampire bat Desmodus rotundus: cloning and expression. Gene, 1991, 105(2): 229−237.

[2] Gardell SJ, Duong LT, Diehl RE, et al. Isolation, characterization, and cDNA cloning of a vampire bat salivary plasminogen activator. J Biol Chem, 1989, 264(30): 17947−17952.

[3] Gohlke M, Nuck R, Kannicht C, et al. Analysis of site-specific N-glycosylation of recombinant Desmodus rotundus salivary plasminogen activator rDSPA alpha 1 expressed in Chinese hamster ovary cells. Glycobiology, 1997, 7(1): 67−77.

[4] Gardell SJ, Hare TR, Berqum PW, et al. Vampire bat salivary plasminogen activator is quiescent in human plasma in the absence of fibrin unlike human tissue plasminogen activator. Blood, 1990, 76(12): 2560−2564.

[5] Gardell SJ, Ramjit DR, Stabilito II, et al. Effective thrombolysis without marked plasminenia after bolus intravenous administration of vampire bat salivary plasminogen activator in rabbits. Circulation, 1991, 84(1): 244−253.

[6] Liberatore GT, Samson A, Bladin C, et al. Vampire bat salivary plasminogen activator (desmoteplase): a unique fibrinolytic enzyme that does not promote neurodegeneration. Stroke, 2003, 34(2): 537−543.

[7] Gulba DC, Praus M, Dechend R, et al. Update on the toxicology and pharmacology of rDSPA alpha 1 (bat-PA) in animals and humans. Fibrinol Proteol, 1997, 11(2): 55−62.

[8] Hacke W, Albers G, Al-Rawi Y, et al. The desmoteplase in acute ischemic stroke trial (DIAS): a phase II MRI-based 9-hour window acute stroke thrombolysis trial with intravenous desmoteplase. Stroke, 2005, 36(1): 66−73.

[9] Krätzschmar J, Haendler B, Bringmann P, et al. High-level secretion of the four salivary plasminogen activators from the vampire bat Desmodus rotundus by stably transfected baby hamster kidney cells. Gene, 1992, 116(2): 281−284. [10] Petri T, Langer G, Bringmann P, et al. Production of vampire bat plasminogen activator DSPAalpha1 in CHO and insect cells. J Biotechnol, 1995, 39(1): 75−83.

[11] Schiermeyer A, Schinkel H, Apel S, et al. Production of Desmodus rotundus salivary plasminogen activator alpha1 (DSPAα1) in tobacco is hampered by proteolysis. Biotechnol Bioeng, 2005, 89(7): 848−858.

[12] Kobayashi K, Kuwae S, Ohya T, et al. High-level expression of recombinant human serum albumin from the methylotrophic yeast Pichia pastoris with minimal protease production and activation. J Biosci Bioeng, 2000, 89(1): 55−61.

[13] Trimble RB, Lubowski C, Hauer CR, et al. Characterization of N- and O-linked glycosylation of recombinant human bile salt-stimulated lipase secreted by Pichia pastoris. Glycobiology, 2004, 14(3): 265−274.

[14] Sreekrishna K, Brankamp RG, Kropp KE, et al. Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris. Gene, 1997, 190(1): 55−62.

[15] Bretthauer RK, Castellino FJ. Glycosylation of Pichia pastoris-derived proteins. Biotechnol Appl Biochem, 1999, 30(3): 193−200.

[16] Hamilton SR, Bobrowicz P, Bobrowicz B, et al. Production of complex human glycoproteins in yeast. Science, 2003, 301(5637): 1244−1246.

[17] Muraki M. Secretory expression of synthetic human Fas ligand extracellular domain gene in Pichia pastoris: influences of tag addition and N-glycosylation site deletion, and development of a purification method. Protein Expr Purif, 2006, 50(2): 137−146.

[18] Koseki T, Miwa Y, Mese Y, et al. Mutational analysis of N-glycosylation recognition sites on the biochemical properties of Aspergillus kawachii α-L-arabinofuranosidase 54. Biochim Biophys Acta, 2006, 1760(9): 1458−1464.

[19] Han YM, Lei XG. Role of Glycosylation in the functional expression of an Aspergillus niger phytase (phyA) in Pichia pastoris. Arch Biochem Biophys, 1999, 364(1): 83−90.

[20] Daniels R, Kurowski B, Johnson AE, et al. N-linked glycans direct the cotranslational folding pathway of influenza hemagglutinin. Mol Cell, 2003, 11(1): 79−90.

[21] Fisher R, Waller EK, Grossi G, et al. Isolation and characterizationo of the human tissue-type plasminogen activator structural gene including its 5′ flanking region. J Biol Chem, 1985, 260(20): 11223−11230.

[22] Cereghino JL, Cregg JM. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev, 2000, 24(1): 45−66.

[23] Macauley-Patrick S, Fazenda ML, McNeil B, et al. Heterologous protein production using the Pichia pastoris expression system. Yeast, 2005, 22(4): 249−270.

[24] Cereghino GP, Cereghino JL, Ilgen C, et al. Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris. Curr Opin Biotechnol, 2002, 13(4): 329−332.

[25] Turner KM, Wright LC, Sorrell TC, et al. N-linked glycosylation sites affect secretion of cryptococcal phospholipase B1, irrespective of glycosylphosphatidylinositol anchoring. Biochim Biophys Acta, 2006, 1760(10): 1569−1579.

[26] Hamilton SR, Gerngross TU. Glycosylation engineering in yeast: the advent of fully humanized yeast. Curr Opin Biotechnol, 2007, 18(5): 387−392.

[27] Bretthauer RK. Genetic engineering of Pichia pastoris to humanize N-glycosylation of proteins. Trends Biotechnol, 2003, 21(11): 459−462.