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Similarity of recombinant human perlecan domain 1 by alternative expression systems bioactive heterogenous recombinant human perlecan D1.

Ellis AL, Pan W, Yang G, Jones K, Chuang C, Whitelock JM, DeCarlo AA - BMC Biotechnol. (2010)

Bottom Line: Additionally, we demonstrate HS-mediated binding of both rhPln.D1 species to several other GFs.Finally, we corroborate the augmentation of FGF-mediated cell activation by rhPln.D1 and demonstrate mitogenic signalling through the FGFR1c receptor.With importance especially to the emerging field of DNA-based therapeutics, we have shown here that proteoglycan synthesis, in different cell lines where GAG profiles typically differ, can be directed by recombinant technology to produce populations of bioactive recombinants with highly similar GAG profiles.

View Article: PubMed Central - HTML - PubMed

Affiliation: Agenta Biotechnologies, Inc., Innovation Depot, Birmingham, AL 35203, USA.

ABSTRACT

Background: Heparan sulfate glycosaminoglycans are diverse components of certain proteoglycans and are known to interact with growth factors as a co-receptor necessary to induce signalling and growth factor activity. In this report we characterize heterogeneously glycosylated recombinant human perlecan domain 1 (HSPG2 abbreviated as rhPln.D1) synthesized in either HEK 293 cells or HUVECs by transient gene delivery using either adenoviral or expression plasmid technology.

Results: By SDS-PAGE analysis following anion exchange chromatography, the recombinant proteoglycans appeared to possess glycosaminoglycan chains ranging, in total, from 6 kDa to >90 kDa per recombinant. Immunoblot analysis of enzyme-digested high Mr rhPln.D1 demonstrated that the rhPln.D1 was synthesized as either a chondroitin sulfate or heparan sulfate proteoglycan, in an approximately 2:1 ratio, with negligible hybrids. Secondary structure analysis suggested helices and sheets in both recombinant species. rhPln.D1 demonstrated binding to rhFGF-2 with an apparent kD of 2 ± 0.2 nM with almost complete susceptibility to digestion by heparinase III in ligand blot analysis but not to chondroitinase digestion. Additionally, we demonstrate HS-mediated binding of both rhPln.D1 species to several other GFs. Finally, we corroborate the augmentation of FGF-mediated cell activation by rhPln.D1 and demonstrate mitogenic signalling through the FGFR1c receptor.

Conclusions: With importance especially to the emerging field of DNA-based therapeutics, we have shown here that proteoglycan synthesis, in different cell lines where GAG profiles typically differ, can be directed by recombinant technology to produce populations of bioactive recombinants with highly similar GAG profiles.

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Western blots of rhPln.D1 glycosidase digests. Pooled anionic exchange fractions (34-52 ml) from HEK 293 culture (Pln.198) or HUVECs (Pln.247) were subjected to digestion with either buffer only, heparinase I (Hep I), heparinase II (Hep II), heparinase III (Hep III), chondroitinase ABC (Chond. abc), all three heparinases plus chondroitinase ABC, or heparanase. Treated samples were analyzed by western blot using mAb CSI 001-71. Similar data were generated using plasmid expression of pln.247 (not shown).
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Figure 5: Western blots of rhPln.D1 glycosidase digests. Pooled anionic exchange fractions (34-52 ml) from HEK 293 culture (Pln.198) or HUVECs (Pln.247) were subjected to digestion with either buffer only, heparinase I (Hep I), heparinase II (Hep II), heparinase III (Hep III), chondroitinase ABC (Chond. abc), all three heparinases plus chondroitinase ABC, or heparanase. Treated samples were analyzed by western blot using mAb CSI 001-71. Similar data were generated using plasmid expression of pln.247 (not shown).

Mentions: To assess the relative contributions of CS and HS to each of the recombinants, aliquots of the high Mr rhPln.D1 preparations were digested with heparinases I, II, or III, or with chondroitinase ABC, or with mammalian heparanase (Figure 5). By western blot, treatment with heparinase I or II produced no measurable change in the staining pattern in either of the recombinant pools (rhPln.198 from HEK 293 cells and rhPln.247 from HUVEC). Treatment with heparinase III resulted in the appearance of low Mr species similar to those seen early in the elution profile (Figure 1). Treatment with chondroitinase ABC resulted in significant loss of the major Pln.D1 immunoreactivity and a more dense low Mr band. Treatment of the recombinant proteoglycans with the combination of heparinases and chondroitinase ABC resulted in further elimination of the higher Mr immunoreactivity, and greater density in the low Mr band. Treatment with mammalian heparanase did not demonstrate a clearly observable specific activity against the recombinant pools by this assay (Pln.247, Figure 5; Pln.198, data not shown). In summary, data from these experiments suggested that recombinant synthesis of different Pln.D1 variants in these two different cell types generated similar HS and CS levels. Also, it is important to note that digestion with either heparinase III or chondroitinase ABC did not produce intermediate bands of immunoreactivity or silver staining between the high Mr rhPln.D1 and the dominant digest products (43 kDa, Pln.247; 33 kDa, Pln.198); this suggested that the core proteins were either fully decorated with HS (approximately 1/3 of total) or fully decorated with CS (approximately 2/3 of total), and that there were negligible hybrids.


Similarity of recombinant human perlecan domain 1 by alternative expression systems bioactive heterogenous recombinant human perlecan D1.

Ellis AL, Pan W, Yang G, Jones K, Chuang C, Whitelock JM, DeCarlo AA - BMC Biotechnol. (2010)

Western blots of rhPln.D1 glycosidase digests. Pooled anionic exchange fractions (34-52 ml) from HEK 293 culture (Pln.198) or HUVECs (Pln.247) were subjected to digestion with either buffer only, heparinase I (Hep I), heparinase II (Hep II), heparinase III (Hep III), chondroitinase ABC (Chond. abc), all three heparinases plus chondroitinase ABC, or heparanase. Treated samples were analyzed by western blot using mAb CSI 001-71. Similar data were generated using plasmid expression of pln.247 (not shown).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2944331&req=5

Figure 5: Western blots of rhPln.D1 glycosidase digests. Pooled anionic exchange fractions (34-52 ml) from HEK 293 culture (Pln.198) or HUVECs (Pln.247) were subjected to digestion with either buffer only, heparinase I (Hep I), heparinase II (Hep II), heparinase III (Hep III), chondroitinase ABC (Chond. abc), all three heparinases plus chondroitinase ABC, or heparanase. Treated samples were analyzed by western blot using mAb CSI 001-71. Similar data were generated using plasmid expression of pln.247 (not shown).
Mentions: To assess the relative contributions of CS and HS to each of the recombinants, aliquots of the high Mr rhPln.D1 preparations were digested with heparinases I, II, or III, or with chondroitinase ABC, or with mammalian heparanase (Figure 5). By western blot, treatment with heparinase I or II produced no measurable change in the staining pattern in either of the recombinant pools (rhPln.198 from HEK 293 cells and rhPln.247 from HUVEC). Treatment with heparinase III resulted in the appearance of low Mr species similar to those seen early in the elution profile (Figure 1). Treatment with chondroitinase ABC resulted in significant loss of the major Pln.D1 immunoreactivity and a more dense low Mr band. Treatment of the recombinant proteoglycans with the combination of heparinases and chondroitinase ABC resulted in further elimination of the higher Mr immunoreactivity, and greater density in the low Mr band. Treatment with mammalian heparanase did not demonstrate a clearly observable specific activity against the recombinant pools by this assay (Pln.247, Figure 5; Pln.198, data not shown). In summary, data from these experiments suggested that recombinant synthesis of different Pln.D1 variants in these two different cell types generated similar HS and CS levels. Also, it is important to note that digestion with either heparinase III or chondroitinase ABC did not produce intermediate bands of immunoreactivity or silver staining between the high Mr rhPln.D1 and the dominant digest products (43 kDa, Pln.247; 33 kDa, Pln.198); this suggested that the core proteins were either fully decorated with HS (approximately 1/3 of total) or fully decorated with CS (approximately 2/3 of total), and that there were negligible hybrids.

Bottom Line: Additionally, we demonstrate HS-mediated binding of both rhPln.D1 species to several other GFs.Finally, we corroborate the augmentation of FGF-mediated cell activation by rhPln.D1 and demonstrate mitogenic signalling through the FGFR1c receptor.With importance especially to the emerging field of DNA-based therapeutics, we have shown here that proteoglycan synthesis, in different cell lines where GAG profiles typically differ, can be directed by recombinant technology to produce populations of bioactive recombinants with highly similar GAG profiles.

View Article: PubMed Central - HTML - PubMed

Affiliation: Agenta Biotechnologies, Inc., Innovation Depot, Birmingham, AL 35203, USA.

ABSTRACT

Background: Heparan sulfate glycosaminoglycans are diverse components of certain proteoglycans and are known to interact with growth factors as a co-receptor necessary to induce signalling and growth factor activity. In this report we characterize heterogeneously glycosylated recombinant human perlecan domain 1 (HSPG2 abbreviated as rhPln.D1) synthesized in either HEK 293 cells or HUVECs by transient gene delivery using either adenoviral or expression plasmid technology.

Results: By SDS-PAGE analysis following anion exchange chromatography, the recombinant proteoglycans appeared to possess glycosaminoglycan chains ranging, in total, from 6 kDa to >90 kDa per recombinant. Immunoblot analysis of enzyme-digested high Mr rhPln.D1 demonstrated that the rhPln.D1 was synthesized as either a chondroitin sulfate or heparan sulfate proteoglycan, in an approximately 2:1 ratio, with negligible hybrids. Secondary structure analysis suggested helices and sheets in both recombinant species. rhPln.D1 demonstrated binding to rhFGF-2 with an apparent kD of 2 ± 0.2 nM with almost complete susceptibility to digestion by heparinase III in ligand blot analysis but not to chondroitinase digestion. Additionally, we demonstrate HS-mediated binding of both rhPln.D1 species to several other GFs. Finally, we corroborate the augmentation of FGF-mediated cell activation by rhPln.D1 and demonstrate mitogenic signalling through the FGFR1c receptor.

Conclusions: With importance especially to the emerging field of DNA-based therapeutics, we have shown here that proteoglycan synthesis, in different cell lines where GAG profiles typically differ, can be directed by recombinant technology to produce populations of bioactive recombinants with highly similar GAG profiles.

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