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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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FGF-2 ligand blot binding of glycosidase-digested rhPln.D1. Pooled anionic exchange fractions of rhPln.198 or rhPln.247 from HEK 293 cell culture were subjected to digestion as designated; 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. Samples were subjected to SDS-PAGE and blotted to nitrocellulose. After blocking, the blot was then incubated with rhFGF-2 overnight, washed, then the bound rhFGF-2 was detected by anti-FGF-2 biotin-conjugates and streptavidin-alkaline phosphatase staining.
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Figure 6: FGF-2 ligand blot binding of glycosidase-digested rhPln.D1. Pooled anionic exchange fractions of rhPln.198 or rhPln.247 from HEK 293 cell culture were subjected to digestion as designated; 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. Samples were subjected to SDS-PAGE and blotted to nitrocellulose. After blocking, the blot was then incubated with rhFGF-2 overnight, washed, then the bound rhFGF-2 was detected by anti-FGF-2 biotin-conjugates and streptavidin-alkaline phosphatase staining.

Mentions: Knowing that a significant proportion of the rhPln.198 and rhPln.247 was glycosylated with HS, we tested the hypothesis that the rhPln.D1 would bind FGF-2. Applying rhFGF-2 in solution to the electrophoretically separated recombinant pools that had been immobilized on a nitrocellulose blot (Figure 6), the FGF-2 bound specifically to the rhPln.198 and rhPln.247. Further, digestion with heparinase III, but not chondroitinase ABC, mostly eliminated FGF binding to the enriched rhPln.D1 in this system.


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)

FGF-2 ligand blot binding of glycosidase-digested rhPln.D1. Pooled anionic exchange fractions of rhPln.198 or rhPln.247 from HEK 293 cell culture were subjected to digestion as designated; 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. Samples were subjected to SDS-PAGE and blotted to nitrocellulose. After blocking, the blot was then incubated with rhFGF-2 overnight, washed, then the bound rhFGF-2 was detected by anti-FGF-2 biotin-conjugates and streptavidin-alkaline phosphatase staining.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: FGF-2 ligand blot binding of glycosidase-digested rhPln.D1. Pooled anionic exchange fractions of rhPln.198 or rhPln.247 from HEK 293 cell culture were subjected to digestion as designated; 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. Samples were subjected to SDS-PAGE and blotted to nitrocellulose. After blocking, the blot was then incubated with rhFGF-2 overnight, washed, then the bound rhFGF-2 was detected by anti-FGF-2 biotin-conjugates and streptavidin-alkaline phosphatase staining.
Mentions: Knowing that a significant proportion of the rhPln.198 and rhPln.247 was glycosylated with HS, we tested the hypothesis that the rhPln.D1 would bind FGF-2. Applying rhFGF-2 in solution to the electrophoretically separated recombinant pools that had been immobilized on a nitrocellulose blot (Figure 6), the FGF-2 bound specifically to the rhPln.198 and rhPln.247. Further, digestion with heparinase III, but not chondroitinase ABC, mostly eliminated FGF binding to the enriched rhPln.D1 in this system.

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.

Show MeSH
Related in: MedlinePlus