Limits...
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.

Show MeSH

Related in: MedlinePlus

Immunoblot analysis of growth factor binding to Pln.198 and Pln.247. 500 ng growth factors were bound to nitrocellulose, the blots were blocked, and then incubated in either 1 ug/ml Pln.198 or Pln.247 (panel A) or rhPln.247 pre-digested with heparinase III (panel B). Detection was performed with anti-perlecan domain 1 primary antibody CSI 001-71. Note that while both the background BSA signal and the internal control rhPln.247 signals are slightly higher in panel B (bottom two samples), a decrease in the growth factor binding to Pln.247 was present after digestion.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2944331&req=5

Figure 8: Immunoblot analysis of growth factor binding to Pln.198 and Pln.247. 500 ng growth factors were bound to nitrocellulose, the blots were blocked, and then incubated in either 1 ug/ml Pln.198 or Pln.247 (panel A) or rhPln.247 pre-digested with heparinase III (panel B). Detection was performed with anti-perlecan domain 1 primary antibody CSI 001-71. Note that while both the background BSA signal and the internal control rhPln.247 signals are slightly higher in panel B (bottom two samples), a decrease in the growth factor binding to Pln.247 was present after digestion.

Mentions: Nitrocellulose immobilization of several growth factors and immunoblot analysis confirmed binding of rhPln.198 and rhPln.247 with the growth factors rhFGF-2, rhBMP-6, rhBMP-7, rhPDGF-BB, and VEGF189 (Figure 8A). A significant reduction in binding signal for each resulted from pre-treating the rhPln.D1 with heparinase III (Figure 8B) suggesting some GF affinity for HS. No binding with rhFGF-7 (Figure 8) or rhEGF, rhIGF, BMP-9, and VEGF165 (not shown) was detected by these methods. Results were similar using the rhPln.198 (not shown).


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)

Immunoblot analysis of growth factor binding to Pln.198 and Pln.247. 500 ng growth factors were bound to nitrocellulose, the blots were blocked, and then incubated in either 1 ug/ml Pln.198 or Pln.247 (panel A) or rhPln.247 pre-digested with heparinase III (panel B). Detection was performed with anti-perlecan domain 1 primary antibody CSI 001-71. Note that while both the background BSA signal and the internal control rhPln.247 signals are slightly higher in panel B (bottom two samples), a decrease in the growth factor binding to Pln.247 was present after digestion.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: Immunoblot analysis of growth factor binding to Pln.198 and Pln.247. 500 ng growth factors were bound to nitrocellulose, the blots were blocked, and then incubated in either 1 ug/ml Pln.198 or Pln.247 (panel A) or rhPln.247 pre-digested with heparinase III (panel B). Detection was performed with anti-perlecan domain 1 primary antibody CSI 001-71. Note that while both the background BSA signal and the internal control rhPln.247 signals are slightly higher in panel B (bottom two samples), a decrease in the growth factor binding to Pln.247 was present after digestion.
Mentions: Nitrocellulose immobilization of several growth factors and immunoblot analysis confirmed binding of rhPln.198 and rhPln.247 with the growth factors rhFGF-2, rhBMP-6, rhBMP-7, rhPDGF-BB, and VEGF189 (Figure 8A). A significant reduction in binding signal for each resulted from pre-treating the rhPln.D1 with heparinase III (Figure 8B) suggesting some GF affinity for HS. No binding with rhFGF-7 (Figure 8) or rhEGF, rhIGF, BMP-9, and VEGF165 (not shown) was detected by these methods. Results were similar using the rhPln.198 (not shown).

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