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
Mitogenic Activity of rhPln.247 and rhPln.198. HUVEC (panel A, C) or Baf32 cells (panel B) demonstrated a dose-dependent mitogenic response to rhPln.247 (panel A, B), or rhPln.198 (panel C). Panel B: Proliferation augmentation of FGFR1C expressing BAF32 cells by Pln.247 in the presence of 0.3 nM rhFGF-2 (solid). No proliferation enhancement was observed with Pln.247 without FGF-2 (crosshatch). In panel C, data represents the net absorbance for varying amounts of Pln.D1 with constant growth factor concentration after subtracting the contribution from Pln.D1 alone (without growth factor). Data are the average of four experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 9: Mitogenic Activity of rhPln.247 and rhPln.198. HUVEC (panel A, C) or Baf32 cells (panel B) demonstrated a dose-dependent mitogenic response to rhPln.247 (panel A, B), or rhPln.198 (panel C). Panel B: Proliferation augmentation of FGFR1C expressing BAF32 cells by Pln.247 in the presence of 0.3 nM rhFGF-2 (solid). No proliferation enhancement was observed with Pln.247 without FGF-2 (crosshatch). In panel C, data represents the net absorbance for varying amounts of Pln.D1 with constant growth factor concentration after subtracting the contribution from Pln.D1 alone (without growth factor). Data are the average of four experiments.

Mentions: A mitogenic dose response to rhPln.247 by HUVEC in low-serum growth medium occurred (Figure 9A). Using Baf32 cells, which have a limited mitogenic response to growth factors and express only the FGFR1c receptor, we demonstrated rhPln.247-dependant mitogenicity through the FGF-2/FGFR1c activation pathway (Figure 9B). Similar results were obtained with rhPln.198 also demonstrating a dose-dependent synergistic response of HUVEC to rhPln.198 in the presence of constant rhFGF-2 levels (Figure 9C).


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)

Mitogenic Activity of rhPln.247 and rhPln.198. HUVEC (panel A, C) or Baf32 cells (panel B) demonstrated a dose-dependent mitogenic response to rhPln.247 (panel A, B), or rhPln.198 (panel C). Panel B: Proliferation augmentation of FGFR1C expressing BAF32 cells by Pln.247 in the presence of 0.3 nM rhFGF-2 (solid). No proliferation enhancement was observed with Pln.247 without FGF-2 (crosshatch). In panel C, data represents the net absorbance for varying amounts of Pln.D1 with constant growth factor concentration after subtracting the contribution from Pln.D1 alone (without growth factor). Data are the average of four experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 9: Mitogenic Activity of rhPln.247 and rhPln.198. HUVEC (panel A, C) or Baf32 cells (panel B) demonstrated a dose-dependent mitogenic response to rhPln.247 (panel A, B), or rhPln.198 (panel C). Panel B: Proliferation augmentation of FGFR1C expressing BAF32 cells by Pln.247 in the presence of 0.3 nM rhFGF-2 (solid). No proliferation enhancement was observed with Pln.247 without FGF-2 (crosshatch). In panel C, data represents the net absorbance for varying amounts of Pln.D1 with constant growth factor concentration after subtracting the contribution from Pln.D1 alone (without growth factor). Data are the average of four experiments.
Mentions: A mitogenic dose response to rhPln.247 by HUVEC in low-serum growth medium occurred (Figure 9A). Using Baf32 cells, which have a limited mitogenic response to growth factors and express only the FGFR1c receptor, we demonstrated rhPln.247-dependant mitogenicity through the FGF-2/FGFR1c activation pathway (Figure 9B). Similar results were obtained with rhPln.198 also demonstrating a dose-dependent synergistic response of HUVEC to rhPln.198 in the presence of constant rhFGF-2 levels (Figure 9C).

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