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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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Growth-factor binding to rhPln.198, rhPln.247, and HSPG in solid phase. Commercial HSPG, Pln.247, or rhPln.198 were bound directly to polystyrene microtiter wells. The proteoglycan samples were incubated with 6-fold dilutions of rhFGF-2. After washing away unbound FGF, the bound FGF was detected with anti-FGF biotin conjugates.
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Figure 7: Growth-factor binding to rhPln.198, rhPln.247, and HSPG in solid phase. Commercial HSPG, Pln.247, or rhPln.198 were bound directly to polystyrene microtiter wells. The proteoglycan samples were incubated with 6-fold dilutions of rhFGF-2. After washing away unbound FGF, the bound FGF was detected with anti-FGF biotin conjugates.

Mentions: Solid phase binding analysis of rhFGF-2 to the rhPln.198 and rhPln.247 demonstrated an estimated kD of 2.0 ± 0.2 nM for each, which was similar to binding of rhFGF-2 with a commercial HSPG (Sigma Aldrich) (Figure 7). Binding was the same when capturing the rhPln.D1 in the high Mr preparations onto a solid-phase anti-perlecan mAb 001-71 ensuring that binding of FGF-2 to the enriched high Mr Pln.D1 was a result of binding to the rhPln.198 and not a contaminant (data 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)

Growth-factor binding to rhPln.198, rhPln.247, and HSPG in solid phase. Commercial HSPG, Pln.247, or rhPln.198 were bound directly to polystyrene microtiter wells. The proteoglycan samples were incubated with 6-fold dilutions of rhFGF-2. After washing away unbound FGF, the bound FGF was detected with anti-FGF biotin conjugates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Growth-factor binding to rhPln.198, rhPln.247, and HSPG in solid phase. Commercial HSPG, Pln.247, or rhPln.198 were bound directly to polystyrene microtiter wells. The proteoglycan samples were incubated with 6-fold dilutions of rhFGF-2. After washing away unbound FGF, the bound FGF was detected with anti-FGF biotin conjugates.
Mentions: Solid phase binding analysis of rhFGF-2 to the rhPln.198 and rhPln.247 demonstrated an estimated kD of 2.0 ± 0.2 nM for each, which was similar to binding of rhFGF-2 with a commercial HSPG (Sigma Aldrich) (Figure 7). Binding was the same when capturing the rhPln.D1 in the high Mr preparations onto a solid-phase anti-perlecan mAb 001-71 ensuring that binding of FGF-2 to the enriched high Mr Pln.D1 was a result of binding to the rhPln.198 and not a contaminant (data 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