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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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Anion Exchange Chromatography of rhPln.247 conditioned media (CM). A: Chromatogram of elution profile for rhPln.198-Ad and rhPln.247-Ad CM expressed from HEK 293 cells; B and C: Western blots of anion exchange fractions with mAb CSI 001-71 recognizing the Pln.D1 core; D and E: Stains-All analysis of HEK 293 fractions; F: Fractions from HUVEC synthesis. Panels B and D represent rhPln.198 fractionation; panels C, E, F represent rhPln.247 fractionation. (M) Markers; (CM) Conditioned medium undiluted from cells. Similar data were generated using plasmid expression of pln.247 (not shown).
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Figure 1: Anion Exchange Chromatography of rhPln.247 conditioned media (CM). A: Chromatogram of elution profile for rhPln.198-Ad and rhPln.247-Ad CM expressed from HEK 293 cells; B and C: Western blots of anion exchange fractions with mAb CSI 001-71 recognizing the Pln.D1 core; D and E: Stains-All analysis of HEK 293 fractions; F: Fractions from HUVEC synthesis. Panels B and D represent rhPln.198 fractionation; panels C, E, F represent rhPln.247 fractionation. (M) Markers; (CM) Conditioned medium undiluted from cells. Similar data were generated using plasmid expression of pln.247 (not shown).

Mentions: HEK 293 cells were infected with the Pln.247-Ad or the Pln.198-Ad and the conditioned medium was subjected to anion exchange chromatography with a linear NaCl gradient to 1 M (Figure 1A). Testing the hypothesis that retention on the anionic exchange column through increasing ionic strength of the elution buffer would be characterized by an immunopositive rhPln.D1 with increasing GAG chain length, we sampled a broad range of immunopositive fractions by Western blot using the anti-Pln.D1 mAb CSI 001-071 (Figure 1B,C). In the eluant, we found that the immunoreactive species with the lower net apparent kDa (shorter GAGs) eluted earliest (up to 26 ml) and that increasing salt eluted rhPln.D1 species with increasing GAG length and greater apparent kDa. The early fractions of each recombinant were characterized by highly immunoreactive, minimally glycosylated species, the lowest approximately where the unglycosylated core of Pln.198 (Figure 1B) and Pln.247 (Figure 1C) was expected to migrate. Beyond 30 ml, eluting from approximately 0.5 M NaCl, these lower Mr species were absent while a broad range of immunoreactivity spanning 30-60 kDa upward (Pln.198) or 50-120 (Pln.247) eluted, gradually increasing in apparent Mr through 52 ml (eluting in 1 M NaCl). The range of apparent kDa in these higher Mr fractions suggested a recombinant species with GAG chains totalling 24 kDa to >90 kDa in rhPln.247, or average individual chain sizes of 8-30 kDa, assuming glycosylation of the 3 SGD linkage sites within rhPln.247. The higher Mr fractions of Pln.198 did not appear to be glycosylated as extensively, with estimated GAG chains of 3-13 kDa again assuming glycosylation at each of the three SGD regions in rhPln.198. The synthesis of rhPln.247 in HUVEC culture resulted in matching data (Figure 1F, Western blot). Similar data were also produced when the HEK 293 cells were transfected instead with a mammalian expression plasmid encoding the Pln.247 transgene (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)

Anion Exchange Chromatography of rhPln.247 conditioned media (CM). A: Chromatogram of elution profile for rhPln.198-Ad and rhPln.247-Ad CM expressed from HEK 293 cells; B and C: Western blots of anion exchange fractions with mAb CSI 001-71 recognizing the Pln.D1 core; D and E: Stains-All analysis of HEK 293 fractions; F: Fractions from HUVEC synthesis. Panels B and D represent rhPln.198 fractionation; panels C, E, F represent rhPln.247 fractionation. (M) Markers; (CM) Conditioned medium undiluted from cells. Similar data were generated using plasmid expression of pln.247 (not shown).
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Figure 1: Anion Exchange Chromatography of rhPln.247 conditioned media (CM). A: Chromatogram of elution profile for rhPln.198-Ad and rhPln.247-Ad CM expressed from HEK 293 cells; B and C: Western blots of anion exchange fractions with mAb CSI 001-71 recognizing the Pln.D1 core; D and E: Stains-All analysis of HEK 293 fractions; F: Fractions from HUVEC synthesis. Panels B and D represent rhPln.198 fractionation; panels C, E, F represent rhPln.247 fractionation. (M) Markers; (CM) Conditioned medium undiluted from cells. Similar data were generated using plasmid expression of pln.247 (not shown).
Mentions: HEK 293 cells were infected with the Pln.247-Ad or the Pln.198-Ad and the conditioned medium was subjected to anion exchange chromatography with a linear NaCl gradient to 1 M (Figure 1A). Testing the hypothesis that retention on the anionic exchange column through increasing ionic strength of the elution buffer would be characterized by an immunopositive rhPln.D1 with increasing GAG chain length, we sampled a broad range of immunopositive fractions by Western blot using the anti-Pln.D1 mAb CSI 001-071 (Figure 1B,C). In the eluant, we found that the immunoreactive species with the lower net apparent kDa (shorter GAGs) eluted earliest (up to 26 ml) and that increasing salt eluted rhPln.D1 species with increasing GAG length and greater apparent kDa. The early fractions of each recombinant were characterized by highly immunoreactive, minimally glycosylated species, the lowest approximately where the unglycosylated core of Pln.198 (Figure 1B) and Pln.247 (Figure 1C) was expected to migrate. Beyond 30 ml, eluting from approximately 0.5 M NaCl, these lower Mr species were absent while a broad range of immunoreactivity spanning 30-60 kDa upward (Pln.198) or 50-120 (Pln.247) eluted, gradually increasing in apparent Mr through 52 ml (eluting in 1 M NaCl). The range of apparent kDa in these higher Mr fractions suggested a recombinant species with GAG chains totalling 24 kDa to >90 kDa in rhPln.247, or average individual chain sizes of 8-30 kDa, assuming glycosylation of the 3 SGD linkage sites within rhPln.247. The higher Mr fractions of Pln.198 did not appear to be glycosylated as extensively, with estimated GAG chains of 3-13 kDa again assuming glycosylation at each of the three SGD regions in rhPln.198. The synthesis of rhPln.247 in HUVEC culture resulted in matching data (Figure 1F, Western blot). Similar data were also produced when the HEK 293 cells were transfected instead with a mammalian expression plasmid encoding the Pln.247 transgene (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.

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