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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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ELISA identifies only Pln.D1 but not Pln.D3-5 in enriched rhPln.198. The enriched high Mr rhPln.198 synthesized by HEK 293 cells was used to coat microtiter wells that were subsequently blocked then incubated with 1.7 μg/ml of different primary antibodies as depicted in the legend. ELISA was completed as described in Methods. Background signal for each antibody binding to uncoated wells was subtracted to produce the net signal. Inset: ELISA data showing negligible mAb A76 reactivity against rhPln.247 and strong reactivity with full-length endothelial perlecan (purified as previously described [53]), while mAb A71 reacted strongly to both the native perlecan and the truncated recombinant. Net absorbance minus buffer-coated wells represented.
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Figure 2: ELISA identifies only Pln.D1 but not Pln.D3-5 in enriched rhPln.198. The enriched high Mr rhPln.198 synthesized by HEK 293 cells was used to coat microtiter wells that were subsequently blocked then incubated with 1.7 μg/ml of different primary antibodies as depicted in the legend. ELISA was completed as described in Methods. Background signal for each antibody binding to uncoated wells was subtracted to produce the net signal. Inset: ELISA data showing negligible mAb A76 reactivity against rhPln.247 and strong reactivity with full-length endothelial perlecan (purified as previously described [53]), while mAb A71 reacted strongly to both the native perlecan and the truncated recombinant. Net absorbance minus buffer-coated wells represented.

Mentions: To support the hypothesis that the CSI-001-71 immunoreactive species eluting in relatively high salt were recombinant and not native Pln.D1, we pooled the fractions from 34-52 ml containing only high Mr rhPln.D1, reduced the salt by buffer exchange, then aliquots were immobilized in microtiter wells and ELISA performed with a panel of anti-perlecan antibodies (Figure 2). These data indicated that the perlecan core was recognized only by the anti-D1 core antibody CSI 001-71 but not by the anti-D3 antibody 7B5 (Invitrogen cat. 13-4400) nor the anti-D4 antibody A7L6 (Millipore mAb1948) nor the anti-D5 antibody CSI 001-74. The recombinant pools were each found to have CS glycosylation recognized by mAb CS56 (Sigma Aldrich) while mAb 10E4 recognized the predominately unsulfated HS epitopes [36]. Interestingly, mAb CSI 001-76 (clone A76), that is known to recognize domain 1 of native perlecan (Figure 2 inset, endothelial perlecan), poorly recognized the truncated recombinants rhPln.198 (Figure 2) and rhPln.247 (inset, Figure 2) relative to mAb CSI 001-71 (A71).


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)

ELISA identifies only Pln.D1 but not Pln.D3-5 in enriched rhPln.198. The enriched high Mr rhPln.198 synthesized by HEK 293 cells was used to coat microtiter wells that were subsequently blocked then incubated with 1.7 μg/ml of different primary antibodies as depicted in the legend. ELISA was completed as described in Methods. Background signal for each antibody binding to uncoated wells was subtracted to produce the net signal. Inset: ELISA data showing negligible mAb A76 reactivity against rhPln.247 and strong reactivity with full-length endothelial perlecan (purified as previously described [53]), while mAb A71 reacted strongly to both the native perlecan and the truncated recombinant. Net absorbance minus buffer-coated wells represented.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2944331&req=5

Figure 2: ELISA identifies only Pln.D1 but not Pln.D3-5 in enriched rhPln.198. The enriched high Mr rhPln.198 synthesized by HEK 293 cells was used to coat microtiter wells that were subsequently blocked then incubated with 1.7 μg/ml of different primary antibodies as depicted in the legend. ELISA was completed as described in Methods. Background signal for each antibody binding to uncoated wells was subtracted to produce the net signal. Inset: ELISA data showing negligible mAb A76 reactivity against rhPln.247 and strong reactivity with full-length endothelial perlecan (purified as previously described [53]), while mAb A71 reacted strongly to both the native perlecan and the truncated recombinant. Net absorbance minus buffer-coated wells represented.
Mentions: To support the hypothesis that the CSI-001-71 immunoreactive species eluting in relatively high salt were recombinant and not native Pln.D1, we pooled the fractions from 34-52 ml containing only high Mr rhPln.D1, reduced the salt by buffer exchange, then aliquots were immobilized in microtiter wells and ELISA performed with a panel of anti-perlecan antibodies (Figure 2). These data indicated that the perlecan core was recognized only by the anti-D1 core antibody CSI 001-71 but not by the anti-D3 antibody 7B5 (Invitrogen cat. 13-4400) nor the anti-D4 antibody A7L6 (Millipore mAb1948) nor the anti-D5 antibody CSI 001-74. The recombinant pools were each found to have CS glycosylation recognized by mAb CS56 (Sigma Aldrich) while mAb 10E4 recognized the predominately unsulfated HS epitopes [36]. Interestingly, mAb CSI 001-76 (clone A76), that is known to recognize domain 1 of native perlecan (Figure 2 inset, endothelial perlecan), poorly recognized the truncated recombinants rhPln.198 (Figure 2) and rhPln.247 (inset, Figure 2) relative to mAb CSI 001-71 (A71).

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