Similarity of recombinant human perlecan domain 1 by alternative expression systems bioactive heterogenous recombinant human perlecan D1.
Similarity of recombinant human perlecan domain 1 by alternative expression systems bioactive heterogenous recombinant human perlecan D1.
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替代表达系统生物活性异源重组人基底膜蛋白 D1 与重组人基底膜蛋白结构域 1 的相似性。
DOI：
10.1186/1472-6750-10-66
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发表时间：
2010-09-09
影响因子：
3.5
通讯作者：
DeCarlo, Arthur A.
中科院分区：
文献类型：
--
作者：
Ellis, April L.;Pan, Wensheng;Yang, Guang;Jones, Kim;Chuang, Christine;Whitelock, John M.;DeCarlo, Arthur A.
硫酸乙酰肝素糖胺聚糖是某些蛋白聚糖的不同组分，并且已知其作为诱导信号传导和生长因子活性所必需的共受体与生长因子相互作用。在这份报告中，我们表征异质糖基化重组人串珠素结构域1（HSPG 2缩写为rhPln.D1）在HEK 293细胞或HUVEC中合成的瞬时基因传递使用腺病毒或表达质粒技术。通过阴离子交换层析后的SDS-PAGE分析，重组蛋白聚糖似乎具有范围为总共6 kDa至>90 kDa/重组体的糖胺聚糖链。酶消化的高Mr rhPln. D1的免疫印迹分析表明，rhPln. D1以约2：1的比例合成为硫酸软骨素或硫酸乙酰肝素蛋白聚糖，杂合体可忽略不计。二级结构分析表明，在两个重组物种的螺旋和片。在配体印迹分析中，rhPln.D1表现出与rhFGF-2的结合，表观kD为2 ± 0.2 nM，对肝素酶III的消化几乎完全敏感，但对软骨素酶消化不敏感。此外，我们证明了HS介导的结合两个rhPln.D1物种的其他几个GF。最后，我们证实了rhPln.D1对FGF介导的细胞活化的增强作用，并证明了通过FGFR 1c受体的促有丝分裂信号传导。特别是对基于DNA的治疗的新兴领域的重要性，我们在这里已经表明，蛋白聚糖合成，在不同的细胞系中，其中GAG概况通常不同，可以通过重组技术来指导，以产生具有高度相似的GAG概况的生物活性重组体的群体。
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. 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. 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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DOI：
10.1016/s1357-2725(99)00123-5
发表时间：
2000-02-01
影响因子：
4
作者：
Nugent, MA;Iozzo, RV
通讯作者：
Iozzo, RV
影响因子：
24.5
作者：
Hull, MA;Knifton, A;Hawkey, CJ
通讯作者：
Hawkey, CJ
影响因子：
4.8
作者：
Knox, S;Merry, C;Whitelock, J
通讯作者：
Whitelock, J
DOI：
10.1006/bbrc.1995.1805
发表时间：
1995-06-06
影响因子：
3.1
作者：
KOKENYESI, R;SILBERT, JE
通讯作者：
SILBERT, JE
影响因子：
37.8
作者：
Losordo, DW;Vale, PR;Kuntz, RE
通讯作者：
Kuntz, RE