Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.341987
Title: A mutagenic study of functional and structural aspects of rat insulin-like growth factor binding protein-5
Author: Song, Hyuk
ISNI:       0000 0001 3470 8937
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 2001
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Abstract:
On going research in our laboratory is focussed on the potential role of Insulin-like growth factor binding protein-5 (IGFBP-5) in apoptosis of mammary epithelial cells. In order to increase our understanding of IGFBP-5 functions, the work described below focussed on the relationship between the structure of the binding protein and its important molecular interactions. Using site-directed mutagenesis, we have mutated two highly conserved amino acids, Gly203 and Gln209 (G203K and Q209A respectively) within the basic amino acid rich region (201- 218) in the C-terminal domain of rat IGFBP-5. After analysis of binding activity using three different methods - IGF ligand blotting, IGF solution phase equilibrium binding and biosensor measurement - we have shown that the mutation of either of these two residues results in a reduction in affinity of the binding protein for both IGF-I and IGF-II by approximately ten-fold. Furthermore, mutation of both amino acids (G203K/Q209A, termed the Double mutant) had a cumulative effect and results in the complete ablation of IGF-I binding and a several thousand-fold reduction in IGF-II binding. This reduction in IGF binding affinity was comparable to that observed for the C-terminally truncated mutant BP550 (residues 1-168), which suggests that Gly203 and Gln209 may be the major determinants for IGF binding in the C-terminal domain. In addition, using heparin ligand blots, we confirm that mutation of basic residues within the C-terminal 201- 218 region (Hep- mutant: R201L, K202E, K206Q and R214A) results in major attenuation of heparin binding, whereas the G203K and Q209A single mutants and the Double mutant have no reduction in heparin binding. Therefore, our data suggests a potential overlap of heparin- and IGF- binding domains in the C-terminal region of IGFBP-5, and based on this, we discuss a potential model to explain the observed lower IGF binding affinity of ECM-bound IGFBP-5. In an attempt to assess the separate contribution of the N- and C-terminal domains to IGF binding, we also made four chimeric IGFBP cDNAs, BP552, BP522, BP255 and BP225, by switching domains between rat IGFBP-5 and rat IGFBP-2. We were consistently unable to detect expression of BP225 protein in the baculovirus/insect cell system, so this chimera had to be excluded from further study. Recognition of BP552 and BP522 proteins by both anti-rat IGFBP-5 and anti-rat IGFBP-2 antisera confirms their identity as chimeric proteins made up of domains from the native binding proteins, while BP255 was only recognised by the anti-rat IGFBP-5. The IGF binding properties of unpurified BP552, BP522 and BP255 proteins were assessed by IGF ligand blotting and IGF solution phase binding assays. These experiments demonstrated that BP552 and BP522 had comparable affinities with native IGFBP, whereas we were unable to detect any binding of BP255 to IGF-I by either technique, and only very weak interaction with IGF-II. Purified BP552 and BP522 were also tested by biosensor analysis, and data confirmed the results from the previous experiments. From this we can conclude that the N-terminal domain of IGFBP-5 can co-operate with the C-terminal domain of IGFBP-2 to produce a high affinity IGF binding species, irrespective of which central domain is present, whereas preliminary evidence would suggest that this cooperation may not occur between the N-terminal domain of IGFBP-2 and the C-terminal domain of lGFBP-5. In addition to IGF binding, the IGFBP-5 protein contains consensus heparin binding motifs in both its C-terminal and central domains, although only the C-terminal site was previously shown to be functional. Using heparin ligand blotting, it was found that the ability of BP552 and BP550 to bind to heparin was equivalent to that of WTIGFBP-5, whereas WTIGFBP-2 and BP522 failed to bind. These results demonstrate that an active heparin binding site in BP552 and BP550 is contained within the central domain of IGFBP-5, and that this site is only active in the absence of the carboxy-terminal domain. We subsequently mutated two basic amino acids (RI36A:R137A) in the central consensus binding sites between residues 132-140. This resulted in the loss of heparin binding for BP550, confirming the importance of these two basic amino acids in the central domain heparin binding activity. In light of these findings, we suggest that C-terminally truncated fragments of IGFBP-5 generated in vivo by proteolysis could retain heparin/extracellular matrix (ECM) binding properties. Finally, we propose that our various IGFBP-5 mutants described above provide an opportunity to test the structure-function relationships of this binding protein in an appropriate biological context.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.341987  DOI: Not available
Keywords: Apoptosis; Mammary epithelial cells
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