| Objective:To explore the relationship between the affinity of human growth hormone (hGH) to its receptor and its biopotency by site-directed singal or doubal residue mutagenesis at site1 (S1), site2 (S2) or both in the hormone's molecule.Methods:1. Preparation and affinity-screening of hGH mutant According to literature,9 amino acid residues that may affect affinity were mutated in hGH S1, S2 or both with site-directed singal or doubal residue mutagenesis. The successful mutants (hGHmut), as well as wild type hGH (hGH-WT) for control were then transformed into the cell strain transetta DE3 to express the target protein. The purity,concentration and molecular weight of each recombinant mutant proteins were identified by SDS-PAGE and ultraviolet spectrophotometer, and their affinity and kinetics to growth hormone binding receptor were analysed by surface plasmon resonance (SPR)2. Detect the biopotency of hGHmuts The biopotency of hGHmuts were analysed at both in vitro and in vivo. In vitro, the cell proliferation ability of hGHmuts was measured by cell proliferation assays with hGH-WT as control. In vivo experiment, different hGHmuts were injected (0.5mg/kg, once a day for 2 weeks) into pituitary GH-deficient Lewis dwarf rats (7-week age). And then the the animals were weighted every other day; the tibia was dissected and weighted after last injection with blood and liver collected for detection of serum IGF-1 by Elisa or liver gene of IGF-1 and GHR expression by Real-Time PCR.3. Analysis of the relationship between affinity and biopotency of hGHmut The ligand-receptor binding response of the second hGHBP to 1:1 hGH-hGHBP intermediate complex was detected by trimolecular SPR and used to represent the formation of 1:2 hGH-hGHBP ternary complex of different hGHmuts. The correlation of the formation of ternary complex to bipotency, and the relationship between kinetics of the mutants with increased affility and 1:2 hGH-hGHBP ternary complex formation were analyzed.Results:1. Identification of hGHmuts and their affinity The sequeneces of 4 S1 mutants,4 S2 mutants and 4 S1+S2 combined mutants were identified by sequencing, which showing a consistency with the experimental design. SDS-PAGE showed that the molecular weight of the 12 hGHmuts were 22kDa, consistent with hGH-WT. And the purity of each mutant protein was more than 90% after purification. Ultraviolet spectrophotometry showed that the OD260/280 nm of hGH-WT and 12 hGHmut recombinant proteins were less than 0.65 and the concentration was greater than 2 mg /ml.By SPR screening,4 single mutants with enhanced affility were selected, including R167XS1, E174AS1 and R64K/F54PS1 with increased affility of S1, and N109XS2, of S2. The other single mutants showed a reduced S1 or S2 affinity. To obtain mutants with enhanced affinity of both S1 and S2, the combined mutants, R167X/N109XS1/S2, E174A/N109XS1/S2, were prepared and identified.The kinetic analysis of the mutants with enhanced affinity showed that there were two different binding modes:In the first model (such as R167XS1, E174AS1, R167X/N109XS1/S2 and E174A/N109XSI/S2), the enhanced affinity was due to an increased association rate (kon) with a reduced dissociation rate (koff) of hormone-receptor molecules. In the second, the kon did not change or slightly decreased, the enhanced affinity is only due to a reduced koff.2. Biological effects of hGHmut In vitro (cell proliferation assay) and in vivo (dwarf rat weight gain) experiments showed that:? The mutants with decreased affinity in any one of S1 or S2 had a reduced biopotency.? The mutants with increased affinity in S1 with S2 unaffected had a increased biopotency to some degree in vitro, but which could not be detected in vivo. ? The mutants with increased affinity in S2 alone had no improving biopotency. ? the combined mutants with increased affinity in both S1 and S2 had a substantially enhanced biopotency both in vitro and in vivo; their in vivo biopotency had no gender difference, and was related to increased serum IGF-1 and local expression of GHR and IGF-1 genes in the liver.3. Relationship between the biopotency and the number of 1:2 hGH-hGHBP ternary complex formation of hGHmut Correlation analysis showed that the ternary complexs binding response had a significant negative correlation with the EC50 in vitro (r=-0.97,p=0.001) and a significant positive correlation with growth percentages of dwarf rats in vivo (r=+0.89,p<0.01).The mutants with increaced affinity in S2 alone (N109XS2) had no elevated binding response of 1:2 hGH-hGHBP ternary complex due to the 1:1 hGH-hGHBP intermediate complex was unchanged. The mutants with increased affinity in S1 and unaffected S2 (E174AS1 and R167XS1) produced an elevation of the ternary complex binding response.The combined mutants with increased affinity in both S1 and S2 (E174A/N109XS1/S2 and R167X/N109XS1/S2) had an elevated ternary complex binding response which was significantly higher than that produced by the mutants with increased affinity in S1 alone.4. Relationship between the kinetics and the number of 1:2 hGH-hGHBP ternary complex formation of hGHmut Analysis of high affinity hGHmuts in the relationship between their binding kinetics and the ternary complexes formation showed that the mutants with enhanced affinity due to an increased kon with a reduced koff (such as R167XS1, E174AS1, R167X/N109XS1/S2 and E174A/N109XS1/S2) produeced an elevated ternary complex binding response. While those with their affinity enhanced by a reduced koff alone did not.Conclusion:1. A decrease in affinity of hGH S1 or S2 reduces the 1:2 hGH-hGHR ternary complex formation and the hormone's biopotency; An increase in hGH S1 affinity alone (S2 affinity unaffected) can promote 1:2 hGH-hGHR ternary complex formation and increase the biopotency of the hormone in vitro, suggesting that the affinity of hGH S1 or S2 to GHR is not surpasses the requirements for the hormone's biopotency.2. An increase in hGH S2 affinity alone can not promote the 1:2 hGH-hGHR ternary complex formation and biopotency due to the 1:1 hGH-hGHR dimer does not change. This suggests that, in comparison to S2, S1 is dominant in affecting on hGH's biopotency.3. An increase in affinity of both S1 and S2 can substancially promote the formation of 1:2 hGH-hGHBP ternary complex formation, and enhance biopotency of hGH both in vitro and in vivo. The enhancement biopotency in vivo is related to the amplifying mechnism of GH-IGF-1 axis system. |
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