Characterisation of the 5 kDa growth hormone isoform.
OBJECTIVE
The 5 kDa N-terminal fragment of 43 amino acids of human growth hormone (GH) shows a specific and significant in-vivo insulin-like activity. This isoform can be easily obtained by solid phase synthesis methods. Our objective in this study is to describe this procedure in detail and to provide structural information of the protein.


METHODS
Solid phase synthesis was employed for the synthesis of the 5 kDa GH isoform. Circular dichroism and limited proteolysis have been carried out to provide structural information about the folded state of the protein in solution. Surface plasmon resonance was used to compare the structural equivalence between the synthetic protein and a proteolytic homologue at an antibody binding level. For this purpose, a murine monoclonal antibody specific for the 5 kDa isoform was generated and characterised employing this and several other GH isoforms.


RESULTS
Circular dichroism and proteolysis results suggested that the C-terminal segment of the 5 kDa protein folds in an alpha-helix. The comparison of the synthetic product to its proteolytic homologue at an antibody binding level suggested structural equivalency. A highly specific antibody against the 5 kDa GH isoform was generated with null cross-reactivity for 17, 20 and 22 kDa isoforms. Kinetic data on the interaction with the synthetic 5 kDa GH was obtained.


CONCLUSIONS
The structure of the protein appears to be different in comparison to when it is included within the 22 kDa GH isoform. Finally, a highly specific antibody has been generated. The possible significance of the 5 kDa protein as a potential agent for obesity-related diseases is discussed.
Pharmacology Research Unit, Municipal Institute for Medical Research (IMIM), Barcelona, Spain.
Gerard
G
Such-Sanmartín
Jaume
J
Bosch
Jordi
J
Segura
Moutian
M
Wu
Hongwu
H
Du
Guangyu
G
Chen
Shan
S
Wang
Miquel
M
Vila-Perelló
David
D
Andreu
Ricardo
R
Gutiérrez-Gallego
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000494:pharmacology; D019382:Human Growth Hormone / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D051379:Mice; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D020349:Surface Plasmon Resonance
D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D010455:Peptides; D020033:Protein Isoforms; D011994:Recombinant Proteins; D019382:Human Growth Hormone