Our previous studies of the gene responsible for the expression of pituitary GH (GH1 gene) have identified 13 novel missense mutations in 52 children with short stature and 154 controls, which occurred more frequently in patients than controls. These mutations result in expression of variant forms of GH with a single amino acid alteration that could lead to conformational changes within the protein, possibly resulting in cleavage sites hidden in wild-type GH becoming exposed, leading to increased susceptibility to proteolytic degradation. To investigate this, GH (WT and variant) was expressed in insect cells, quantified by IRMA, the concentration adjusted to 60nM and incubated at 37 degC for 1 hour with trypsin or chymotrypsin, both 0.1 micrograms per millilitre (the lowest concentration of the proteases that produced detectable degradation of WT). The reaction was stopped by the addition of trypsin-chymotrypsin inhibitor and the amount of intact GH remaining following proteolysis was analysed by western blotting. Samples were run on a 12% polyacrylamide gel and electroblotted onto PVDF membrane, probed with an anti-human GH antibody and developed using enhanced chemiluminescence. The resulting films were analysed using imaging densitometry and the results expressed as a percentage of intact protein remaining compared to control (n=3). Of the 13 variants, 5 were significantly more degraded than WT by both proteases e.g. Asp11Asn, Thr27Ile, Ser71Phe and Gln91Leu which, following proteolysis, only 64%, 40%, 56% and 44% respectively, remained as intact GH compared to WT (100%), 4 showed no difference compared to WT in their susceptibility to degradation by either protease e.g. Lys41Arg (96%), Val110Ile (99%) and Thr175Ala (90%) and 3 showed increased susceptibility to only one of the proteases e.g. Ser108Cys (trypsin 90%, chymotrypsin 38%). In conclusion, GH variants show differential susceptibility to proteolysis, which may be an indication of conformational changes within the variant protein.
22 - 24 Mar 2004
British Endocrine Societies