IDENTIFICATION OF ASP(95) AS THE SITE OF SUCCINIMIDE FORMATION IN RECOMBINANT HUMAN GLIAL-CELL LINE-DERIVED NEUROTROPHIC FACTOR

Human glial cell line-derived neurotrophic factor is a single polypept ide of 134 amino acids and functions as a disulfide-linked dimer. Incu bation of the protein in pH 5.0 and at 37 degrees C for 1 week showed that 5% of the material was converted to a form that eluted after the major protein peak on a cation-exchange column. The modified component gave an average molecular mass of 30367.0 u (theoretical = 30384.8 u) , Within measurement error, this 17.8-u decrease in mass indicated the loss of a water molecule. This observation, together with the protein 's behavior on cation-exchange chromatography and the mode of incubati on used to generate the modification, was consistent with cyclic imide (succinimide) formation at an aspartyl residue. Hence, only a monomer of the dimeric protein was modified. The modified monomer was purifie d and subjected to peptic degradation. By a combination of N-terminal analysis and mass spectrometry, the region containing Asp(95)-Lys(96) was identified to be modified. This was further confirmed by carboxype ptidase Y digestion of the modified peptide where the modified region was found to be resistant to further enzymatic degradation. Furthermor e, incubation of the modified monomer in pH 8.5 for 2 h yielded two pe aks, in agreement with the succinimide model where the cyclic imide wa s hydrolyzed into a mixture of isoaspartate and aspartate. Tryptic map ping of the isoaspartyl-containing protein showed that Asp(95) was ref ractory to Edman degradation, confirming it was in the isoaspartate fo rm. Hence, the modification observed was due to succinimide formation at Asp95. This is the first report of succinimide formation at an Asp- Lys linkage. (C) 1998 Academic Press.