Derived amino acid sequence of shark (Squalus acanthias) liver pre-glutamine synthetase
Laud, Purnima R.
Campbell, J. W.
Doctor of Philosophy
The enzyme glutamine synthetase occurs as species- and as tissue-specific isozymes in elasmobranchs. In liver tissue, glutamine synthetase is mitochondrial where it functions to provide glutamine to carbamyl phosphate synthetase-III for urea biosynthesis. In both dogfish shark and in stingray liver glutamine synthetase mRNA is translated as a 3 kDa larger precursor than in the mature matrix form. This indicates targeting of the pre-glutamine synthetase to liver mitochondria via a classical N-terminal signal that is cleaved during the import process. In order to examine this, degenerate oligomer primers flanking a highly conserved sequence in avian and mammalian glutamine synthetases were prepared and this primer fragment was amplified by the reverse transcriptase-polymerase chain reaction (RT-PCR) technique. The resulting PCR product was used to screen a $\lambda$ZapII shark liver mRNA-cDNA library. Tertiary screening yielded 5 clones, one, of 1.3 kb (pSGS-6), contained the complete coding sequence plus 5$\sp\prime$ and 3$\sp\prime$ flanking nucleotides. pSGS-6 coded for a peptide of 403 amino acids whose sequence was approximately 83% identical to that of other vertebrate sequences. The calculated molecular weight of this peptide was 45.4 kDa. Compared with other vertebrate glutamine synthetases the shark peptide has an additional 29 amino acids at the N-terminus. Removal of 14 of these would result in a 43.6 kDa peptide which is consistent with the observed differences between pre-glutamine synthetase and mitochondrial glutamine synthetase in shark liver. This putative targeting signal contained several basic (R and K), hydroxyl (S and T) and no acidic amino acids. The seven most N-terminal amino acids can be projected to form a very strong amphipathic alpha helix with a hydrophobic moment/residue of 0.779 and is thus presumably an important component of the targeting signal.
Molecular biology; Biology; Cell biology