Novel Aspects of Tetramer Assembly and N-terminal Domain Structure and Function Are Revealed by Recombinant Expression of Human AMP Deaminase Isoforms

Abstract

AMP deaminase isoforms purified from endogenous sources display smaller than predicted subunit molecular masses, whereas baculoviral expression of human AMPD1 (isoform M) and AMPD3 (isoform E) cDNAs produces full-sized recombinant enzymes. However, nearly 100 N-terminal amino acid residues are cleaved from each recombinant polypeptide during storage at 4°C. Expression of N-truncated cDNAs (⌬L96AMPD1 and ⌬M90AMPD3) produces stable recombinant enzymes exhibiting subunit molecular masses and kinetic properties that are similar to those reported for purified isoforms M and E. Conversely, wild type recombinant isoforms display significantly higher K m(app) values in the absence of ATP. Gel filtration analysis demonstrates native tetrameric structures for all recombinant proteins, except the wild type AMPD1 enzyme, which forms aggregates of tetramers that disperse upon cleavage of N-terminal residues at 4°C. These data: 1) confirm that available literature on AMP deaminase is likely derived from N-truncated enzymes and 2) are inconsistent with a new model proposing native trimeric structure of an N-truncated rabbit skeletal muscle AMP deaminase (Ranieri-Raggi, M., Montali, U., Ronca, F., Sabbatini, A., Brown, P. E., Moir, A. J. G., and Raggi, A. (1997) Biochem. J. 326, 641-648). N-terminal residues also influence actomyosin-binding properties of the enzyme, which are enhanced and suppressed by AMPD1 and AMPD3 sequences, respectively. Finally, co-expression of AMPD1 and AMPD3 recombinant polypeptides produces tetrameric enzymes with either isoform-specific or mixed subunits, and also reveals that tetramer assembly is driven by relative polypeptide abundance with no apparent preference for like subunits.

N-terminal Sequence and Distal Histidine Residues Are Responsible for pH-regulated Cytoplasmic Membrane Binding of Human AMP Deaminase Isoform E

“…Expression and Purification of Recombinant AMPD3 Enzymes-Human AMPD3 recombinant (wild type 1b and all N-truncated) enzymes and an N-truncated AMPD1 (⌬M54) enzyme were expressed in Sf9 (Spodoptera frugiperda) cells using baculoviral technology as previously described (20,21). All N-truncated constructs were produced by oligonucleotide-directed mutagenesis using wild type cDNAs (AMPD1[exon 2ϩ] and AMPD3[1b]) as templates.…”

“…However, extreme N-terminal regions in mammalian AMPD polypeptides are highly sensitive to proteolysis during purification and subsequent storage of the enzyme at 4°C (16 -19). Recombinant technology provides a means to overexpress AMPD enzymes that can be purified with subunits predominantly intact, although these are also subject to proteolysis during storage at 4°C (20). The availability of purified AMPD enzymes with subunits predominantly intact has stimulated interest in the structural and functional significance of extreme N-terminal sequences that were likely missing from previously characterized enzyme preparations.…”

“…Conversely, divergent N-terminal residues may play an important role in the intracellular distribution of AMPD as evidenced by their effects on contractile protein binding behavior (20,21). Although a more C-terminal conserved contractile protein binding domain has been identified in all AMPD isoforms, a stretch of sequence in the unique N-terminal region of the AMPD1 polypeptide is required for the high actomyosin binding capacity of isoform M (21).…”

“…Recombinant technology provides a means to overexpress AMPD enzymes that can be purified with subunits predominantly intact, although these are also subject to proteolysis during storage at 4°C (20). The availability of purified AMPD enzymes with subunits predominantly intact has stimulated interest in the structural and functional significance of extreme N-terminal sequences that were likely missing from previously characterized enzyme preparations.Recent studies have shown that extreme N-terminal sequences only subtly influence the catalytic and regulatory properties of human AMPD isoforms (20,21). Conversely, divergent N-terminal residues may play an important role in the intracellular distribution of AMPD as evidenced by their effects on contractile protein binding behavior (20,21).…”

“…Recent studies have shown that extreme N-terminal sequences only subtly influence the catalytic and regulatory properties of human AMPD isoforms (20,21). Conversely, divergent N-terminal residues may play an important role in the intracellular distribution of AMPD as evidenced by their effects on contractile protein binding behavior (20,21).…”