Carboxy-terminal sequence divergence and processing of the polyprotein antigen from Dirofilaria immitis.
A polyprotein composed of multiple units arranged in direct tandem arrays has been identified in parasitic and free living nematodes. Analysis of previously cloned units from the Dirofilaria immitis polyprotein antigen (DiPA) indicated the units were nearly identical but here we demonstrate that they segregate into two related families. The consensus repeats, DiPA-CR1 and CR2, derived for each family are 80% identical. However, the repeats at the C-terminus of the polyprotein have diverged from DiPA-CR1 and CR2. This was shown by DNA sequence and Southern blot analysis of a 1.9 kb cDNA clone that encodes 4.4 C-terminal repeats (DiPA-TR1 through TR5). DiPA-TR3 through TR5 show 27-52% amino acid identity with the consensus repeats and 31-35% amino acid identity with one another. Metabolic labeling studies have shown that cleavage of DiPA generates a protein "ladder' from 14 to > 200 kDa. RRKR, a cleavage motif of subtilisin-like proprotein convertases, was identified as the natural cleavage site. In vitro digestion experiments with proteinase K suggest a structural model for DiPA consisting of protease resistant cores joined by protease sensitive linkers containing the RRKR site. This motif is absent between DiPA-TR3 and TR4 and has been altered to KR between DiPA-TR4 and TR5. An immunoblot of D. immitis extract probed with anti-DiPA-TR4/5 serum demonstrates the absence of cleavage at these sites. These divergent repeats provide an opportunity to investigate processing of the D. immitis polyprotein in vivo.
Poole
C B
CB
Molecular Parasitology Division, New England Biolabs, Beverly, MA 01915, USA.
Hornstra
L J
LJ
Benner
J S
JS
Fink
J R
JR
McReynolds
L A
LA
D000595:Amino Acid Sequence; D000818:Animals; D000947:Antigens, Helminth / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D016384:Consensus Sequence; D004183:Dirofilaria immitis / Q000737:chemistry*; D015801:Helminth Proteins / Q000737:chemistry* / Q000378:metabolism; D015151:Immunoblotting; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D012091:Repetitive Sequences, Nucleic Acid; D016415:Sequence Alignment
D000947:Antigens, Helminth; C104085:DIPA protein, Dirofilaria immitis; D015801:Helminth Proteins; D011506:Proteins; D011994:Recombinant Proteins