Identification and functional characterization of a novel acetylcholine-binding protein from the marine annelid Capitella teleta.
We identified a homologue of the molluscan acetylcholine-binding protein (AChBP) in the marine polychaete Capitella teleta, from the annelid phylum. The amino acid sequence of C. teleta AChBP (ct-AChBP) is 21-30% identical with those of known molluscan AChBPs. Sequence alignments indicate that ct-AChBP has a shortened Cys loop compared to other Cys loop receptors, and a variation on a conserved Cys loop triad, which is associated with ligand binding in other AChBPs and nicotinic ACh receptor (nAChR) alpha subunits. Within the D loop of ct-AChBP, a conserved aromatic residue (Tyr or Trp) in nAChRs and molluscan AChBPs, which has been implicated directly in ligand binding, is substituted with an isoleucine. Mass spectrometry results indicate that Asn122 and Asn216 of ct-AChBP are glycosylated when expressed using HEK293 cells. Small-angle X-ray scattering data suggest that the overall shape of ct-AChBP in the apo or unliganded state is similar to that of homologues with known pentameric crystal structures. NMR experiments show that acetylcholine, nicotine, and alpha-bungarotoxin bind to ct-AChBP with high affinity, with K(D) values of 28.7 microM, 209 nM, and 110 nM, respectively. Choline bound with a lower affinity (K(D) = 163 microM). Our finding of a functional AChBP in a marine annelid demonstrates that AChBPs may exhibit variations in hallmark motifs such as ligand-binding residues and Cys loop length and shows conclusively that this neurotransmitter binding protein is not limited to the phylum Mollusca.
McCormack
Thomas
T
Laboratory of Neurobiology, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, P.O. Box 12233, Research Triangle Park, North Carolina 27709, USA.
Petrovich
Robert M
RM
Mercier
Kelly A
KA
DeRose
Eugene F
EF
Cuneo
Matthew J
MJ
Williams
Jason
J
Johnson
Katina L
KL
Lamb
Patricia W
PW
London
Robert E
RE
Yakel
Jerrel L
JL
D000109:Acetylcholine / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D019295:Computational Biology; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011077:Polychaeta; D011487:Protein Conformation; D053838:Scattering, Small Angle; D014961:X-Ray Diffraction
D002352:Carrier Proteins; D000109:Acetylcholine