STRUCTURAL DISTINCTIONS AMONG ACETYLCHOLINESTERASES

  • Rosenberry, Terrone L, (PI)

Project: Research project

Description

Long-term objectives are to characterize the biochemical features of
distinct acetyl-cholinesterase (AChE) forms in synapses: their
structures, processes of assembly and localization, and functions in
synaptic transmission. The current program focuses on the structures
that attach AChEs to cell membranes. Previous work has demonstrated three
distinct classes of attachment structures, each rather unusual for
membrane proteins; 1) Dodecameric (A12) AChE is localized in skeletal
neuromuscular junctions by collagen-like subunits. 2) Dimeric (G2) AChE
in mammalian erythrocytes, nerve endings in torpedo electric organ, and
insect heads in anchored by a glycoinositol phospholipid covalently
linked to the C-terminus. 3) Tetrameric (G4) AChE in mammalian brain
appears to bind to membranes through a small hydrophobic noncatalytic
subunit. The proposed Specific Aims involve the latter two AChE classes. 1. A 20-kDa subunit in G4 bovine brain AChE has been identified that
appears responsible for membrane interaction, and its structure will be
determined to establish whether it is a peptide a lipid, or both. The
subunit amino acid sequence will be pursued, and antisera against this
subunit will be obtained 2. Additional structural features of the
glycoinositol phospholipid anchor of human erythrocyte AChE will be
defined. These include the hexose and hexose phosphate components and the
positions of hexose and inositol linkage. Peptides that include the C-
terminus of this AChE will sequenced. 3. Palmitoylation of inositol
renders the anchor of human erythrocyte AChE resistant to a
characteristic phospholipase C. Evidence of this modification will be
sought in a variety of cell lines by examining both endogenous G2 AChE
and transfected Drosophila G2 AChE. Free glycoinositol phospholipid
precursors of AChE anchors will be investigated. 4. A nucleotide sequence
corresponding to a stable transmembrane peptide will be substituted for
the 3' coding region that directs the glycoinositol phospholipid anchor
addition in Drosophila AChE. Our long-term goal in this aim is to produce
a transgenic fly in which AChE is anchored only by peptide and assess the
effects of this changes on fly development and AChE location. Because 30-
40 membrane proteins currently are known to be anchored by glycoinositol
phospholipids, further information about these anchors is vital to a
better understanding of membrane protein function.
StatusFinished
Effective start/end date7/1/805/31/07

Funding

  • National Institutes of Health
  • National Institutes of Health: $75,000.00
  • National Institutes of Health: $261,013.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $243,442.00
  • National Institutes of Health: $261,013.00
  • National Institutes of Health
  • National Institutes of Health: $261,013.00
  • National Institutes of Health: $189,487.00
  • National Institutes of Health: $195,171.00
  • National Institutes of Health
  • National Institutes of Health: $284,463.00
  • National Institutes of Health: $307,675.00
  • National Institutes of Health
  • National Institutes of Health: $261,013.00
  • National Institutes of Health
  • National Institutes of Health

Fingerprint

Acetylcholinesterase
Cholinesterases
Catalytic Domain
Acylation
Pain
Ligands
Peptides
Phospholipids
Hexoses
Membranes
Electric Organ
Collagen
Membrane Proteins
Glycosaminoglycans
Gel Chromatography
Erythrocytes
Synapses
Brain
Acetylthiocholine
Fluorescence

ASJC

  • Medicine(all)
  • Neuroscience(all)