Crosslinking spectrin

The neurotoxic effects of -hexane have been associated with the pyrrolidation of protein (Graham et al. 1995) by 2,5-hexanedione. Therefore, the development of analytical methods to determine this potential biomarker of effect of -hexane in hair and the subsequent crosslinking of the blood proteins, spectrin and hemoglobin, would be useful. 

Spectrin/fodrin Actin crosslinking protein in cortical cytoskeleton (Wl)... 

Significant contributors to cell structure are those proteins that crosslink actin filaments or connect actin filaments to the cell membrane. Examples of such proteins can be found within the spectrin superfamily of cytoskeletal proteins. This discrete group is principally composed of the actin crosslinking protein o-actinin, and the membrane-associated actin-binding proteins spectrin and dystrophin. 

N-terminal actin-binding domains and in the spectrin repeats that form the rod domains (Davison and Critchley, 1988). The spectrin repeats are found in distinct multiples in each protein, resulting in a characteristic actin crosslinking distance. a-Actinin contains four repeats, /3-spectrin contains 17, a-spectrin contains 20, and dystrophin contains 24. The sequences of some spectrin repeats of a- and /3-spectrin are similar in many ways to the four repeats present in a-actinin (Dubreuil, 1991). Within the cell, a-actinin and spectrin dimerize, although the spectrins interact further to generate a functional tetramer (Fig. 1). Most notable is that the ends of the native spectrin tetramer involved in the dimerization event show remarkable similarity to the rod domain repeats of a-actinin that also mediate dimer formation. 

Fig. 11. The structure of a-actinin and the two vertebrate Z-band lattices. (A) The ubiquitous protein a-actinin is an anti-parallel homodimer. Each 100 KDa monomer comprises four central spectrin repeats (SI to S4) an EF-hand domain and two calponin homology domains (CH) at the N-terminus. The EF-hand domains bind calcium in non-muscle cells. One a-actinin molecule binds two actin filaments via the calponin homology domains. a-Actinin binds titin via EF-hand domains. (B, C) The Z-band is the site where actin filaments from adjacent sarcomeres overlap in a tetragonal lattice and are crosslinked by a-actinin molecules. The polarity and origin of the actin filaments is indicated by U (up) and D (down). The appearance of the Z-band in cross-section is typically basketweave-like (B) or small square-like (G). The appearance is reported to transform between the two appearances depending on the state of the muscle.

Fig. 5.6. Two-dimensional gel electrophoresis of reversibly crosslinked human erythrocyte membranes. Ghosts were crosslinked with methyl 3-(4-azidophenyldithio)propionimidate and electrophoresed on a 0.5% agarose-1.75% polyacrylamide tube gel. The gel was treated with P-mercaptoethanol (10 mM), and electrophoresed in the second dimension on a 4 to 12 % gradient polyacrylamide slab gel. Some of the crosslinked components (mostly oligomers of spectrin) were incompletely cleaved. (Ji et al., 1980).

Valentine et al. (1993) have shown that the initial dithiocarbamate protein adduct decomposes to isothiocyanate derivatives which then react with protein nucleophiles resulting in crosslinking. The crosslinking of protein in the nerve axons to cause their ultimate degeneration is correlated with the crosslinking of spectrin, a blood cell membrane protein. This suggests that the latter can be used as a biomarker of adverse effect of nerve damage. 

The concentration of crosslinked red blood cell spectrin has been suggested as a marker of nerve protein crosslinking damage that leads to slower conduction velocities and abnormal nerves due to protein adduct formation initially with dithiocarbamates which decompose to form isothiocyanate adducts (Valentine et al. 1993). These latter adducts can then cause the actual crosslinking of both spectrin and nerve protein. 

As new red blood cells must be made to replace the damaged spectrin, the crosslinking of this protein may serve as a longer term biomarker of carbon disulfide exposure. 

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