Studies on the antigenic sites of actin: a comparative study of the immunogenic crossreactivity of invertebrate actins

Abstract

The structural homologies of invertebrate actins with cytoplasmic vertebrate actins have recently been substantiated by comparative sequence analyses. This suggests that cytoplasmic actin is the ancestral precursor of smooth and striated muscle actin in vertebrates. We have raised antibodies in rabbits against a number of invertebrate muscle actins and have characterized the antisera by means of the highly sensitive ELISA method, which allows quantitation of nanomolar amounts of actin. Despite the fact that the invertebrate actins examined are very similar in primary structure, our results indicate that antibodies raised against them clearly distinguish between only a few amino-acid substitutions, and that the immunoreactivities quantitatively reflect the genetic divergence of this ubiquitous conservative protein. Examination of several proteolytic fragments of scallop actin for immunoreactivity with the homologous antiserum suggests that the major antigenic sites of actin are located within the amino terminal region of the molecule, while a carboxy terminal fragment comprising residues 69-372 exhibits very weak crossreactivity. Immunoadsorption experiments further indicate that species-specific antibodies are directed to antigenic determinants in the N-terminal region. This finding is supported by an examination of the effects of chemical modifications to Tyr, His, Arg, and Cys residues on the immunoreactivity of actin. Interaction with DNAase I markedly decreases the immunoreactivity of actin. This is consonant with the finding that the amino terminal peptide comprising residues 1-207 inhibits DNAase I, whilst a tryptic fragment fails to bind to the enzyme. The interaction is abolished by EDTA and the removal of the tightly bound cation is accompanied by a conformational change, shown by shifts in circular dichroic spectra. The possible involvement of the amino terminal peptide of actin in cation binding is discussed.

Membrane-associated actin in the rhabdomeral microvilli of crayfish photoreceptors.

“…15) . Preabsorption of antiserum with insect myofibrils was not sufficient to completely abolish labeling, probably due to the marked species-specific properties of the antibody (21). Nonimmune sera were occasionally found to label the basement membrane weakly even after extensive blocking procedures, probably owing to the presence of autoimmune antibodies to actin which are frequently present in human and in rabbit sera.…”

Comparative study of invertebrate actins: Antigenic cross-reactivity versus sequence variability

“…There have been previous attempts to induce antibodies to invertebrate actins (De Couet, 1983;Kleeve et al, 1979) but the sera were used without antibody fractionation and presented more or less broad specifity, impeding discrimination between direct effects due to amino-acid substitutions and those linked to related conformational changes. The use of chemical derivatives of actin and proteolytic fragments as immunogens to diversify serum specificity, and the purification of induced antibody populations on insolubilized derivatives thereby localizing the corresponding antigenic epitopes, made it possible to detect substituted sites in actins among the invertebrate phyla.…”

“…In invertebrates, the situation is not well documented (Vandekerckhove & Weber, 1984). Only few complete sequences have been determined (Fryberg et al, 1981;Vandekerckhove & Weber, 1984) and comparisons between actin isoforms are generally based on the variable acidic sequence 1-7 which allows electrophoretic separation by electrofocusing (De Couet, 1983;Huang et al, 1984;Margulis et al, 1982). This short sequence has been determined in numerous vertebrates, but only in some phyla in invertebrates (Vandekerckhove & Weber, 1984).…”

Structural and functional variations in skeletal-muscle and scallop muscle actins

“…The proposed involvement (De Couet, 1983;Hambly et al, 1986) of this variable extremity (1-12 sequence) of actins in the reported functional variation has not been confirmed Strzelecka-Golaszewska et al, 1985). This raises questions as to the location and the nature of the amino acid substitutions along the primary structure and the related consequences for the biological role of the isoactin expressed in a particular tissue.…”

“…Skeletal-muscle and cardiac striated-muscle actins are of the a-type, smooth-muscle actins of the a-and y-types and cytoplasmic actins of the fi-and y-types (Bulinski et al, 1983). Invertebrate actins, which have been less well analysed (De Couet, 1983), share homologous sequences with cytoplasmic actins (Vandekerckhove et al, 1983). For instance, scallop adductor-muscle actin has been characterized as a fl-like actin (Khaitlina, 1986), in contrast with its location in a striated muscle (Millman & Bennett, 1976).…”