Kettin, a large modular protein in the Z-disc of insect muscles.

Abstract

Z‐discs of insect flight muscle contain a large protein of 500–700 kDa. Monoclonal antibodies label an epitope in the molecule at the Z‐disc in Drosophila and Lethocerus (waterbug). A partial cDNA of 1.6 kb from the Drosophila gene has been cloned and sequenced. The corresponding amino acid sequence has a modular structure composed of four conserved repeats of 95 amino acids homologous to immunoglobulin C2 domains (called class II domains in muscle proteins), separated by less conserved linker sequences of 35 amino acids. An expressed class II domain with flanking linker sequences binds to actin and alpha‐actinin but not to myosin. Single molecules of the protein would be large enough to span the Z‐disc. We suggest that the protein acts as scaffolding in the Z‐disc and we call the protein kettin. The Ca2+ activated protease, calpain, disrupts the Z‐disc of striated muscle, releasing alpha‐actinin intact. Calpain digests kettin to a series of peptides of between 30 and 170 kDa which are released from the myofibril. Digestion of kettin may cause disintegration of the Z‐disc and alpha‐actinin release which lead to disassembly of the myofibril.

Caenorhabditis elegansKettin, a Large Immunoglobulin-like Repeat Protein, Binds to Filamentous Actin and Provides Mechanical Stability to the Contractile Apparatuses in Body Wall Muscle

“…Localization of Ce-kettin on the thin filaments was proximal to the dense bodies and was similar to that of kettins from insects (Lakey et al, 1990(Lakey et al, , 1993van Straaten et al, 1999) and crayfish (Maki et al, 1995;Fukuzawa et al, 2001), which are localized to the side of the Z-discs. In addition, the N terminus of insect kettin is located within the Z-discs (van Straaten et al, 1999).…”

“…Previously, the full-length kettin has been demonstrated to bind to actin filaments with high affinity (Maki et al, 1995;van Straaten et al, 1999), and the stoichiometry has suggested that one Ig-repeat may bind one actin monomer in the filament (van Straaten et al, 1999). This was also supported by the report that a single Ig-repeat of kettin is capable of binding to actin filaments (Lakey et al, 1993). However, our results show that the four C-terminal Ig-repeats (KETN-CT1) bind to actin at lower stoichiometry than one repeat per one actin monomer, and, interestingly, that the adjacent non-Ig region augments both affinity and stoichiometry for binding to actin.…”

“…In vitro studies have shown that insect kettin binds to ␣-actinin, whereas it competes with tropomyosin for binding to actin (Lakey et al, 1993;van Straaten et al, 1999), but their functional relationship in vivo is not understood. We examined how Ce-kettin interacts with tropomyosin and ␣-acti- nin in the C. elegans body wall muscle.…”

“…Kettin directly binds to actin filaments with high-affinity (Lakey et al, 1993;Maki et al, 1995;van Straaten et al, 1999). Proteolytic removal of kettin by calpain from the Z-discs causes disintegration of the Z-discs (Lakey et al, 1993) and decrease in stiffness of the myofibrils (Kulke et al, 2001b).…”

“…Kettin directly binds to actin filaments with high-affinity (Lakey et al, 1993;Maki et al, 1995;van Straaten et al, 1999). Proteolytic removal of kettin by calpain from the Z-discs causes disintegration of the Z-discs (Lakey et al, 1993) and decrease in stiffness of the myofibrils (Kulke et al, 2001b). Kettin is one of the first proteins to colocalize with actin during the early stages of myofibrillogenesis (Ayme-Southgate et al, 2004), and genetic analysis has shown that it is essential for myofibril assembly in the fruit fly (Hakeda et al, 2000).…”