NEBULETTE - A 107 KD NEBULIN-LIKE PROTEIN IN CARDIAC-MUSCLE

A 107-kD protein has been identified in primary cultures of chicken em bryonic cardiomyocytes by immunoprecipitations with certain anti-nebul in monoclonal antibodies (mAbs). These mAbs, prepared against a fragme nt of human skeletal muscle nebulin located near the carboxyl terminus , detect a 107-kD protein in extracts of adult chicken heart, adult mo use heart, and adult rabbit heart by immunoblot analysis. A partial cD NA corresponding to this protein has been isolated by immunological sc reening of a chicken heart cDNA expression vector library. The partial cDNA encodes a 380-amino acid open reading frame composed entirely of nebulin-like 35-residue modules marked by the highly conserved sequen ce motifs: SXXXYK and TPD. The open reading frame exhibits 60-85% homo logy with skeletal muscle nebulins from a variety of species. This cDN A recognizes an similar to 8-kb transcript in cardiac RNA and does not hybridize to skeletal muscle RNAs by northern analysis. Immunofluores cence localization of this nebulin-like protein in primary cultures of chicken cardiomyocytes and embryonic chicken cardiac myofibrils indic ates that the protein is localized to the I-Z-I complex of the myofibr ils, extending approximately 25% of the thin filament length. Comparis ons of the distribution of this protein relative to actin, myosin, and titin in spreading cardiomyocytes suggest that the cardiac nebulin-li ke protein becomes aligned with the nascent myofibrils early during my ofibrillogenesis. To distinguish this petite nebulin-like protein from the 600-900 kD skeletal muscle nebulin, we have named it nebulette. ( C) 1995 Wiley-Liss, Inc.