Identification and characterization of two huge protein components of the brush border cytoskeleton: evidence for a cellular isoform of titin.
Two extremely high molecular weight proteins were found to be components of the intestinal epithelial cell brush border cytoskeleton. The largest brush border protein, designated T-protein, migrated on SDS gels as a doublet of polypeptides with molecular weights similar to muscle titin T I and T II. The other large brush border protein, designated N-protein, was found to have a polypeptide molecular weight similar to muscle nebulin. In Western analysis, a polyclonal antibody raised against brush border T-protein reacted specifically with T-protein in isolated brush borders and cross-reacted with titin in pectoralis and cardiac muscle samples. T-protein was distinguished from the muscle titins by an anti-cardiac titin mAb. A polyclonal antibody raised against N-protein was specific for N-protein in brush borders and cross-reacted with nothing in pectoralis muscle. Immunolocalization in cryosections of intestinal epithelia and SDS-PAGE analysis of fractionated brush borders revealed that both T-protein and N-protein are concentrated distinctly in the brush border terminal web region subjacent to the microvilli, but absent from the microvilli. EM of rotary-replicated T-protein samples revealed many of the molecules to be long (912 +/- 40 nm) and fibrous with a globular head on one end. In some of the molecules, the head domain appeared to be extended in a fibrous conformation yielding T-protein up to 1,700-nm long. The brush border N-protein was found as long polymers with a repeating structural unit of approximately 450 nm. Our findings indicate that brush border T-protein is a cellular isoform of titin and suggest that both T-protein and N-protein play structural roles in the brush border terminal web.
Eilertsen
K J
KJ
Department of Biological Science, Florida State University, Tallahassee 32306-3050.
Keller
T C
TC
D000818:Animals; D000906:Antibodies; D002458:Cell Fractionation; D002645:Chickens; D064211:Connectin; D003599:Cytoskeleton / Q000737:chemistry / Q000648:ultrastructure*; D004591:Electrophoresis, Polyacrylamide Gel; D004848:Epithelium / Q000648:ultrastructure; D015151:Immunoblotting; D007422:Intestines / Q000737:chemistry / Q000648:ultrastructure; D008565:Membrane Proteins / Q000032:analysis* / Q000302:isolation & purification; D008854:Microscopy, Electron; D008871:Microvilli / Q000737:chemistry / Q000648:ultrastructure*; D008970:Molecular Weight; D009124:Muscle Proteins / Q000032:analysis* / Q000302:isolation & purification; D011494:Protein Kinases
D000906:Antibodies; D064211:Connectin; D008565:Membrane Proteins; D009124:Muscle Proteins; D011494:Protein Kinases