A novel epitope of entactin is present at the mammalian neuromuscular junction.
The extracellular matrix (ECM) at the neuromuscular junction (NMJ) is biochemically and functionally specialized, and bears molecules that can regulate both the formation and function of this peripheral synapse. We have previously purified one synaptic component of the muscle ECM--a unique laminin isoform named s-laminin--from a rat schwannoma cell line (Chiu et al., 1992). To develop new probes for the ECM, monoclonal antibodies were generated against other components produced by this cell line. One of these new antibodies, 9H6, binds selectively at the synaptic cleft of NMJs in adult rats, but not at extrasynaptic sites on the muscle surface. On Western blots, 9H6 recognizes a 150 kDa band that colocalizes, and copurifies with the laminin-binding, ECM glycoprotein entactin under both reducing and nonreducing conditions. N-terminal sequence analysis also indicates that the 9H6 antigen is related to entactin. However, polyclonal antibodies to entactin stain both synaptic and extrasynaptic sites. Thus, 9H6 appears to identify an entactin epitope with a very restricted distribution. Treatment with N-glycanase reduces the molecular mass of entactin and eliminates 9H6 binding, suggesting that the 9H6 epitope at synapses is dependent on glycosylation. Recent studies have shown that novel isoforms of laminin, collagen IV, agrin, and AChE are selectively sequestered at the NMJ. Our results indicate that the entactin present at the synaptic cleft also differs from entactin present outside the synapse. The synaptic form of entactin may contribute to the unique functions of the ECM at the neuromuscular synapse.
Chiu
A Y
AY
Division of Neurosciences, Beckman Research Institute of the City of Hope, Duarte, California 91010.
Ko
J
J
D000581:Amidohydrolases; D000818:Animals; D000906:Antibodies; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000032:analysis*; D005109:Extracellular Matrix / Q000737:chemistry / Q000648:ultrastructure; D007150:Immunohistochemistry; D007668:Kidney / Q000294:innervation; D008562:Membrane Glycoproteins / Q000032:analysis* / Q000276:immunology; D009442:Neurilemmoma; D009469:Neuromuscular Junction / Q000737:chemistry* / Q000648:ultrastructure*; D009928:Organ Specificity; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D010525:Peripheral Nerves / Q000737:chemistry / Q000166:cytology; D051381:Rats; D014407:Tumor Cells, Cultured
D000906:Antibodies; D000911:Antibodies, Monoclonal; D000939:Epitopes; D008562:Membrane Glycoproteins; C029967:nidogen; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase