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.