Purification and Lectin‐Binding Properties of s‐Laminin, A Synaptic Isoform of the Laminin B1 Chain

The extracellular matrix (ECM) at the vertebrate neuromuscular junction is a repository of functionally important molecules, some of which can regulate the formation of synapses during regeneration. One candidate molecule is s‐laminin, a 185‐kDa homologue of the laminin B1 chain. Whereas several members of the laminin family are present throughout the ECM ensheathing muscle fibers, immunoreactivity for s‐laminin is found selectively at synaptic sites in adult and embryonic rats, and is detectable at a time when synaptogenesis is taking place during development. We have reported previously that a rat schwannoma cell line, D6P2T, produces and releases large amounts of s‐laminin in culture. We have now purified s‐laminin from medium conditioned by these cells by using a simple three‐step procedure. Serum‐free, conditioned medium is separated by ion‐exchange chromatography on DEAE‐Sephacel, followed by size‐exclusion chromatography on 500 HR‐Seph‐acryl. Finally, s‐laminin is dissociated from other ECM components by agarose gel electrophoresis under reducing conditions and recovered in solution by extracting slices of agarose gel. The purified preparation displays one silver‐stained band that is recognized by three monoclonal antibodies known to bind to different epitopes on s‐laminin. Lectin‐binding studies demonstrate that s‐laminin is a glycoprotein and bears many of the carbohydrate moieties present on the B1 and B2 chains of laminin. Thus, the three 185–220‐kDa members of the laminin family are related in both their protein and carbohydrate domains.