Molecular characterization of a heparan sulfate proteoglycan that interacts with the neural cell adhesion molecule (NCAM)

Development of the nervous system during embryogenesis consists of a complex series of elaborately regulated events, including cell proliferation, migration, differentiation, neurite extension, and synaptogenesis. This regulation largely depends on complex interaction of cells with their environment and neighboring cells. Molecules involved in these processes include proteins and proteoglycans, both on the cell surface and in the extracellular matrix (ECM).;Previous studies in our laboratory have identified a heparan sulfate proteoglycan (HSPG) that interacts with the neural cell adhesion molecule (NCAM) in embryonic chick retinal cells. A high molecular weight HSPG was later coimmunoprecipitated with NCAM from chick brain extracts. Independently, the same HSPG was also identified in immunostaining studies searching for proteoglycans in the chick nervous system with possible functions of regulating developing axonal pathfinding. This HSPG is not related to other identified HSPGs, as suggested by immunological studies.;A random-primed E9 chick brain cDNA expression library was screened using two monoclonal antibodies and a polyclonal antiserum, all of which were generated against the core protein of this HSPG. 10 cDNA clones were isolated, and sequencing analysis and immunoreactivity studies revealed that they were identical to the ECM protein agrin. Immunocytochemistry located the HSPG to the synaptic site of the neuromuscular junction. In addition, the localization of this agrin HSPG mRNA expression in CNS was demonstrated by in situ hybridization. Since previous studies from our laboratory showed that this HSPG interacts with NCAM, this finding suggests that the interaction between agrin and NCAM might be involved in a variety of processes associated wi h neural development, including cell adhesion and synaptogenesis.;A novel alternative splicing site of agrin mRNA was identified based on the isolation of an agrin cDNA clone from the same E9 chick brain cDNA library. This site, named site C, contains a 21 bp fragment in the amino-terminal encoding region of agrin mRNA. RT-PCR (reverse transcription polymerase chain reaction) analysis confirmed the existence of this splicing site. In brain the agrin isoform containing the 21 bp insert is the predominant species, but the isoform without the insert is prevalent in non-neuronal cell populations. The splicing of site C is developmentally regulated. In brain agrin isoform with the splicing site is down-regulated, while the isoform without the insert is up-regulated, consistent with the increase in glial number during brain development. Both isoforms are down-regulated in heart during development. Although the splicing of site C appears to be restricted to neuronal cells, its function has yet to be determined.;A human agrin cDNA was also isolated by screening a human brain cDNA library. This cDNA is 1,310 bp long with one open reading frame. Its nucleotide and predicted amino acid sequence are highly homologous to both rat and chick agrin. The isolation of this human agrin cDNA helps to obtain the complete cDNA sequence of human agrin, and the generation of a polyclonal antiserum to its fusion protein will facilitate the studies of agrin functions in human. (Abstract shortened by UMI.).