Association of laminin with heparan and chondroitin sulfate-bearing proteoglycans in neurite-promoting factor complexes from rat schwannoma cells

The present studies were undertaken to confirm the presence and identity of a putative proteoglycan associated with laminin in neurite-promoting factor complexes isolated from rat schwannoma cell conditioned medium. Sucrose density gradient centrifugation of the complex resolved two laminin-associated Na₂[³⁵S]O₄-labeled peaks which were termed Pools A and B. Both pools had nearly all their [³⁵S] cpms associated with glycosaminoglycan, contained heparan sulfate-proteoglycan core protein antigen and displayed a similarly high neurite promoting potency relative to their laminin contents. However, Pool A contained about twice as many [³⁵S] cpms and twice as much proteoglycan core protein per laminin than Pool B. Seventy percent of Pool A cpms was associated with heparan sulfate and 30% with chondroitin sulfate whereas the inverse was true for Pool B. Treatment with heparitinase and/or chondroitinase ABC caused laminin in either pool to elute at lower salt concentrations from DEAE cellulose. In SDS-PAGE the [³⁵S] cpms of both pools ran with the same mobility as laminin but could be separated from laminin under reducing conditions. The Pool A cpms remained at 900 KD and the Pool B cpms spread over the 200–900 KD range. By rotary shadowing electron microscopy, Pool B fractions contained primarily cross-shaped laminin images, often associated with proteoglycan-like images. Pool A fractions contained i) dense, aggregated images including intact laminin from which emanated proteoglycan-like strands, ii) circular images bearing globular domains and less commonly, iii) distorted cross-shaped laminin-like images. These studies support the existence of at least two forms of laminin-proteoglycan complexes which differ in biochemical, immunochemical and ultrastructural characteristics.Abbreviations HSPG heparan sulfate proteoglycan - ELISA enzyme-linked immunoassay - Pool A Fractions 10–13 in sucrose gradient (Figure 1, lower panel) - Pool B Fractions 14–16 in sucrose gradient (Figure 1, lower panel) - HDPG High density proteoglycan, Fractions 1–3 in cesium chloride gradient, Figure 1, middle panel - CPC cetylpyridinium chloride - GAG glycosaminoglycanSpecial issue dedicated to Dr. E. M. Shooter and Dr. S. Varon.