Structural analysis of a human glial variant laminin.
Astrocytes secrete laminin-like molecules in culture and may represent a major source of laminin in the developing central nervous system, yet these laminins have not been extensively characterized. We previously reported the presence of an astrocyte-derived variant laminin in media conditioned by human U251 MG astrocytoma cells. This laminin was partially purified in a highly anionic Mono Q fraction with strong adhesion activity for fibroblasts and glial cells (Aukhil et al. (1990) Matrix 10: 98-111). We now show that glial laminin could be dissociated from an anionic species, perhaps an approximately 400-kDa keratan sulfate proteoglycan present in the preparation, by a second round of Mono Q anion exchange chromatography in the presence of 6 M urea. Cell adhesion activity remained tightly associated with laminin-containing fractions, suggesting that glial laminin was responsible for the adhesion activity in the original preparation. Immunochemical and SDS-PAGE gel analyses of laminin heterotrimers demonstrated that glial laminin contained the beta 2 and gamma 1 chains in disulfide-bonded heterotrimeric complexes with a 360-kDa chain, a 320-kDa chain, or a postulated approximately 200-kDa chain. While these chains were not recognized by antibodies directed against the alpha 1-, alpha 2-, or alpha 3-related laminin chains, rotary shadowed glial laminin molecules appeared to contain alpha chains, as judged by the presence of an apparent G-domain terminating the long arm of each laminin molecule. These findings suggest that glial laminin contains one or more variant alpha chains, perhaps related to one of the more recently described alpha chains, alpha 3B, alpha 4, or alpha 5. Together our results implicate human U251 MG glial laminin as a previously uncharacterized laminin isoform with strong adhesive activity for fibroblasts and glial cells.
LeMosy
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Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA. ellen.lemosy@yale.edu
Lightner
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Erickson
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D000837:Anion Exchange Resins; D001253:Astrocytes / Q000737:chemistry* / Q000166:cytology; D001254:Astrocytoma; D002448:Cell Adhesion; D011508:Chondroitin Sulfate Proteoglycans; D002852:Chromatography, Ion Exchange / Q000379:methods; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D006801:Humans; D007632:Keratan Sulfate; D007797:Laminin / Q000737:chemistry* / Q000302:isolation & purification / Q000648:ultrastructure; D000071234:Lumican; D008970:Molecular Weight; D011487:Protein Conformation; D012117:Resins, Synthetic; D014407:Tumor Cells, Cultured
D000837:Anion Exchange Resins; D011508:Chondroitin Sulfate Proteoglycans; C000604306:LUM protein, human; D007797:Laminin; D000071234:Lumican; D012117:Resins, Synthetic; C042855:Mono Q; D007632:Keratan Sulfate