TY - JOUR
T1 - Structural analysis of a human glial variant laminin
AU - LeMosy, Ellen K.
AU - Lightner, Virginia A.
AU - Erickson, Harold P.
N1 - Funding Information:
We thank Robert Burgeson, Bruce Caterson, Eva Engvall, and Joshua Sanes for generous provision of antibodies and Henry Tan for excellent photography. This work was supported by NIH Grant CA 47056.
PY - 1996/8/25
Y1 - 1996/8/25
N2 - 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 ~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 β2 and γ1 chains in disulfide-bonded heterotrimeric complexes with a 360-kDa chain, a 320-kDa chain, or a postulated ~200-kDa chain. While these chains were not recognized by antibodies directed against the α1-, α2-, or α3-related laminin chains, rotary shadowed glial laminin molecules appeared to contain α 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 α chains, perhaps related to one of the more recently described α chains, α3B, α4, or α5. Together our results implicate human U251 MG glial laminin as a previously uncharacterized laminin isoform with strong adhesive activity for fibroblasts and glial cells.
AB - 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 ~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 β2 and γ1 chains in disulfide-bonded heterotrimeric complexes with a 360-kDa chain, a 320-kDa chain, or a postulated ~200-kDa chain. While these chains were not recognized by antibodies directed against the α1-, α2-, or α3-related laminin chains, rotary shadowed glial laminin molecules appeared to contain α 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 α chains, perhaps related to one of the more recently described α chains, α3B, α4, or α5. Together our results implicate human U251 MG glial laminin as a previously uncharacterized laminin isoform with strong adhesive activity for fibroblasts and glial cells.
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U2 - 10.1006/excr.1996.0252
DO - 10.1006/excr.1996.0252
M3 - Article
C2 - 8806454
AN - SCOPUS:0030601334
SN - 0014-4827
VL - 227
SP - 80
EP - 88
JO - Experimental Cell Research
JF - Experimental Cell Research
IS - 1
ER -