Posttranslational modifications and β/γ chain associations of human laminin α1 and laminin α5 chains:: purification of laminin-3 from placenta

Laminins assemble into trimers composed of alpha, beta, and gamma chains which posttranslationally are glycosylated and sometimes proteolytically cleaved. In the current paper we set out to characterize posttranslational modifications and the laminin isoforms formed by laminin alpha 1 and alpha 5 chains. Comparative pulse-chase experiments and deglycosylation studies in JAR cells established that the M-r 360,000 laminin al chain is glycosylated into a mature M-r 400,000 band while the M-r 370,000 laminin alpha 5 chain is glycosylated into a M-r 390,000 form that upon secretion is further processed into a M-r 380,000 form. Hence, despite the shorter peptide length of alpha 1 chain in comparison with the alpha 5 chain, secreted alpha 1 assumes a larger size in SDS-PAGE due to a higher degree of N-linked glycosylation and due to the lack of proteolytic processing. Immunoprecipitations and Western blotting of JAR laminins identified laminin alpha 1 and laminin alpha 5 chains in laminin-1 and laminin-10. In placenta laminin alpha 1 chain (M-r 400,000) and laminin alpha 5 chain (M-r 380,000/370,000 doublet) were found in laminin-1/-3 and laminin-10/-11. Immunohistochemically we could establish that the laminin alpha 1 chain in placenta is deposited in the developing villous and trophoblast basement membrane, also found to contain laminin beta 2 chains. Surprisingly, a fraction of the laminin alpha 1 chain from JAR cells and placenta could not be precipitated by antibodies to laminin beta 1-beta 3 chains, possibly pointing to an unexpected complexity in the chain composition of alpha 1-containing laminin isoforms. (C) 2000 Academic Press.