Posttranslational modifications and beta/gamma chain associations of human laminin alpha1 and laminin alpha5 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 alpha1 and alpha5 chains. Comparative pulse-chase experiments and deglycosylation studies in JAR cells established that the M(r) 360,000 laminin alpha1 chain is glycosylated into a mature M(r) 400,000 band while the M(r) 370,000 laminin alpha5 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 alpha1 chain in comparison with the alpha5 chain, secreted alpha1 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 alpha1 and laminin alpha5 chains in laminin-1 and laminin-10. In placenta laminin alpha1 chain (M(r) 400,000) and laminin alpha5 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 alpha1 chain in placenta is deposited in the developing villous and trophoblast basement membrane, also found to contain laminin beta2 chains. Surprisingly, a fraction of the laminin alpha1 chain from JAR cells and placenta could not be precipitated by antibodies to laminin beta1-beta3 chains, possibly pointing to an unexpected complexity in the chain composition of alpha1-containing laminin isoforms.
Champliaud
M F
MF
The Cutaneous Biology Research Center, Massachusetts General Hospital, Charlestown, Massachusetts, 02129, USA.
Virtanen
I
I
Tiger
C F
CF
Korhonen
M
M
Burgeson
R
R
Gullberg
D
D
D015153:Blotting, Western; D002822:Choriocarcinoma; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007536:Isomerism; D007797:Laminin / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D010920:Placenta / Q000737:chemistry*; D011233:Precipitin Tests; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D013462:Sulfur Radioisotopes; D014407:Tumor Cells, Cultured; D014594:Uterine Neoplasms
D007797:Laminin; D009842:Oligopeptides; D013462:Sulfur Radioisotopes; C119013:laminin 10; C097043:laminin alpha5; C067029:cystinyl-aspartyl-prolyl-glycyl-tyrosyl-isoleucyl-glycyl-seryl-arginyl-NH2; C084098:laminin A; C095567:laminin alpha 3