Human laminin M chain: epitope analysis of its monoclonal antibodies by immunoscreening of cDNA clones and tissue expression.
We have produced four monoclonal antibodies (mAbs) against human placenta laminin purified by immunoaffinity chromatography. Three clones, 2D9, 3DM, and 4F1, recognized 320 kDa (M) chain of the laminin and the other one, 3DB, recognized B2 chain. One cDNA clone (HLM-1, 3.5 kb) was immunoscreened from human placenta cDNA library using 2D9, and three further overlapping cDNA clones (HLM-2, HLM-3, and HLM-4) covering 2.0 kb were isolated. Nucleotide sequencing and fusion protein analysis demonstrated that the amino acid sequence deduced from HLM-1 coincided with that of the G-domain of human merosin chain, and HLM-2, HLM-3, and HLM-4 encoded the long-arm and EGF-like domain of M chain. The binding regions of 2D9 and 3DM to M chain were defined as homologous repeating sequences of G2-G3 region of G-domain and the carboxy-terminal region of the long-arm, respectively. The extents of identity of amino acid sequences of the long-arm and EGF-like domains between human M chain and A chain were about 37% and 52%, respectively, which were lower than between mouse and human A chains. Northern blot analysis revealed that M chain mRNA, 8.6 kb, was highly expressed in heart and placenta, but less highly expressed in skeletal muscle, brain, and lung. Immunostaining showed selective distributions of M chain in nerve fibers in the dermis and mesangial matrix of the kidney, and B2 chain in subepidermal and kidney glomerular basement membranes.
Hori
H
H
Department of Tissue Physiology, Tokyo Medical and Dental University.
Kanamori
T
T
Mizuta
T
T
Yamaguchi
N
N
Liu
Y
Y
Nagai
Y
Y
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000276:immunology*; D001665:Binding Sites; D015152:Blotting, Northern; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D000939:Epitopes / Q000032:analysis*; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D007797:Laminin / Q000032:analysis / Q000235:genetics* / Q000276:immunology*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010920:Placenta / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D000939:Epitopes; D007797:Laminin; D046911:Macromolecular Substances