A NEWLY IDENTIFIED 105-KD LOWER LAMINA-LUCIDA AUTOANTIGEN IS AN ACIDIC PROTEIN DISTINCT FROM THE 105-KD GAMMA-2 CHAIN OF LAMININ-5

A 105-kD lower lamina lucida antigen (p105) has been detected by autoantibodies (anti-p105) from patients with a novel immunobullous disease, To distinguish p105 from other known lamina lucida components, we performed comparative immunoblotting on purified human amniotic laminin-5 (kalinin), 804G matrix (enriched in laminin-5), and keratinocyte and fibroblast proteins using anti-804G matrix antibody (J-18) and anti-p105, J-18 labeled the truncated laminin-5 gamma 2 chain in amniotic laminin-5, 8046 matrix, and keratinocyte conditioned medium, but did not label fibroblast cytosol, Conversely, anti-p105 did not label amniotic laminin-5 or 804G matrix, but did label p105 in both keratinocyte conditioned medium and fibroblast cytosol. J-18 labeled the 105-kD laminin-5 gamma 2 chain in reduced keratinocyte proteins and a 400-kD laminin-5 complex under non-reducing conditions, In contrast, anti-p105 labeled p105 under both reducing and non-reducing conditions but did not label a 400-kD protein complex, Similarly, comparative immunoblotting on keratinocyte proteins using anti-p105 and anti-laminin-1 revealed no commonly labeled protein bands. Electrophoretic fractionations by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting of these fractions revealed that the peak fractions of keratinocyte proteins reactive with anti-p105 are different from those reactive with J-18, Furthermore, keratinocyte proteins fractionated by Mono Q anion-exchange chromatography revealed fractions immunoreactive with anti-p105, whereas J-18 showed no reactivity with these fractions, Two-dimensional gel electrophoresis and immunoblotting with anti-p105 revealed p105 to be an acidic protein with isoelectric points between 5.7 and 6.3, distinct from the isoelectric points of laminin-5 gamma 2 chain. We conclude that p105 is an acidic protein located in the lamina lucida and distinct from the truncated laminin-5 gamma 2 chain and the laminin-1 family.