FIGURE 3.

Identification of hepsin-cleaved product as laminin-332 β3 chain. A, Western blot was performed on Ln-332 alone (0.1 μm) and in combination with hepsin (0.7 μm) and probed with a polyclonal antibody specific for the β3 chain. Application of β3 antibody identified the ∼100-kDa cleaved product of Ln-332 β3 chain and the uncleaved β3 chain. B, trypsin digestion of the ∼100 kDa band was performed for 2 h at 37°C before subjection to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The mass spectral data were used to examine protein data bases to generate statistically significant candidate identifications using GPS Explorer software running the Mascot data base search algorithm. Seventeen individual peptides (highlighted in gray) were identified from the digestion, which directly aligned with the sequence of rat Ln-332 β3 chain. N-terminal sequencing was also applied to the digested band, which generated the sequence LQGSCF (underlined). C, schematic representation of Ln-332 including the specific hepsin cleavage site determined to be between the Arg²⁴⁵-Leu²⁴⁶ residues of the rat Ln-332 β3 chain. Note that cysteine residues (underlined) are not detected by Edman sequencing and were deduced from cDNA. The three chains (α3, β3, and γ2), including domains I–VI and LG domains of Ln-332, are also indicated. One-letter amino acid codes are shown.