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Characerization of a novel pore‐forming protein in macrophages
Author(s) -
Armas Lesley R,
Ramos Jahir,
Lyapichev Kirill,
Shiratsuchi Motoaki,
Deyev Vadim,
Podack Eckhard
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.672.30
Subject(s) - perforin , cytoplasm , trypsin , chemistry , cleavage (geology) , transmembrane protein , transfection , serine protease , microbiology and biotechnology , proteases , lysis , biochemistry , biophysics , protease , biology , gene , in vitro , enzyme , cytotoxic t cell , paleontology , receptor , fracture (geology)
Perforin 2 (P2) is the predicted protein of Macrophage specific gene 1 (Mpg‐1) expressed in differentiated macrophages. The function of Perforin 2 is not known. When P2 is overexpressed in 293 cells it forms polymeric pore complexes similar to poly C9 and poly Perforin1 in membranes that are observed by negative staining and electron microscopy. The pore structure of poly P2 suggests cytolytic function similar to Perforin1 and C9. P2 has a predicted transmembrane domain followed by a short cytoplasmic domain containing a cluster of lysine residues followed by a conserved tyrosine and serine residue, suggesting functional roles in P2 polymerization and expression of cytolytic activity. In this study, we isolated enriched subcellular fractions containing P2 from 293 cells transfected with eGFP tagged P2. The fractions were digested with TPCK‐treated trypsin for various amounts of time to determine P2 susceptibility to proteolytic cleavage. We found that with increasing exposure to trypsin, P2 was cleaved into 2 pieces whose molecular weights by SDS‐PAGE corresponded to cleavage at the cluster of lysines in the cytoplasmic domain of P2. The electronmicroscopic pore structure of poly P2 however remained intact indicating protease resistance of the pore complex. The proteolytic cleavage of P2 is likely important for P2 function in macrophages. Supported by NIH CA039201