Expression of recombinant human Met-ase-1: a NK cell-specific granzyme.
Human Met-ase-1 is a member of a family of cytotoxic lymphocyte serine proteases (granzymes), but is expressed specifically in CD3- large granular lymphocytes with natural killer cell activity. We have devised a polymerase chain reaction strategy to delete the predicted hexapropeptide of human Met-ase-1 (Ser-6 to Gln-1), to enable its expression and activation in mammalian COS cells. In addition, using peptide immunization we have derived a unique and specific monoclonal antibody detecting human Met-ase-1. Western blot analysis and protease assays of transfected COS cell lysates against a panel of thiobenzyl ester substrates formally demonstrated that the human Met-ase-1 gene encodes a serine proteinase that specifically hydrolyzes substrates containing a methionine (Met-) side chain at P1. The expression of active human Met-ase-1 and the generation of a specific anti-human Met-ase-1 monoclonal antibody will now enable a detailed structure/function analysis of key amino acids that confer this unusual serine protease specificity.
Smyth
M J
MJ
Cellular Cytotoxicity Laboratory, Austin Research Institute, Austin Hospital, Heidelberg, Victoria, Australia.
O'Connor
M D
MD
Kelly
J M
JM
Ganesvaran
P
P
Thia
K Y
KY
Trapani
J A
JA
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D017931:DNA Primers / Q000737:chemistry; D006801:Humans; D007694:Killer Cells, Natural / Q000201:enzymology*; D008715:Methionine; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins; D012697:Serine Endopeptidases / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D013379:Substrate Specificity
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D011994:Recombinant Proteins; D008715:Methionine; C084504:Hu-Met-1; D012697:Serine Endopeptidases