Biochemical properties of the matrix metalloproteinase NtMMP1 from Nicotiana tabacum cv. BY-2 suspension cells.
A zinc-dependent matrix metalloproteinase (NtMMP1) found in the plasma membrane of Nicotiana tabacum cv. Bright Yellow 2 (BY-2) suspension cells is thought to be responsible for the degradation of recombinant proteins secreted into the culture supernatant. We have characterized the proteolytic activity of NtMMP1 by expressing a recombinant derivative lacking the C-terminal transmembrane domain in yeast. After purifying the protein by affinity chromatography, its autocatalytic activity was analyzed using monoclonal antibodies raised against its N-terminal and C-terminal portions. Both the unprocessed and processed forms of NtMMP1 displayed caseinolytic activity and N-terminal sequencing identified an autocatalytic cleavage site within the sequence motif HFSFFP, which is similar to the corresponding sequences of the human matrix metalloproteinases stromelysin-1 (MMP-3) and stromelysin-2 (MMP-10). Unlike all other matrix metalloproteinases investigated so far, NtMMP1 contains a disulfide bond within its propeptide thus rendering the proenzyme catalytically active. Kinetic analysis of NtMMP1 with a synthetic substrate revealed a K(m) of 10.55 +/- 0.9 microM, a k(cat) of 0.6 +/- 0.01 s(-1) and maximum activity at pH 7.5. We found that NtMMP1 degrades Desmodus rotundus salivary plasminogen activator alpha 1 (DSPAalpha1), a biopharmaceutical protein, that has proven difficult to produce in tobacco BY-2 cells. This provides a likely explanation for the frequent instability of secreted recombinant biopharmaceuticals produced in plant suspension cell cultures. Our data suggest new avenues that can be explored to improve the production of pharmaceutical proteins in plants and plant cells.
Mandal
Manoj K
MK
Institute for Molecular Biotechnology, RWTH Aachen University, Aachen, Germany.
Fischer
Rainer
R
Schillberg
Stefan
S
Schiermeyer
Andreas
A
D000911:Antibodies, Monoclonal; D015151:Immunoblotting; D020782:Matrix Metalloproteinases / Q000378:metabolism*; D010940:Plant Proteins / Q000378:metabolism*; D014026:Nicotiana / Q000166:cytology* / Q000201:enzymology*
D000911:Antibodies, Monoclonal; D010940:Plant Proteins; D020782:Matrix Metalloproteinases