An amino acid switch (Gly281-->Arg) within the "hinge" region of the tryptophan synthase beta subunit creates a novel cleavage site for the OmpT protease and selectively diminishes affinity toward a specific monoclonal antibody.
The in vitro susceptibility to endogenous proteases of the beta subunit of Escherichia coli tryptophan synthase was studied immunochemically. Whereas the wild-type beta subunit was apparently very stable, the missense mutant beta(B8), carrying an amino acid switch from Gly to Arg at residue 281, underwent specific proteolytic cleavage. Polyclonal chicken antibodies and monoclonal antibodies specific for the N terminus (monoclonal antibody (mAb) 15-1), the C terminus (mAb 93-6), and the "hinge" region (mAb 164-2) were used to study the hydrolysis of the beta(B8) polypeptide. Cleavage products of 30 kDa, from the N terminus, and 13 kDa, from the C terminus, were observed. These two polypeptides correspond to the well characterized F1 (N-terminal) and F2 (C-terminal) fragments that are generated during the limited tryptic proteolysis of the wild-type beta subunit. The outer membrane-associated protease OmpT was shown to be responsible for the cleavage of the beta(B8) mutant protein. Proteolytic cleavage, observed only under neutral non-denaturing conditions, was specific for the peptide bond between Arg281 and Met282. The Arg-Met peptide bond has not previously been reported to be susceptible to cleavage by the OmpT protease. The beta(B8) polypeptide had dramatically reduced affinity for mAb 164-2. This antibody interacted more strongly with the OmpT-generated F1-like fragment than with the intact beta(B8) protein. These results strongly suggest that the G281R mutation alters the conformation of the hinge region of the mutant beta subunit, particularly the beta-turn around Gly281. The implications with respect to the epitope recognized by mAb 164-2 are discussed.
Zhao
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GP
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907.
Somerville
R L
RL
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000915:Antibody Affinity; D001120:Arginine / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology; D005998:Glycine / Q000737:chemistry*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D011487:Protein Conformation; D012697:Serine Endopeptidases / Q000378:metabolism*; D014367:Tryptophan Synthase / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D001120:Arginine; D012697:Serine Endopeptidases; C057840:omptin outer membrane protease; D014367:Tryptophan Synthase; D005998:Glycine