Antibodies against active-site peptides common to glucosyltransferases of mutans streptococci.
Polyclonal antibodies were raised against peptides derived from an active-site sequence common to the family of mutans streptococcal glucosyltransferases (GTFs). The sequence contains an aspartic acid residue that functions in formation of the enzyme transition state in catalysis. Two GTFs were targeted with similar but not identical sequences in this region: one that synthesizes an alpha-1,3-linked water-insoluble glucan and a homologous GTF that synthesizes an alpha-1,6-linked water-soluble glucan. For each enzyme, an 8-mer and 22-mer peptide were prepared. The two peptide lengths were chosen in order to increase the likelihood of the peptides folding in a conformation similar to that of the native enzyme. Each peptide immunogen produced high titers of antibody in rabbits, and all antisera cross-reacted with all peptides, albeit to various degrees. Native enzyme showed weak interaction with antisera, which, on the basis of enzyme denaturation experiments, likely reflects binding to a small but finite population of denatured enzyme in the sample. GTF was assayed for inhibition in the presence of protein A-purified immunoglobulin G from each antiserum. Given the mass of the antibody and catalytic importance of the peptide, any enzyme-antibody complex formation would result in enzyme inhibition. No significant inhibition was observed, which demonstrates that either polyclonal antibodies raised against each of the four peptides cannot access this active-site region, or antibodies do not recognize the native enzyme conformation. The advantages and challenges of generating antibodies against enzyme active-site peptides are discussed in the context of the crystal structure of Aspergillus oryzae alpha-amylase, which has a homologous peptide segment which serves the same catalytic function.
Cope
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Department of Basic Sciences, School of Dentistry, University of Southern California, Los Angeles 90089-0641.
Mooser
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D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology*; D001665:Binding Sites; D003429:Cross Reactions; D005964:Glucosyltransferases / Q000276:immunology*; D007106:Immune Sera / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000276:immunology*; D011817:Rabbits; D013291:Streptococcus / Q000201:enzymology*
D000907:Antibodies, Bacterial; D007106:Immune Sera; D007074:Immunoglobulin G; D010446:Peptide Fragments; D005964:Glucosyltransferases