title: Targeting the active site of the placental isozyme of alkaline phosphatase by phage-displayed scFv antibodies selected by a specific uncompetitive inhibitor.
abstract: BACKGROUND
  The isozymes of alkaline phosphatase, the tissue non-specific, intestinal and placental, have similar properties and a high degree of identity. The placental isozyme (PLAP) is an oncofetal antigen expressed in several malignancies including choriocarcinoma, seminoma and ovarian carcinoma. We had earlier attempted to isolate PLAP-specific scFv from a synthetic human immunoglobulin library but were unable to do so, presumably because of the similarity between the isozymes. In this work, we have employed a PLAP-specific uncompetitive inhibitor, L-Phe-Gly-Gly, to select isozyme specific scFvs. An uncompetitive inhibitor binds to the enzyme in the presence of substrate and stabilizes the enzyme-substrate complex. Several uncompetitive inhibitors have varying degrees of isozyme specificity for human alkaline phosphatase isozymes. A specific uncompetitive inhibitor would be able to unmask conformational differences between the otherwise very similar molecules. Also, such inhibitors would be directed to regions at/close to the active site of the enzyme. In this work, the library was first incubated with PLAP and the bound clones then eluted by incubation with L-Phe-Gly-Gly along with the substrate, para-nitro phenyl phosphate (pNPP). The scFvs were then studied with regard to the biochemical modulation of their binding, isozyme specificity and effect on enzyme activity.
  
  
  RESULTS
  Of 13 clones studied initially, the binding of 9 was inhibited by L-Phe-Gly-Gly (with pNPP) and 2 clones were inhibited by pNPP alone. Two clones had absolute and 2 clones had partial specificity to PLAP. Two clones were cross-reactive with only one other isozyme. Three scFv clones, having an accessible His6-tag, were purified and studied for their modulation of enzyme activity. All the three scFvs inhibited PLAP activity with the kinetics of competitive inhibition. Cell ELISA could demonstrate binding of the specific scFvs to the cell surface expressed PLAP.
  
  
  CONCLUSIONS
  The results demonstrate the biochemical modulation of scFv binding. Also, the scFvs bound to the active site and denied the access to the substrate. The selection strategy could generate specific anti-enzyme antibodies to PLAP that can potentially be used for targeting, for modulating enzyme activity in in vitro and in vivo and as probes for the active site. This strategy also has a general application in selecting antibodies from combinatorial libraries to closely related molecules and conformations.
authors.affiliation: Department of Biochemistry, All India Institute of Medical Sciences, New Delhi-110029, India. deeptisaini@gmail.com
authors.forename: Deepti
authors.initials: D
authors.lastname: Saini
authors.forename: Mrinalini
authors.initials: M
authors.lastname: Kala
authors.forename: Vishal
authors.initials: V
authors.lastname: Jain
authors.forename: Subrata
authors.initials: S
authors.lastname: Sinha
mesh_terms: D000469:Alkaline Phosphatase / Q000037:antagonists & inhibitors / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001665:Binding Sites; D016503:Drug Delivery Systems / Q000379:methods*; D004791:Enzyme Inhibitors / Q000276:immunology; D018390:Gene Targeting / Q000379:methods; D007128:Immunoglobulin Fragments / Q000276:immunology*; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000276:immunology; D019151:Peptide Library; D010455:Peptides / Q000276:immunology; D010920:Placenta / Q000201:enzymology* / Q000276:immunology*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods
chemical_list: D000911:Antibodies, Monoclonal; D004791:Enzyme Inhibitors; D007128:Immunoglobulin Fragments; D007527:Isoenzymes; D019151:Peptide Library; D010455:Peptides; D000469:Alkaline Phosphatase