Construction of antibody mimics from a noncatalytic enzyme-detection of polysialic acid.

We have used a conceptually novel way to construct antibody mimics based on the binding of a noncatalytic enzyme to its substrate. Bacteriophage-derived endosialidase cleaves polysialic acid (polySia), an important oncofetal and bacterial antigen, which is poorly immunogenic. We fused to green fluorescent protein (GFP) a catalytically inactive endosialidase known to bind but not degrade polysialic acid. The fusion protein is a convenient single-step reagent in fluorescence microscopy, binding assays and immunoblots. It efficiently and specifically detected polysialic acid in developing brain, neuroblastoma cells and bacteria causing meningitis. Enzyme-substrate interactions represent an unexploited source of molecular recognition events. Some of these could be used in designing well-defined substitute antibodies for the study of target molecules which are difficult to purify, available in low quantities, are unstable or have poor immunogenity.

[1]  F. Lottspeich,et al.  Molecular cloning and functional expression of bacteriophage PK1E‐encoded endoneuraminidase Endo NE , 1995, Molecular microbiology.

[2]  K. Rajewsky,et al.  Inactivation of the N-CAM gene in mice results in size reduction of the olfactory bulb and deficits in spatial learning , 1994, Nature.

[3]  Peter W Taylor,et al.  Expression, mutagenesis and kinetic analysis of recombinant K1E endosialidase to define the site of proteolytic processing and requirements for catalysis , 2002, Molecular microbiology.

[4]  J. Pellequer,et al.  Affinity of monoclonal antibodies to large multivalent antigens: influence of steric hindrance on antibody affinity constants calculated from Scatchard plots. , 1993, Molecular immunology.

[5]  C. Cordova,et al.  TYPE-III HYPERUPOPROTEINÆMIA , 1977, The Lancet.

[6]  E. Vimr,et al.  Biosynthesis of the polysialic acid capsule inEscherichia coli K1 , 1995, Journal of Industrial Microbiology.

[7]  D. Figarella-Branger,et al.  A monoclonal antibody against Meningococcus group B polysaccharides used to immunocapture and quantify polysialylated NCAM in tissues and biological fluids. , 1995, Journal of immunological methods.

[8]  T. Grogan,et al.  Plasma cells in multiple myeloma express a natural killer cell- associated antigen: CD56 (NKH-1; Leu-19) , 1990 .

[9]  Introduction to Antibody Engineering and Phage Display , 2000, Vox Sanguinis.

[10]  J. Luzio,et al.  Complete nucleotide sequence of the gene encoding bacteriophage E endosialidase: implications for K1E endosialidase structure and function. , 1995, The Biochemical journal.

[11]  E. Vimr,et al.  Complete nucleotide sequence of the bacteriophage K1F tail gene encoding endo-N-acylneuraminidase (endo-N) and comparison to an endo-N homolog in bacteriophage PK1E , 1993, Journal of bacteriology.

[12]  C. Goridis,et al.  Occurrence of α2–8 linked polysialosyl units in a neural cell adhesion molecule , 1983 .

[13]  A. Goldman,et al.  Expression, purification and crystallization of a collagen-binding fragment of Yersinia adhesin YadA. , 2002, Acta crystallographica. Section D, Biological crystallography.

[14]  M. Miettinen,et al.  Neural cell adhesion molecule distribution in soft tissue tumors. , 1993, Human pathology.

[15]  D. Siegel.  Recombinant monoclonal antibody technology. , 2002, Transfusion clinique et biologique : journal de la Societe francaise de transfusion sanguine.

[16]  U. Rutishauser.  Polysialic acid at the cell surface: Biophysics in service of cell interactions and tissue plasticity , 1998, Journal of cellular biochemistry.

[17]  H. Rahmann,et al.  Polysialic acid on the neural cell adhesion molecule correlates with expression of polysialyltransferases and promotes neuroblastoma cell growth. , 1998, Cancer research.

[18]  Janet M. Thornton,et al.  The Catalytic Site Atlas: a resource of catalytic sites and residues identified in enzymes using structural data , 2004, Nucleic Acids Res..

[19]  L. Garcia-Segura,et al.  Gonadal hormone regulation of neuronal-glial interactions in the developing neuroendocrine hypothalamus , 1995, The Journal of Steroid Biochemistry and Molecular Biology.

[20]  R. Gerardy-Schahn,et al.  Polysialic Acid Directs Tumor Cell Growth by Controlling Heterophilic Neural Cell Adhesion Molecule Interactions , 2003, Molecular and Cellular Biology.

[21]  C. Figdor,et al.  Expression of neural cell adhesion molecule-related sialoglycoprotein in small cell lung cancer and neuroblastoma cell lines H69 and CHP-212. , 1990, Cancer research.

[22]  J. Shiloach,et al.  Identity between polysaccharide antigens of Moraxella nonliquefaciens, group B Neisseria meningitidis, and Escherichia coli K1 (non-O acetylated) , 1991, Infection and immunity.

[23]  T. Cheasty,et al.  Isolation of bacteriophages specific for the K1 polysaccharide antigen of Escherichia coli , 1977, Journal of clinical microbiology.

[24]  C. O’Sullivan.  Aptasensors – the future of biosensing? , 2002, Analytical and bioanalytical chemistry.

[25]  B. Barrell,et al.  Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence , 1998, Nature.

[26]  E. Hoiczyk,et al.  Structure and sequence analysis of Yersinia YadA and Moraxella UspAs reveal a novel class of adhesins , 2000, The EMBO journal.

[27]  C. Bult,et al.  Functional annotation of a full-length mouse cDNA collection , 2001, Nature.

[28]  D. Figarella-Branger,et al.  Correlation between polysialic-neural cell adhesion molecule levels in CSF and medulloblastoma outcomes. , 1996, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[29]  J. Nakayama,et al.  Developmental regulation of polysialic acid synthesis in mouse directed by two polysialyltransferases, PST and STX. , 1998, Glycobiology.

[30]  Valery A Petrenko,et al.  Phage display for detection of biological threat agents. , 2003, Journal of microbiological methods.

[31]  J. Finne,et al.  Differential activities of bacteriophage depolymerase on bacterial polysaccharide: binding is essential but degradation is inhibitory in phage infection of K1-defective Escherichia coli , 1992, Journal of bacteriology.

[32]  D. Bitter‐Suermann,et al.  Reexpression of poly(sialic acid) units of the neural cell adhesion molecule in Wilms tumor. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[33]  G. Rougon,et al.  Cell biology of polysialic acid , 1997, Current Opinion in Neurobiology.

[34]  M. Leinonen,et al.  ANTIGENIC SIMILARITIES BETWEEN BRAIN COMPONENTS AND BACTERIA CAUSING MENINGITIS Implications for Vaccine Development and Pathogenesis , 1983, The Lancet.

[35]  F. dall’Olio,et al.  Sialyltransferases in cancer , 2001, Glycoconjugate Journal.

[36]  J. Lindon,et al.  Production of colominic acid by Pasteurella haemolytica serotype A2 organisms , 1987 .

[37]  R. Gerardy-Schahn,et al.  Proteolytic Processing and Oligomerization of Bacteriophage-derived Endosialidases* , 2003, The Journal of Biological Chemistry.

[38]  H. Kalimo,et al.  Mutant bacteriophage with non-catalytic endosialidase binds to both bacterial and eukaryotic polysialic acid and can be used as probe for its detection , 2001, Glycoconjugate Journal.

[39]  K. Timmis,et al.  NZB mouse system for production of monoclonal antibodies to weak bacterial antigens: isolation of an IgG antibody to the polysaccharide capsules of Escherichia coli K1 and group B meningococci. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[40]  C. Sato,et al.  Characterization of the Antigenic Specificity of Four Different Anti-(α2→8-Linked Polysialic Acid) Antibodies Using Lipid-conjugated Oligo/Polysialic Acids (*) , 1995, The Journal of Biological Chemistry.

[41]  V. Petrenko,et al.  Phages from landscape libraries as substitute antibodies. , 2000, Protein engineering.

[42]  C. MacKenzie,et al.  Analysis by Surface Plasmon Resonance of the Influence of Valence on the Ligand Binding Affinity and Kinetics of an Anti-carbohydrate Antibody (*) , 1996, The Journal of Biological Chemistry.

[43]  N. Sharon,et al.  Lectins: Carbohydrate-Specific Proteins That Mediate Cellular Recognition. , 1998, Chemical reviews.

[44]  M. Valtonen,et al.  Serotypes, hemolysin production, and receptor recognition of Escherichia coli strains associated with neonatal sepsis and meningitis , 1985, Infection and immunity.

[45]  D. Kasper,et al.  Structure, conformation and immunology of sialic acid-containing polysaccharides of human pathogenic bacteria , 1984 .

[46]  M. Fukuda.  Possible roles of tumor-associated carbohydrate antigens. , 1996, Cancer research.

[47]  P. Talaga,et al.  High affinity binding of long-chain polysialic acid to antibody, and modulation by divalent cations and polyamines. , 2002, Molecular immunology.

[48]  M. Lipinski,et al.  Characterization of neural cell adhesion molecules (NCAM) expressed by ewing and neuroblastoma cell lines , 1987, International journal of cancer.