Identification of polypeptides with selective affinity to intact mouse cerebellar granule neurons from a random peptide-presenting phage library.
Targeting of postmitotic neurons selectively for gene delivery poses a challenge. One way to achieve such a selective targeting is to link the gene delivery vector with small ligand-binding polypeptides which have selective affinity to intact neurons. In order to identify such novel neuron selective polypeptides, we screened a phage-display library displaying random 12-mer polypeptides and subtractively bio-panned for clones having selectivity towards cultured mouse cerebellar granule neurons. The selected phage clones were amplified and sequenced. Affinities of these clones to neurons were determined by the visible presence or absence of fluorescence of phage particles as detected by immunocytochemistry using an antibody to M-13 phage. This affinity was further qualified by how much phage was bound, and where in or on the cell it tended to accumulate. The selectivity of binding to neurons was determined by the negative binding of these clones to several cultured non-neuronal cells, including, primary glial cells, NT2 cells, human embryonic kidney 293 cells, neuroblastoma cells, and mouse 3T3 cells. Among the 46 clones that we have sequenced and characterized, four clones appeared to have excellent selectivity in binding to neurons. Homology comparison of these polypeptides revealed that three of them contained a consensus D(E)-W(F)-I(N)-D-W motif. This motif was also present in the Bdm1 gene product which was predominantly expressed in postnatal brains. Further characterizations of these polypeptides are required to reveal the utilities of these peptides to function as an effective linker to facilitate gene transfer selectively to neurons.
Hou
Sheng T
ST
Experimental Stroke Group, NRC Institute for Biological Sciences, National Research Council Canada, 1500 Montreal Road Labs, Bldg. M-54, Ottawa, Ont., Canada K1A 0R6. sheng.hou@nrc-cnrc.gc.ca
Dove
Mike
M
Anderson
Erica
E
Zhang
Jiangbing
J
MacKenzie
C Roger
CR
D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D001426:Bacterial Proteins / Q000378:metabolism; D017104:Bacteriophage M13 / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology*; D003001:Cloning, Molecular; D004277:DNA, Single-Stranded / Q000138:chemical synthesis / Q000737:chemistry; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D051379:Mice; D018716:Molecular Mimicry / Q000502:physiology; D008969:Molecular Sequence Data; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D019151:Peptide Library; D010455:Peptides / Q000302:isolation & purification / Q000494:pharmacology*; D011485:Protein Binding / Q000502:physiology; D014760:Viral Fusion Proteins / Q000235:genetics
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D019151:Peptide Library; D010455:Peptides; D014760:Viral Fusion Proteins