title: Expression, purification and fluorine-18 radiolabeling of recombinant S100A4: a potential probe for molecular imaging of receptor for advanced glycation endproducts in vivo?
abstract: Data concerning the pathophysiological role of extracellular S100A4, a member of the multigenic family of Ca(2+)-modulated S100 proteins, and its interaction with the receptor for advanced glycation endproducts (RAGE) or other putative receptors in tumorigenesis, metastasis, and inflammatory processes in vivo are scarce. One reason is the shortage of suitable radiotracer methods. We report a novel methodology using recombinant human S100A4 as potential probe for molecular imaging and functional characterization of this interaction. Therefore, human S100A4 was cloned as GST fusion protein in the bacterial expression vector pGEX-6P-1 and expressed in E. coli strain BL21. Purified recombinant human S100A4 was radiolabeled with the positron emitter fluorine-18 ((18)F) by conjugation with N-succinimidyl-4-[(18)F]fluorobenzoate ([(18)F]SFB). The radioligand [(18)F]fluorobenzoyl-S100A4 ((18)F-S100A4) was used in cell binding experiments in RAGE-bearing human melanoma cells and endothelial cells in vitro, and in both biodistribution experiments and small animal positron emission tomography (PET) studies in normal rats in vivo. The cellular association and tissue-specific distribution of (18)F-S100A4 in vitro and in vivo correlated well with the protein expression and anatomical localization of RAGE, e.g., in the vascular system and in lung. Compared to other S100 RAGE radioligands, the overall findings of this study indicate that extracellular S100A4 in vivo shows only a moderate interaction with RAGE and, furthermore, exhibits a substantially faster metabolic degradation. On the other hand, the approach allows the use of quantitative small animal PET and provides a novel probe to both delineate functional expression and differentiate multiligand interaction of RAGE under normal and pathophysiological conditions in rodent models of disease.
authors.affiliation: Department of Radiopharmaceutical Biology, Institute of Radiopharmacy, Research Center Dresden-Rossendorf, POB 51 01 19, 01314, Dresden, Germany.
authors.forename: Susann
authors.initials: S
authors.lastname: Wolf
authors.forename: Cathleen
authors.initials: C
authors.lastname: Haase-Kohn
authors.forename: Jens
authors.initials: J
authors.lastname: Lenk
authors.forename: Susan
authors.initials: S
authors.lastname: Hoppmann
authors.forename: Ralf
authors.initials: R
authors.lastname: Bergmann
authors.forename: Joerg
authors.initials: J
authors.lastname: Steinbach
authors.forename: Jens
authors.initials: J
authors.lastname: Pietzsch
mesh_terms: D000818:Animals; D001565:Benzoates / Q000737:chemistry; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D005462:Fluorine Radioisotopes / Q000737:chemistry; D017127:Glycation End Products, Advanced; D006801:Humans; D008297:Male; D008545:Melanoma / Q000378:metabolism; D049268:Positron-Emission Tomography / Q000379:methods; D051381:Rats; D017208:Rats, Wistar; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic / Q000032:analysis* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D013388:Succinimides / Q000737:chemistry
chemical_list: D001565:Benzoates; D005462:Fluorine Radioisotopes; D017127:Glycation End Products, Advanced; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic; D011993:Recombinant Fusion Proteins; D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins; D013388:Succinimides; C075713:N-succinimidyl-4-fluorobenzoate; C077591:S100A4 protein, human