Article Figures & Data
Figures
- Fig. 1.
A, construction of geneVIII-HBDt fusion insertion into M13 phage genome. The DNA sequence of the VEGF165 HBDt (Cys137 to Cys160 (1) ) was generated from synthetic oligonucleotides representing the 5′- and 3′-sequences with an overlap in the middle and adapter sequences containing NheI and HindIII restriction sites (underlined) at the respective ends. The oligonucleotides were hybridized and the sequence was extended using PCR technology. The restriction-digested HBDt insert and the CIAP-treated vector were gel-purified, ligated, and transformed into Escherichia coli XL1-blue. M13 phage plaque assay was performed in the presence of IPTG and X-Gal. Replicative form DNA was prepared from colorless plaques, and the desired DNA constructs were identified by PCR amplification, restriction digest analysis, and DNA sequencing. B, predicted structure of the HBDt subdomain. Using the structure solution for the complete HBD domain (2) , the truncated form was subjected to computational prediction by molecular dynamics using Discover and energy minimization in InsightII.
- Fig. 2.
HBDt.Phage Binds to Heparin Matrix. A, equal quantities of HBDt.Phage (VhbdCSC), wild-type (insertless) phage, or irrelevant control (AL4-15) M13 phage were each mixed separately with heparin matrix beads. Two additional mutant forms of HBDt.Phage were created and analyzed. One has a transversion of T to G at codon Cys24 to encode Gly in place of Cys (VhbdCSG). The second has the additional change of G to A at codon 8 to encode Asp in place of Ser8 (VhbdCNG). The binding of the correct HBDt.Phage VhbdCSC to heparin beads was significantly greater (>37-fold) than the control wild-type insertless phage or the irrelevant AL4-15 phage. However, when Cys24 was mutated to Gly in VhbdCSG and this as well as the Ser8→Asp mutation in VhbdCNG, heparin binding decreased to only ∼10-fold greater than control phage. Results are from three independent experiments (mean ± SE). Differences between experimental groups were significant by nonparametric one-way ANOVA, Kruskal-Wallis statistics (P < 0.02), and by Dunn’s Multiple Comparison post tests. B, the graph illustrates analysis of competitive binding of HBDt VhbdCSG phage to heparin beads. HBDt VhbdCSG phage was mixed with wild-type phage at 1:1 ratio, and the quantity of phage bound to the heparin beads was assayed. In these experiments, the wild-type phage was quantitated by X-Gal substrate blue conversion. The HBDt.Phage was not competed by irrelevant wild-type phage and composed of 77% of total phage recovered from the heparin matrix.
- Fig. 3.
Binding of HBDt.Phage to cellular GAGs. CHO-k1 cells normally expressing surface GAGs (A and C), and CHO-GAG-deficient cells pgsA-745 (B and D) adherent to plastic culture plates were briefly fixed with paraformaldehyde, washed, and overlaid with equal amounts of HBDt.Phage (A and B) and control DB3.Phage (C and D). After 60 min, the plates were thoroughly washed with saline and cell-bound phage was visualized with antiphage antibody followed by FITC-conjugated antirabbit IgG antibody (green). The signal visualized at 488 nm in the confocal microscope was superimposed over the DIC image of the cells (gray).
- Fig. 4.
HBDt.Phage selectively accumulates in CT26 and MATB-III tumors. HBDt.Phage was infused in the tail vein of BALB/c mice (A) bearing CT26 tumors and Fischer 344 rats (C) with MATB-III tumors. The animals were sacrificed 30 or 60 min later by perfusion with saline at arterial pressure with saline. The organs and tumors were harvested, weighed, and assayed for phage content. The graph shows data at 30 and 60 min. No phage was detected after 3-h circulation (data not shown). Each column represents the mean of three different experiments (mean ± SE). The differences among the experimental groups were found statistically significant using nonparametric one-way ANOVA, Kruskal-Wallis statistics (P < 0.01), with Dunn’s Multiple Comparison as post tests. The phage titer in the blood was also assayed after 30 and 60 min in mouse (B) and rat (D). Also, HBDt.Phage was mixed with the wild-type counterpart at 1:1 ratio and infused in Fischer 344 rats carrying MATB-III tumors and left to circulate for 60 min, after which the animals were rapidly perfused with saline at arterial pressure and sacrificed. The graph illustrates the ratio MAT HBDt.Phage to the wild-type insertless phage in different organs. Differentiation between the HBDt.Phage and wild-type phage was based on white versus blue plaque generation, respectively. Each column represents the mean and SE of three independent experiments. The differences between groups were significant by nonparametric one-way ANOVA, Kruskal-Wallis statistics (P < 0.05), with Dunn’s Multiple Comparison post tests.
- Fig. 5.
Localization of the HBDt.Phage in vivo. Frozen sections from each of the organs assayed for phage by plaque titer (Fig. 4C) <$REFLINK> were fixed in acetone and incubated with rabbit antiphage IgG followed by a FITC-conjugated antirabbit IgG antibody and also with biotin-conjugated mouse antirat anti-CD31 monoclonal antibody followed by Streptavidin-conjugated Texas Red. The phage-positive signals (FITC) and CD31-positive ECs (Texas Red) were visualized under 488 and 568 nm light by confocal microscopy (power ×63 oil lens, scale shown in μm). A, C, E, and G show localization of HBDt.Phage (green) with respect to the CD31-positive ECs (red) in the MATB-III tumor, brain, kidney glomerulus, and a renal arterial vessel, respectively. B, D, F, and H show the H&E stain for adjacent serial sections
Tables
- Table 1
Conventional heparin-binding domainsa
Molecule Group HBD sequence Motif aFGF (17) GFb PLGNYKKPKLLYCSNG XBBXBX bFGF (22) GF GHFKDPKRLYCKNGGF ?(XBBXXXBX) β-TG (23) GF PDAPRIKKIVQKKLAG XBXBBX FGFR-1 (23) GF receptor AAPVAHLKKEMKN ?(XBBXXBX) Noggin (9) TGF-β binding protein AQGKKQRLSKKLRRKLQM XBBXBX, ?(B)xN IGFBP-3 (23) GF binding protein GFYKKKQCRPSKG XBBBXXBX Apo B (23) Human plasma LDL FIIPSPKRPVKLLSG ?(XBBXXBX) Apo B (17) Human plasma LDL ALLKKTKNSEEF XBBXBX Apo E (23) Human plasma LDL SHLRKLRKRLLRDA XBBBXXBX Apo E (17) Human plasma LDL AWGERLRARMEEMGSRTRDRL ?(XBX)xN TFPI (14) Tissue factor pathway inhibitor GGLIKTKRKRKKQRVKIAY ?(B)xN PVC-21 MuLV (12) Murine leukemia virus PGSHRPRKAKSC XBBXBX PRV-Be (11) Pseudorabies virus SRRKPPRN…YRRGRF XBBBXXBX, XBBXBX α4N(V)-NTD (24) Adhesion molecule RRRKGKGKKKDRGRKGKG (XBBXB)x3, ?(B)xN α4 Type V collagen (24) Collagen PRRRKGKGKKKDRGRKGKGRKKNKT XXBBXBX, ?(B)xN Laminin α1 (25) Adhesion molecule DVRKRLQVQLSIRT ?(XBBBX) Annexin II tetramer, p36 (23) Adhesion molecule LKIRSEFKKKYGKSLYY XBBBXXBX Pro-islet amyloid polypeptide (16) Regulatory peptide TPIESHQVEKRKCNTATC ?(XBBBX) Heparin affin regulatory peptide (HARP) (15) Regulatory peptide GKLTKPKQAESKKKKKE ?(B)xN -
a HBDs have been designated by common motifs XBBXBX or XBBBXXBX (17). The ? indicates suggested potential motifs.
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b GF, growth factor; TGF, transforming growth factor.
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Volume 62, Issue 23
